Coordinate up-regulation of low-density lipoprotein receptor and cyclo-oxygenase-2 gene expression in human colorectal cells and in colorectal adenocarcinoma biopsies

NASA Technical Reports Server (NTRS)

Lum, D. F.; McQuaid, K. R.; Gilbertson, V. L.; Hughes-Fulford, M.

1999-01-01

Many colorectal cancers have high levels of cyclo-oxygenase 2 (COX-2), an enzyme that metabolizes the essential fatty acids into prostaglandins. Since the low-density lipoprotein receptor (LDLr) is involved in the uptake of essential fatty acids, we studied the effect of LDL on growth and gene regulation in colorectal cancer cells. DiFi cells grown in lipoprotein-deficient sera (LPDS) grew more slowly than cells with LDL. LDLr antibody caused significant inhibition of tumor cell growth but did not affect controls. In addition, LDL uptake did not change in the presence of excess LDL, suggesting that ldlr mRNA lacks normal feedback regulation in some colorectal cancers. Analysis of the ldlr mRNA showed that excess LDL in the medium did not cause down-regulation of the message even after 24 hr. The second portion of the study examined the mRNA expression of ldlr and its co-regulation with cox-2 in normal and tumor specimens from patients with colorectal adenocarcinomas. The ratio of tumor:paired normal mucosa of mRNA expression of ldlr and of cox-2 was measured in specimens taken during colonoscopy. ldlr and cox-2 transcripts were apparent in 11 of 11 carcinomas. There was significant coordinate up-regulation both of ldlr and of cox-2 in 6 of 11 (55%) tumors compared with normal colonic mucosa. There was no up-regulation of cox-2 without concomitant up-regulation of ldlr. These data suggest that the LDLr is abnormally regulated in some colorectal tumors and may play a role in the up-regulation of cox-2. Copyright 1999 Wiley-Liss, Inc.

cDNA cloning and expression analysis of a phosphopyruvate hydratase gene from the chinese shrimp Fenneropenaeus chinensis.

PubMed

Li, Xupeng; Meng, Xianhong; Luo, Kun; Luan, Sheng; Cao, Baoxiang; Kong, Jie

2017-04-01

In the present study a cDNA encoding a phosphopyruvate hydratase (enolase) was cloned from the muscle of the Chinese shrimp (Fenneropenaeus chinensis) and named as FcEnolase. The cDNA of FcEnolase encoded a protein of 434 amino acid residues with a molecular mass 47.22 kDa. The residues 342-355 constituted the signature motif "LLLKVNQIGSVTES". A SNP locus (C96T) in the ORF at 96 bp was identified. The results showed that the FcEnolase was a conserved gene. In the normal F. chinensis, the mRNA level in the muscle was much higher (P < 0.05) than the mRNA level in the gill and hepatopancreas. To verify the mRNA level of FcEnolase in the F. chinensis post WSSV infection, a real-time RT-PCR was performed. In the WSSV-infected F. chinensis, the FcEnolase mRNA level was significantly (P < 0.05) up-regulated in the muscle at 12 and 24 h post challenge (hpc) to approximately 2.7-fold and 2.7-fold the mRNA level in the controls, respectively. The FcEnolase mRNA level in the gill was significantly (P < 0.05) down-regulated at 6 hpc to approximately 0.3-fold the mRNA level in the control, followed by a significant (P < 0.05) up-regulation at 12 hpc to approximately 2.8-fold the mRNA level in the control. There was no obvious change of FcEnolase mRNA level in the hepatopancreas during the infection process. The expression profile coincided with the fact that WSSV primarily infects the tissues of muscle and gill, but hardly infects hepatopancreas. To verify the protein level of FcEnolase post WSSV infection, a Western blot was performed. The FcEnolase protein level in the muscle at 24 hpc significantly (P < 0.05) increased to approximately 2.1-fold the level in the control. These results showed the characterization of FcEnolase and suggested that the FcEnolase might be involved in the response of F. chinensis to WSSV infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

NASA Technical Reports Server (NTRS)

Cubano, L. A.; Lewis, M. L.

2001-01-01

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat).

PubMed

Cubano, L A; Lewis, M L

2001-05-01

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

PubMed Central

2011-01-01

Background Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). High amounts of NO are synthesized following up-regulation of inducible NO synthase (iNOS). The expression of iNOS is tightly regulated by complex molecular mechanisms, involving both transcriptional and post-transcriptional processes. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. mTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. It is also a key regulator of intracellular processes in glial cells. However, with respect to iNOS expression, both stimulatory and inhibitory actions involving the mTOR pathway have been described. In this study the effects of mTOR inhibition on iNOS regulation were evaluated in astrocytes. Methods Primary cultures of rat cortical astrocytes were activated with different proinflammatory stimuli, namely a mixture of cytokines (TNFα, IFNγ, and IL-1β) or by LPS plus IFNγ. Rapamycin was used at nM concentrations to block mTOR activity and under these conditions we measured its effects on the iNOS promoter, mRNA and protein levels. Functional experiments to evaluate iNOS activity were also included. Results In this experimental paradigm mTOR activation did not significantly affect astrocyte iNOS activity, but mTOR pathway was involved in the regulation of iNOS expression. Rapamycin did not display any significant effects under basal conditions, on either iNOS activity or its expression. However, the drug significantly increased iNOS mRNA levels after 4 h incubation in presence of pro-inflammatory stimuli. This stimulatory effect was transient, since no differences in either iNOS mRNA or protein levels were detected after 24 h. Interestingly, reduced levels of iNOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability. Conclusion The present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies. PMID:21208419

Chronic exposure to low levels of inorganic arsenic causes alterations in locomotor activity and in the expression of dopaminergic and antioxidant systems in the albino rat.

PubMed

Rodríguez, Verónica Mireya; Limón-Pacheco, Jorge Humberto; Carrizales, Leticia; Mendoza-Trejo, María Soledad; Giordano, Magda

2010-01-01

Several studies have associated chronic arsenicism with decreases in IQ and sensory and motor alterations in humans. Likewise, studies of rodents exposed to inorganic arsenic ((i)As) have found changes in locomotor activity, brain neurochemistry, behavioral tasks, oxidative stress, and in sensory and motor nerves. In the current study, male Sprague-Dawley rats were exposed to environmentally relevant doses of (i)As (0.05, 0.5 mg (i)As/L) and to a high dose (50 mg (i)As/L) in drinking water for one year. Hypoactivity and increases in the striatal dopamine content were found in the group treated with 50 mg (i)As/L. Exposure to 0.5 and 50 mg (i)As/L increased the total brain content of As. Furthermore, (i)As exposure produced a dose-dependent up-regulation of mRNA for Mn-SOD and Trx-1 and a down-regulation of DAR-D₂ mRNA levels in the nucleus accumbens. DAR-D₁ and Nrf2 mRNA expression were down-regulated in nucleus accumbens in the group exposed to 50 mg (i)As/L. Trx-1 mRNA levels were up-regulated in the cortex in an (i)As dose-dependent manner, while DAR-D₁ mRNA expression was increased in striatum in the 0.5 mg (i)As/L group. These results show that chronic exposure to low levels of arsenic causes subtle but region-specific changes in the nervous system, especially in antioxidant systems and dopaminergic elements. These changes became behaviorally evident only in the group exposed to 50 mg (i)As/L. Copyright © 2010 Elsevier Inc. All rights reserved.

SEIZURE ACTIVITY INVOLVED IN THE UP-REGULATION OF BDNF mRNA EXPRESSION BY ACTIVATION OF CENTRAL MU OPIOID RECEPTORS

PubMed Central

ZHANG, H. N.; KO, M. C.

2009-01-01

Chemical-induced seizures up-regulated brain-derived neurotrophic factor (BDNF) mRNA expression. Intracerebroventricular (i.c.v.) administration of endogenous opioids preferentially activating μ opioid receptor (MOR) could also increase BDNF mRNA expression. The aim of this study was to determine to what extent i.c.v. administration of synthetic MOR-selective agonists in rats can modulate both seizure activity and up-regulation of BDNF mRNA expression. Effects and potencies of i.c.v. administration of morphine and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), were directly investigated by scoring behavioral seizures and measuring BDNF mRNA expression. In addition, effects of the opioid receptor antagonist naloxone and antiepileptic drugs, diazepam, phenobarbital, and valproate, on i.c.v. MOR agonist-induced behavioral seizures and up-regulation of BDNF mRNA expression were determined. A single i.c.v. administration of morphine (10–100 μg) or DAMGO (0.15–1.5 μg) dose-dependently elicited behavioral seizures and increased BDNF mRNA expression in the widespread brain regions. However, subcutaneous administration of MOR agonists neither produced behavioral seizures nor increased BDNF mRNA expression. Pretreatment with naloxone 1 mg/kg significantly reduced behavioral seizure scores and the up-regulation of BDNF mRNA expression elicited by i.c.v. morphine or DAMGO. Similarly, diazepam 10 mg/kg and phenobarbital 40 mg/kg significantly blocked i.c.v. MOR agonist-induced actions. Pretreatment with valproate 300 mg/kg only attenuated behavioral seizures, but it did not affect morphine-induced increase of BDNF mRNA expression. This study provides supporting evidence that seizure activity plays an important role in the up-regulation of BDNF mRNA expression elicited by central MOR activation and that decreased inhibitory action of GABAergic system through the modulation on GABA receptor synaptic function by central MOR activation is involved in its regulation of BDNF mRNA expression. PMID:19303919

Transcriptomic Analysis Reveals Wound Healing of Morus alba Root Extract by Up-Regulating Keratin Filament and CXCL12/CXCR4 Signaling.

PubMed

Kim, Kang-Hoon; Chung, Won-Seok; Kim, Yoomi; Kim, Ki-Suk; Lee, In-Seung; Park, Ji Young; Jeong, Hyeon-Soo; Na, Yun-Cheol; Lee, Chang-Hun; Jang, Hyeung-Jin

2015-08-01

Facilitation of the wound healing process is important because a prolonged wound site increases pain and the risk of infection. In oriental medicine, an extract of Morus alba root (MA) has usually been prescribed as traditional treatment for accelerating wound healing, and it has been proven to be safe for centuries. To study the molecular mechanism of MA-mediated skin wound healing, we performed a primary cell culture and a skin explant culture and observed significant difference between the groups with and without MA extract. In the cellular system, a real-time cell analysis and real-time quantitative PCR were performed. It was found that MA extract enhanced proliferation in a dose-dependent manner on Kera-308 cell line, and up-regulated keratin expression including wound-induced Krt6a. In skin explant culture, the mRNA level derived from cell outgrowth displayed a tendency toward more up-regulated mRNA associated keratin filaments and toward a more up-regulated mRNA level of C-X-C motif chemokine 12 (CXCL12) and a chemokine receptor 4 (CXCR4) axis signaling pathway downstream. In this process, we concluded that MA extract had a scientific possibility of wound repair by increasing intracellular and extracellular supports and by inducing a CXCL12/CXCR4 signaling pathway. Copyright © 2015 John Wiley & Sons, Ltd.

Exposure to butachlor causes thyroid endocrine disruption and promotion of metamorphosis in Xenopus laevis.

PubMed

Li, Shuying; Li, Meng; Wang, Qiangwei; Gui, Wenjun; Zhu, Guonian

2016-06-01

Butachlor is extensively applied in rice paddy ecosystem in china, and has been widespread contaminant in the aquatic environment. Here, Xenopus laevis was used for the evaluation of teratogenesis developmental toxicity, and disruption of thyroid system when exposure to different concentrations of butachlor by window phase exposure. Acute toxicity investigation shown that 96 h-LC50 value of butachlor was 1.424 mg L(-1) and 0.962 mg L(-1) for tadpoles (starting from stages 46/47) and embryos (starting from stages 8/9), respectively. Exposure to butachlor caused malformation, including abnormal eye, pericardial edema, enlarged proctodaeum and bent tail. Window phase exposure test indicated that butachlor significantly promote the contents of whole-body thyroid hormones (THs, T3 and T4) at higher levels, indicating thyroid endocrine disruption. At 7 days, exposure to butachlor up-regulated the mRNA expression of genes involved in THs synthesis and metabolism (tshα, tg, tpo and dio1) and THs receptors (trα and trβ). At 14 days, up-regulation of the mRNA expression of genes related to THs synthesis and metabolism (tshα, tshβ, tg, tpo, dio1, dio2 and ttr) and THs receptors (trβ) were also observed after the exposure to butachlor. At 21 days, butachlor up-regulated the mRNA expression of tshα, tg, tpo genes and down-regulated the mRNA expression of tshβ, tg, dio1, ttr and trα genes. These results showed that butachlor could change the mRNA expression of genes involved in the HPT axis and increase whole-body thyroid hormones levels of X. laevis tadpoles in a dose- and time-dependent manner, causing thyroid endocrine disruption and developmental toxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

L-carnitine protects against testicular dysfunction caused by gamma irradiation in mice.

PubMed

Ahmed, Mohamed Mohamed; Ibrahim, Zein Shaban; Alkafafy, Mohamed; El-Shazly, Samir Ahmed

2014-07-01

This study was conducted on mice to evaluate the radioprotective role of L-carnitine against γ-ray irradiation-induced testicular damage. Adult male mice were exposed to whole body irradiation at a total dose of 1 Gy. Radiation exposure was continued 24 h a day (0.1 Gy/day) throughout the 10 days exposure period either in the absence and/or presence of L-carnitine at an i.p. dose of 10 mg/kg body weight/day. Results revealed that γ-rays irradiation suppressed the expression of ABP and CYP450SCC mRNA, whereas treatment with L-carnitine prior and throughout γ-rays irradiation exposure inhibited this suppression. Treatment with γ-ray irradiation or L-carnitine down-regulated expression of aromatase mRNA. With combined treatment, L-carnitine significantly normalized aromatase expression. γ-Ray irradiation up-regulated expression of FasL and Cyclin D2 mRNA, while L-carnitine inhibited these up-regulations. Results also showed that γ-ray-irradiation up-regulated TNF-α, IL1-β and IFN-γ mRNA expressions compared to either controls or the L-carnitine treated group. Moreover, γ-irradiation greatly reduced serum testosterone levels, while L-carnitine, either alone or in combination with irradiation, significantly increased serum testosterone levels compared to controls. In addition, γ-irradiation induced high levels of sperm abnormalities (43%) which were decreased to 12% in the presence of L-carnitine. In parallel with these findings, histological examination showed that γ-irradiation induced severe tubular degenerative changes, which were reduced by L-carnitine pre-treatment. These results clarified the immunostimulatory effects of L-carnitine and its radioprotective role against testicular injury. Copyright © 2014 Elsevier GmbH. All rights reserved.

Intermittent fasting up-regulates Fsp27/Cidec gene expression in white adipose tissue.

PubMed

Karbowska, Joanna; Kochan, Zdzislaw

2012-03-01

Fat-specific protein of 27 kDa (FSP27) is a novel lipid droplet protein that promotes triacylglycerol storage in white adipose tissue (WAT). The regulation of the Fsp27 gene expression in WAT is largely unknown. We investigated the nutritional regulation of FSP27 in WAT. The effects of intermittent fasting (48 d, eight cycles of 3-d fasting and 3-d refeeding), caloric restriction (48 d), fasting-refeeding (3-d fasting and 3-d refeeding), and fasting (3 d) on mRNA expression of FSP27, peroxisome proliferator-activated receptor γ (PPARγ2), CCAAT/enhancer binding protein α (C/EBPα), and M isoform of carnitine palmitoyltransferase 1 (a positive control for PPARγ activation) in epididymal WAT and on serum triacylglycerol, insulin, and leptin levels were determined in Wistar rats. We also determined the effects of PPARγ activation by rosiglitazone or pioglitazone on FSP27 mRNA levels in primary rat adipocytes. Long-term intermittent fasting, in contrast to other dietary manipulations, significantly up-regulated Fsp27 gene expression in WAT. Moreover, in rats subjected to intermittent fasting, serum insulin levels were elevated; PPARγ2 and C/EBPα mRNA expression in WAT was increased, and there was a positive correlation of Fsp27 gene expression with PPARγ2 and C/EBPα mRNA levels. FSP27 mRNA expression was also increased in adipocytes treated with PPARγ agonists. Our study demonstrates that the transcription of the Fsp27 gene in adipose tissue may be induced in response to nutritional stimuli. Furthermore, PPARγ2, C/EBPα, and insulin may be involved in the nutritional regulation of FSP27. Thus intermittent fasting, despite lower caloric intake, may promote triacylglycerol deposition in WAT by increasing the expression of genes involved in lipid storage, such as Fsp27. Copyright © 2012 Elsevier Inc. All rights reserved.

Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella.

PubMed

Aye, Tin Tin; Shim, Jae-Kyoung; Rhee, In-Koo; Lee, Kyeong-Yeoll

2008-08-01

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in naïve insects and in insects manipulated with bacterial and hormonal treatments.

The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Shuai; Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, 8 Gongti South Rd, Beijing; Zou, Lihui

Background: Different types of pulmonary hypertension (PH) share the same process of pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play an important role in this process. Objectives: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration. Methods: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured humanmore » PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated. Results: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level. In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly, and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased PASMCs proliferation and migration, inhibited the apoptosis. Conclusions: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2. - Highlights: • sGC activator BAY41-2272 up regulated PAI-2 in PASMCs, on the mRNA and protein level. • PAI-2 overexpression inhibits the proliferation and migration of human PASMCs. • PAI-2 overexpression promotes the apoptosis of human PASMCs. • sGC activator might alleviate the vascular remodeling in pulmonary hypertension.« less

Differential Regulation of Sorbitol and Sucrose Loading into the Phloem of Plantago major in Response to Salt Stress1[OA

PubMed Central

Pommerrenig, Benjamin; Papini-Terzi, Flavia Stal; Sauer, Norbert

2007-01-01

Several plant families generate polyols, the reduced form of monosaccharides, as one of their primary photosynthetic products. Together with sucrose (Suc) or raffinose, these polyols are used for long-distance allocation of photosynthetically fixed carbon in the phloem. Many species from these families accumulate these polyols under salt or drought stress, and the underlying regulation of polyol biosynthetic or oxidizing enzymes has been studied in detail. Here, we present results on the differential regulation of genes that encode transport proteins involved in phloem loading with sorbitol and Suc under salt stress. In the Suc- and sorbitol-translocating species Plantago major, the mRNA levels of the vascular sorbitol transporters PmPLT1 and PmPLT2 are rapidly up-regulated in response to salt treatment. In contrast, mRNA levels for the phloem Suc transporter PmSUC2 stay constant during the initial phase of salt treatment and are down-regulated after 24 h of salt stress. This adaptation in phloem loading is paralleled by a down-regulation of mRNA levels for a predicted sorbitol dehydrogenase (PmSDH1) in the entire leaf and of mRNA levels for a predicted Suc phosphate synthase (PmSPS1) in the vasculature. Analyses of Suc and sorbitol concentrations in leaves, in enriched vascular tissue, and in phloem exudates of detached leaves revealed an accumulation of sorbitol and, to a lesser extent, of Suc within the leaves of salt-stressed plants, a reduced rate of phloem sap exudation after NaCl treatment, and an increased sorbitol-to-Suc ratio within the phloem sap. Thus, the up-regulation of PmPLT1 and PmPLT2 expression upon salt stress results in a preferred loading of sorbitol into the phloem of P. major. PMID:17434995

Effect of dietary α-lipoic acid on the mRNA expression of genes involved in drug metabolism and antioxidation system in rat liver.

PubMed

Ide, Takashi

2014-08-14

In the present study, the mRNA levels of hepatic proteins involved in the drug metabolism of rats fed α-lipoic acid were evaluated by DNA microarray and real-time PCR analyses. Experimental diets containing 0, 0·1, 0·25 and 0·5 % (w/w) α-lipoic acid were fed to four groups of rats consisting of seven animals each for 21 d. DNA microarray analysis revealed that the diet containing 0·5 % α-lipoic acid significantly (P< 0·05) increased the mRNA levels of various phase I drug-metabolising enzymes up to 15-fold and phase II enzymes up to 52-fold in an isoenzyme-specific manner. α-Lipoic acid also up-regulated the mRNA levels of some members of the ATP-binding cassette transporter superfamily, presumed to be involved in the exportation of xenobiotics, up to 6·6-fold. In addition, we observed that α-lipoic acid increased the mRNA levels of many proteins involved in antioxidation, such as members of the thiol redox system (up to 5·5-fold), metallothioneins (up to 12-fold) and haeme oxygenase 1 (1·5-fold). These results were confirmed using real-time PCR analysis, and α-lipoic acid dose dependently increased the mRNA levels of various proteins involved in drug metabolism and antioxidation. Consistent with these observations, α-lipoic acid dose dependently increased the hepatic concentration of glutathione and the activities of glutathione reductase and glutathione transferase measured using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, but decreased the hepatic and serum concentrations of malondialdehyde. In conclusion, the present study unequivocally demonstrated that α-lipoic acid increases the mRNA expression of proteins involved in drug metabolism and antioxidation in the liver.

[Houttuynia Cordata induces expression of human beta-defensin-2 mRNA in pulmonary epithelial cells in vitro].

PubMed

Luo, Li; Dong, Bi-rong; Teng, Li-hua

2008-07-01

To explore the effects of Houttuynia Cordata on expression of human beta-defensin-2 (HBD-2) in pulmonary epithelial cells (SPC-A-1) in vitro; and to observe the correlationship between the level of HBD-2 mRNA and the concentrations or treatment times of Houttuynia Cordata. The SPC-A-1 cells were cultured with different concentrations of Houttuynia Cordata in vitro, including 0, 12.5, 25, 50, 100 and 200 microg/ml. And then, the SPC-A-1 cells were cultured with the optimal concentration of Houttuynia Cordata in different lengths of time, including 1, 2, 4, 8, 16 and 24 hours. After the treatment, the mRNA level of HBD-2 in pulmonary epithelial cells was detected by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). After being cultured with Houttuynia Cordata, the expression of HBD-2 mRNA had positive correlation with the stimulus concentrations (rs=0.829, P=0.042) and stimulus time (rs=0.914, P=0.003). The highest expression of HBD-2 mRNA was induced by 100 microg/ml Houttuynia Cordata after 8-hour treatment. In comparison with the normal control group and the interleukin-1beta group, 100 microg/ml Houttuynia Cordata could significantly up-regulate the expression of HBD-2 mRNA in SPC-A-1 cells after 8-hour treatment (P<0.01). Houttuynia Cordata can up-regulate expression of HBD-2 mRNA in SPC-A-1 cells, and the highest expression level of HBD-2 mRNA can be obtained by culture with 100 microg/ml Houttuynia Cordata for 8 hours.

Up-regulated EMMPRIN/CD147 protein expression might play a role in colorectal carcinogenesis and its subsequent progression without an alteration of its glycosylation and mRNA level.

PubMed

Zheng, Hua-chuan; Wang, Wei; Xu, Xiao-yan; Xia, Pu; Yu, Miao; Sugiyama, Toshiro; Takano, Yasuo

2011-04-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of colorectal carcinomas (CRC). EMMPRIN expression was examined on tissue microarray containing colorectal carcinomas, adenoma and non-neoplastic mucosa (NNM) by immunohistochemistry and in situ hybridization (ISH). Colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) and tissues were studied for EMMPRIN expression by Western blot or RT-PCR, followed by sequencing. All carcinoma cell lines showed EMMPRIN expression at both mRNA and protein levels. Two synonymous mutations were found in carcinoma cell lines at codon109 (GCT → GCC: Ala) or 179 (GAT → GAC: Asp). Frozen CRC tissues displayed higher EMMPRIN expression than paired NNM (P < 0.05). EMMPRIN expression was immunohistochemically stronger in colorectal high-grade adenoma, adenocarcinoma and metastatic carcinoma than non-neoplastic superficial epithelium and low-grade adenoma (P < 0.05). In contrast, its mRNA level was similar from colorectal NNM, adenoma to adenocarcinoma by ISH, in line with the findings of RT-PCR (P > 0.05). Immunohistochemically, EMMPRIN expression was positively correlated with tumor size, depth of invasion, vascular or lymphatic invasion, grade of infiltration (INF), ki-67 and VEGF expression of CRCs (P < 0.05). Among them, depth of invasion was an independent associated factor for EMMPRIN expression in CRCs (P < 0.05). Up-regulated EMMPRIN protein expression might contribute to colorectal carcinogenesis without the alteration of its glycosylation and mRNA level. Aberrant EMMPRIN protein expression might promote growth or invasion of CRCs possibly through increased ki-67 expression and inducible angiogenesis via up-regulating VEGF expression.

The Expression of Three Opsin Genes from the Compound Eye of Helicoverpa armigera (Lepidoptera: Noctuidae) Is Regulated by a Circadian Clock, Light Conditions and Nutritional Status

PubMed Central

Yan, Shuo; Zhu, Jialin; Zhu, Weilong; Zhang, Xinfang; Li, Zhen; Liu, Xiaoxia; Zhang, Qingwen

2014-01-01

Visual genes may become inactive in species that inhabit poor light environments, and the function and regulation of opsin components in nocturnal moths are interesting topics. In this study, we cloned the ultraviolet (UV), blue (BL) and long-wavelength-sensitive (LW) opsin genes from the compound eye of the cotton bollworm and then measured their mRNA levels using quantitative real-time PCR. The mRNA levels fluctuated over a daily cycle, which might be an adaptation of a nocturnal lifestyle, and were dependent on a circadian clock. Cycling of opsin mRNA levels was disturbed by constant light or constant darkness, and the UV opsin gene was up-regulated after light exposure. Furthermore, the opsin genes tended to be down-regulated upon starvation. Thus, this study illustrates that opsin gene expression is determined by multiple endogenous and exogenous factors and is adapted to the need for nocturnal vision, suggesting that color vision may play an important role in the sensory ecology of nocturnal moths. PMID:25353953

Comparison of prostaglandin F2alpha, bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression.

PubMed

Liang, Yanbin; Li, Chen; Guzman, Victor M; Evinger, Albert J; Protzman, Charles E; Krauss, Achim H-P; Woodward, David F

2003-07-18

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.

Nucleus-specific expression in the multinuclear mushroom-forming fungus Agaricus bisporus reveals different nuclear regulatory programs.

PubMed

Gehrmann, Thies; Pelkmans, Jordi F; Ohm, Robin A; Vos, Aurin M; Sonnenberg, Anton S M; Baars, Johan J P; Wösten, Han A B; Reinders, Marcel J T; Abeel, Thomas

2018-04-24

Many fungi are polykaryotic, containing multiple nuclei per cell. In the case of heterokaryons, there are different nuclear types within a single cell. It is unknown what the different nuclear types contribute in terms of mRNA expression levels in fungal heterokaryons. Each cell of the mushroom Agaricus bisporus contains two to 25 nuclei of two nuclear types originating from two parental strains. Using RNA-sequencing data, we assess the differential mRNA contribution of individual nuclear types and its functional impact. We studied differential expression between genes of the two nuclear types, P1 and P2, throughout mushroom development in various tissue types. P1 and P2 produced specific mRNA profiles that changed through mushroom development. Differential regulation occurred at the gene level, rather than at the locus, chromosomal, or nuclear level. P1 dominated mRNA production throughout development, and P2 showed more differentially up-regulated genes in important functional groups. In the vegetative mycelium, P2 up-regulated almost threefold more metabolism genes and carbohydrate active enzymes (cazymes) than P1, suggesting phenotypic differences in growth. We identified widespread transcriptomic variation between the nuclear types of A. bisporus Our method enables studying nucleus-specific expression, which likely influences the phenotype of a fungus in a polykaryotic stage. Our findings have a wider impact to better understand gene regulation in fungi in a heterokaryotic state. This work provides insight into the transcriptomic variation introduced by genomic nuclear separation. Copyright © 2018 the Author(s). Published by PNAS.

Differential modulation of leukotriene B4 synthesis and degradation in human bronchoalveolar lavage cells by lipopolysaccharide and tobacco smoke.

PubMed

Mao, Jenny T; Tashkin, Donald P; Tsu, I-Hsien; Serio, Kenneth J

2008-09-01

Leukotrienes have been implicated to play a prominent inductive role in carcinogenesis. We previously reported that bronchoalveolar lavage (BAL) cells from smokers manifested higher levels of leukotriene B4 (LTB4) production than ex-smokers. This study aims to elucidate the underlying mechanism(s). BAL cells from current and former smokers were exposed to lipopolysaccharide (LPS) for up to 7 days. LPS induced the release of LTB4 from BAL cells and down-regulated 5-lipoxygenase (5-LOX) mRNA expression in a dose-dependent manner, followed by a decrease in 5-LOX protein production and normalization of LTB4 levels. Exogenous LTB4 inhibited LPS-induced 5-LOX activity and accentuated the down-regulation of 5-LOX mRNA, whereas suppression of 5-LOX abrogated the LPS-induced changes, suggesting a negative feedback mechanism. LPS concomitantly induced expression and activity of the LTB4 metabolizing enzyme LTB4 omega-hydroxylase (LTB4OH) in ex-smokers' BAL cells, but not in smokers' BAL cells. In vitro smoke exposure of ex-smokers' BAL cells also abrogated the LPS-induced up-regulation of LTB4OH mRNA expression. Furthermore, ex-smokers' BAL cells expressed significantly higher LTB4OH mRNA levels than smokers' BAL cells. Such differential modulation of LTB4 synthesis and degradation by LPS in the setting of tobacco smoke exposure suggests that mechanisms responsible for sustained elevation of LTB4 levels in the lung microenvironment may contribute to the pathogenesis of tobacco-related respiratory diseases such as lung cancer. By regulating the balance of LTB4 in the lung, LTB4OH may function as a suppressor of lung carcinogenesis.

A nonimprinted Prader-Willi Syndrome (PWS)-region gene regulates a different chromosomal domain in trans but the imprinted pws loci do not alter genome-wide mRNA levels.

PubMed

Stefan, Mihaela; Portis, Toni; Longnecker, Richard; Nicholls, Robert D

2005-05-01

Prader-Willi syndrome (PWS) is a complex neurobehavioral disorder that results from loss of function of 10 clustered, paternally expressed genes in a 1.5-Mb region of chromosome 15q11-q13. Many of the primary PWS region genes appear to have nuclear RNA regulatory functions, suggesting that multiple genetic pathways could be secondarily affected in PWS. Using a transgenic mouse model of PWS (TgPWS) with an approximately 4-Mb chromosome 7C deletion of paternal origin that models the neonatal phenotype of the human syndrome we compared by oligonucleotide microarrays expression levels of approximately 12,000 genes and ESTs in TgPWS and wild-type brain. Hybridization data were processed with two distinct statistical algorithms and revealed a dramatically reduced expression of 4 imprinted genes within the deletion region in TgPWS mice, with 2 nonimprinted, codeleted genes reduced twofold. However, only 3 genes outside the deletion were significantly altered in TgPWS mouse brain, with approximately 1.5-fold up-regulation of mRNA levels. Remarkably, these genes map to a single chromosome domain (18B3), and by quantitative RT-PCR we show that 8 genes in this domain are up-regulated in TgPWS brain. These 18B3 genes were up-regulated in an equivalent manner in Angelman syndrome mouse (TgAS) brain, which has the same deletion but of maternal origin. Therefore, the trans-regulation of the chromosome 18B3 domain is due to decreased expression of a nonimprinted gene within the TgPWS/AS mouse deletion in mouse chromosome 7C. Most surprisingly, since 48-60% of the genome was screened, it appears that the imprinted mouse PWS loci do not widely regulate mRNA levels of other genes and may regulate RNA structure.

Urinary bladder organ hypertrophy is partially regulated by Akt1-mediated protein synthesis pathway.

PubMed

Qiao, Li-Ya; Xia, Chunmei; Shen, Shanwei; Lee, Seong Ho; Ratz, Paul H; Fraser, Matthew O; Miner, Amy; Speich, John E; Lysiak, Jeffrey J; Steers, William D

2018-05-15

The present study aims to investigate the role of Akt in the regulation of urinary bladder organ hypertrophy caused by partial bladder outlet obstruction (pBOO). Male rats were surgically induced for pBOO. Real-time PCR and western blot were used to examine the levels of mRNA and protein. A phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was used to inhibit the activity of endogenous Akt. The urinary bladder developed hypertrophy at 2 weeks of pBOO. The protein but not mRNA levels of type I collagen and α-smooth muscle actin (αSMA) were increased in pBOO bladder when compared to sham control. The phosphorylation (activation) levels of Akt1 (p-Ser 473 ), mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and 4E-BP1 were also increased in pBOO bladder. LY294002 treatment reduced the phosphorylation levels of Akt1 and 4E-BP1, and the protein levels of type I collagen and αSMA in pBOO bladder. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) were increased in pBOO bladder, and PCNA up-regulation occurred in urothelial not muscular layer. LY294002 treatment had no effect on the mRNA and protein levels of PCNA in pBOO bladder. LY294002 treatment partially reduced the bladder weight caused by pBOO. pBOO-induced urinary bladder hypertrophy is attributable to fibrosis, smooth muscle cellular hypertrophy, and urothelium cell hyper-proliferation. Akt1-mediated protein synthesis in pBOO bladder contributes to type I collagen and αSMA but not PCNA up-regulation. Target of Akt1 is necessary but not sufficient in treatment of urinary bladder hypertrophy following pBOO. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantitative proteomics reveals that peroxidases play key roles in post-flooding recovery in soybean roots.

PubMed

Khan, Mudassar Nawaz; Sakata, Katsumi; Hiraga, Susumu; Komatsu, Setsuko

2014-12-05

Soybean is an important legume crop that exhibits markedly reduced growth and yields under flooding conditions. To unravel the mechanisms involved in recovery after flooding in soybean root, gel-free proteomic analysis was performed. Morphological analysis revealed that growth suppression was more severe with increased flooding duration. Out of a total of 1645 and 1707 identified proteins, 73 and 21 proteins were changed significantly during the recovery stage following 2 and 4 days flooding, respectively. Based on the proteomic, clustering, and in silico protein-protein interaction analyses, six key enzymes were analyzed at the mRNA level. Lipoxygenase 1, which was increased at the protein level during the recovery period, was steadily down-regulated at the mRNA level. The peroxidase superfamily protein continuously increased in abundance during the course of recovery and was up-regulated at the mRNA level. HAD acid phosphatase was decreased at the protein level and down-regulated at the transcript level, while isoflavone reductase and an unknown protein were increased at both the protein and mRNA levels. Consistent with these findings, the enzymatic activity of peroxidase was decreased under flooding stress but increased significantly during the recovery sage. These results suggest that peroxidases might play key roles in post-flooding recovery in soybean roots through the scavenging of toxic radicals.

Green tea (Camellia sinensis) administration induces expression of immune relevant genes and biochemical parameters in rainbow trout (Oncorhynchus mykiss).

PubMed

Nootash, Shahab; Sheikhzadeh, Najmeh; Baradaran, Behzad; Oushani, Ali Khani; Maleki Moghadam, Mohammad Reza; Nofouzi, Katayoon; Monfaredan, Amir; Aghebati, Leili; Zare, Fatemeh; Shabanzadeh, Sadigheh

2013-12-01

Present study elucidates the efficacy of green tea (Camellia sinensis) on growth performance, immune and antioxidant systems and cytokine gene expression in rainbow trout tissues. Green tea was supplemented at 20, 100, and 500 mg kg(-1) diet and fed to fish (average weight: 23.5 g) for 35 days. No remarkable changes in growth performance were observed among all test groups. Lower lipid peroxidation product and higher superoxide dismutase activity were noted in fish received the medium dose of green tea. Significant increase in serum bactericidal activity and total protein were recorded in all treatment groups. All doses of green tea up-regulated Interleukin-1β transcription in the spleen, while Interleukin-1β mRNA level decreased significantly in the kidney of low dose of green tea. Interleukin-6 mRNA level was up-regulated in the spleen of high dose of green tea and liver of middle and high doses of green tea. High dose and medium dose of green tea up-regulated the interleukin-8 transcription in the kidney and liver, respectively. Meanwhile, green tea inhibited the production of interleukin-10 in all treatment groups compared with control group. Medium dose of green tea up-regulated tumor necrosis factor-α transcription in all fish tissues, while high dose and low dose of green tea enhanced tumor necrosis factor-α mRNA levels in the kidney and spleen, respectively. Present study suggests that green tea especially at 100 mg kg(-1) feed may effectively enhance the antioxidant system and immune system in rainbow trout. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inhibition of hepatic gluconeogenesis and enhanced glucose uptake contribute to the development of hypoglycemia in mice bearing interleukin-1beta- secreting tumor.

PubMed

Metzger, Shulamit; Nusair, Samir; Planer, David; Barash, Varda; Pappo, Orit; Shilyansky, Joel; Chajek-Shaul, Tova

2004-11-01

Mice bearing IL-1beta-secreting tumor were used to study the chronic effect of IL-1beta on glucose metabolism. Mice were injected with syngeneic tumor cells transduced with the human IL-1beta gene. Serum IL-1beta levels increased exponentially with time. Secretion of IL-1beta from the developed tumors was associated with decreased food consumption, reduced body weight, and reduced blood glucose levels. Body composition analysis revealed that IL-1beta caused a significant loss in fat tissue without affecting lean body mass and water content. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities and mRNA levels of these enzymes were reduced, and 2-deoxy-glucose uptake by peripheral tissues was enhanced. mRNA levels of glucose transporters (Gluts) in the liver were determined by real-time PCR analysis. Glut-3 mRNA levels were up-regulated by IL-1beta. Glut-1 and Glut-4 mRNA levels in IL-1beta mice were similar to mRNA levels in pair-fed mice bearing nonsecreting tumor. mRNA level of Glut-2, the major Glut of the liver, was down-regulated by IL-1beta. We concluded that both decreased glucose production by the liver and enhanced glucose disposal lead to the development of hypoglycemia in mice bearing IL-1beta-secreting tumor. The observed changes in expression of hepatic Gluts that are not dependent on insulin may contribute to the increased glucose uptake.

Antiepileptic drugs affect neuronal androgen signaling via a cytochrome P450-dependent pathway.

PubMed

Gehlhaus, Marcel; Schmitt, Nina; Volk, Benedikt; Meyer, Ralf P

2007-08-01

Recent data imply an important role for brain cytochrome P450 (P450) in endocrine signaling. In epileptic patients, treatment with P450 inducers led to reproductive disorders; in mouse hippocampus, phenytoin treatment caused concomitant up-regulation of CYP3A11 and androgen receptor (AR). In the present study, we established specific in vitro models to examine whether CYP3A isoforms cause enhanced AR expression and activation. Murine Hepa1c1c7 cells and neuronal-type rat PC-12 cells were used to investigate P450 regulation and its effects on AR after phenytoin and phenobarbital administration. In both cell lines, treatment with antiepileptic drugs (AEDs) led to concomitant up-regulation of CYP3A (CYP3A11 in Hepa1c1c7 and CYP3A2 in PC-12) and AR mRNA and protein. Inhibition of CYP3A expression and activity by the CYP3A inhibitor ketoconazole or by CYP3A11-specific short interfering RNA molecules reduced AR expression to basal levels. The initial up-regulation of AR signal transduction, measured by an androgen-responsive element chloramphenicol-acetyltransferase reporter gene assay, was completely reversed after specific inhibition of CYP3A11. Withdrawal of the CYP3A11 substrate testosterone prevented AR activation, whereas AR mRNA expression remained up-regulated. In addition, recombinant CYP3A11 was expressed heterologously in PC-12 cells, thereby eliminating any direct drug influence on the AR. Again, the initial up-regulation of AR mRNA and activity was reduced to basal levels after silencing of CYP3A11. In conclusion, we show here that CYP3A2 and CYP3A11 are crucial mediators of AR expression and signaling after AED application. These findings point to an important and novel function of P450 in regulation of steroid hormones and their receptors in endocrine tissues such as liver and brain.

Identification and Function Analysis of enolase Gene NlEno1 from Nilaparvata lugens (Stål) (Hemiptera:Delphacidae)

PubMed Central

Wang, Wei-Xia; Li, Kai-Long; Chen, Yang; Lai, Feng-Xiang; Fu, Qiang

2015-01-01

The enolase [EC 4.2.1.11] is an essential enzyme in the glycolytic pathway catalyzing the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP). In this study, a full-length cDNA encoding α-enolase was cloned from rice brown planthopper (Nilaparvata lugens) and is provisionally designated as NlEno1. The cDNA sequence of NlEno1 was 1,851 bp with an open reading frame (ORF) of 1,305 bp and encoding 434 amino acids. The deduced protein shares high identity of 80–87% with ENO1-like protein from Hemiptera, Diptera, and Lepidoptera speices. The NlEno1 showed the highest mRNA expression level in hemolymph, followed by fat body, salivary gland, ovaries and egg, and showed trace mRNA levels in testis. The mRNA of NlEno1 showed up-regulated level in virulent N. lugens population Mudgo, IR56 and IR42 when compared with TN1 population. Injection of double-stranded RNA (dsRNA) of NlEno1 into the adults significantly down-regulated the NlEno1 mRNA level along with decreased eggs and offspring. Moreover, injection of NlEno1-dsRNA decreased mRNA level of Vitellogenin (Vg) gene. These results showed that the NlEno1, as a key glycolytic enzyme, may play roles in regulation of fecundity and adaptation of N. lugens to resistant rice varieties. PMID:26056319

Soluble soy protein peptic hydrolysate stimulates adipocyte differentiation in 3T3-L1 cells.

PubMed

Goto, Tsuyoshi; Mori, Ayaka; Nagaoka, Satoshi

2013-08-01

The molecular mechanisms underlying the potential health benefit effects of soybean proteins on obesity-associated metabolic disorders have not been fully clarified. In this study, we investigated the effects of soluble soybean protein peptic hydrolysate (SPH) on adipocyte differentiation by using 3T3-L1 murine preadipocytes. The addition of SPH increased lipid accumulation during adipocyte differentiation. SPH increased the mRNA expression levels of an adipogenic marker gene and decreased that of a preadipocyte marker gene, suggesting that SPH promotes adipocyte differentiation. SPH induced antidiabetic and antiatherogenic adiponectin mRNA expression and secretion. Moreover, SPH increased the mRNA expression levels of insulin-responsive glucose transporter 4 and insulin-stimulated glucose uptake. The expression levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, during adipocyte differentiation were up-regulated in 3T3-L1 cells treated with SPH, and lipid accumulation during adipocyte differentiation induced by SPH was inhibited in the presence of a PPARγ antagonist. However, SPH did not exhibit PPARγ ligand activity. These findings indicate that SPH stimulates adipocyte differentiation, at least in part, via the up-regulation of PPARγ expression levels. These effects of SPH might be important for the health benefit effects of soybean proteins on obesity-associated metabolic disorders. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sex hormone-binding globulin and corticosteroid-binding globulin mRNA levels in infertile women with luteal phase deficiency.

PubMed

Misao, R; Nakanishi, Y; Fujimoto, J; Tamaya, T

1995-09-01

This study was designed to investigate the biological significance in intracellular expression of sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) mRNA in uterine endometrium with luteal phase deficiency (designated as out-of-phase endometrium or low serum progesterone level). The levels of such mRNAs were measured by the quantitative reverse transcription-polymerase chain reaction. Under the normal serum 17 beta-estradiol and progesterone levels in the mid-luteal phase, the levels of SHBG and CBG mRNAs in the out-of-phase endometria were not significantly different from those in the normal endometria. On the other hand, SHBG and CBG mRNA levels in the endometria of low serum midluteal progesterone level were significantly (p < 0.05) reduced and raised, respectively, compared with normal levels. These findings suggest that the synthesis of endometrial steroid-binding proteins in the out-of-phase endometrium is conserved, as that in the in-phase endometrium, whereas the decreased progesterone level might up-regulate CBG expression with down-regulation of SHBG expression.

Cyclooxygenase system contributes to the maintenance of post convulsive period of epileptic phenomena in the genetically epileptic El mice.

PubMed

Okada, Kazumasa; Yamashita, Uki; Tsuji, Sadatoshi

2006-09-01

Recent studies have shown that cytokines and cyclooxygenase (COX)-2 are up-regulated in the brain of human epilepsy patients and animal models of epilepsy. We investigated the effect of inflammatory responses induced by intramuscular injection of turpentine on the epileptic phenomenon in genetically epileptic El mice. As parameters of epileptic seizure, seizure threshold (number of toss-ups to induce convulsion), duration of actual convulsion and duration of post actual convulsive period (period from the offset of convulsion to full recovery) were evaluated. The post actual convulsive period was prolonged without any change of seizure threshold or duration of actual convulsion 24 h after turpentine injection. Although pretreatment with indomethacin for one week did not change the seizure parameters, indomethacin suppressed the prolongation of the post actual convulsive period induced by turpentine. The mRNA expression of IL-1beta, IL-6 and COX-2 in the cerebral cortex was detected by RT-PCR. There was no difference in the mRNA expression in the cerebral cortex before and 24 h after seizure. The mRNA levels of IL-1beta, IL-6 and COX-2 in the cerebral cortex were up-regulated 24 h after turpentine injection. On the other hand, the up-regulated mRNA levels of IL-1beta, IL-6 and COX-2 in the cerebral cortex after turpentine treatment were not suppressed by indomethacin. These results suggest that prostaglandins induced with COX-2 in the cerebral cortex seem to play an important role in the maintenance of the post convulsive period, but not in induction and maintenance of the actual convulsive state.

Thyroid hormone receptor beta2 is strongly up-regulated at all levels of the hypothalamo-pituitary-thyroidal axis during late embryogenesis in chicken.

PubMed

Grommen, Sylvia V H; Arckens, Lutgarde; Theuwissen, Tim; Darras, Veerle M; De Groef, Bert

2008-03-01

In this study, we tried to elucidate the changes in thyroid hormone (TH) receptor beta2 (TRbeta2) expression at the different levels of the hypothalamo-pituitary-thyroidal (HPT) axis during the last week of chicken embryonic development and hatching, a period characterized by an augmented activity of the HPT axis. We quantified TRbeta2 mRNA in retina, pineal gland, and the major control levels of the HPT axis - brain, pituitary, and thyroid gland - at day 18 of incubation, and found the most abundant mRNA content in retina and pituitary. Thyroidal TRbeta2 mRNA content increased dramatically between embryonic day 14 and 1 day post-hatch. In pituitary and hypothalamus, TRbeta2 mRNA expression rose gradually, in parallel with increases in plasma thyroxine concentrations. Using in situ hybridization, we have demonstrated the presence of TRbeta2 mRNA throughout the diencephalon and confirmed the elevation in TRbeta2 mRNA expression in the hypophyseal thyrotropes. In vitro incubation with THs caused a down-regulation of TRbeta2 mRNA levels in embryonic but not in post-hatch pituitaries. The observed expression patterns in pituitary and diencephalon may point to substantial changes in TRbeta2-mediated TH feedback active during the perinatal period. The strong rise in thyroidal TRbeta2 mRNA content could be indicative of an augmented modulation of thyroid development and/or function by THs toward and after hatching. Finally, THs proved to exert an age-dependent effect on pituitary TRbeta2 mRNA expression.

Quantitative Expression of C-Type Lectin Receptors in Humans and Mice

PubMed Central

Lech, Maciej; Susanti, Heni Eka; Römmele, Christoph; Gröbmayr, Regina; Günthner, Roman; Anders, Hans-Joachim

2012-01-01

C-type lectin receptors and their adaptor molecules are involved in the recognition of glycosylated self-antigens and pathogens. However, little is known about the species- and organ-specific expression profiles of these molecules. We therefore determined the mRNA expression levels of Dectin-1, MR1, MR2, DC-SIGN, Syk, Card-9, Bcl-10, Malt-1, Src, Dec-205, Galectin-1, Tim-3, Trem-1, and DAP-12 in 11 solid organs of human and mice. Mouse organs revealed lower mRNA levels of most molecules compared to spleen. However, Dec-205 and Galectin-1 in thymus, Src in brain, MR2, Card-9, Bcl-10, Src, and Dec-205 in small intestine, MR2, Bcl-10, Src, Galectin-1 in kidney, and Src and Galectin-1 in muscle were at least 2-fold higher expressed compared to spleen. Human lung, liver and heart expressed higher mRNA levels of most genes compared to spleen. Dectin-1, MR1, Syk and Trem-1 mRNA were strongly up-regulated upon ischemia-reperfusion injury in murine kidney. Tim3, DAP-12, Card-9, DC-SIGN and MR2 were further up-regulated during renal fibrosis. Murine kidney showed higher DAP-12, Syk, Card-9 and Dectin-1 mRNA expression during the progression of lupus nephritis. Thus, the organ-, and species-specific expression of C-type lectin receptors is different between mice and humans which must be considered in the interpretation of related studies. PMID:22949850

Early life stress stimulates hippocampal reelin gene expression in a sex-specific manner: evidence for corticosterone-mediated action.

PubMed

Gross, Claus M; Flubacher, Armin; Tinnes, Stefanie; Heyer, Andrea; Scheller, Marie; Herpfer, Inga; Berger, Mathias; Frotscher, Michael; Lieb, Klaus; Haas, Carola A

2012-03-01

Early life stress predisposes to the development of psychiatric disorders. In this context the hippocampal formation is of particular interest, because it is affected by stress on the structural and cognitive level. Since little is known how early life stress is translated on the molecular level, we mimicked early life stress in mouse models and analyzed the expression of the glycoprotein Reelin, a master molecule for development and differentiation of the hippocampus. From postnatal day 1 (P1) to P14, mouse pups were subjected to one of the following treatments: nonhandling (NH), handling (H), maternal separation (MS), and early deprivation (ED) followed by immediate (P15) or delayed (P70) real time RT-PCR analysis of reelin mRNA expression. We show that at P15, reelin mRNA levels were significantly increased in male H and ED groups when compared with the NH group. In contrast, no stress-induced alterations of reelin mRNA expression were found in female animals. This sex difference in stress-mediated stimulation of reelin expression was maintained into adulthood, since at P70 intergroup differences were still found in male, but not in female mice. On the cellular level, however, we did not find any significant differences in cell densities of Reelin-immunolabeled neurons between treatment groups or sexes, but an overall reduction of Reelin-expressing neurons in the adult hippocampus when compared to P15. To address the question whether corticosterone mediates the stress-induced up-regulation of reelin gene expression, we used age-matched hippocampal slice cultures derived from male and female mouse pups. Quantitative determination of mRNA levels revealed that corticosterone treatment significantly up-regulated reelin mRNA expression in male, but not in female hippocampi. Taken together, these results show a sex-specific regulation of reelin gene expression by early life experience, most likely mediated by corticosterone. Copyright © 2010 Wiley Periodicals, Inc.

Trans fatty acids exacerbate dextran sodium sulphate-induced colitis by promoting the up-regulation of macrophage-derived proinflammatory cytokines involved in T helper 17 cell polarization.

PubMed

Okada, Y; Tsuzuki, Y; Sato, H; Narimatsu, K; Hokari, R; Kurihara, C; Watanabe, C; Tomita, K; Komoto, S; Kawaguchi, A; Nagao, S; Miura, S

2013-12-01

Numerous reports have shown that a diet containing large amounts of trans fatty acids (TFAs) is a major risk factor for metabolic disorders. Although recent studies have shown that TFAs promote intestinal inflammation, the underlying mechanisms are unknown. In this study, we examined the effects of dietary fat containing TFAs on dextran sodium sulphate (DSS)-induced colitis. C57 BL/6 mice were fed a diet containing 1·3% TFAs (mainly C16:1, C18:1, C18:2, C20:1, C20:2 and C22:1), and then colitis was induced with 1·5% DSS. Colonic damage was assessed, and the mRNA levels of proinflammatory cytokines and major regulators of T cell differentiation were measured. The TFA diet reduced survival and exacerbated histological damage in mice administered DSS compared with those fed a TFA-free diet. The TFA diet significantly elevated interleukin (IL)-6, IL-12p40, IL-23p19 and retinoic acid-related orphan receptor (ROR)γt mRNA levels in the colons of DSS-treated animals. Moreover, IL-17A mRNA levels were elevated significantly by the TFA diet, with or without DSS treatment. We also examined the expression of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and peritoneal macrophages. These cells were exposed to TFAs (linoelaidic acid or elaidic acid) with or without LPS and the mRNA levels of various cytokines were measured. IL-23p19 mRNA levels were increased significantly by TFAs in the absence of LPS. Cytokine expression was also higher in LPS-stimulated cells exposed to TFAs than in unexposed LPS-stimulated cells. Collectively, our results suggest that TFAs exacerbate colonic inflammation by promoting Th17 polarization and by up-regulating the expression of proinflammatory cytokines in the inflamed colonic mucosa. © 2013 British Society for Immunology.

Oxidative stress, endocrine disruption, and malformation of Bufo gargarizans embryo exposed to sub-lethal cadmium concentrations.

PubMed

Wu, Chao; Zhang, Yuhui; Chai, Lihong; Wang, Hongyuan

2017-01-01

Thyroid hormone (TH) is critical for vertebrate postembryonic development as well as embryonic development. Chinese toad (Bufo gargarizans) embryos were exposed to different concentrations of cadmium (5, 50, 100, 200 and 500μg Cd L -1 ) for 7days. Malformations were monitored daily, and growth and development of embryos were measured at day 4 and 7, and type 2 and 3 iodothyronine deiodinase (Dio2 and Dio3), thyroid hormone receptors (TRα and TRβ) mRNA levels were also measured to assess disruption of TH synthesis. In addition, superoxide dismutase (SOD), glutathione peroxidase (GPx) and heat shock proteins (HSPs) mRNA expression were examined to evaluate the ability of scavenging ROS. Our results demonstrated a bimodal inhibitory effect of Cd on the embryo growth and development of Bufo gargarizans. Reduced mean stage, total length and weight were observed at 5, 50, 200 and 500, but not at 100μg Cd L -1 . Embryos malformation occurred in all cadmium treatments. Morphological abnormalities of embryos are characterized by axial flexures, abdominal edema, stunted growth and fin flexure. Real-time PCR results show that exposure to cadmium down-regulated TRα and Dio3 mRNA expression and up-regulated Dio2 mRNA level. SOD and GPx mRNA expression was significantly up-regulated after cadmium exposure. We concluded that cadmium could change mRNA expression of TRα, Dio2 and Dio3 leading the inhibition of growth and development of B. gargarizans embryo, which suggests that cadmium might have the endocrine-disrupting effect in embryos. Moreover, the reduced ability of scavenging ROS induced by cadmium might be responsible for the teratogenic effects of cadmium. Copyright © 2016 Elsevier B.V. All rights reserved.

Distinct Protein Expression Profiles of Solid-Pseudopapillary Neoplasms of the Pancreas.

PubMed

Park, Minhee; Lim, Jong-Sun; Lee, Hyoung-Joo; Na, Keun; Lee, Min Jung; Kang, Chang Moo; Paik, Young-Ki; Kim, Hoguen

2015-08-07

Solid-pseudopapillary neoplasm (SPN) is an uncommon pancreatic tumor with mutation in CTNNB1 and distinct clinical and pathological features. We compared the proteomic profiles of SPN to mRNA expression. Pooled SPNs and pooled non-neoplastic pancreatic tissues were examined with high-resolution mass spectrometry. We identified 329 (150 up-regulated and 179 down-regulated) differentially expressed proteins in SPN. We identified 191 proteins (58.1% of the 329 dysregulated proteins) with the same expression tendencies in SPN based on mRNA data. Many overexpressed proteins were related to signaling pathways known to be activated in SPNs. We found that several proteins involved in Wnt signaling, including DKK4 and β-catenin, and proteins that bind β-catenin, such as FUS and NONO, were up-regulated in SPNs. Molecules involved in glycolysis, including PKM2, ENO2, and HK1, were overexpressed in accordance to their mRNA levels. In summary, SPN showed (1) distinct protein expression changes that correlated with mRNA expression, (2) overexpression of Wnt signaling proteins and proteins that bind directly to β-catenin, and (3) overexpression of proteins involved in metabolism. These findings may help develop early diagnostic biomarkers and molecular targets.

Targeting FASN for Breast Cancer Treatment by Repositioning PPIs

DTIC Science & Technology

2017-01-01

the sole cytosolic enzyme responsible for de novo synthesis of palmitate. Recently, it was found that FASN up-regulation contributes to drug and...increased whereas Flag-p65 (NF-κB) over-expression significantly reduced PARP-1 protein and mRNA levels. Knocking down p65 using shRNA significantly...increased PARP-1 protein and mRNA levels (Fig. 2C), whereas activation of p65 with TNF-α had contrary effect (Fig. 2D). Taken together, these results

Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in the female mouse brain.

PubMed

Kumari, Anita; Singh, Padmanabh; Baghel, Meghraj Singh; Thakur, M K

2016-05-01

Adverse early life experience is prominent risk factors for numerous psychiatric illnesses, including mood and anxiety disorders. It imposes serious long-term costs on the individual as well as health and social systems. Hence, developing therapies that prevent the long-term consequences of early life stress is of utmost importance, and necessitates a better understanding of the mechanisms by which early life stress triggers long-lasting alterations in gene expression and behavior. Post-weaning isolation rearing of rodents models the behavioral consequences of adverse early life experiences in humans and it is reported to cause anxiety like behavior which is more common in case of females. Therefore, in the present study, we have studied the impact of social isolation of young female mice for 8weeks on the anxiety like behavior and the underlying molecular mechanism. Elevated plus maze and open field test revealed that social isolation caused anxiety like behavior. BDNF, a well-known molecule implicated in the anxiety like behavior, was up-regulated both at the message and protein level in cerebral cortex by social isolation. CREB-1 and CBP, which play a crucial role in BDNF transcription, were up-regulated at mRNA level in cerebral cortex by social isolation. HDAC-2, which negatively regulates BDNF expression, was down-regulated at mRNA and protein level in cerebral cortex by social isolation. Furthermore, BDNF acts in concert with Limk-1, miRNA-132 and miRNA-134 for the regulation of structural and morphological plasticity. Social isolation resulted in up-regulation of Limk-1 mRNA and miRNA-132 expression in the cerebral cortex. MiRNA-134, which inhibits the translation of Limk-1, was decreased in cerebral cortex by social isolation. Taken together, our study suggests that social isolation mediated anxiety like behavior is associated with up-regulation of BDNF expression and concomitant increase in the expression of CBP, CREB-1, Limk-1 and miRNA-132, and decrease in the expression of HDAC-2 and miRNA-134 in the cerebral cortex. Copyright © 2016. Published by Elsevier Inc.

The Regulation of Alfalfa Saponin Extract on Key Genes Involved in Hepatic Cholesterol Metabolism in Hyperlipidemic Rats

PubMed Central

Shi, Yinghua; Guo, Rui; Wang, Xianke; Yuan, Dedi; Zhang, Senhao; Wang, Jie; Yan, Xuebing; Wang, Chengzhang

2014-01-01

To investigate the cholesterol-lowering effects of alfalfa saponin extract (ASE) and its regulation mechanism on some key genes involved in cholesterol metabolism, 40 healthy 7 weeks old male Sprague Dawley (SD) rats were randomly divided into four groups with 10 rats in each group: control group, hyperlipidemic group, ASE treatment group, ASE prevention group. The body weight gain, relative liver weight and serum lipid 1evels of rats were determined. Total cholesterol (TC) and total bile acids (TBA) levels in liver and feces were also measured. Furthermore, the activity and mRNA expressions of Hmgcr, Acat2, Cyp7a1 and Ldlr were investigated. The results showed the following: (1) The abnormal serum lipid levels in hyperlipidemic rats were ameliorated by ASE administration (both ASE prevention group and treatment group) (P<0.05). (2) Both ASE administration to hyperlipidemic rats significantly reduced liver TC and increased liver TBA level (P<0.05). TC and TBA levels in feces of hyperlipidemic rats were remarkably elevated by both ASE administration (P<0.05). (3) mRNA expressions of Hmgcr and Acat2 in the liver of hyperlipidemic rats were remarkably down-regulated (P<0.05), as well as mRNA expressions of Cyp7a1 and Ldlr were dramatically up-regulated by both ASE administration (P<0.05). The activities of these enzymes also paralleled the observed changes in mRNA levels. (4) There was no significant difference between ASE treatment and ASE prevention group for most parameters evaluated. Our present study indicated that ASE had cholesterol-lowering effects. The possible mechanism could be attributed to (1) the down-regulation of Hmgcr and Acat2, as well as up-regulation of Cyp7a1 and Ldlr in the liver of hyperlipidemic rats, which was involved in cholesterol biosynthesis, uptake, and efflux pathway; (2) the increase in excretion of cholesterol. The findings in our study suggested ASE had great potential usefulness as a natural agent for treating hyperlipidemia. PMID:24505463

Identification of differentially expressed genes in human breast cancer cells induced by 4-hydroxyltamoxifen and elucidation of their pathophysiological relevance and mechanisms

PubMed Central

Fang, Qi; Yao, Shuang; Luo, Guanghua; Zhang, Xiaoying

2018-01-01

While tamoxifen (TAM) is used for treating estrogen receptor (ER)a-positive breast cancer patients, its anti-breast cancer mechanisms are not completely elucidated. This study aimed to examine effects of 4-hydroxyltamoxifen (4-OH-TAM) on ER-positive (ER+) breast cancer MCF-7 cell growth and gene expression profiles. MCF-7 cell growth was inhibited by 4-OH-TAM dose-dependently with IC50 of 29 μM. 332 genes were up-regulated while 320 genes were down-regulated. The mRNA levels of up-regulated genes including STAT1, STAT2, EIF2AK2, TGM2, DDX58, PARP9, SASH1, RBL2 and USP18 as well as down-regulated genes including CCDN1, S100A9, S100A8, ANXA1 and PGR were confirmed by quantitative real-time PCR (qRT-PCR). In human breast tumor tissues, mRNA levels of EIF2Ak2, USP18, DDX58, RBL2, STAT2, PGR, S1000A9, and CCND1 were significantly higher in ER+- than in ER--breast cancer tissues. The mRNA levels of EIF2AK2, TGM2, USP18, DDX58, PARP9, STAT2, STAT1, PGR and CCND1 were all significantly higher in ER+-tumor tissues than in their corresponding tumor-adjacent tissues. These genes, except PGR and CCND1 which were down-regulated, were also up-regulated in ER+ MCF-7 cells by 4-OH-TAM. Total 14 genes mentioned above are involved in regulation of cell proliferation, apoptosis, cell cycles, and estrogen and interferon signal pathways. Bioinformatics analysis also revealed other novel and important regulatory factors that are associated with these genes and involved in the mentioned functional processes. This study has paved a foundation for elucidating TAM anti-breast cancer mechanisms in E2/ER-dependent and independent pathways. PMID:29416786

Identification of differentially expressed genes in human breast cancer cells induced by 4-hydroxyltamoxifen and elucidation of their pathophysiological relevance and mechanisms.

PubMed

Fang, Qi; Yao, Shuang; Luo, Guanghua; Zhang, Xiaoying

2018-01-05

While tamoxifen (TAM) is used for treating estrogen receptor (ER)a-positive breast cancer patients, its anti-breast cancer mechanisms are not completely elucidated. This study aimed to examine effects of 4-hydroxyltamoxifen (4-OH-TAM) on ER-positive (ER + ) breast cancer MCF-7 cell growth and gene expression profiles. MCF-7 cell growth was inhibited by 4-OH-TAM dose-dependently with IC 50 of 29 μM. 332 genes were up-regulated while 320 genes were down-regulated. The mRNA levels of up-regulated genes including STAT1, STAT2, EIF2AK2, TGM2, DDX58, PARP9, SASH1, RBL2 and USP18 as well as down-regulated genes including CCDN1, S100A9, S100A8, ANXA1 and PGR were confirmed by quantitative real-time PCR (qRT-PCR). In human breast tumor tissues, mRNA levels of EIF2Ak2, USP18, DDX58, RBL2, STAT2, PGR, S1000A9, and CCND1 were significantly higher in ER + - than in ER - -breast cancer tissues. The mRNA levels of EIF2AK2, TGM2, USP18, DDX58, PARP9, STAT2, STAT1, PGR and CCND1 were all significantly higher in ER + -tumor tissues than in their corresponding tumor-adjacent tissues. These genes, except PGR and CCND1 which were down-regulated, were also up-regulated in ER + MCF-7 cells by 4-OH-TAM. Total 14 genes mentioned above are involved in regulation of cell proliferation, apoptosis, cell cycles, and estrogen and interferon signal pathways. Bioinformatics analysis also revealed other novel and important regulatory factors that are associated with these genes and involved in the mentioned functional processes. This study has paved a foundation for elucidating TAM anti-breast cancer mechanisms in E2/ER-dependent and independent pathways.

The 70 kDa Heat Shock Protein Assists during the Repair of Chilling Injury in the Insect, Pyrrhocoris apterus

PubMed Central

Koštál, Vladimír; Tollarová-Borovanská, Michaela

2009-01-01

Background The Pyrrhocoris apterus (Insecta: Heteroptera) adults attain high levels of cold tolerance during their overwintering diapause. Non-diapause reproducing adults, however, lack the capacity to express a whole array of cold-tolerance adaptations and show relatively low survival when exposed to sub-zero temperatures. We assessed the competence of non-diapause males of P. apterus for responding to heat- and cold-stresses by up-regulation of 70 kDa heat shock proteins (Hsps) and the role of Hsps during repair of heat- and cold-induced injury. Principal Findings The fragments of P. apterus homologues of Hsp70 inducible (PaHsp70) and cognate forms (PaHsc70) were cloned and sequenced. The abundance of mRNA transcripts for the inducible form (qPCR) and corresponding protein (Western blotting) were significantly up-regulated in response to high and low temperature stimuli. In the cognate form, mRNA was slightly up-regulated in response to both stressors but very low or no up-regulation of protein was apparent after heat- or cold-stress, respectively. Injection of 695 bp-long Pahsp70 dsRNA (RNAi) caused drastic suppression of the heat- and cold-stress-induced Pahsp70 mRNA response and the up-regulation of corresponding protein was practically eliminated. Our RNAi predictably prevented recovery from heat shock and, in addition, negatively influenced repair of chilling injuries caused by cold stress. Cold tolerance increased when the insects were first exposed to a mild heat shock, in order to trigger the up-regulation of PaHsp70, and subsequently exposed to cold stress. Conclusion Our results suggest that accumulation of PaHsp70 belongs to a complex cold tolerance adaptation in the insect Pyrrhocoris apterus. PMID:19229329

Induction of the plasticity-associated immediate early gene Arc by stress and hallucinogens: role of brain-derived neurotrophic factor.

PubMed

Benekareddy, Madhurima; Nair, Amrita R; Dias, Brian G; Suri, Deepika; Autry, Anita E; Monteggia, Lisa M; Vaidya, Vidita A

2013-03-01

Exposure to stress and hallucinogens in adulthood evokes persistent alterations in neurocircuitry and emotional behaviour. The structural and functional changes induced by stress and hallucinogen exposure are thought to involve transcriptional alterations in specific effector immediate early genes. The immediate early gene, activity regulated cytoskeletal-associated protein (Arc), is important for both activity and experience dependent plasticity. We sought to examine whether trophic factor signalling through brain-derived neurotrophic factor (BDNF) contributes to the neocortical regulation of Arc mRNA in response to distinct stimuli such as immobilization stress and the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI). Acute exposure to either immobilization stress or DOI induced Arc mRNA levels within the neocortex. BDNF infusion into the neocortex led to a robust up-regulation of local Arc transcript expression. Further, baseline Arc mRNA expression in the neocortex was significantly decreased in inducible BDNF knockout mice with an adult-onset, forebrain specific BDNF loss. The induction of Arc mRNA levels in response to both acute immobilization stress or a single administration of DOI was significantly attenuated in the inducible BDNF knockout mice. Taken together, our results implicate trophic factor signalling through BDNF in the regulation of cortical Arc mRNA expression, both under baseline conditions and following stress and hallucinogen exposure. These findings suggest the possibility that the regulation of Arc expression via BDNF provides a molecular substrate for the structural and synaptic plasticity observed following stimuli such as stress and hallucinogens.

Uridine 5'-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia

PubMed

Santoso; Thornburg

1998-02-01

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5'-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.

Dietary betaine supplementation in hens modulates hypothalamic expression of cholesterol metabolic genes in F1 cockerels through modification of DNA methylation.

PubMed

Idriss, Abdulrahman A; Hu, Yun; Hou, Zhen; Hu, Yan; Sun, Qinwei; Omer, Nagmeldin A; Abobaker, Halima; Ni, Yingdong; Zhao, Ruqian

2018-03-01

Betaine is widely used in animal nutrition to promote growth, development and methyl donor during methionine metabolism through nutritional reprogramming via regulation of gene expression. Prenatal betaine exposure is reported to modulate hypothalamic cholesterol metabolism in chickens, yet it remains unknown whether feeding hens with betaine-supplemented diet may affect hypothalamic cholesterol metabolism in F1 offspring. In this study, hens were fed with basal or betaine-supplemented (0.5%) for 30days, and the eggs were collected for incubation. The hatchlings were raised under the same condition up to 56days of age. Betaine-treated group showed significantly (P<0.05) higher plasma concentration of total cholesterol and HDL-cholesterol, together with increased hypothalamic content of total cholesterol and cholesterol ester. Concordantly, hypothalamic gene expression of SREBP2, HMGCR, and LDLR was significantly up regulated (P<0.05). Also, mRNA abundances of SREBP1, ACAT1 and APO-A1 were up-regulated, while that of CYP46A1 was significantly down-regulated (P<0.05). These changes coincided with a significant down-regulation of BDNF and CRH, and a significant up-regulation of NPY mRNA expression. Moreover, genes involved in methyl transfer cycle were also modulated. DNMT1 and BHMT were up-regulated (P<0.05) at both mRNA and protein levels, which was associated with significant modifications of CpG methylation on the promoter of SREBP-1, SREBP-2 and APO-A1 genes as detected by bisulfate sequencing. These results indicate that feeding betaine to hens modulates hypothalamic expression of genes involved in cholesterol metabolism and brain functions in F1 cockerels with modification of promoter DNA methylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Dietary choline regulates antibacterial activity, inflammatory response and barrier function in the gills of grass carp (Ctenopharyngodon idella).

PubMed

Zhao, Hua-Fu; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Wu, Pei; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

2016-05-01

An 8-week feeding trial was conducted to determine the effects of graded levels of choline (197-1795 mg/kg) on antibacterial properties, inflammatory status and barrier function in the gills of grass carp. The results showed that optimal dietary choline supplementation significantly improved lysozyme and acid phosphatase activities, complement component 3 (C3) content, and the liver expressed antimicrobial peptide 2 and Hepcidin mRNA levels in the gills of fish (P < 0.05). In addition, appropriate dietary choline significantly decreased the oxidative damage, which might be partly due to increase copper, zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) activities and increased glutathione content in the gills of fish (P < 0.05). Moreover, appropriate dietary choline significantly up-regulated the mRNA levels of interleukin 10 and transforming growth factor β1, Zonula occludens 1, Occludin, Claudin-b, c, 3 and 12, inhibitor of κBα, target of rapamycin, Cu/Zn-SOD, CAT, GR, GPx, GST and NF-E2-related factor 2 in the gills of fish (P < 0.05). Conversely, appropriate dietary choline significantly down-regulated the mRNA levels of pro-inflammatory cytokines, tumor necrosis factor α, interleukin 8, interferon γ, interleukin 1β, and related signaling factors, nuclear factor kappa B p65, IκB kinase β, IκB kinase γ, myosin light chain kinase and Kelch-like-ECH-associated protein 1a (Keap1a) in the gills of fish (P < 0.05). However, choline did not have a significant effect on the mRNA levels of IκB kinase α, Claudin-15 and Keap1b in the gills of fish. Collectively, appropriate dietary choline levels improved gill antibacterial properties and relative gene expression levels of tight junction proteins, and decreased inflammatory status, as well as up-regulated the mRNA levels of related signaling molecules in the gills of fish. Based on gill C3 content and AHR activity, the dietary choline requirements for young grass carp (266.5-787.1 g) were estimated to be 1191.0 and 1555.0 mg/kg diet, respectively. Copyright © 2016. Published by Elsevier Ltd.

Drug-activated multiple pathways of defensin mRNA regulation in HL-60 cells are defined by reversed roles of participating protein kinases.

PubMed

Herwig, S; Su, Q; Tempst, P

1998-10-01

Defensin transcription in HL-60 promyelocytic leukemia cells is greatly enhanced during retinoic acid (RA)-induced differentiation. We have probed this regulatory pathway by selective modulation of various kinase activities. Induction was potentiated by elevated cAMP and attenuated by protein kinase C inhibition, entirely correlated to enhanced or blocked morphological differentiation, respectively. Yet, defensin mRNA was also induced in undifferentiated HL-60 cells, but not in others, by cAMP alone. By contrast, modulators that cooperated with RA had adverse effects on the normal capacity of dimethyl sulfoxide to up regulate these transcripts as well. Thus, defensin mRNA accumulation can be selectively uncoupled from maturation stage; and transcript levels may be regulated by multiple pathways, each independently acted upon by different chemical inducers.

GnRH mRNA levels in male three-spined sticklebacks, Gasterosteus aculeatus, under different reproductive conditions.

PubMed

Shao, Yi Ta; Tseng, Yung Che; Chang, Chia-Hao; Yan, Hong Young; Hwang, Pung Pung; Borg, Bertil

2015-02-01

In vertebrates, reproduction is regulated by the brain-pituitary-gonad (BPG) axis, where the gonadotropin-releasing hormone (GnRH) is one of the key components. However, very little is known about the possible role of GnRH in the environmental and feedback control of fish reproduction. To investigate this, full-length gnrh2 (chicken GnRH II) and gnrh3 (salmon GnRH) sequences of male three-spined sticklebacks (Gasterosteus aculeatus), which are clustered with the taxa of the same GnRH type as other Euteleostei, were cloned and annotated. gnrh1 is absent in this species. The mRNA levels of gnrh2 and gnrh3 in the sticklebacks' brain were measured under breeding and post-breeding conditions as well as in castrated and sham-operated breeding fish and castrated/sham-operated fish kept under long-day (LD 16:8) and short-day (LD 8:16) conditions. Fully breeding males had considerably higher mRNA levels of gnrh2 and gnrh3 in the thalamus (Th) and in the telencephalon and preoptic area (T+POA), respectively, than post-breeding males. Sham-operated breeding males have higher gnrh3 mRNA levels than the corresponding castrated males. Moreover, higher gnrh2 mRNA levels in the Th and higher gnrh3 mRNA levels in the T+POA and hypothalamus (HypTh) were also found in long-day sham-operated males than in sham-operated fish kept under an inhibitory short day photoperiod. Nevertheless, gnrh2 and gnrh3 mRNA levels were not up-regulated in castrated males kept under long-day photoperiod, which suggests that positive feedbacks on the brain-pituitary-gonad axis are necessary for this response. Copyright © 2014 Elsevier Inc. All rights reserved.

The role of leptin and ghrelin in appetite regulation in the Australian Spinifex hopping mouse, Notomys alexis, during long-term water deprivation.

PubMed

Donald, John A; Hamid, Noor Khalidah Abdul; McLeod, Janet L

2017-04-01

Water deprivation of the Spinifex hopping mouse, Notomys alexis, induced a biphasic pattern of food intake with an initial hypophagia that was followed by an increased, and then sustained food intake. The mice lost approximately 20% of their body mass and there was a loss of white adipose tissue. Stomach ghrelin mRNA was significantly higher at day 2 of water deprivation but then returned to the same levels as water-replete (day 0) mice for the duration of the experiment. Plasma ghrelin was unaffected by water deprivation except at day 10 where it was significantly increased. Plasma leptin levels decreased at day 2 and day 5 of water deprivation, and then increased significantly by the end of the water deprivation period. Water deprivation caused a significant decrease in skeletal muscle leptin mRNA expression at days 2 and 5, but then it returned to day 0 levels by day 29. In the hypothalamus, water deprivation caused a significant up-regulation in both ghrelin and neuropeptide Y mRNA expression, respectively. In contrast, hypothalamic GHSR1a mRNA expression was significantly down-regulated. A significant increase in LepRb mRNA expression was observed at days 17 and 29 of water deprivation. This study demonstrated that the sustained food intake in N. alexis during water deprivation was uncoupled from peripheral appetite-regulating signals, and that the hypothalamus appears to play an important role in regulating food intake; this may contribute to the maintenance of fluid balance in the absence of free water. Copyright © 2016 Elsevier Inc. All rights reserved.

Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice

PubMed Central

Buckley, Jill; Willingham, Emily; Agras, Koray; Baskin, Laurence S

2006-01-01

Background Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels. Methods We gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors α and β, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females), we measured the lengths of the casts and performed ANOVA analysis on these data. Results Our morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias) and masculinizing females (longer urethras). Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen α and β, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor α mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor α and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl estradiol. Conclusion The results suggest that vinclozolin virilizes females and directly or indirectly affects progesterone receptor expression. It also affects estrogen receptor expression in a sex-based manner. We found no in vivo effect of vinclozolin on androgen receptor expression. We propose that vinclozolin, which has been designated an anti-androgen, may also exert its effects by involving additional steroid-signaling pathways. PMID:16504050

Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice.

PubMed

Buckley, Jill; Willingham, Emily; Agras, Koray; Baskin, Laurence S

2006-02-21

Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels. We gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors alpha and beta, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females), we measured the lengths of the casts and performed ANOVA analysis on these data. Our morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias) and masculinizing females (longer urethras). Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen alpha and beta, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor alpha mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor alpha and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl estradiol. The results suggest that vinclozolin virilizes females and directly or indirectly affects progesterone receptor expression. It also affects estrogen receptor expression in a sex-based manner. We found no in vivo effect of vinclozolin on androgen receptor expression. We propose that vinclozolin, which has been designated an anti-androgen, may also exert its effects by involving additional steroid-signaling pathways.

Corticosteroids reduce IL-6 in ASM cells via up-regulation of MKP-1.

PubMed

Quante, Timo; Ng, Yee Ching; Ramsay, Emma E; Henness, Sheridan; Allen, Jodi C; Parmentier, Johannes; Ge, Qi; Ammit, Alaina J

2008-08-01

The mechanisms by which corticosteroids reduce airway inflammation are not completely understood. Traditionally, corticosteroids were thought to inhibit cytokines exclusively at the transcriptional level. Our recent evidence, obtained in airway smooth muscle (ASM), no longer supports this view. We have found that corticosteroids do not act at the transcriptional level to reduce TNF-alpha-induced IL-6 gene expression. Rather, corticosteroids inhibit TNF-alpha-induced IL-6 secretion by reducing the stability of the IL-6 mRNA transcript. TNF-alpha-induced IL-6 mRNA decays at a significantly faster rate in ASM cells pretreated with the corticosteroid dexamethasone (t(1/2) = 2.4 h), compared to vehicle (t(1/2) = 9.0 h; P < 0.05) (results are expressed as decay constants [k] [mean +/- SEM] and half-life [h]). Interestingly, the underlying mechanism of inhibition by corticosteroids is via the up-regulation of an endogenous mitogen-activated protein kinase (MAPK) inhibitor, MAPK phosphatase-1 (MKP-1). Corticosteroids rapidly up-regulate MKP-1 in a time-dependent manner (44.6 +/- 10.5-fold increase after 24 h treatment with dexamethasone; P < 0.05), and MKP-1 up-regulation was temporally related to the inhibition of TNF-alpha-induced p38 MAPK phosphorylation. Moreover, TNF-alpha acts via a p38 MAPK-dependent pathway to stabilize the IL-6 mRNA transcript (TNF-alpha, t(1/2) = 9.6 h; SB203580 + TNF-alpha, t(1/2) = 1.5 h), exogenous expression of MKP-1 significantly inhibits TNF-alpha-induced IL-6 secretion and MKP-1 siRNA reverses the inhibition of TNF-alpha-induced IL-6 secretion by dexamethasone. Taken together, these results suggest that corticosteroid-induced MKP-1 contributes to the repression of IL-6 secretion in ASM cells.

Impact of DBP on histology and expression of HSP 70 in gill and liver tissue of Cyprinus carpio.

PubMed

Agus, Hizlan H; Erkmen, Belda; Sümer, Sibel; Sepici-Dinçel, Aylin; Erkoç, Figen

2015-09-01

Di-n-butyl phthalate (DBP) widely used plasticizer in the plastic industry, affects regulation of the endocrine system and causes toxicity in animals. In the present study, the aim was to study the toxic effects/damages of DBP exposure using Hsp70 levels and histopathological changes in Carp liver and gill. Hsp70 expression levels were assessed as specific biomarker of in vivo ecotoxicological stress. Carp (Cyprinus carpio) were exposed to sub-lethal concentration of DBP (di-n-butyl phthalate, 1 mg/L) for 4, 24 and 96 h. Gill and liver tissues were evaluated histopathologically and RNA quantifications for Hsp70 expression levels were carried out using a two-step real-time RT-PCR. In liver, a rapid but non-significant increase in mRNA levels in the first 4 h was observed. mRNA levels significantly increased up to 2-3 fold after 24 and 96 h (p < 0.05). However, irregular mRNA level changes were also recorded: Gill specific and time-dependent regulation of Hsp70 expression were 4-5 fold inhibition after 4 and 24 h (p < 0.05), then increased up to 4 fold after 96 h (p < 0.05). Histopathological findings support altered transcription results as: Epithelial lifting, hyperplasia, fusion of secondary lamellae, telangiectasis, passive hyperemia and hydropic degeneration. Significant alterations of Hsp70 levels were likely due to a tissue specific response against chemical stress, cellular damage and lesions due to DBP. Carp was found to be a suitable experimental model for toxicology, and Hsp70 mRNA levels are reliable, specific biomarkers.

Changes in liver PPARalpha mRNA expression in response to two levels of high-safflower-oil diets correlate with changes in adiposity and serum leptin in rats and mice.

PubMed

Hsu, Shan-Ching; Huang, Ching-jang

2007-02-01

The ligand-dependent transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) is known to be activated by common fatty acids and to regulate the expression of genes of various lipid oxidation pathways and transport. High-fat diets provide more fatty acids, which presumably could enhance lipid catabolism through up-regulation of PPARalpha signaling. However, high intake of fat could also lead to obesity. To examine PPARalpha signaling in high-fat feeding and obesity, this study examined the hepatic mRNA expression of PPARalpha and some of its target genes in Wistar rats and C57BL/6J mice fed two levels (20% or 30% wt/wt) of high-safflower-oil (SFO; oleic-acid-rich) diets until animals showed significantly higher body weight (13 weeks for rats and 22 weeks for mice) than those of control groups fed a 5% SFO diet. At the end of these respective feeding periods, only the rats fed 30% SFO and the mice fed 20% SFO among the two groups fed high-fat diets showed significantly higher body weight, white adipose tissue weight, serum leptin and mRNA expression of PPARalpha (P<.05) compared to the respective control groups. Despite elevated acyl-CoA (a PPARalpha target gene) protein and activity in both groups fed high-fat diets, the mRNA expression level of most PPARalpha target genes examined correlated mainly to PPARalpha mRNA levels and not to fat intake or liver lipid levels. The observation that the liver PPARalpha mRNA expression in groups fed high-fat diets was significantly higher only in obese animals with elevated serum leptin implied that obesity and associated hyperleptinemia might have a stronger impact than dietary SFO intake per se on PPARalpha-regulated mRNA expression in the liver.

Effects of antihistamine on up-regulation of histamine H1 receptor mRNA in the nasal mucosa of patients with pollinosis induced by controlled cedar pollen challenge in an environmental exposure unit.

PubMed

Kitamura, Yoshiaki; Nakagawa, Hideyuki; Fujii, Tatsuya; Sakoda, Takema; Enomoto, Tadao; Mizuguchi, Hiroyuki; Fukui, Hiroyuki; Takeda, Noriaki

2015-11-01

In the present study, we examined the effects of antihistamine on the up-regulation of H1R mRNA in the nasal mucosa of patients with pollinosis induced by controlled exposure to pollen using an environmental exposure unit. Out of 20 patients, we designated 14 responders, whose levels of H1R mRNA in the nasal mucosa were increased after the first pollen exposure and excluded 6 non-responders. Accordingly, the first exposure to pollen without treatment significantly induced both nasal symptoms and the up-regulation of H1R mRNA in the nasal mucosa of the responders. Subsequently, prophylactic administration of antihistamine prior to the second pollen exposure significantly inhibited both of the above effects in the responders. Moreover, the nasal expression of H1R mRNA before the second pollen exposure in the responders pretreated with antihistamine was significantly decreased, as compared with that before the first pollen exposure without treatment. These findings suggest that antihistamines suppressed histamine-induced transcriptional activation of H1R gene in the nasal mucosa, in addition to their blocking effect against histamine on H1R, resulting in a decrease of nasal symptoms. These findings further suggest that by their inverse agonistic activity, antihistamines suppress the basal transcription of nasal H1R in the absence of histamine in responders. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

C/EBPα regulates CRL4Cdt2-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint

PubMed Central

Hall, Jonathan R; Bereman, Michael S; Nepomuceno, Angelito I; Thompson, Elizabeth A; Muddiman, David C; Smart, Robert C

2014-01-01

The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4Cdt2. Cdt2 is the substrate recognition subunit of CRL4Cdt2 and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21. PMID:25483090

Identification of CRASH, a gene deregulated in gynecological tumors.

PubMed

Evtimova, Vesna; Zeillinger, Robert; Kaul, Sepp; Weidle, Ulrich H

2004-01-01

We have identified CRASH, a human asparaginase-like protein which is composed of 308 amino acids and exhibits 32% homology to human aspartylglucosaminadase at the amino acid level. Database analysis revealed that the gene corresponding to CRASH is composed of 7 exons and 6 introns. Steady-state level of CRASH mRNA was found to be increased in 5 cell lines derived from metastatic lesions compared with 2 cell lines derived from primary mammary carcinoma and HMEC (human mammary epithelial cells). We found that the mRNA level of CRASH correlates with the metastatic propensity of several isogenic human colon cancer and pancreatic carcinoma cell lines. CRASH corresponds to a recently identified sperm autoantigen and furthermore we have demonstrated inducibility of CRASH mRNA by androgen and progesterone. Investigation of several types of human cancers and their corresponding normal tissues revealed high levels of CRASH mRNA in uterine, mammary and ovarian tumors compared with the corresponding normal tissues. CRASH mRNA expression was analysed in breast cancer samples with disclosed clinico-pathological features and corresponding normal tissues. The levels of CRASH mRNA were significantly up-regulated in tumors compared with normal breast tissues and correlate with lack of estrogen receptor expression of the tumors.

[Effects of Electroacupunctrue Combined with Dietary Control on Peroxisome Proliferator-activa- ted Receptor-α, and Liver Fatty Acid-binding Protein Levels in Non-alcoholic Fatty Liver Disease Rats].

PubMed

Zhang, Yi; Tang, Cheng-lin; Tian, Yuan; Yuan, Hai-zhou; Yang, Hui; Tang, Nian-zhen; Gao, Rui-qi; Cao, Jing

2015-10-01

To observe the effect of electroacupunctrue (EA) intervention or EA combined with dietary control on peroxisome proliferator-activated receptor (PPAR)-α, and liver fatty acid-binding protein (L-FABP) levels in non-alcoholic fatty liver disease (NAFLD) rats, so as to reveal its mechanism underlying improvement of NAFLD. Sixty SD male rats were randomly divided into common diet (control) group (n = 10) and high-fat diet group (n = 45). The NAFLD model was established by feeding the animals with high-fat forage (HFF, including cholesterol, sodium cholate, propylthiouracil, sucrose, lard and common forage) for 5 weeks. Forty NAFLD rats were then randomized into model, EA + HFF, low-fat forage (LFF) and EA+ LFF groups (n = 10 rats in each group). EA (4 Hz/20 Hz, 3 mA) was applied to ipsilateral "Zusanli" (ST 36),"Sanyinjiao" (SP 6) and "Taichong" (LR 3) for 20 min, once daily for 4 weeks. The pathologic changes of the hepatic tissue were detected by H. E. staining. Serum total cholesterol (TC) and triglyceride (TG) contents were determined by using enzymatic methods, serum free fat acids (FFA) content was detected by colorimetry. The expression levels of PPAR-α and L-FABP protein and gene of the liver tissue were determined by Western blot and RT-PCR, respectively. H. E. staining showed that the hepatocytes presented moderate or severe bullous adipose degeneration in rats of the model group, vesicular steatosis in the EA + HFF and LFF groups, turned to almost normal but with small amount of lipid droplets in the EA + LFF group. The contents of serum TC, TG and FFA were significantly higher in the model group than in the control group (P < 0.05), and were obviously decreased in the EA + HFF, LFF and EA + LFF groups in comparison with the model group (P < 0.05). Compared to the control group, hepatic PPAR-α protein and mRNA were markedly down-regulated in the model group, and hepatic L-FABP protein and mRNA considerably up-regulated in the model group (P < 0.05). Following EA intervention and low fat diet feeding, PPAR-α protein and mRNA were markedly up-regulated while L-FABP protein and mRNA remarkably down-regulated in the EA + HFF, LFF and EA + LFF groups (P < 0.05). EA intervention or EA combined with dietary control can relieve hepatic pathological changes in NAFLD rats, which may be related to its effects in down-regulating blood lipid level and hepatic L-FABP protein and mRNA expression and in up-regulating PPAR-α protein and mRNA expression, and the effect of combined treatment was relatively better than simple EA or LFF treatment.

Genomic analysis of wig-1 pathways.

PubMed

Sedaghat, Yalda; Mazur, Curt; Sabripour, Mahyar; Hung, Gene; Monia, Brett P

2012-01-01

Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain. Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes, and a number of wig-1 regulated genes were identified that potentially links wig-1 various signaling pathways and diseases. Antisense oligonucleotides can effectively reduce wig-1 levels in mouse liver and brain, which results in specific changes in gene expression for pathways relevant to both the nervous system and cancer.

Genomic Analysis of wig-1 Pathways

PubMed Central

Sedaghat, Yalda; Mazur, Curt; Sabripour, Mahyar; Hung, Gene; Monia, Brett P.

2012-01-01

Background Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain. Methods and Results Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes, and a number of wig-1 regulated genes were identified that potentially links wig-1 various signaling pathways and diseases. Conclusion Antisense oligonucleotides can effectively reduce wig-1 levels in mouse liver and brain, which results in specific changes in gene expression for pathways relevant to both the nervous system and cancer. PMID:22347364

MicroRNA networks in mouse lung organogenesis.

PubMed

Dong, Jie; Jiang, Guoqian; Asmann, Yan W; Tomaszek, Sandra; Jen, Jin; Kislinger, Thomas; Wigle, Dennis A

2010-05-26

MicroRNAs (miRNAs) are known to be important regulators of both organ development and tumorigenesis. MiRNA networks and their regulation of messenger RNA (mRNA) translation and protein expression in specific biological processes are poorly understood. We explored the dynamic regulation of miRNAs in mouse lung organogenesis. Comprehensive miRNA and mRNA profiling was performed encompassing all recognized stages of lung development beginning at embryonic day 12 and continuing to adulthood. We analyzed the expression patterns of dynamically regulated miRNAs and mRNAs using a number of statistical and computational approaches, and in an integrated manner with protein levels from an existing mass-spectrometry derived protein database for lung development. In total, 117 statistically significant miRNAs were dynamically regulated during mouse lung organogenesis and clustered into distinct temporal expression patterns. 11,220 mRNA probes were also shown to be dynamically regulated and clustered into distinct temporal expression patterns, with 3 major patterns accounting for 75% of all probes. 3,067 direct miRNA-mRNA correlation pairs were identified involving 37 miRNAs. Two defined correlation patterns were observed upon integration with protein data: 1) increased levels of specific miRNAs directly correlating with downregulation of predicted mRNA targets; and 2) increased levels of specific miRNAs directly correlating with downregulation of translated target proteins without detectable changes in mRNA levels. Of 1345 proteins analyzed, 55% appeared to be regulated in this manner with a direct correlation between miRNA and protein level, but without detectable change in mRNA levels. Systematic analysis of microRNA, mRNA, and protein levels over the time course of lung organogenesis demonstrates dynamic regulation and reveals 2 distinct patterns of miRNA-mRNA interaction. The translation of target proteins affected by miRNAs independent of changes in mRNA level appears to be a prominent mechanism of developmental regulation in lung organogenesis.

Protective role for ovarian glutathione S-transferase isoform pi during 7,12-dimethylbenz[a]anthracene-induced ovotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

2012-04-15

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all developmental stages. This study investigated a role for the glutathione S-transferase (Gst) isoforms alpha (a), mu (m) and pi (p) and the transcription factors, Ahr and Nrf2, during DMBA-induced ovotoxicity, and their regulation by phosphatidylinositol-3 kinase (PI3K) signaling. Negative regulation of JNK by GSTP during DMBA exposure was also studied. Post-natal day (PND) 4 Fischer 344 rat ovaries were exposed to vehicle control (1% DMSO) ± DMBA (1 μM) or vehicle control (1% DMSO) ± LY294002 (PI3K inhibitor; 20 μM) for 1, 2, 4, or 6 days. Total RNA or protein was isolated,more » followed by RT-PCR or Western blotting to determine mRNA or protein level, respectively. Immunoprecipitation using an anti-GSTP antibody was performed to determine interaction between GSTP and JNK, followed by Western blotting to determine JNK and p-c-Jun protein level. DMBA had no impact on Gsta, Gstm or Nrf2 mRNA level, but increased Gstp mRNA and protein after 2 days. Ahr mRNA and protein increased after 2 and 4 days of DMBA exposure, respectively and DMBA increased NRF2 protein level after 4 days. JNK bound to GSTP was increased during DMBA exposure, with a concomitant decrease in unbound JNK and p-c-Jun. Ahr and Gstp mRNA were decreased (2 days) and increased (4 days) by PI3K inhibition, while Gstm mRNA increased (P < 0.05) after both time points, and there was no effect on Nrf2 mRNA. PI3K inhibition increased AHR, NRF2 and GSTP protein level. These findings support involvement of ovarian GSTP during DMBA exposure, and indicate a regulatory role for the PI3K signaling pathway on ovarian xenobiotic metabolism gene expression. -- Highlights: ► Ovarian GSTP is activated in response to DMBA exposure. ► AhR and Nrf2 transcription factors are up-regulated by DMBA. ► PI3K signaling regulates Ahr, Nrf2 and Gstp expression. ► GSTP negatively regulates ovarian JNK in response to DMBA exposure.« less

Differential neonatal imprinting and regulation by estrogen of estrogen receptor subtypes alpha and beta and of the truncated estrogen receptor product (TERP-1) mRNA expression in the male rat pituitary.

PubMed

Tena-Sempere, M; Barreiro, M L; González, L C; Pinilla, L; Aguilar, E

2001-11-01

Two distinct nuclear estrogen receptors (ERs) have been identified, the classical one, renamed ERalpha, and the more recently cloned ERbeta. In a variety of tissues, gene expression of both receptor subtypes results in the generation of multiple transcripts encoding the full-length as well as several alternately spliced isoforms. In the rat pituitary, a truncated, tissue-specific variant of ERalpha, called TERP-1, has been identified and found able to modulate ERalpha and ERbeta activity. So far, its pattern of expression and hormonal regulation have been mostly studied in females. The present study was designed to analyze the pattern of expression of TERP-1 mRNA in the male rat pituitary at different stages of postnatal development, and to evaluate the impact of neonatal imprinting and estrogen treatment upon TERP-1 expression in the male pituitary. Assessment of TERP-1 mRNA levels by semi-quantitative RT-PCR, using a variant-specific primer pair, revealed that TERP-1 is also expressed in the male rat pituitary. Relative mRNA expression levels changed markedly during postnatal development, with moderate expression of the TERP-1 transcript at birth, barely detectable levels during the infantile-prepubertal period, and maximal values in adulthood. Expression of TERP-1 was sensitive to neonatal estrogen exposure, which resulted in a significant, persistent increase in mRNA levels from the infantile period until puberty. This phenomenon was not mimicked by neonatal blockade of endogenous GnRH. In addition, estrogen was able to acutely up-regulate pituitary TERP-1 mRNA expression levels in prepubertal (30-day-old) and adult (75-day-old) males. Interestingly, neonatal imprinting as well as acute estrogen treatment resulted in opposite effects on TERP-1 and full-length ERalpha and ERbeta transcripts, the latter being decreased under both conditions. In conclusion, our data indicate that TERP-1 mRNA is expressed in a developmentally regulated manner in the male rat pituitary, and is affected by neonatal estrogen imprinting and acute estrogen treatment. Regulation of TERP-1 expression by neonatal or acute estrogen treatment may thus represent an additional tuning mechanism for estrogen actions in the male rat pituitary. Copyright 2001 S. Karger AG, Basel

Impact of fasting followed by short-term exposure to interleukin-6 on cytochrome P450 mRNA in mice.

PubMed

Rasmussen, Martin Krøyer; Bertholdt, Lærke; Gudiksen, Anders; Pilegaard, Henriette; Knudsen, Jakob G

2018-01-05

The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification. Copyright © 2017 Elsevier B.V. All rights reserved.

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells.

PubMed

Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Van Seuningen, Isabelle; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

2017-03-06

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3'UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3 -/- mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally.

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells

PubMed Central

Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Seuningen, Isabelle Van; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

2017-01-01

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3′UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3−/− mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally. PMID:28262838

Effect of lipoic acid on paraoxonase-1 and paraoxonase-3 protein levels, mRNA expression and arylesterase activity in liver hepatoma cells.

PubMed

Ozgun, Eray; Sayilan Ozgun, Gulben; Tabakcioglu, Kiymet; Suer Gokmen, Selma; Sut, Necdet; Eskiocak, Sevgi

2017-10-01

Paraoxonase-1 (PON1) and PON3 (PON3) are anti-atherosclerotic enzymes, synthesized primarily in liver and bound to HDL in circulation. The aim of the present study was to investigate the effects of therapeutic doses of lipoic acid on PON1 and PON3 protein levels, mRNA expression and arylesterase activity in liver. We treated HepG2 cells with 10, 40 and 200 μM lipoic acid for 72 h. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. PON1 and PON3 protein levels were measured by Western blotting, their mRNA expression was measured by quantitative PCR and arylesterase activity was measured spectrophotometrically. 200 µM lipoic acid caused a significant increase on PON1 and PON3 protein levels and arylesterase activity as compared with control, 10 µM and 40 µM lipoic acid-treated cells. 200 µM lipoic acid also caused a significant decrease on PON1 mRNA expression whereas on a significant increase PON3 mRNA expression as compared with control, 10 µM and 40 µM lipoic acid-treated cells. Our study showed that although lipoic acid up-regulates PON3 but down-regulates PON1 mRNA expression, it increases both PON1 and PON3 protein levels and arylesterase activity in HepG2 cells. We can report that lipoic acid may be useful for preventing atherosclerosis at therapeutic doses.

TREM-1 SNP rs2234246 regulates TREM-1 protein and mRNA levels and is associated with plasma levels of L-selectin

PubMed Central

Aldasoro Arguinano, Alex-Ander; Dadé, Sébastien; Stathopoulou, Maria; Derive, Marc; Coumba Ndiaye, Ndeye; Xie, Ting; Masson, Christine; Gibot, Sébastien

2017-01-01

High levels of TREM-1 are associated with cardiovascular and inflammatory diseases risks and the most recent studies have showed that TREM-1 deletion or blockade is associated with up to 60% reduction of the development of atherosclerosis. So far, it is unknown whether the levels of TREM-1 protein are genetically regulated. Moreover, TREM family receptors have been suggested to regulate the cellular adhesion process. The goal of this study was to investigate whether polymorphisms within TREM-1 are regulating the variants of serum TREM-1 levels and the expression levels of their mRNA. Furthermore, we aimed to point out associations between polymorphisms on TREM-1 and blood levels of selectins. Among the 10 SNPs studied, the minor allele T of rs2234246, was associated with increased sTREM-1 in the discovery population (p-value = 0.003), explaining 33% of its variance, and with increased levels of mRNA (p-value = 0.007). The same allele was associated with increased soluble L-selectin levels (p-value = 0.011). The higher levels of sTREM-1 and L-selectin were confirmed in the replication population (p-value = 0.0007 and p-value = 0.018 respectively). We demonstrated for the first time one SNP on TREM-1, affecting its expression levels. These novel results, support the hypothesis that TREM-1 affects monocytes extravasation and accumulation processes leading to atherogenesis and atherosclerotic plaque progression, possibly through increased inflammation and subsequent higher expression of sL-selectin. PMID:28771614

Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia1

PubMed Central

Santoso, Djoko; Thornburg, Robert

1998-01-01

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5′-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells. PMID:9490773

Lifestyle intervention up-regulates gene and protein levels of molecules involved in insulin signaling in the endometrium of overweight/obese women with polycystic ovary syndrome.

PubMed

Ujvari, D; Hulchiy, M; Calaby, A; Nybacka, Å; Byström, B; Hirschberg, A L

2014-07-01

Does lifestyle intervention aiming at weight loss influence endometrial insulin signaling in overweight/obese women with polycystic ovary syndrome (PCOS)? Lifestyle intervention up-regulates, both at the mRNA and protein levels, components of insulin signaling in the endometrium of overweight/obese PCOS women, in relation to an improved menstrual pattern. PCOS is a multifactorial endocrine disorder diagnosed by two of the following three criteria: chronic anovulation, hyperandrogenism and polycystic ovaries. Many women with PCOS also have insulin resistance and obesity. The syndrome is furthermore associated with endometrial cancer and possible alterations in endometrial function and receptivity. This study assessed the effects of a combined diet and exercise lifestyle intervention for 3 months. A group of 20 overweight/obese PCOS women with anovulation, hyperandrogenism and polycystic ovaries were subjected to a combined diet and exercise program for 3 months. Ten body mass index (BMI)-matched regularly menstruating overweight/obese controls, nine normal-weight PCOS women and ten normal-weight controls were also included in the study. In an academic clinical setting, women were examined in mid-follicular phase for endocrine assessment and determination of endometrial levels of mRNA and immunohistochemical staining of insulin signaling molecules (the insulin receptor, insulin receptor substrate-1 (IRS1) and glucose transporter (GLUT) 1 and 4). Women with PCOS exhibited lower levels of IRS1 (P < 0.01) and GLUT4 (P < 0.01) mRNA in their proliferative endometrium than BMI-matched controls. After lifestyle intervention, weight loss averaged 4.7% and the menstrual pattern improved in 65% of the overweight/obese women with PCOS. Levels of IRS1 (P < 0.01) and GLUT1 (P < 0.05) mRNA were significantly up-regulated in the endometrium of those women with improved menstrual function, as were the protein expression levels of pY612IRS1 (the activated IRS1 form, P < 0.05), pS312IRS1 (the inhibitory form of IRS1, P < 0.05) and GLUT1 (P < 0.05). Improvement in the menstrual function of women in the obese/overweight group following the lifestyle intervention was positively correlated with the increase in the endometrial level of IRS1 mRNA (r = 0.63, P < 0.01) and negatively correlated with the change in BMI (r = -0.50, P < 0.05). The number of women in each group was limited, although the power calculation indicated that the number of patients subjected to the lifestyle intervention was sufficient. We propose that up-regulation of endometrial IRS1 and GLUT1 in overweight/obese women with PCOS following lifestyle intervention improves the glucose homeostasis and thereby restores the functioning of the endometrium in these women. This study was supported financially by the Swedish Research Council (A.L.H., 20324), Karolinska Institutet and the Stockholm County Council. None of the authors has any conflict of interest to declare.

5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability.

PubMed

Lee, Jee Hoon; Kim, Hyunmi; Woo, Joo Hong; Joe, Eun-hye; Jou, Ilo

2012-02-18

The peroxisome proliferator-activated receptor (PPAR)-α activator, 5,8,11,14-eicosatetraynoic acid (ETYA), is an arachidonic acid analog. It is reported to inhibit up-regulation of pro-inflammatory genes; however, its underlying mechanism of action is largely unknown. In the present study, we focused on the inhibitory action of ETYA on the expression of the chemokine, CCL2/MCP-1, which plays a key role in the initiation and progression of inflammation. To determine the effect of ETYA, primary cultured rat astrocytes and microglia were stimulated with IFN-γ in the presence of ETYA and then, expression of CCL2/MCP-1 and MAPK phosphatase (MKP-1) were determined using RT-PCR and ELISA. MKP-1 mRNA stability was evaluated by treating actinomycin D. The effect of MKP-1 and human antigen R (HuR) was analyzed by using specific siRNA transfection system. The localization of HuR was analyzed by immunocytochemistry and subcellular fractionation experiment. We found that ETYA suppressed CCL2/MCP-1 transcription and secretion of CCL2/MCP-1 protein through up-regulation of MKP-1mRNA levels, resulting in suppression of c-Jun N-terminal kinase (JNK) phosphorylation and activator protein 1 (AP1) activity in IFN-γ-stimulated brain glial cells. Moreover, these effects of ETYA were independent of PPAR-α. Experiments using actinomycin D revealed that the ETYA-induced increase in MKP-1 mRNA levels reflected an increase in transcript stability. Knockdown experiments using small interfering RNA demonstrated that this increase in MKP-1 mRNA stability depended on HuR, an RNA-binding protein known to promote enhanced mRNA stability. Furthermore, ETYA-induced, HuR-mediated mRNA stabilization resulted from HuR-MKP-1 nucleocytoplasmic translocation, which served to protect MKP-1 mRNA from the mRNA degradation machinery. ETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism revealed here suggests eicosanoids as potential therapeutic modulators of inflammation that act through a novel target.

Estrogen-related receptor {alpha} modulates the expression of adipogenesis-related genes during adipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ijichi, Nobuhiro; Ikeda, Kazuhiro; Horie-Inoue, Kuniko

2007-07-06

Estrogen-related receptor {alpha} (ERR{alpha}) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in fatty acid oxidation and mitochondrial biogenesis in brown adipose tissue. However, the physiological role of ERR{alpha} in adipogenesis and white adipose tissue development has not been well studied. Here, we show that ERR{alpha} and ERR{alpha}-related transcriptional coactivators, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) coactivator-1{alpha} (PGC-1{alpha}) and PGC-1{beta}, can be up-regulated in 3T3-L1 preadipocytes at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. Gene knockdown by ERR{alpha}-specific siRNA results in mRNA down-regulation of fatty acidmore » binding protein 4, PPAR{gamma}, and PGC-1{alpha} in 3T3-L1 cells in the adipogenesis medium. ERR{alpha} and PGC-1{beta} mRNA expression can be also up-regulated in another preadipocyte lineage DFAT-D1 cells and a pluripotent mesenchymal cell line C3H10T1/2 under the differentiation condition. Furthermore, stable expression of ERR{alpha} in 3T3-L1 cells up-regulates adipogenic marker genes and promotes triglyceride accumulation during 3T3-L1 differentiation. These results suggest that ERR{alpha} may play a critical role in adipocyte differentiation by modulating the expression of various adipogenesis-related genes.« less

L-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway.

PubMed

Tanaka, Yuma; Shimada, Masaya; Nagaoka, Satoshi

2014-02-14

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that L-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that L-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the L-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling. Copyright © 2014 Elsevier Inc. All rights reserved.

Stronger activation of SREBP-1a by nucleus-localized HBx

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Qi; Qiao, Ling; Yang, Jian

2015-05-08

We previously showed that hepatitis B virus (HBV) X protein activates the sterol regulatory element-binding protein-1a (SREBP-1a). Here we examined the role of nuclear localization of HBx in this process. In comparison to the wild-type and cytoplasmic HBx, nuclear HBx had stronger effects on SREBP-1a and fatty acid synthase transcription activation, intracellular lipid accumulation and cell proliferation. Furthermore, nuclear HBx could activate HBV enhancer I/X promoter and was more effective on up-regulating HBV mRNA level in the context of HBV replication than the wild-type HBx, while the cytoplasmic HBx had no effect. Our results demonstrate the functional significance of themore » nucleus-localized HBx in regulating host lipogenic pathway and HBV replication. - Highlights: • Nuclear HBx is more effective on activating SREBP-1a and FASN transcription. • Nuclear HBx is more effective on enhancing intracellular lipid accumulation. • Nuclear HBx is more effective on enhancing cell proliferation. • Nuclear HBx up-regulates HBV enhancer I/X promoter activity. • Nuclear HBx increases HBV mRNA level in the context of HBV replication.« less

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

PubMed

Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

2001-12-21

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

Impact of exogenous lipase supplementation on growth, intestinal function, mucosal immune and physical barrier, and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella).

PubMed

Liu, Sen; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Wu, Pei; Zeng, Yun-Yun; Xu, Shu-De; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2016-08-01

This study investigated the effects of exogenous lipase supplementation on the growth performance, intestinal growth and function, immune response and physical barrier function, and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella). A total of 450 grass carp (255.02 ± 0.34 g) were fed five diets for 60 days. There were 5 dietary treatments that included a normal protein and lipid diet containing 30% crude protein (CP) with 5% ether extract (EE), and the low-protein and high-lipid diets (28% CP, 6% EE) supplemented with graded levels of exogenous lipase supplementation activity at 0, 1193, 2560 and 3730 U/kg diet. The results indicated that compared with a normal protein and lipid diet (30% CP, 5% EE), a low-protein and high-lipid diet (28% CP, 6% EE) (un-supplemented lipase) improved lysozyme activities and complement component 3 contents in the distal intestine (DI), interleukin 10 mRNA expression in the proximal intestine (PI), and glutathione S-transferases activity and glutathione content in the intestine of young grass carp. In addition, in low-protein and high-lipid diets, optimal exogenous lipase supplementation significantly increased acid phosphatase (ACP) activities and complement component 3 (C3) contents (P < 0.05), up-regulated the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and hepcidin) and anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1) and signaling molecules inhibitor protein-κBα (IκBα) and target of rapamycin (TOR) (P < 0.05), down-regulated the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1β), and signaling molecules (nuclear factor kappa B p65, IκB kinase β, IκB kinase γ) (P < 0.05) in the intestine of young grass carp. Moreover, optimal exogenous lipase supplementation significantly decreased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents (P < 0.05), improved the activities of anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR), glutathione content, and the activities and mRNA levels of antioxidant enzymes (copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase) (P < 0.05), up-regulated signaling molecule NF-E2-related factor 2 (Nrf2) (P < 0.05), down-regulated signaling molecules (Kelch-like-ECH-associated protein 1a, Kelch-like-ECH-associated protein 1b) (P < 0.05) in the intestine of young grass carp. Furthermore, optimal exogenous lipase supplementation significantly elevated the mRNA levels of tight junction proteins (Occludin, zonula occludens 1, Claudin b, Claudin c and Claudin 3) (P < 0.05), down-regulated the mRNA levels of tight junction proteins (Claudin 12 and Claudin 15a) (P < 0.05), down-regulated signaling molecules myosin light chain kinase (P < 0.05) in the intestine of young grass carp. In conclusion, dietary lipid could partially spare protein, and the low-protein and high-lipid diet could improve growth, intestinal growth and function, immune response and antioxidant capability of fish. Meanwhile, in high-fat and low-protein diets, optimal exogenous lipase supplementation improved growth, intestinal growth and function, intestinal immunity, physical barrier, and regulated the mRNA expression of related signal molecules of fish. The optimal level of exogenous lipase supplementation in young grass carp (255-771 g) was estimated to be 1193 U kg(-1) diet. Copyright © 2016. Published by Elsevier Ltd.

Mechanisms of Neuroprotection from Hypoxia-Ischemia (HI) Brain Injury by Up-regulation of Cytoglobin (CYGB) in a Neonatal Rat Model*

PubMed Central

Tian, Shu-Feng; Yang, Han-Hua; Xiao, Dan-Ping; Huang, Yue-Jun; He, Gu-Yu; Ma, Hai-Ran; Xia, Fang; Shi, Xue-Chuan

2013-01-01

This study was designed to investigate the expression profile of CYGB, its potential neuroprotective function, and underlying molecular mechanisms using a model of neonatal hypoxia-ischemia (HI) brain injury. Cygb mRNA and protein expression were evaluated within the first 36 h after the HI model was induced using RT-PCR and Western blotting. Cygb mRNA expression was increased at 18 h in a time-dependent manner, and its level of protein expression increased progressively in 24 h. To verify the neuroprotective effect of CYGB, a gene transfection technique was employed. Cygb cDNA and shRNA delivery adenovirus systems were established (Cygb-cDNA-ADV and Cygb-shRNA-ADV, respectively) and injected into the brains of 3-day-old rats 4 days before they were induced with HI treatment. Rats from different groups were euthanized 24 h post-HI, and brain samples were harvested. 2,3,5-Triphenyltetrazolium chloride, TUNEL, and Nissl staining indicated that an up-regulation of CYGB resulted in reduced acute brain injury. The superoxide dismutase level was found to be dependent on expression of CYGB. The Morris water maze test in 28-day-old rats demonstrated that CYGB expression was associated with improvement of long term cognitive impairment. Studies also demonstrated that CYGB can up-regulate mRNA and protein levels of VEGF and increase both the density and diameter of the microvessels but inhibits activation of caspase-2 and -3. Thus, this is the first in vivo study focusing on the neuroprotective role of CYGB. The reduction of neonatal HI injury by CYGB may be due in part to antioxidant and antiapoptotic mechanisms and by promoting angiogenesis. PMID:23585565

Stress- and Rho-activated ZO-1–associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival

PubMed Central

Nie, Mei; Balda, Maria S.; Matter, Karl

2012-01-01

A central component of the cellular stress response is p21WAF1/CIP1, which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1–associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB’s activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3′-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival. PMID:22711822

Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma messenger ribonucleic acid levels.

PubMed

Yoshiga, Daigo; Sato, Naoichi; Torisu, Takehiro; Mori, Hiroyuki; Yoshida, Ryoko; Nakamura, Seiji; Takaesu, Giichi; Kobayashi, Takashi; Yoshimura, Akihiko

2007-05-01

Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by activating their receptor tyrosine kinase. SH2-B is an adaptor protein containing pleckstrin homology and Src homology 2 (SH2) domains that have been implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic genes including peroxisome proliferator-activated receptor gamma (PPARgamma) were reduced in white adipose tissue of SH2-B-/- mice. Reduced adipocyte differentiation of SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response to insulin and dexamethasone, whereas retroviral SH2-B overexpression enhanced differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced mRNA level of PPARgamma in 3T3-L1 cells, whereas PPARgamma levels were reduced in SH2-B-deficient MEFs in response to insulin. SH2-B-mediated up-regulation of PPARgamma mRNA was blocked by a phosphatidylinositol 3-kinase inhibitor, but not by a MAPK kinase inhibitor. Insulin-induced Akt activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a negative regulator of PPARgamma transcription, were up-regulated by SH2-B overexpression, but reduced in SH2-B-deficient MEFs. These data indicate that SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-Akt-Foxo1-PPARgamma pathway.

Shift from posttranscriptional to predominant transcriptional control of the expression of the GM-CSF gene during activation of human Jurkat cells.

PubMed

Razanajaona, D; Maroc, C; Lopez, M; Mannoni, P; Gabert, J

1992-05-01

The expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is differentially regulated in various cell types. We investigated the mechanisms controlling its expression in 12-O-tetradecanoylphorbol-13-acetate plus phytohemagglutinin-stimulated Jurkat cells, a human T-cell line. In unstimulated cells, GM-CSF mRNA was undetectable by Northern blot. Upon activation, it was detected from 3 h onward, with a progressive increase in the levels of the transcript up to 24 h of stimulation. Whereas cycloheximide treatment at the time of stimulation blocked mRNA induction, its addition at later times resulted in a marked increase in transcript levels. Run-on analysis showed that transcription of the GM-CSF gene was low to undetectable in unstimulated cells; stimulation led to transcriptional activation, which was weak at 6 h but had increased 16-fold at 24 h. In addition, the mRNA half-life decreased during activation, from 2.5 h at 6 h down to 45 min at 24 h. Cycloheximide treatment increased GM-CSF mRNA half-life (3- and 4-fold, respectively). Our results show: (a) both transcriptional and posttranscriptional signals regulate GM-CSF mRNA levels in activated Jurkat cells, (b) de novo protein synthesis is required for mRNA induction, whereas destabilizing labile proteins control the transcript stability, and (c) a shift from a posttranscriptional to a predominant transcriptional control of GM-CSF gene expression occurs during activation.

Identification, Characterization, and Functional Validation of Drought-responsive MicroRNAs in Subtropical Maize Inbreds

PubMed Central

Aravind, Jayaraman; Rinku, Sharma; Pooja, Banduni; Shikha, Mittal; Kaliyugam, Shiriga; Mallikarjuna, Mallana Gowdra; Kumar, Arun; Rao, Atmakuri Ramakrishna; Nepolean, Thirunavukkarasu

2017-01-01

MicroRNA-mediated gene regulation plays a crucial role in controlling drought tolerance. In the present investigation, 13 drought-associated miRNA families consisting of 65 members and regulating 42 unique target mRNAs were identified from drought-associated microarray expression data in maize and were subjected to structural and functional characterization. The largest number of members (14) was found in the zma-miR166 and zma-miR395 families, with several targets. However, zma-miR160, zma-miR390, zma-miR393, and zma-miR2275 each showed a single target. Twenty-three major drought-responsive cis-regulatory elements were found in the upstream regions of miRNAs. Many drought-related transcription factors, such as GAMYB, HD-Zip III, and NAC, were associated with the target mRNAs. Furthermore, two contrasting subtropical maize genotypes (tolerant: HKI-1532 and sensitive: V-372) were used to understand the miRNA-assisted regulation of target mRNA under drought stress. Approximately 35 and 31% of miRNAs were up-regulated in HKI-1532 and V-372, respectively. The up-regulation of target mRNAs was as high as 14.2% in HKI-1532 but was only 2.38% in V-372. The expression patterns of miRNA-target mRNA pairs were classified into four different types: Type I- up-regulation, Type II- down-regulation, Type III- neutral regulation, and Type IV- opposite regulation. HKI-1532 displayed 46 Type I, 13 Type II, and 23 Type III patterns, whereas V-372 had mostly Type IV interactions (151). A low level of negative regulations of miRNA associated with a higher level of mRNA activity in the tolerant genotype helped to maintain crucial biological functions such as ABA signaling, the auxin response pathway, the light-responsive pathway and endosperm expression under stress conditions, thereby leading to drought tolerance. Our study identified candidate miRNAs and mRNAs operating in important pathways under drought stress conditions, and these candidates will be useful in the development of drought-tolerant maize hybrids. PMID:28626466

Gemfibrozil and fenofibrate, Food and Drug Administration-approved lipid-lowering drugs, up-regulate tripeptidyl-peptidase 1 in brain cells via peroxisome proliferator-activated receptor α: implications for late infantile Batten disease therapy.

PubMed

Ghosh, Arunava; Corbett, Grant T; Gonzalez, Frank J; Pahan, Kalipada

2012-11-09

The classical late infantile neuronal ceroid lipofuscinosis (LINCLs) is an autosomal recessive disease, where the defective gene is Cln2, encoding tripeptidyl-peptidase I (TPP1). At the molecular level, LINCL is caused by accumulation of autofluorescent storage materials in neurons and other cell types. Currently, there is no established treatment for this fatal disease. This study reveals a novel use of gemfibrozil and fenofibrate, Food and Drug Administration-approved lipid-lowering drugs, in up-regulating TPP1 in brain cells. Both gemfibrozil and fenofibrate up-regulated mRNA, protein, and enzymatic activity of TPP1 in primary mouse neurons and astrocytes as well as human astrocytes and neuronal cells. Because gemfibrozil and fenofibrate are known to activate peroxisome proliferator-activated receptor-α (PPARα), the role of PPARα in gemfibrozil- and fenofibrate-mediated up-regulation of TPP1 was investigated revealing that both drugs up-regulated TPP1 mRNA, protein, and enzymatic activity both in vitro and in vivo in wild type (WT) and PPARβ(-/-), but not PPARα(-/-), mice. In an attempt to delineate the mechanism of TPP1 up-regulation, it was found that the effects of the fibrate drugs were abrogated in the absence of retinoid X receptor-α (RXRα), a molecule known to form a heterodimer with PPARα. Accordingly, all-trans-retinoic acid, alone or together with gemfibrozil, up-regulated TPP1. Co-immunoprecipitation and ChIP studies revealed the formation of a PPARα/RXRα heterodimer and binding of the heterodimer to an RXR-binding site on the Cln2 promoter. Together, this study demonstrates a unique mechanism for the up-regulation of TPP1 by fibrate drugs via PPARα/RXRα pathway.

Gemfibrozil and Fenofibrate, Food and Drug Administration-approved Lipid-lowering Drugs, Up-regulate Tripeptidyl-peptidase 1 in Brain Cells via Peroxisome Proliferator-activated Receptor α

PubMed Central

Ghosh, Arunava; Corbett, Grant T.; Gonzalez, Frank J.; Pahan, Kalipada

2012-01-01

The classical late infantile neuronal ceroid lipofuscinosis (LINCLs) is an autosomal recessive disease, where the defective gene is Cln2, encoding tripeptidyl-peptidase I (TPP1). At the molecular level, LINCL is caused by accumulation of autofluorescent storage materials in neurons and other cell types. Currently, there is no established treatment for this fatal disease. This study reveals a novel use of gemfibrozil and fenofibrate, Food and Drug Administration-approved lipid-lowering drugs, in up-regulating TPP1 in brain cells. Both gemfibrozil and fenofibrate up-regulated mRNA, protein, and enzymatic activity of TPP1 in primary mouse neurons and astrocytes as well as human astrocytes and neuronal cells. Because gemfibrozil and fenofibrate are known to activate peroxisome proliferator-activated receptor-α (PPARα), the role of PPARα in gemfibrozil- and fenofibrate-mediated up-regulation of TPP1 was investigated revealing that both drugs up-regulated TPP1 mRNA, protein, and enzymatic activity both in vitro and in vivo in wild type (WT) and PPARβ−/−, but not PPARα−/−, mice. In an attempt to delineate the mechanism of TPP1 up-regulation, it was found that the effects of the fibrate drugs were abrogated in the absence of retinoid X receptor-α (RXRα), a molecule known to form a heterodimer with PPARα. Accordingly, all-trans-retinoic acid, alone or together with gemfibrozil, up-regulated TPP1. Co-immunoprecipitation and ChIP studies revealed the formation of a PPARα/RXRα heterodimer and binding of the heterodimer to an RXR-binding site on the Cln2 promoter. Together, this study demonstrates a unique mechanism for the up-regulation of TPP1 by fibrate drugs via PPARα/RXRα pathway. PMID:22989886

Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

NASA Astrophysics Data System (ADS)

Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

2015-10-01

Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

Phloretin enhances adipocyte differentiation and adiponectin expression in 3T3-L1 cells.

PubMed

Hassan, Meryl; El Yazidi, Claire; Landrier, Jean-François; Lairon, Denis; Margotat, Alain; Amiot, Marie-Josèphe

2007-09-14

Adipocyte dysfunction is strongly associated with the development of cardiovascular risk factors and diabetes. It is accepted that the regulation of adipogenesis or adipokines expression, notably adiponectin, is able to prevent these disorders. In this report, we show that phloretin, a dietary flavonoid, enhances 3T3-L1 adipocyte differentiation as evidenced by increased triglyceride accumulation and GPDH activity. At a molecular level, mRNA expression levels of both PPARgamma and C/EBPalpha, the master adipogenic transcription factors, are markedly increased by phloretin. Moreover, mRNA levels of PPARgamma target genes such as LPL, aP2, CD36 and LXRalpha are up-regulated by phloretin. We also show that phloretin enhances the expression and secretion of adiponectin. Co-transfection studies suggest the induction of PPARgamma transcriptional activity as a possible mechanism underlying the phloretin-mediated effects. Taken together, these results suggest that phloretin may be beneficial for reducing insulin resistance through its potency to regulate adipocyte differentiation and function.

Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1.

PubMed

Song, Chun-Li; Liu, Bin; Diao, Hong-Ying; Shi, Yong-Feng; Zhang, Ji-Chang; Li, Yang-Xue; Liu, Ning; Yu, Yun-Peng; Wang, Guan; Wang, Jin-Peng; Li, Qian

2016-06-28

Insulin-like growth factor-1 (IGF-1) is an important regulator of cardiomyocyte homeostasis and cardiac structure, and the prosurvival and antiapoptotic effects of IGF-1 have been investigated. However, the effect of microRNA-320 (miR-320) in ischemia and reperfusion (I/R) by targeting IGF-1 is rarely discussed. We investigated the role of miR-320 in I/R injury. A total of 192 healthy female Wistar rats were divided into eight groups (n = 24). Rat heart I/R model was established. Hemodynamics, infarct size weight (ISW), heart function, and rat cardiomyocyte apoptosis were measured. Hypoxia-reoxygenation (H/R) in rat cardiomyocyte was used to simulate the I/R process. The mRNA levels of miR-320 and IGF-1, and proteins levels of IGF-1, IGF-1R, p-IGF-1R, p-ASK1, p-JNK, p-p38, Bcl-2, Bax and Caspase-3 were measured. In vivo inhibition of miR-320 expression significantly increased IGF-1 and IGF-1R mRNA levels, elevated the absolute values of SBP, DBP, MAP, ± dp/dtmax, LVEF and LVFS, decreased ISW, LVESD and LVEDd and the number of TUNEL positive cells, lowered the levels of p-ASK1, p-JNK, p-p38, Bax and Caspase-3 and increased expression of Bcl-2 compared to the I/R + NC group. Compared to H/R + NC group in vitro, miR-320 inhibition increased IGF-1 mRNA levels, inhibited cardiomyocyte apoptosis, down-regulated p-ASK, p-JNK, p-p38, Bax and Caspase-3 levels, and up-regulated Bcl-2 level. MiR-320 inhibition target elevated IGF-1 mRNA and protein levels, suppress early cardiomyocyte apoptosis of I/R, and inhibited ASK1-JNK/p38 pathway, which provides a new target for clinical study of I/R injury.

Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1

PubMed Central

Song, Chun-Li; Liu, Bin; Diao, Hong-Ying; Shi, Yong-Feng; Zhang, Ji-Chang; Li, Yang-Xue; Liu, Ning; Yu, Yun-Peng; Wang, Guan; Wang, Jin-Peng; Li, Qian

2016-01-01

Insulin-like growth factor-1 (IGF-1) is an important regulator of cardiomyocyte homeostasis and cardiac structure, and the prosurvival and antiapoptotic effects of IGF-1 have been investigated. However, the effect of microRNA-320 (miR-320) in ischemia and reperfusion (I/R) by targeting IGF-1 is rarely discussed. We investigated the role of miR-320 in I/R injury. A total of 192 healthy female Wistar rats were divided into eight groups (n = 24). Rat heart I/R model was established. Hemodynamics, infarct size weight (ISW), heart function, and rat cardiomyocyte apoptosis were measured. Hypoxia-reoxygenation (H/R) in rat cardiomyocyte was used to simulate the I/R process. The mRNA levels of miR-320 and IGF-1, and proteins levels of IGF-1, IGF-1R, p-IGF-1R, p-ASK1, p-JNK, p-p38, Bcl-2, Bax and Caspase-3 were measured. In vivo inhibition of miR-320 expression significantly increased IGF-1 and IGF-1R mRNA levels, elevated the absolute values of SBP, DBP, MAP, ± dp/dtmax, LVEF and LVFS, decreased ISW, LVESD and LVEDd and the number of TUNEL positive cells, lowered the levels of p-ASK1, p-JNK, p-p38, Bax and Caspase-3 and increased expression of Bcl-2 compared to the I/R + NC group. Compared to H/R + NC group in vitro, miR-320 inhibition increased IGF-1 mRNA levels, inhibited cardiomyocyte apoptosis, down-regulated p-ASK, p-JNK, p-p38, Bax and Caspase-3 levels, and up-regulated Bcl-2 level. MiR-320 inhibition target elevated IGF-1 mRNA and protein levels, suppress early cardiomyocyte apoptosis of I/R, and inhibited ASK1-JNK/p38 pathway, which provides a new target for clinical study of I/R injury. PMID:27175593

Low intracellular iron increases the stability of matriptase-2.

PubMed

Zhao, Ningning; Nizzi, Christopher P; Anderson, Sheila A; Wang, Jiaohong; Ueno, Akiko; Tsukamoto, Hidekazu; Eisenstein, Richard S; Enns, Caroline A; Zhang, An-Sheng

2015-02-13

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Low Intracellular Iron Increases the Stability of Matriptase-2*

PubMed Central

Zhao, Ningning; Nizzi, Christopher P.; Anderson, Sheila A.; Wang, Jiaohong; Ueno, Akiko; Tsukamoto, Hidekazu; Eisenstein, Richard S.; Enns, Caroline A.; Zhang, An-Sheng

2015-01-01

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation. PMID:25550162

Mechanical tensile stress effects on the expression of bone sialoprotein in bovine cementoblasts.

PubMed

Yu, Hongyou; Ren, Yijin; Sandham, Andrew; Ren, Aishu; Huang, Lan; Bai, Ding

2009-03-01

To develop a new cementoblast culture method and to detect bone sialoprotein (BSP) expression in response to high and low mechanical tensile stress in cementoblast in vitro. Cementoblasts were collected from the roots of newborn bovine teeth and were identified with cementum-derived attachment protein (CAP) antibody 3G9. Cell proliferation was evaluated by MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, and mineralization was confirmed by von Kossa staining. Mechanical tensile stress was applied in vitro to the cementoblast with the use of a uniaxial four-point bending system with 2000 or 4000 microstrains, at a frequency of 0.5 Hz for 3, 6, 12, 24, or 36 hours. BSP mRNA level was quantified by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). A large amount of cementoblast was observed to be expressing CAP. Cementoblasts had a proliferation tendency similar to that of osteoblasts but different from that of periodontal ligament (PDL) cells. Cementoblasts had the ability to become mineralized between osteoblasts and PDL cells. The mechanical tensile stress significantly up-regulated BSP mRNA expression, which reached a peak at 24 hours in both 2000 and 4000 microstrain groups (P < .01) and was tenfold and sixfold higher than that of controls, respectively. BSP expression dropped toward baseline levels at 36 hours in both groups. Mechanical tensile stress up-regulated the expression of BSP. Low mechanical tensile stress induced earlier and more intensive up-regulation of BSP mRNA; this might represent the optimal stimuli for cementoblast activity.

l-Arginine Attenuates Cardiac Dysfunction, But Further Down-Regulates α-Myosin Heavy Chain Expression in Isoproterenol-Induced Cardiomyopathy.

PubMed

Kralova, Eva; Doka, Gabriel; Pivackova, Lenka; Srankova, Jasna; Kuracinova, Kristina; Janega, Pavol; Babal, Pavel; Klimas, Jan; Krenek, Peter

2015-10-01

In view of previously reported increased capacity for nitric oxide production, we suggested that l-arginine (ARG), the nitric oxide synthase (NOS) substrate, supplementation would improve cardiac function in isoproterenol (ISO)-induced heart failure. Male Wistar rats were treated with ISO for 8 days (5 mg/kg/day, i.p.) or vehicle. ARG was given to control (ARG) and ISO-treated (ISO+ARG) rats in water (0.4 g/kg/day). ISO administration was associated with 40% mortality, ventricular hypertrophy, decreased heart rate, left ventricular dysfunction, fibrosis and ECG signs of ischaemia. RT-PCR showed increased mRNA levels of cardiac hypertrophy marker atrial natriuretic peptide, but not BNP, decreased expression of myosin heavy chain isoform MYH6 and unaltered expression of pathological MYH7. ISO increased the protein levels of endothelial nitric oxide synthase, but at the same time it markedly up-regulated mRNA and protein levels of gp91phox, a catalytical subunit of superoxide-producing NADPH oxidase. Fibrosis was markedly increased by ISO. ARG treatment moderately ameliorated left ventricular dysfunction, but was without effect on cardiac hypertrophy and fibrosis. Combination of ISO and ARG led to a decrease in cav-1 expression, a further increase in MYH7 expression and a down-regulation of MYH6 that inversely correlated with gp91phox mRNA levels. Although ARG, at least partially, improved ISO-impaired basal left ventricular systolic function, it failed to reduce cardiac hypertrophy, fibrosis, oxidative stress and mortality. The protection of contractile performance might be related to increased capacity for nitric oxide production and the up-regulation of MYH7 which may compensate for the marked down-regulation of the major MYH6 isoform. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Experimental study on inhibitory effects of histone deacetylase inhibitor MS-275 and TSA on bladder cancer cells.

PubMed

Qu, Wei; Kang, Yin-Dong; Zhou, Mei-Sheng; Fu, Li-Li; Hua, Zhen-Hao; Wang, Li-Ming

2010-01-01

To investigate the inhibitory effect of histone deacetylase (HDAC) inhibitors (MS-275 and TSA) on T24 human bladder cancer cells in vitro, and explore the possible mechanism. The MTT assay was employed to evaluate the inhibitory effect of MS-275 and TSA on T24 cell growth. FCM was used to analyze the variation of T24 cell cycle distribution and the apoptotic ratio after T24 cells were treated with MS-275 and TSA. Histone acetylation level was detected by Western blot. mRNA expression of p21 WAF1/CIP1, cyclin A, and cyclin E was measured by FQ-PCR. Dynamic changes of Bcl-2 and bax expression were detected by FCM. MS-275 and TSA inhibited T24 cell growth in a concentration and time-dependent manner. Treatment with 4 μmol/l MS-275 or 0.4 μmol/l TSA blocked cell cycling in the G0/G1 phase and induced a significant increase in cell apoptosis. MS-275 and TSA significantly increased the level of histone acetylation, induced p21CIP1WAF1 mRNA expression, and inhibited cyclin A mRNA expression, though no significant effect was observed on cyclin E. Bcl-2 expression was down-regulated, while bax expression was up-regulated. HDAC inhibitors can block bladder cancer cell cycle in vitro and induce apoptosis. The molecular mechanism may be associated with increased level of histone acetylation, down-regulation of p21WAF1/CIP1 expression, up-regulation of cyclin A expression, and dynamic change of bcl-2 and bax expression. Copyright © 2010 Elsevier Inc. All rights reserved.

Butyric acid regulates progesterone and estradiol secretion via cAMP signaling pathway in porcine granulosa cells.

PubMed

Lu, Naisheng; Li, Mengjiao; Lei, Hulong; Jiang, Xueyuan; Tu, Weilong; Lu, Yang; Xia, Dong

2017-09-01

Butyric acid (BA), one of the short chain fatty acids (SCFAs), has positive actions on the metabolism, inflammation, etc. However, whether it influences the reproductive physiology and if so the detail mechanism involved has not yet been determined. In this study, the porcine granulosa cells (PGCs) were treated with gradient concentrations of BA. After 24h culture, 0.05mM BA significantly stimulated the progesterone (P 4 ) secretion (P<0.05), 5mM and 10mM BA significantly inhibited the P 4 secretion (P<0.05). Simultaneously, BA up-regulated the estradiol (E 2 ) secretion in a dose dependent manner, 5mM and 10mM BA significantly promoted the E 2 level (P<0.05). In addition, 10mM BA significantly promoted the G-protein-coupled receptor 41/43 mRNA (P<0.05). Interestingly, 5mM BA treatment significantly down-regulated cyclic adenosine monophosphate (cAMP) content (P<0.05), steroidogenic acute regulatory (StAR), steroidogenic factor 1 (SF1), P450scc in the mRNA and/or protein level (P<0.05), and these actions were reversed by cAMP activator forskolin (FK). Moreover, the co-treatment of 5mM BA and bupivacaine (BPC, the cAMP inhibitor) significantly accumulated the inhibition action of BPC on cAMP, the secretion of P 4 , and the abundance of StAR mRNA (P<0.05), inhibited the up-regulation of 5mM BA on the E 2 secretion (P<0.05). Further, the Global Proteome and KEGG pathway analysis found that 5mM BA significantly up-regulated the I3LM80 proteins (P<0.05), which is involved in the steroid biosynthesis signaling pathway. 5mM BA significantly decreased the F2Z5G3 protein level (P<0.05), and the cAMP signaling pathway. In conclusion, present findings for the first time demonstrated that BA could regulate the P 4 and E 2 hormone synthesis in PGCs via the cAMP signaling pathway. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Antidiabetic activity of Ganoderma lucidum polysaccharides F31 down-regulated hepatic glucose regulatory enzymes in diabetic mice.

PubMed

Xiao, Chun; Wu, Qingping; Zhang, Jumei; Xie, Yizhen; Cai, Wen; Tan, Jianbin

2017-01-20

Ganoderma lucidum (Lin Zhi) has been used to treat diabetes in Chinese folk for centuries. Our laboratory previously demonstrated that Ganoderma lucidum polysaccharides (GLPs) had hypoglycemic effects in diabetic mice. Our aim was to identify the main bioactives in GLPs and corresponding mechanism of action. Four polysaccharide-enriched fraction were isolated from GLPs and the antidiabetic activities were evaluated by type 2 diabetic mice. Fasting serum glucose (FSG), fasting serum insulin (FSI) and epididymal fat/BW ratio were measured at the end of the experiment. In liver, the mRNA levels of hepatic glucose regulatory enzymes were determined by quantitative polymerase chain reaction (qPCR) and the protein levels of phospho-AMP-activated protein kinase (p-AMPK)/AMPK were determined by western blotting test. In epididymal fat tissue, the mRNA and protein levels GLUT4, resistin, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC1) were determined by qPCR and immuno-histochemistry. The structure of polysaccharide F31 was obtained from GPC, FTIR NMR and GC-MS spectroscopy, RESULTS: F31 significantly decreased FSG (P<0.05), FSI and epididymal fat/BW ratio (P<0.01). In liver, F31 decreased the mRNA levels of hepatic glucose regulatory enzymes, and up-regulated the ratio of phospho-AMP-activated protein kinase (p-AMPK)/AMPK. In epididymal fat tissue, F31 increased the mRNA levels of GLUT4 but decreased fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC1) and resistin. Immuno-histochemistry results revealed F31 increased the protein levels of GLUT4 and decreased resistin. Data suggested that the main bioactives in GLPs was F31, which was determined to be a β-heteropolysaccharide with the weight-average molecular weight of 15.9kDa. The possible action mechanism of F31 may be associated with down-regulation of the hepatic glucose regulated enzyme mRNA levels via AMPK activation, improvement of insulin resistance and decrease of epididymal fat/BW ratio. These results strongly suggest that F31 has antidiabetic potential. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Quantitative assessment of hTERT mRNA expression in dysplastic nodules of HBV-related hepatocarcinogenesis.

PubMed

Oh, Bong-Kyeong; Kim, Young-Joo; Park, Young Nyun; Choi, Jinsub; Kim, Kyung Sik; Park, Chanil

2006-04-01

Telomerase reverse transcriptase (hTERT) is the rate-limiting determinant of telomerase, which is critical for carcinogenesis. Dysplastic nodules (DNs) appear to be preneoplastic lesions of hepatocellular carcinomas (HCCs). In this study, in order to characterize DNs, hTERT mRNA, hTERT gene dosage, and mRNA for c-myc, a transcriptional activator of hTERT were studied in human multi-step hepatocarcinogenesis. Fifty four hepatic nodules including 5 large regenerative nodules, 14 low-grade DNs, 7 high-grade DNs, 11 DNs with HCC foci and 17 HCCs, 23 livers with chronic hepatitis/cirrhosis, and 6 normal livers were examined. Transcript levels were measured by real-time quantitative RT-PCR and gene dosages by real-time PCR and Southern blotting. The hTERT mRNA levels increased with the progression of hepatocarcinogenesis, and a significant induction in the transition between low- and high-grade DNs was seen. Most high-grade DNs strongly expressed hTERT mRNA at levels similar to those of HCCs. Twenty-one percent of low-grade DNs had high levels of hTERT mRNA, up to those of high-grade DNs and there was no difference in the pathological features between low-grade DNs with and without increased hTERT mRNA levels. No correlation was found between hTERT mRNA levels, hTERT gene dosage, and c-myc mRNA levels. These results suggest that the induction of hTERT mRNA is an important early event and that its measurement by real-time quantitative RT-PCR is a useful tool to detect premalignant/malignant tendencies in hepatic nodules. However, hTERT gene dosage and c-myc expression are not the main mechanisms regulating hTERT expression in hepatocarcinogenesis.

Menisci of the rabbit knee require mechanical loading to maintain homeostasis: cyclic hydrostatic compression in vitro prevents derepression of catabolic genes.

PubMed

Natsu-Ume, Takashi; Majima, Tokifumi; Reno, Carol; Shrive, Nigel G; Frank, Cyril B; Hart, David A

2005-07-01

The purpose of this study was to examine the influence of removing menisci from their in vivo loading environment on gene expression patterns and to determine whether in vitro loading can maintain the tissues in their in vivo phenotype. Lateral and medial rabbit meniscal explants from one leg were cultured in vitro and subjected to intermittent cyclic hydrostatic pressure (CHP) of 1 MPa at 0.5 Hz for 1 min and a rest period of 14 min (4 h of culture). The contralateral menisci were incubated at atmospheric pressure for 4 h. Menisci from both legs of another set of rabbits were frozen immediately to yield time zero values reflective of in vivo mRNA levels. Total RNA was isolated from all groups and processed for reverse transcription-polymerase chain reaction analysis for a subset of relevant genes (matrix molecules, cytokines, proteinases and inhibitors, enzymes). It was found that mRNA levels for MMP-1, MMP-3, TIMPs, iNOS, COX-2, interleukin-1beta in both menisci, and interleukin-6 in medial menisci were significantly elevated in tissues cultured under nonloading conditions compared to the time zero controls. Subjecting menisci to CHP significantly prevented these increases in mRNA levels for nearly all of the indicated molecules. In contrast, there were no significant differences in mRNA levels for collagens, biglycan, MMP-13, or TIMP-4 between the time zero values and those cultured under either nonloading or loading conditions. These studies demonstrate that removing rabbit menisci from their normal in vivo mechanical environment leads to an apparent up-regulation of a subset of potent effector molecules that could mediate catabolic activities, and that in vitro CHP can largely prevent this apparent up-regulation.

[Changes of FoxP3, CD4(+)CD25(+) regulatory T cells, TLR2 and TLR9 in children with infectious mononucleosis].

PubMed

Wang, Qiang; Wang, Zuo-Feng; Cao, Mei; Wang, Zhi-Ying

2013-04-01

The aim of this study was to investigate the effects of TLR2, TLR9, CD4(+)CD25(+) regulatory T cells (Treg) and transcription factor FoxP3 in the pathogenesis of children with infectious mononucleosis (IM). Thirty-five acute IM patients admitted in our hospital from April 2010 to January 2011 were enrolled in this study. Thirty-five healthy subjects were taken as control. The thirty-five patients before treatment were considered as patients in acute stage, after treatment and without clinical symptom they were thought as patients in recovery stage. The expression levels of TLR2, TLR9 and FoxP3 mRNA were detected by real time PCR using SYBR Green I. The expression of T lymphocyte subset CD4(+)CD25(+) in peripheral blood mononuclear cells was detected by flow cytometry. The results showed that the relative levels of TLR2 mRNA (4.03 ± 0.56), TLR9 mRNA (8.88 ± 1.56) in peripheral blood mononuclear cells of IM patients in acute stage were significantly higher than those of the controls [TLR2 mRNA (2.22 ± 0.57), TLR9 mRNA (3.63 ± 1.30)] and IM patients in recovery stage [TLR2 mRNA (2.76 ± 0.83), TLR9 mRNA (5.34 ± 1.60)] (P < 0.01). The result of CD4(+)CD25(+) (2.38 ± 1.32%) and relative level of FoxP3 mRNA(2.82 ± 0.90) in peripheral blood mononuclear cells of IM patients in acute stage were lower than those of the control [CD4(+)CD25(+) (7.85 ± 1.97%), FoxP3 mRNA (4.65 ± 1.23) ] (P < 0.01). There was no significant difference in CD4(+)CD25(+) (6.81 ± 1.84%), FoxP3 mRNA(4.11 ± 1.37) levels between IM patients in recovery stage and the controls (P > 0.05). It is concluded that the expression of CD4(+)CD25(+)regulatory T cells is reduced, and its special transcription factor FoxP3 mRNA is down-regulated, but expression levels of TLR2 mRNA, TLR9 mRNA are up-regulated in IM patients of acute stage.

Cinnamaldehyde up-regulates the mRNA expression level of TRPV1 receptor potential ion channel protein and its function in primary rat DRG neurons in vitro.

PubMed

Sui, Feng; Lin, Na; Guo, Jian-You; Zhang, Chang-Bin; Du, Xin-Liang; Zhao, Bao-Sheng; Liu, Hong-Bin; Yang, Na; Li, Lan-Fang; Guo, Shu-Ying; Huo, Hai-Ru; Jiang, Ting-Liang

2010-01-01

Cinnamaldehyde (1) is a pharmacologically active ingredient isolated from cassia twig (Ramulus Cinnamomi), which is commonly used in herbal remedies to treat fever-related diseases. Both TRPV1 and TRPM8 ion channel proteins are abundantly expressed in sensory neurons, and are assumed to act as a thermosensor, with the former mediating the feeling of warmth and the latter the feeling of cold in the body. Both of them have recently been reported to be involved in thermoregulation. The purpose of this paper is to further uncover the antipyretic mechanisms of 1 by investigating its effects on the mRNA expression levels and functions of both TRPV1 and TRPM8. The results showed that 1 could up-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and its calcium-mediating function was significantly increased at 39 degrees C, all of which could not be blocked by pretreatment of the neuronal cells with ruthenium red, a general transient receptor potential (TRP) blocker, indicating that the action of 1 was achieved through a non-TRPA1 channel pathway. In conclusion, the findings in our in vitro studies might account for part of the peripheral molecular mechanisms for the antipyretic action of 1.

5-hydroxytryptamine level and 5-HT2A receptor mRNA expression in the guinea pigs eyes with spectacle lens-induced myopia

PubMed Central

Yang, Ji-Wen; Xu, Yan-Chun; Sun, Lin; Tian, Xiao-Dan

2010-01-01

AIM To investigate 5-hydroxytryptamine (5-HT) function and 5-HT receptor 2A (5-HT2A) mRNA expression in the formation of lens-induced myopia (LIM). METHODS Lens-induced myopia construction method was applied to generate myopia on guinea pig right eye (LIM eye). RESULTS LIM eyes formed significant myopia with longer axial length. 5-HT level in retina, choroids and sclera from LIM eyes was significantly higher than that in control group. 5-HT2A mRNA expression was also significantly up-regulated. CONCLUSION Refraction lens could induce myopia in guinea pig and 5-HT may play an important role in the formation of myopia by binding with 5-HT2A receptor. PMID:22553578

[Up regulation of phenylacetate to glioma homeobox gene expression].

PubMed

Tian, Yu; Yang, Chaohua; Zhao, Conghai

2002-03-01

Even though phenylacetate (PA) bas been shown to inhibit the growth and induce differentiation in rat C6 glioma cell line, its mechanisms are still poorly understood. This study is aimed to identify which Hox gene is related to glioma and to observe the change in expression on mRNA level as treated by phenylasetate. Twenty-two kinds of Hox gene were divided into 3 groups according to their primer sequence. Semiquantitative reverse transcription- polymerase chain reaction (RT-PCR) was used to investigate the mRNA expression of Hox gene groups and some Hox gene in rat C6 glioma cell line following differentiation induced by PA. The level of Hox gene expression was expressed as ratio expression rate (RER) of Hox gene/beta-actin according to computer image analysis and the difference between C6 cells and PA treated C6 cells was analyzed by student t-test. It was found that Hox genes matching to primers P2 were mildly expressed in C6 cells and the expression of HoxB2 mRNA was significantly up-regulated in PA treated C6 cells (P < 0.001). The weak expression of HoxB2 may be involved in glioma origin and the mechanisms of PA action are correlated with transcription process in the glioma cells.

Cytokine expression in response to root canal infection in gnotobiotic mice.

PubMed

Maciel, K F; Neves de Brito, L C; Tavares, W L F; Moreira, G; Nicoli, J R; Vieira, L Q; Ribeiro Sobrinho, A P

2012-04-01

To examine cytokine expression profiles during periapical lesion development in response to synergetic human pathogens in a gnotobiotic mouse model. Human strains of Fusobacterium nucleatum and Peptostreptococcus prevotii were inoculated into the root canals of germ-free mice in either mono- or bi-association. Animals were killed 7 and 14 days after infection, and periapical tissues were collected. mRNA expression of the cytokines IFN-γ, TNF-α, Receptor activator of nuclear factor kappa-B ligand (RANKL), IL-10, IL-4 and transforming growth factor β (TGF-β) was assessed using real-time PCR. Levene's test was used to assess the equality of variance of the data, whereas a t-test for independent samples was used to evaluate the significance of the differences between groups (P < 0.05). The mRNA expression of IFN-γ and TNF-α was up-regulated by F. nucleatum during the acute (day 7) and chronic phase (day 14) of periapical lesion development. However, in bi-infection the expression of IFN-γ and TNF-α were effectively absent at both time-points. RANKL mRNA expression was down-regulated during dual infection at the chronic phase. As IL-4 expression was similar at both time-points, IL-4 does not appear to be involved in the periapical response to these bacterial strains. IL-10 was up-regulated during the chronic phase by mono-infection with either F. nucleatum or P. prevotii. Dual infection increased TGF-β mRNA expression on day 7, which paralleled the decrease in IFN-γ and TNF-α mRNA levels at the same time-point. F. nucleatum increased TGF-β mRNA expression during the chronic phase. Cytokine profiles depend on the nature of the bacterial challenge. Both TGF-β and IL-10 appeared to be regulating the proinflammatory cytokine responses at both time-points of the periapical immune response. © 2012 International Endodontic Journal.

Organ-specific proteomics analysis for identification of response mechanism in soybean seedlings under flooding stress.

PubMed

Khatoon, Amana; Rehman, Shafiq; Hiraga, Susumu; Makino, Takahiro; Komatsu, Setsuko

2012-10-22

Flooding is one of the severe environmental factors which impair growth and yield in soybean plant. To investigate the organ specific response mechanism of soybean under flooding stress, changes in protein species were analyzed using a proteomics approach. Two-day-old soybeans were subjected to flooding for 5 days. Proteins were extracted from root, hypocotyl and leaf, and separated by two-dimensional polyacrylamide gel electrophoresis. In root, hypocotyl and leaf, 51, 66 and 51 protein species were significantly changed, respectively, under flooding stress. In root, metabolism related proteins were increased; however these proteins were decreased in hypocotyl and leaf. In all 3 organs, cytoplasm localized proteins were decreased, and leaf chloroplastic proteins were also decreased. Isoflavone reductase was commonly decreased at protein level in all 3 organs; however, mRNA of isoflavone reductase gene was up-regulated in leaf under flooding stress. Biophoton emission was increased in all 3 organs under flooding stress. The up-regulation of isoflavone reductase gene at transcript level; while decreased abundance at protein level indicated that flooding stress affected the mRNA translation to proteins. These results suggest that concurrence in expression of isoflavone reductase gene at mRNA and protein level along with imbalance in other disease/defense and metabolism related proteins might lead to impaired growth of root, hypocotyl and leaf of soybean seedlings under flooding stress. Copyright © 2012 Elsevier B.V. All rights reserved.

Disruption of sex-hormone levels and steroidogenic-related gene expression on Mongolia Racerunner (Eremias argus) after exposure to triadimefon and its enantiomers.

PubMed

Li, Jitong; Chang, Jing; Li, Wei; Guo, Baoyuan; Li, Jianzhong; Wang, Huili

2017-03-01

Triadimefon (TF) is a widely used chiral fungicide with one chiral centre and two enantiomers (TF 1 and TF 2 ). However, little is reported about the ecological toxicity of reptiles on an enantioselective level. TF is a potential endocrine disruptor that may interfere with sex steroid hormones, such as testosterone (T) and 17beta-estradiol (E 2 ). In our study, the lizards Mongolia Racerunner (Eremias argus) were orally exposed to TF and its enantiomers for 21 days. Plasma sex steroid hormones and steroidogenic-related genes, including 17-beta-hydroxysteroid (hsd17β), cytochrome P450 enzymes (cyp19 and cyp17), and steroid hormone receptors (erα and Ar) were evaluated. After exposure, the plasma testosterone level in the 100 mg/kg bw group was elevated, while the oestradiol level was reduced. This phenomenon may be caused by the transformation of cyp19, which may inhibit the conversion of testosterone to oestradiol and affect sexual behaviour. In addition, the two enantiomers have different effects on hormone levels, which testified to the previously reported biotoxic dissimilarity between TF 1 and TF 2 in organisms. Furthermore, the cyp19 mRNA level in liver and gonad of the TF 2 and TF group (100 mg/kg bw ) were significantly down-regulated, while the cyp17 and hsd17β mRNA levels were up-regulated. The expression of erα and Ar mRNA levels were up-regulated in males but not in females, which may indicate that TF has sex differences on these two genes. As seen from the above results, TF and its enantiomers may have endocrine-disrupting effects on lizards (E. argus) by acting sensitively on sex steroid hormones and steroidogenic-related genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Amyloid precursor protein controls cholesterol turnover needed for neuronal activity

PubMed Central

Pierrot, Nathalie; Tyteca, Donatienne; D'auria, Ludovic; Dewachter, Ilse; Gailly, Philippe; Hendrickx, Aurélie; Tasiaux, Bernadette; Haylani, Laetitia El; Muls, Nathalie; N'Kuli, Francisca; Laquerrière, Annie; Demoulin, Jean-Baptiste; Campion, Dominique; Brion, Jean-Pierre; Courtoy, Pierre J; Kienlen-Campard, Pascal; Octave, Jean-Noël

2013-01-01

Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full-length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG-CoA reductase (HMGCR)-mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co-immunoprecipitated and co-localized in the Golgi. This interaction prevented Site-2 protease-mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24-hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity. PMID:23554170

Effect of raclopride on dopamine D2 receptor mRNA expression in rat brain.

PubMed

Kopp, J; Lindefors, N; Brené, S; Hall, H; Persson, H; Sedvall, G

1992-01-01

Prolonged treatment with dopamine D2 receptor antagonists is known to elevate the density of dopamine D2 receptor binding sites in caudate-putamen and nucleus accumbens in rat and human brain. In this study we used the dopamine D2 receptor antagonist raclopride (3 mumol/kg, s.c.) to determine if a single injection or daily administration of this drug for up to 18 days changed the expression of dopamine D2 receptor mRNA in rat caudate-putamen and accumbens as measured by in situ hybridization. A single injection of raclopride did not significantly change the numerical density of dopamine D2 receptor mRNA-expressing neurons in any of the regions examined. A daily administration of raclopride for 18 days resulted in a 31% increase in the number of cells expressing detectable amounts of dopamine D2 receptor mRNA in dorsolateral caudate-putamen and in a 20% increase in the area of silver grains over individual hybridization-positive neurons in this brain region measured on emulsion-dipped slides. The region-specific increase in the D2 receptor mRNA level in dorsolateral caudate-putamen was confirmed by measurement of the hybridization signal on X-ray film autoradiograms. The levels of D2 receptor mRNA remained unchanged in medial caudate-putamen and accumbens after 18 days' treatment. The region-selective increase in dopamine D2 receptor mRNA expression in dorsolateral caudate-putamen indicates a differential regulation of dopamine D2 receptor mRNA expression in a subpopulation of caudate-putamen neurons by this neuroleptic. We suggest that the increase in dopamine D2 receptor density in caudate-putamen known to follow prolonged dopamine D2 receptor blockade to some extent is regulated at the level of gene expression.

Effects of dietary omega-3/omega-6 fatty acid ratios on reproduction in the young breeder rooster.

PubMed

Feng, Yun; Ding, Yu; Liu, Juan; Tian, Ye; Yang, Yanzhou; Guan, Shuluan; Zhang, Cheng

2015-03-21

Polyunsaturated fatty acids (PUFAs) are necessary for the body's metabolism, growth and development. Although PUFAs play an important role in the regulation of reproduction, their role in testis development in the rooster is unknown. The present study was conducted to investigate the effects of omega-3/omega-6 (n-3/n-6, PUFAs) ratios on reproductive performance in young breeder roosters. Plasma levels of reproductive hormones, testis development, and reproductive hormone receptor and StAR mRNA expression were also assessed. Although PUFAs (n-3/n-6: 1/4.15) had no significant effect on the testis index (P > 0.05), the spermatogonial development and germ cell layers were increased. Moreover, serum levels of hormones (GnRH, FSH, LH and T) on day 35 were also significantly increased by PUFAs (n-3/n-6: 1/4.15). To investigate whether PUFAs regulate the expression of hormone receptors and StAR, real time-PCR was used to measure GnRHR, FSHR, LHR and StAR mRNA levels. PUFAs significantly increased the mRNA levels of all of these genes. These results indicate that PUFAs enhance the reproductive performance of young roosters by increasing hormone secretion and function, the latter by up-regulating receptor expression. These findings provide a sound basis for a balanced n-3/n-6 PUFA ratio being beneficial to young rooster reproduction.

Regulation of insulin preRNA splicing by glucose

PubMed Central

Wang, Juehu; Shen, Luping; Najafi, Habiba; Kolberg, Janice; Matschinsky, Franz M.; Urdea, Mickey; German, Michael

1997-01-01

Glucose tightly regulates the synthesis and secretion of insulin by β cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single β cell (βTC3 cells or mouse islets), whereas 1 million non-insulin-producing α cells (αTC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from β cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up ≈2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription. PMID:9113994

Altered expression of glial markers, chemokines, and opioid receptors in the spinal cord of type 2 diabetic monkeys.

PubMed

Kiguchi, Norikazu; Ding, Huiping; Peters, Christopher M; Kock, Nancy D; Kishioka, Shiroh; Cline, J Mark; Wagner, Janice D; Ko, Mei-Chuan

2017-01-01

Neuroinflammation is a pathological condition that underlies diabetes and affects sensory processing. Given the high prevalence of pain in diabetic patients and crosstalk between chemokines and opioids, it is pivotal to know whether neuroinflammation-associated mediators are dysregulated in the central nervous system of diabetic primates. Therefore, the aim of this study was to investigate whether mRNA expression levels of glial markers, chemokines, and opioid receptors are altered in the spinal cord and thalamus of naturally occurring type 2 diabetic monkeys (n=7) compared with age-matched non-diabetic monkeys (n=6). By using RT-qPCR, we found that mRNA expression levels of both GFAP and IBA1 were up-regulated in the spinal dorsal horn (SDH) of diabetic monkeys compared with non-diabetic monkeys. Among all chemokines, expression levels of three chemokine ligand-receptor systems, i.e., CCL2-CCR2, CCL3-CCR1/5, and CCL4-CCR5, were up-regulated in the SDH of diabetic monkeys. Moreover, in the SDH, seven additional chemokine receptors, i.e., CCR4, CCR6, CCR8, CCR10, CXCR3, CXCR5, and CXCR6, were also up-regulated in diabetic monkeys. In contrast, expression levels of MOP, KOP, and DOP, but not NOP receptors, were down-regulated in the SDH of diabetic monkeys, and the thalamus had fewer changes in the glial markers, chemokines and opioids. These findings indicate that neuroinflammation, manifested as glial activation and simultaneous up-regulation of multiple chemokine ligands and receptors, seems to be permanent in type 2 diabetic monkeys. As chemokines and opioids are important pain modulators, this first-in-primate study provides a translational bridge for determining the functional efficacy of spinal drugs targeting their signaling cascades. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of Glutamine Transporter Slc38a3 (SNAT3) During Acidosis is Mediated by a Different Mechanism than Tissue-Specific Expression

PubMed Central

Balkrishna, Sarojini; Bröer, Angelika; Welford, Scott M.; Hatzoglou, Maria; Bröer, Stefan

2015-01-01

Background Despite homeostatic pH regulation, systemic and cellular pH changes take place and strongly influence metabolic processes. Transcription of the glutamine transporter SNAT3 (Slc38a3) for instance is highly up-regulated in the kidney during metabolic acidosis to provide glutamine for ammonia production. Methods Slc38a3 promoter activity and messenger RNA stability were measured in cultured cells in response to different extracellular pH values. Results Up-regulation of SNAT3 mRNA was mediated both by the stabilization of its mRNA and by the up-regulation of gene transcription. Stabilisation of the mRNA involved a pH-response element, while enhanced transcription made use of a second pH-sensitive Sp1 binding site in addition to a constitutive Sp1 binding site. Transcriptional regulation dominated the early response to acidosis, while mRNA stability was more important for chronic adaptation. Tissue-specific expression of SNAT3, by contrast, appeared to be controlled by promoter methylation and histone modifications. Conclusions Regulation of SNAT3 gene expression by extracellular pH involves post-transcriptional and transcriptional mechanisms, the latter being distinct from the mechanisms that control the tissue-specific expression of the gene. PMID:24854847

Rapamycin protects against paraquat-induced pulmonary fibrosis: Activation of Nrf2 signaling pathway.

PubMed

Xu, Yiheng; Tai, Wenlin; Qu, Xiaoyuan; Wu, Wenjuan; Li, ZhenKun; Deng, Shuhao; Vongphouttha, Chanthasone; Dong, Zhaoxing

2017-08-19

Paraquat (PQ) is a widely used herbicide indeveloping countries worldwide, and pulmonary fibrosis is one of the most typical features of PQ poisoning. The molecular mechanism of PQ toxicity especially how to treat PQ-induced pulmonary fibrosis is still largely unknown. In animal model of pulmonary fibrosis, we used HE staining, western blotting assay and Real-time PCR assay to analyze the effects of rapamycin on the PQ-induced epithelial mesenchymal transition (EMT). We found that PQ induced the pulmonary fibrosis using HE staining and Masson's staining, and up-regulated the activity of HYP and the mRNA expressions of Collagen I and III (COL-1and COL-3) in pulmonary tissues. We also found that rapamycin down-regulated the mesenchymal cell marker Vimentin and up-regulated the epithelial cell marker E-cadherin both in mRNA and protein levels compared with PQ group. And the EMT associated transcription factor Snail was decreased by rapamycin treatment compared with PQ group. And PQ decreased the Nrf2 expression both in mRNA and protein levels, and rapamycin inhibited these effects of PQ. SFN, a activator of Nrf2, could inhibit the EMT and the expression of Snail. And knockdowon of Nrf2 could abolish the inhibitory effects of rapamycin of PQ-induced EMT. In conclusion, rapamycin protects against paraquat-induced pulmonary fibrosis by activation of Nrf2 signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

PABPN1-Dependent mRNA Processing Induces Muscle Wasting

PubMed Central

Raz, Yotam; van Putten, Maaike; Paniagua-Soriano, Guillem; Krom, Yvonne D.; Florea, Bogdan I.; Raz, Vered

2016-01-01

Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3’-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting. PMID:27152426

Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading

NASA Technical Reports Server (NTRS)

Howard, G.; Steffen, J. M.; Geoghegan, T. E.

1989-01-01

The regulatory role of transcriptional alterations in unloaded skeletal muscles was investigated by determining levels of total muscle RNA and mRNA fractions in soleus, gastrocnemius, and extensor digitorum longus (EDL) of rats subjected to whole-body suspension for up to 7 days. After 7 days, total RNA and mRNA contents were lower in soleus and gastrocnemius, compared with controls, but the concentrations of both RNAs per g muscle were unaltered. Alpha-actin mRNA (assessed by dot hybridization) was significantly reduced in soleus after 1, 3, and 7 days of suspension and in gastrocnemius after 3 and 7 days, but was unchanged in EDL. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked alteration in mRNAs coding for several small proteins. Results suggest that altered transcription and availability of specific mRNAs contribute significantly to the regulation of protein synthesis during skeletal muscle unloading.

Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells

PubMed Central

Chen, Yu-Ching; Chang, Hsun-Ming; Cheng, Jung-Chien; Tsai, Horng-Der; Wu, Cheng-Hsuan; Leung, Peter C.K.

2015-01-01

STUDY QUESTION Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell–cell communication in human granulosa cells? SUMMARY ANSWER TGF-β1 up-regulates Cx43 and increases gap junction intercellular communication activities (GJIC) in human granulosa cells, and this effect occurs via the activin receptor-like kinase (ALK)5-mediated Sma- and Mad-related protein (SMAD)2/3-SMAD4-dependent pathway. WHAT IS KNOWN ALREADY TGF-β1 and its receptors are expressed in human granulosa cells, and follicular fluid contains TGF-β1 protein. In human granulosa cells, Cx43 gap junctions play an important role in the development of follicles and oocytes. STUDY DESIGN, SIZE, DURATION This is an experimental study which was performed over a 1-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing IVF in an academic research center were used as the study models. Cx43 mRNA and protein expression levels were examined after exposure of SVOG cells to recombinant human TGF-β1. An activin/TGF-β type I receptor inhibitor, SB431542, and small interfering RNAs targeting ALK4, ALK5, SMAD2, SMAD3 and SMAD4 were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. MAIN RESULTS AND THE ROLE OF CHANCE TGF-β1 treatment increased phosphorylation of SMAD2/3 (P < 0.0001) and up-regulated Cx43 mRNA and protein levels (P < 0.001) in SVOG cells and these stimulatory effects were abolished by the TGF-β type I receptor inhibitor SB431542. In addition, the up-regulatory effect of TGF-β1 on Cx43 expression (mRNA and protein) was confirmed in primary cultures of human granulosa-lutein cells (P < 0.05). The small interfering RNA-mediated knockdown of ALK5, but not ALK4, abolished the TGF-β1-induced phosphorylation of SMAD2/3 and the up-regulation of Cx43. Furthermore, knockdown of SMAD2/3 or the common SMAD, SMAD4, abolished the stimulatory effects of TGF-β1 on Cx43 expression in SVOG cells. The TGF-β1-induced up-regulation of Cx43 contributed to the increase of GJIC between SVOG cells (P < 0.001). LIMITATIONS, REASONS FOR CAUTION The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo. WIDER IMPLICATIONS OF THE FINDINGS Our studies represent the first comprehensive research of molecular mechanisms of TGF-β1 in the regulation of Cx43 expression and GJIC in human granulosa cells and demonstrate that TGF-β1 may play a crucial role in the local modulation of cell–cell communication. Deepening our understanding of the molecular determinants will offer important insights into ovarian physiology and lead to the development of potential therapeutic methods for fertility regulation. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by an operating grant from the Canadian Institutes of Health Research to P.C.K.L. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER NA. PMID:26202915

Urotensin II contributes to collagen synthesis and up-regulates Egr-1 expression in cultured pulmonary arterial smooth muscle cells through the ERK1/2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wei; Cai, Zhifeng; Liu, Mengmeng

Aim: The objective of this study was to investigate the effects of urotensin II (UII) treatment on the proliferation and collagen synthesis of cultured rat pulmonary arterial smooth muscle cells (PASMCs) and to explore whether these effects are mediated by mitogen-activated protein kinase (MAPK) signaling pathways and early growth response 1 (Egr-1). Methods: The proliferation of cultured PASMCs stimulated with different doses of UII was detected by BrdU incorporation. The mRNA expression levels of procollagen I (procol I), procollagen III (procol III), extracellular regulated protein kinase 1/2 (ERK1/2), stress-stimulated protein kinase (Sapk), p38 MAPK (p38), and Egr-1 mRNA in culturedmore » PASMCs after treatment with UII, the UII-specific antagonist urantide, and the ERK1/2 inhibitor PD98059 were detected by real-time polymerase chain reaction (PCR), and the protein expression levels of procol I, procol III, phosphorylated (p)-ERK1/2, p-Sapk, p-p38, and Egr-1 were detected by Western blotting. Results: Treatment with UII increased the proliferation of cultured PASMCs in a dose-dependent manner (P < 0.05). However, treatment with urantide and PD98059 inhibited the promoting effect of UII on PASMC proliferation (P < 0.05). Real-time PCR analysis showed that UII up-regulated the expression of procol I, procol III, ERK1/2, Sapk, and Egr-1 mRNA (P < 0.05), but not p38 mRNA. However, the up-regulating effect of UII was inhibited by PD98059 and urantide. Western blotting analysis showed that UII increased the synthesis of collagen I, collagen III, p-ERK1/2, p-Sapk, and Egr-1, and these effects also were inhibited by PD98059 and urantide (P < 0.05). Conclusions: Egr-1 participates in the UII-mediated proliferation and collagen synthesis of cultured rat PASMCs via activation of the ERK1/2 signaling pathway.« less

Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics

PubMed Central

Velazquez, J R; Lacy, P; Moqbel, R

2000-01-01

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins. PMID:10792507

A platelet-activating factor (PAF) receptor deficiency exacerbates diet-induced obesity but PAF/PAF receptor signaling does not contribute to the development of obesity-induced chronic inflammation.

PubMed

Yamaguchi, Masahiko; Matsui, Masakazu; Higa, Ryoko; Yamazaki, Yasuhiro; Ikari, Akira; Miyake, Masaki; Miwa, Masao; Ishii, Satoshi; Sugatani, Junko; Shimizu, Takao

2015-02-15

Platelet-activating factor (PAF) is a well-known phospholipid that mediates acute inflammatory responses. In the present study, we investigated whether PAF/PAF receptor signaling contributed to chronic inflammation in the white adipose tissue (WAT) of PAF receptor-knockout (PAFR-KO) mice. Body and epididymal WAT weights were higher in PAFR-KO mice fed a high-fat diet (HFD) than in wild-type (WT) mice. TNF-α mRNA expression levels in epididymal WAT and the infiltration of CD11c-positive macrophages into epididymal WAT, which led to chronic inflammation, were also elevated in HFD-fed PAFR-KO mice. HFD-fed PAFR-KO mice had higher levels of fasting serum glucose than HFD-fed WT mice as well as impaired glucose tolerance. Although PAF receptor signaling up-regulated the expression of TNF-α and lipopolysaccharide induced the expression of acyl-CoA:lysophosphatidylcholine acyltransferase 2 (LPCAT2) mRNA in bone marrow-derived macrophages, no significant differences were observed in the expression of LPCAT2 mRNA and PAF levels in epididymal WAT between HFD-fed mice and normal diet-fed mice. In addition to our previous finding in which energy expenditure in PAF receptor (PAFR)-deficient mice was low due to impaired brown adipose tissue function, the present study demonstrated that PAF/PAF receptor signaling up-regulated the expression of Ucp1 mRNA, which is essential for cellular thermogenesis, in 3T3-L1 adipocytes. We concluded that the marked accumulation of abdominal fat due to HFD feeding led to more severe chronic inflammation in WAT, which is associated with glucose metabolism disorders, in PAFR-KO mice than in WT mice, and PAF/PAF receptor signaling may regulate energy expenditure and adiposity. Copyright © 2015 Elsevier Inc. All rights reserved.

Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

USGS Publications Warehouse

Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

2002-01-01

Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl2 mRNA

PubMed Central

Otake, Yoko; Soundararajan, Sridharan; Sengupta, Tapas K.; Kio, Ebenezer A.; Smith, James C.; Pineda-Roman, Mauricio; Stuart, Robert K.; Spicer, Eleanor K.

2007-01-01

B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells that are resistant to apoptosis as a result of bcl2 oncogene overexpression. Studies were done to determine the mechanism for the up-regulation of bcl-2 protein observed in CD19+ CLL cells compared with CD19+ B cells from healthy volunteers. The 11-fold higher level of bcl-2 protein in CLL cells was positively correlated with a 26-fold elevation in the cytosolic level of nucleolin, a bcl2 mRNA–stabilizing protein. Measurements of the bcl2 heterogeneous nuclear/bcl2 mRNA (hnRNA)/mRNA ratios and the rates of bcl2 mRNA decay in cell extracts indicated that the 3-fold higher steady-state level of bcl2 mRNA in CLL cells was the result of increased bcl2 mRNA stability. Nucleolin was present throughout the nucleus and cytoplasm of CLL cells, whereas in normal B cells nucleolin was only detected in the nucleus. The addition of recombinant human nucleolin to extracts of normal B cells markedly slowed the rate of bcl2 mRNA decay. SiRNA knockdown of nucleolin in MCF-7 cells resulted in decreased levels of bcl2 mRNA and protein but no change in β-actin. These results indicate that bcl-2 overexpression in CLL cells is related to stabilization of bcl2 mRNA by nucleolin. PMID:17179226

Coupled Evolution of Transcription and mRNA Degradation

PubMed Central

Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

2011-01-01

mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins.

PubMed

Kessels, Jana Elena; Wessels, Inga; Haase, Hajo; Rink, Lothar; Uciechowski, Peter

2016-09-01

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2'-deoxycytidine (AZA) increased intracellular (after 24 and 48h) and total cellular zinc levels (after 48h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48h. MT mRNA was significantly enhanced after 24h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

Deleted in breast cancer 1 (DBC1) protein regulates hepatic gluconeogenesis.

PubMed

Nin, Veronica; Chini, Claudia C S; Escande, Carlos; Capellini, Verena; Chini, Eduardo N

2014-02-28

Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.

Up-regulation of the G3PDH 'housekeeping' gene by estrogen.

PubMed

Galal, Nadia; El-Beialy, Waleed; Deyama, Yoshiaki; Yoshimura, Yoshitaka; Tei, Kanchu; Suzuki, Kuniaki; Totsuka, Yasunori

2010-01-01

Proteomic and genomic studies commonly involve the assessment of mRNA levels using reverse transcription-polymerase chain reaction (PCR) and real-time quantitative PCR. An internal standard RNA is fundamentally analyzed along with the investigated mRNA to document the specificity of the effect(s) on mRNA and to correct for inter-sample variations. In our studies implementing estrogen treatments on different cell lines, we initially used glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as an internal standard. However, the results of PCR amplification demonstrated that 17β-estradiol enhanced the expression of the G3PDH gene, rendering it impossible to use G3PDH as an unbiased comparative control.

Sugar Allocation to Metabolic Pathways is Tightly Regulated and Affects the Virulence of Streptococcus mutans

PubMed Central

Kawada-Matsuo, Miki; Oogai, Yuichi; Komatsuzawa, Hitoshi

2016-01-01

Bacteria take up and metabolize sugar as a carbohydrate source for survival. Most bacteria can utilize many sugars, including glucose, sucrose, and galactose, as well as amino sugars, such as glucosamine and N-acetylglucosamine. After entering the cytoplasm, the sugars are mainly allocated to the glycolysis pathway (energy production) and to various bacterial component biosynthesis pathways, including the cell wall, nucleic acids and amino acids. Sugars are also utilized to produce several virulence factors, such as capsule and lipoteichoic acid. Glutamine-fructose-6-phosphate aminotransferase (GlmS) and glucosamine-6-phosphate deaminase (NagB) have crucial roles in sugar distribution to the glycolysis pathway and to cell wall biosynthesis. In Streptococcus mutans, a cariogenic pathogen, the expression levels of glmS and nagB are coordinately regulated in response to the presence or absence of amino sugars. In addition, the disruption of this regulation affects the virulence of S. mutans. The expression of nagB and glmS is regulated by NagR in S. mutans, but the precise mechanism underlying glmS regulation is not clear. In Staphylococcus aureus and Bacillus subtilis, the mRNA of glmS has ribozyme activity and undergoes self-degradation at the mRNA level. However, there is no ribozyme activity region on glmS mRNA in S. mutans. In this review article, we summarize the sugar distribution, particularly the coordinated regulation of GlmS and NagB expression, and its relationship with the virulence of S. mutans. PMID:28036052

Regulation of Egr-1, VIP, and Shh mRNA and Egr-1 protein in the mouse retina by light and image quality.

PubMed

Brand, Christine; Burkhardt, Eva; Schaeffel, Frank; Choi, Jeong Won; Feldkaemper, Marita Pauline

2005-04-28

To analyze mRNA expression changes of Egr-1, VIP, and Shh under different light and treatment conditions in mice. The mRNA expression levels of the three genes and additionally the Egr-1 protein expression were compared in form deprived eyes and eyes with normal vision. Moreover, the influence of dark to light and light to dark transitions and of changes in retinal illumination on mRNA levels was investigated. Form deprivation of mice was induced by fitting frosted diffusers over one eye and an attentuation matched neutral density (ND) filter over the other eye. To measure the effects of retinal illumination changes on mRNA expression, animals were bilaterally fitted with different ND filters. Semiquantitative real-time RT-PCR was used to measure the mRNA levels and immunohistochemistry was applied to localize and detect Egr-1 protein. The expression levels of both Egr-1 mRNA and protein were reduced in form deprived eyes compared to their fellow eyes after 30 min and 1 h, respectively. Egr-1 mRNA was strikingly upregulated both after dark to light and light to dark transitions, whereas minor changes in retinal illumination by covering the eyes with neutral density filters did not alter Egr-1 mRNA expression. In mice, the mRNA levels of VIP and Shh were not affected by form deprivation, but they were found to be regulated depending on the time of day. Both Egr-1 mRNA and protein expression levels were strongly regulated by light, especially by transitions between light and darkness. Image contrast may exert an additional influence on mRNA and protein expression of Egr-1, particularly in the cells in the ganglion cell layer and in bipolar cells.

Expression of FSH receptor in the hamster ovary during perinatal development

PubMed Central

Chakraborty, Prabuddha; Roy, Shyamal K.

2014-01-01

FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression. PMID:25462586

Ginsenoside Rg3 up-regulates the expression of vascular endothelial growth factor in human dermal papilla cells and mouse hair follicles.

PubMed

Shin, Dae Hyun; Cha, Youn Jeong; Yang, Kyeong Eun; Jang, Ik-Soon; Son, Chang-Gue; Kim, Bo Hyeon; Kim, Jung Min

2014-07-01

Crude Panax ginseng has been documented to possess hair growth activity and is widely used to treat alopecia, but the effects of ginsenoside Rg3 on hair growth have not to our knowledge been determined. The aim of the current study was to identify the molecules through which Rg3 stimulates hair growth. The thymidine incorporation for measuring cell proliferation was determined. We used DNA microarray analysis to measure gene expression levels in dermal papilla (DP) cells upon treatment with Rg3. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) in human DP cells were measured by real-time polymerase chain reaction and immunohistochemistry, respectively. We also used immunohistochemistry assays to detect in vivo changes in VEGF and 3-stemness marker expressions in mouse hair follicles. Reverse transcription polymerase chain reaction showed dose-dependent increases in VEGF mRNA levels on treatment with Rg3. Immunohistochemical analysis showed that expression of VEGF was significantly up-regulated by Rg3 in a dose-dependent manner in human DP cells and in mouse hair follicles. In addition, the CD8 and CD34 were also up-regulated by Rg3 in the mouse hair follicles. It may be concluded that Rg3 might increase hair growth through stimulation of hair follicle stem cells and it has the potential to be used in hair growth products. Copyright © 2013 John Wiley & Sons, Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murooka, Thomas T.; Rahbar, Ramtin; Department of Immunology, University of Toronto, Ont.

The proliferative capacity of cancer cells is regulated by factors intrinsic to cancer cells and by secreted factors in the microenvironment. Here, we investigated the proto-oncogenic potential of the chemokine receptor, CCR5, in MCF-7 breast cancer cell lines. At physiological levels, CCL5, a ligand for CCR5, enhanced MCF-7.CCR5 proliferation. Treatment with the mTOR inhibitor, rapamycin, inhibited this CCL5-inducible proliferation. Because mTOR directly modulates mRNA translation, we investigated whether CCL5 activation of CCR5 leads to increased translation. CCL5 induced the formation of the eIF4F translation initiation complex through an mTOR-dependent process. Indeed, CCL5 initiated mRNA translation, shown by an increase inmore » high-molecular-weight polysomes. Specifically, we show that CCL5 mediated a rapid up-regulation of protein expression for cyclin D1, c-Myc and Dad-1, without affecting their mRNA levels. Taken together, we describe a mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs, thereby providing CCR5-positive breast cancer cells with a proliferative advantage.« less

Codon-Resolution Analysis Reveals a Direct and Context-Dependent Impact of Individual Synonymous Mutations on mRNA Level

PubMed Central

Chen, Siyu; Li, Ke; Cao, Wenqing; Wang, Jia; Zhao, Tong; Huan, Qing; Yang, Yu-Fei; Wu, Shaohuan; Qian, Wenfeng

2017-01-01

Abstract Codon usage bias (CUB) refers to the observation that synonymous codons are not used equally frequently in a genome. CUB is stronger in more highly expressed genes, a phenomenon commonly explained by stronger natural selection on translational accuracy and/or efficiency among these genes. Nevertheless, this phenomenon could also occur if CUB regulates gene expression at the mRNA level, a hypothesis that has not been tested until recently. Here, we attempt to quantify the impact of synonymous mutations on mRNA level in yeast using 3,556 synonymous variants of a heterologous gene encoding green fluorescent protein (GFP) and 523 synonymous variants of an endogenous gene TDH3. We found that mRNA level was positively correlated with CUB among these synonymous variants, demonstrating a direct role of CUB in regulating transcript concentration, likely via regulating mRNA degradation rate, as our additional experiments suggested. More importantly, we quantified the effects of individual synonymous mutations on mRNA level and found them dependent on 1) CUB and 2) mRNA secondary structure, both in proximal sequence contexts. Our study reveals the pleiotropic effects of synonymous codon usage and provides an additional explanation for the well-known correlation between CUB and gene expression level. PMID:28961875

Gene specific epigenetic regulation of hepatic folate transport system is responsible for perturbed cellular folate status during aging and exogenous modulation.

PubMed

Ahmad Najar, Rauf; Rahat, Beenish; Hussain, Aashiq; Thakur, Shilpa; Kaur, Jaspreet; Kaur, Jyotdeep; Hamid, Abid

2016-06-01

The present study was designed to identify the molecular mechanism of folate modulation and aging on aberrant liver folate transporter system. An in vivo rat model was used, in which weanling, young and adult rats were given folate deficient diet for 3 and 5 months and after 3 months of folate deficiency, one group received physiological folate repletion (2 mg/kg diet) and another group received over supplemented folate diet (8 mg/kg diet) for another 2 months. In adult group, 3 and 5 months of folate deficiency decreased serum and tissue folate levels with decreased uptake of folate, further associated with decreased expression levels of reduced folate carrier (RFC) and increased expression levels of folate exporter (ABCG2) at both mRNA and protein levels, which in turn regulated by promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 gene. Promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 may be attributed to the down regulation of RFC and up regulation of ABCG2 at mRNA and protein levels in conditions of 3 and 5 months of folate deficiency in the adult group. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Type of carbohydrate in feed affects the expression of small leucine-rich proteoglycans (SLRPs), glycosaminoglycans (GAGs) and interleukins in skeletal muscle of Atlantic cod (Gadus morhua L.).

PubMed

Tingbø, M G; Pedersen, M E; Grøndahl, F; Kolset, S O; Veiseth-Kent, E; Enersen, G; Hannesson, K O

2012-09-01

Aquaculture requires feed that ensures rapid growth and healthy fish. Higher inclusion of plant ingredients is desirable, as marine resources are limited. In this study we investigated the effects of higher starch inclusion in feed on muscular extracellular matrix and interleukin expression in farmed cod. Starch was replaced by complex fibers in the low-starch diet to keep total carbohydrate inclusion similar. Blood glucose and fructosamine levels were elevated in the high-starch group. The group fed a high-starch diet showed up-regulation on mRNA level of proteoglycans biglycan and decorin. ELISA confirmed the real-time PCR results on protein level for biglycan and also showed increase of lumican. For decorin the protein levels were decreased in the high-starch group, in contrast to real-time PCR results. Disaccharide analyses using HPLC showed reduction of glycosaminoglycans. Further, there was up-regulation of interleukin-1β and -10 on mRNA level in muscle. This study shows that the muscular extracellular matrix composition is affected by diet, and that a high-starch diet results in increased expression of pro-inflammatory genes similar to diabetes in humans. Copyright © 2012 Elsevier Ltd. All rights reserved.

LIN28 expression in malignant germ cell tumors down-regulates let-7 and increases oncogene levels

PubMed Central

Murray, Matthew J.; Saini, Harpreet K.; Siegler, Charlotte A.; Hanning, Jennifer E.; Barker, Emily M.; van Dongen, Stijn; Ward, Dawn M.; Raby, Katie L.; Groves, Ian J.; Scarpini, Cinzia G.; Pett, Mark R.; Thornton, Claire M.; Enright, Anton J.; Nicholson, James C.; Coleman, Nicholas

2013-01-01

Despite their clinico-pathologic heterogeneity, malignant germ-cell-tumors (GCTs) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of down-regulation of the let-7 family of tumor-suppressor microRNAs in malignant-GCTs. Microarray results from pediatric and adult samples (n=45) showed that LIN28, the negative-regulator of let-7 biogenesis, was abundant in malignant-GCTs, regardless of patient age, tumor site or histologic subtype. Indeed, a strong negative-correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, since the sequence complementary to the 2-7nt common let-7 seed ‘GAGGUA’ was enriched in the 3′untranslated regions of mRNAs up-regulated in pediatric and adult malignant-GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were up-regulated in malignant-GCT cells, confirming significant negative-correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by qRT-PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67 and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant-GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and down-regulate MYCN, AURKB and LIN28, the latter via a double-negative feedback loop. We concluded that the LIN28/let-7 pathway has a critical pathobiological role in malignant-GCTs and therefore offers a promising target for therapeutic intervention. PMID:23774216

Up-regulation of mRNA ventricular PRNP prion protein gene expression in air pollution highly exposed young urbanites: endoplasmic reticulum stress, glucose regulated protein 78, and nanosized particles.

PubMed

Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

2013-11-28

Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.

Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

PubMed Central

Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

2013-01-01

Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role. PMID:24287918

Identification and Characterization of MYC Regulatory Elements: Links to Prostate Cancer

DTIC Science & Technology

2011-06-01

cell proliferation and growth,MYC is up-regulated at both the mRNA and protein levels in aggressive prostate cancers ( DeMarzo et al. 2003). In...evolutionary synthesis: A genetic theory of morphological evolution. Cell 134: 25–36. DeMarzo AM, Nelson WG, Isaacs WB, Epstein JI. 2003. Pathological and

The effect of prolonged treatment with imipramine on the biosynthesis and functional characteristics of D2 dopamine receptors in the rat caudate putamen

PubMed Central

Dziedzicka-Wasylewska, Marta; Rogoż, Renata

1998-01-01

The present study shows the effects of imipramine in a single dose (10 mg kg−1, p.o.) or following repeated (14 days, twice a day) treatment on the level of mRNA coding for D2 dopamine receptors in the rat caudate putamen (CP). Repeated administration of imipramine resulted in the increase of the level of mRNA coding for D2 dopamine receptors. Radioligand binding studies with the D2 receptor agonist, [3H]-N-0437, indicated, that following imipramine administration, the affinity of the agonist for the D2 dopamine receptor significantly increased, though without any alterations in the Bmax. Pharmacological manipulations (by use of forskolin, GppNHp and quinpirole) of the cyclic AMP generating system, ex vivo following administration of imipramine indicated that an up-regulation of factors inhibiting cyclic GMP formation takes place. Most probably it is the D2 dopamine receptor which undergoes functional up-regulation, resulting from the enhancement of its biosynthesis. PMID:9535010

The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

PubMed

García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

2016-05-05

We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Parvalbumin Interneurons of Central Amygdala Regulate the Negative Affective States and the Expression of Corticotrophin-Releasing Hormone During Morphine Withdrawal

PubMed Central

Wang, Li; Shen, Minjie; Jiang, Changyou

2016-01-01

Background: The central nucleus of the amygdala (CeA) is a crucial component of the neuronal circuitry mediating aversive emotion. Its role in the negative affective states during drug withdrawal includes changes in opioidergic, GABAergic, and corticotropin-releasing factor neurotransmission. However, the modulation of the neurobiological interconnectivity in the CeA and its effects in the negative reinforcement of drug dependents are poorly understood. Method: We performed electrophysiological recordings to assess the membrane excitability of parvalbumin (PV)+ interneurons in the CeA during chronic morphine withdrawal. We tested the morphine withdrawal–induced negative affective states, such as the aversive (assessed by conditioned place aversion), anxiety (assessed by elevated plus maze), and anhedonic-like (assessed by saccharin preference test) behaviors, as well as the mRNA level of corticotropin-releasing hormone (CRH) via optogenetic inhibition or activation of PV+ interneurons in the CeA. Result: Chronic morphine withdrawal increased the firing rate of CeA PV+ interneurons. Optogenetic inhibition of the activity of CeA PV+ interneurons attenuated the morphine withdrawal–induced negative affective states, such as the aversive, anxiety, and anhedonic-like behaviors, while direct activation of CeA PV+ interneurons could trigger those negative affective-like behaviors. Optogenetic inhibition of the CeA PV+ interneurons during the morphine withdrawal significantly attenuated the elevated CRH mRNA level in the CeA. Conclusion: The activity of PV+ interneurons in the CeA was up-regulated during chronic morphine withdrawal. The activation of PV+ interneurons during morphine withdrawal was crucial for the induction of the negative emotion and the up-regulation of CRH mRNA levels in the CeA. PMID:27385383

Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

PubMed Central

Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

2015-01-01

Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

Puerarin blocks the signaling transmission mediated by P2X3 in SG and DRG to relieve myocardial ischemic damage.

PubMed

Liu, Shuangmei; Zhang, Chunping; Shi, Qingming; Li, Guilin; Song, Miaomiao; Gao, Yun; Xu, Changshui; Xu, Hong; Fan, Bo; Yu, Shicheng; Zheng, Chaoran; Zhu, Qicheng; Wu, Bing; Peng, Lichao; Xiong, Huangui; Wu, Qin; Liang, Shangdong

2014-02-01

P2X₃ receptors in stellate ganglia (SG) and cervical dorsal root ganglia (DRG) neurons are involved in sympathoexcitatory reflex induced by myocardial ischemic damage. Puerarin, a major active ingredient extracted from the traditional Chinese plant medicine Ge-gen, has been widely used in treatment of myocardial and cerebral ischemia. The present study is aimed to observe the effects of puerarin on the signaling transmission mediated by P2X₃ receptor in SG and DRG after myocardial ischemic damage. Our results showed that systolic blood pressure and heart rate increased, and the expression levels of P2X₃ mRNA and protein in SG and DRG were up-regulated after myocardial ischemic damage. Puerarin reduced systolic blood pressure and heart rate, relieved pain and decreased up-regulated expression of P2X₃ mRNA and protein in SG and DRG after myocardial ischemia. Puerarin inhibited the up-regulated ATP-activated currents in DRG neurons after myocardial ischemia. Thus, puerarin can relieve myocardial ischemic damage through blocking the P2X₃ signaling transmission and then depressed the aggravated sympathoexcitatory reflex. Copyright © 2014 Elsevier Inc. All rights reserved.

Estradiol In Females May Negate Skeletal Muscle Myostatin Mrna Expression And Serum Myostatin Propeptide Levels After Eccentric Muscle Contractions

PubMed Central

Willoughby, Darryn S.; Wilborn, Colin D.

2006-01-01

Eccentric contractions produce a significant degree of inflammation and muscle injury that may increase the expression of myostatin. Due to its anti- oxidant and anti-flammatory effects, circulating 17-β estradiol (E2) may attenuate myostatin expression. Eight males and eight females performed 7 sets of 10 reps of eccentric contractions of the knee extensors at 150% 1-RM. Each female performed the eccentric exercise bout on a day that fell within her mid-luteal phase (d 21-23 of her 28-d cycle). Blood and muscle samples were obtained before and 6 and 24 h after exercise, while additional blood samples were obtained at 48 and 72 h after exercise. Serum E2 and myostatin LAP/propeptide (LAP/pro) levels were determined with ELISA, and myostatin mRNA expression determined using RT-PCR. Data were analyzed with two-way ANOVA and bivariate correlations (p < 0.05). Females had greater levels of serum E2 throughout the 72- h sampling period (p < 0.05). While males had greater body mass and fat-free mass, neither was correlated to the pre-exercise levels of myostatin mRNA and LAP/pro for either gender (p > 0.05). Compared to pre-exercise, males had significant increases (p < 0.05) in LAP/propetide and mRNA of 78% and 28%, respectively, at 24 h post-exercise, whereas females underwent respective decreases of 10% and 21%. E2 and LAP/propeptide were correlated at 6 h (r = -0.804, p = 0.016) and 24 h post- exercise (r = -0.841, p = 0.009) in males, whereas in females E2 levels were correlated to myostatin mRNA at 6 h (r =0.739, p = 0.036) and 24 h (r = 0.813, p = 0.014) post-exercise and LAP/propeptide at 6 h (r = 0.713, p = 0.047) and 24 h (r = 0.735, p = 0.038). In females, myostatin mRNA expression and serum LAP/propeptide levels do not appear to be significantly up-regulated following eccentric exercise, and may be due to higher levels of circulating E2. Key Points The pre-exercise levels of myostatin mRNA and propeptide were not significantly different between genders, and even though the total body mass and fat-free mass of males were significantly greater than females, neither was correlated to myostatin mRNA or LAP/propeptide. Myostatin mRNA expression in females is less than in males 24 h after a single bout of eccentric exercise. Myostatin LAP/propeptide levels in females are lower in females than in males 24 h after a single bout of eccentric exercise, thereby suggesting a gender-specific mechanism in which females may be less responsive to eccentric exercise than males. Myostatin mRNA expression in females is attenuated, possibly due to inhibition in myostatin signaling, and appears to be more related to the presence of a higher level of circulating E2 rather than body composition. Due to their higher level of E2, females seem to be less susceptible to the mechanism by which eccentric exercise apparently up-regulates myostatin mRNA expression in males. PMID:24357964

Effect of dietary fat and the circadian clock on the expression of brain-derived neurotrophic factor (BDNF).

PubMed

Genzer, Yoni; Dadon, Maayan; Burg, Chen; Chapnik, Nava; Froy, Oren

2016-07-15

Brain-derived neurotrophic factor (BDNF) is the most abundant neurotrophin in the brain and its decreased levels are associated with the development of obesity and neurodegeneration. Our aim was to test the effect of dietary fat, its timing and the circadian clock on the expression of BDNF and associated signaling pathways in mouse brain and liver. Bdnf mRNA oscillated robustly in brain and liver, but with a 12-h shift between the tissues. Brain and liver Bdnf mRNA showed a 12-h phase shift when fed ketogenic diet (KD) compared with high-fat diet (HFD) or low-fat diet (LFD). Brain or liver Bdnf mRNA did not show the typical phase advance usually seen under time-restricted feeding (RF). Clock knockdown in HT-4 hippocampal neurons led to 86% up-regulation of Bdnf mRNA, whereas it led to 60% down-regulation in AML-12 hepatocytes. Dietary fat in mice or cultured hepatocytes and hippocampal neurons led to increased Bdnf mRNA expression. At the protein level, HFD increased the ratio of the mature BDNF protein (mBDNF) to its precursor (proBDNF). In the liver, RF under LFD or HFD reduced the mBDNF/proBDNF ratio. In the brain, the two signaling pathways related to BDNF, mTOR and AMPK, showed reduced and increased levels, respectively, under timed HFD. In the liver, the reverse was achieved. In summary, Bdnf expression is mediated by the circadian clock and dietary fat. Although RF does not affect its expression phase, in the brain, when combined with high-fat diet, it leads to a unique metabolic state in which AMPK is activated, mTOR is down-regulated and the levels of mBDNF are high. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Elevated Hippocampal Cholinergic Neurostimulating Peptide precursor protein (HCNP-pp) mRNA in the amygdala in major depression.

PubMed

Bassi, Sabrina; Seney, Marianne L; Argibay, Pablo; Sibille, Etienne

2015-04-01

The amygdala is innervated by the cholinergic system and is involved in major depressive disorder (MDD). Evidence suggests a hyper-activate cholinergic system in MDD. Hippocampal Cholinergic Neurostimulating Peptide (HCNP) regulates acetylcholine synthesis. The aim of the present work was to investigate expression levels of HCNP-precursor protein (HCNP-pp) mRNA and other cholinergic-related genes in the postmortem amygdala of MDD patients and matched controls (females: N = 16 pairs; males: N = 12 pairs), and in the mouse unpredictable chronic mild stress (UCMS) model that induced elevated anxiety-/depressive-like behaviors (females: N = 6 pairs; males: N = 6 pairs). Results indicate an up-regulation of HCNP-pp mRNA in the amygdala of women with MDD (p < 0.0001), but not males, and of UCMS-exposed mice (males and females; p = 0.037). HCNP-pp protein levels were investigated in the human female cohort, but no difference was found. There were no differences in gene expression of acetylcholinesterase (AChE), muscarinic (mAChRs) or nicotinic receptors (nAChRs) between MDD subjects and controls or UCMS and control mice, except for an up-regulation of AChE in UCMS-exposed mice (males and females; p = 0.044). Exploratory analyses revealed a baseline expression difference of cholinergic signaling-related genes between women and men (p < 0.0001). In conclusion, elevated amygdala HCNP-pp expression may contribute to mechanisms of MDD in women, potentially independently from regulating the cholinergic system. The differential expression of genes between women and men could also contribute to the increased vulnerability of females to develop MDD. Copyright © 2015 Elsevier Ltd. All rights reserved.

Translational repression of mouse mu opioid receptor expression via leaky scanning

PubMed Central

Song, Kyu Young; Hwang, Cheol Kyu; Kim, Chun Sung; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2007-01-01

Mu opioid receptor (MOR) expression is under temporal and spatial controls, but expression levels of the MOR gene are relatively low in vivo. In addition to transcriptional regulations, upstream AUGs (uAUGs) and open reading frames (uORFs) profoundly affect the translation of the primary ORF and thus the protein levels in several genes. The 5′-untranslated region (UTR) of mouse MOR mRNA contains three uORFs preceding the MOR main initiation codon. In MOR-fused EGFP or MOR promoter/luciferase reporter constructs, mutating each uAUG individually or in combinations increased MOR transient heterologous expression in neuroblastoma NMB and HEK293 cells significantly. Translation of such constructs increased up to 3-fold without altering the mRNA levels if either the third uAUG or both the second and third AUGs were mutated. Additionally, these uAUG-mediated translational inhibitions were independent of their peptide as confirmed by internal mutation analyses in each uORF. Translational studies indicated that protein syntheses were initiated at these uAUG initiation sites, with the third uAUG initiating the highest translation level. These results support the hypothesis that uORFs in mouse MOR mRNA act as negative regulators through a ribosome leaky scanning mechanism. Such leaky scanning resulted in the suppression of mouse MOR under normal conditions. PMID:17284463

[Wnt/β-catenin pathway involved in the regulation of rat mesangial cell proliferation by adipose-derived mesenchymal stem cells].

PubMed

Li, Zhi; Zhang, Mengying; Li, Xueqin; Lu, Jinming; Xu, Liang

2016-11-01

Objective To investigate the effect of adipose-derived mesenchymal stem cells (ADSCs) on glomerular mesangial cell proliferation via Wnt/β-catenin pathway. Methods The rat glomerular mesangial cells (HBZY-1) were incubated in conditioned ADSC medium. Cell cycle was analyzed with flow cytometry; the proliferation rate of HBZY-1 and the expression levels of relative genes and proteins of Wnt signaling pathway were measured using RNA interference, quantitative real-time PCR and Western blotting, respectively. Results HBZY-1 proliferation was significantly inhibited under the action of conditioned ADSC medium, whereas dickkopf WNT signaling pathway inhibitor 1 (DKK1) mRNA level was up-regulated. Fibronectin and TGF-β1 mRNA expression as well as β-catenin and Bcl-2 protein levels of HBZY-1 were significantly down-regulated. DKK1 gene expression level in ADSCs was significantly higher than that of HBZY-1. After RNA interference, DKK1 expression level in ADSCs was markedly inhibited, yet the β-catenin protein level was notably elevated. The β-catenin and Bcl-2 protein levels of HBZY-1 were also significantly raised in HBZY-1 after cultured with conditioned medium containing ADSCs treated with RNA interference. Conclusion Wnt/β-catenin may be a potential signaling pathway involved in the regulative effect of ADSCs on glomerular mesangial cell proliferation.

Biology in the Dry Seed: Transcriptome Changes Associated with Dry Seed Dormancy and Dormancy Loss in the Arabidopsis GA-Insensitive sleepy1-2 Mutant

PubMed Central

Nelson, Sven K.; Ariizumi, Tohru; Steber, Camille M.

2017-01-01

Plant embryos can survive years in a desiccated, quiescent state within seeds. In many species, seeds are dormant and unable to germinate at maturity. They acquire the capacity to germinate through a period of dry storage called after-ripening (AR), a biological process that occurs at 5–15% moisture when most metabolic processes cease. Because stored transcripts are among the first proteins translated upon water uptake, they likely impact germination potential. Transcriptome changes associated with the increased seed dormancy of the GA-insensitive sly1-2 mutant, and with dormancy loss through long sly1-2 after-ripening (19 months) were characterized in dry seeds. The SLY1 gene was needed for proper down-regulation of translation-associated genes in mature dry seeds, and for AR up-regulation of these genes in germinating seeds. Thus, sly1-2 seed dormancy may result partly from failure to properly regulate protein translation, and partly from observed differences in transcription factor mRNA levels. Two positive regulators of seed dormancy, DELLA GAI (GA-INSENSITIVE) and the histone deacetylase HDA6/SIL1 (MODIFIERS OF SILENCING1) were strongly AR-down-regulated. These transcriptional changes appeared to be functionally relevant since loss of GAI function and application of a histone deacetylase inhibitor led to decreased sly1-2 seed dormancy. Thus, after-ripening may increase germination potential over time by reducing dormancy-promoting stored transcript levels. Differences in transcript accumulation with after-ripening correlated to differences in transcript stability, such that stable mRNAs appeared AR-up-regulated, and unstable transcripts AR-down-regulated. Thus, relative transcript levels may change with dry after-ripening partly as a consequence of differences in mRNA turnover. PMID:29312402

Hepcidin regulation in wild-type and Hfe knockout mice in response to alcohol consumption: evidence for an alcohol-induced hypoxic response.

PubMed

Heritage, Mandy L; Murphy, Therese L; Bridle, Kim R; Anderson, Gregory J; Crawford, Darrell H G; Fletcher, Linda M

2009-08-01

Expression of Hamp1, the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice (Hfe(-/-)). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe(-/-) mice. Hfe(-/-) and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was measured by western blot. Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe(-/-) mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1alpha protein levels were elevated in alcohol-fed wild-type animals compared with controls. Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.

Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

PubMed

Nakazato, K; Ishibashi, T; Nagata, K; Seino, Y; Wada, Y; Sakamoto, T; Matsuoka, R; Teramoto, T; Sekimata, M; Homma, Y; Maruyama, Y

2001-04-01

Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

SND1, a component of RNA-induced silencing complex, is up-regulated in human colon cancers and implicated in early stage colon carcinogenesis.

PubMed

Tsuchiya, Naoto; Ochiai, Masako; Nakashima, Katsuhiko; Ubagai, Tsuneyuki; Sugimura, Takashi; Nakagama, Hitoshi

2007-10-01

Colon cancers have been shown to develop after accumulation of multiple genetic and epigenetic alterations with changes in global gene expression profiles, contributing to the establishment of widely diverse phenotypes. Transcriptional and posttranscriptional regulation of gene expression by small RNA species, such as the small interfering RNA and microRNA and the RNA-induced silencing complex (RISC), is currently drawing major interest with regard to cancer development. SND1, also called Tudor-SN and p100 and recently reported to be a component of RISC, is among the list of highly expressed genes in human colon cancers. In the present study, we showed remarkable up-regulation of SND1 mRNA in human colon cancer tissues, even in early-stage lesions, and also in colon cancer cell lines. When mouse Snd1 was stably overexpressed in IEC6 rat intestinal epithelial cells, contact inhibition was lost and cell growth was promoted, even after the cells became confluent. Intriguingly, IEC6 cells with high levels of Snd1 also showed an altered distribution of E-cadherin from the cell membrane to the cytoplasm, suggesting loss of cellular polarity. Furthermore, the adenomatous polyposis coli (Apc) protein was coincidentally down-regulated, with no significant changes in the Apc mRNA level. Immunohistochemical analysis using chemically induced colonic lesions developed in rats revealed overexpression of Snd1 not only in colon cancers but also in aberrant crypt foci, putative precancerous lesions of the colon. Up-regulation of SND1 may thus occur at a very early stage in colon carcinogenesis and contribute to the posttranscriptional regulation of key players in colon cancer development, including APC and beta-catenin.

[Expression and clinical significance of KNSL4 in breast cancer].

PubMed

Feng, Yu-Mei; Wan, Yan-Fang; Li, Xiao-Qing; Cao, Xu-Chen; Li, Xi

2006-06-01

Previous screening of breast cancer metastasis-related genes found that the mRNA level of kinesin-like 4 (KNSL4) gene is down-regulated in metastatic lymph nodes as compared with the paired primary breast cancer. This study was to clarify the correlations of KNSL4 mRNA expression to metastasis and prognosis of breast cancer, and explore the correlation of KNSL4 expression to c-erbB-2 expression to explore potential mechanisms of promoting metastasis by KNSL4. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the mRNA level of KNSL4 in 108 specimens of primary breast cancer. The correlations of KNSL4 mRNA level to metastasis and prognosis of the 108 cases were analyzed. Immunohistochemistry was used to assess c-erbB-2 protien expression in 76 out of the 108 cases, and the correlation of KNSL4 expression to c-erbB-2 expression was analyzed. The mRNA level of KNSL4 was significantly lower in the cases at stages iii-iv than in the cases at stages iii-iv (P<0.001), significantly lower in the cases with more than 3 metastatic lymph nodes than in the cases with 0-3 metastatic positive lymph nodes (P<0.01), slightly lower in the cases with negative estrogen receptor or prognesterone receptor than in the cases with positive receptors (P>0.05), lower in the 6 cases with distant metastasis than in the rest cases without distant metastasis within 24 month follow up, lower in the 3 cases with bilateral breast cancer than in other cases with unilateral breast cancer, and significantly lower in c-erbB-2-positive group than in c-erB-2-negative group (P<0.01). The down-regulation of KNSL4 mRNA level is correlated to prognosis of primary breast cancer. It may enhance metastatic ability of breast cancer cells through promoting c-erbB-2 transcription and translation.

RGS2 is regulated by angiotensin II and functions as a negative feedback of aldosterone production in H295R human adrenocortical cells.

PubMed

Romero, Damian G; Plonczynski, Maria W; Gomez-Sanchez, Elise P; Yanes, Licy L; Gomez-Sanchez, Celso E

2006-08-01

Regulator of G protein signaling (RGS) proteins interact with Galpha-subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. Angiotensin (Ang) II interacts with its G protein-coupled receptor in zona glomerulosa adrenal cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. We studied Ang II-mediated regulation of RGS2, the role of RGS2 in steroidogenesis, and the intracellular signal events involved in H295R human adrenal cells. We report that both H295R cells and human adrenal gland express RGS2 mRNA. In H295R cells, Ang II caused a rapid and transient increase in RGS2 mRNA levels quantified by real-time RT-PCR. Ang II effects were mimicked by calcium ionophore A23187 and blocked by calcium channel blocker nifedipine. Ang II effects also were blocked by calmodulin antagonists (W-7 and calmidazolium) and calcium/calmodulin-dependent kinase antagonist KN-93. RGS2 overexpression by retroviral infection in H295R cells caused a decrease in Ang II-stimulated aldosterone secretion but did not modify cortisol secretion. In reporter assays, RGS2 decreased Ang II-mediated aldosterone synthase up-regulation. These results suggest that Ang II up-regulates RGS2 mRNA by the calcium/calmodulin-dependent kinase pathway in H295R cells. RGS2 overexpression specifically decreases aldosterone secretion through a decrease in Ang II-mediated aldosterone synthase-induced expression. In conclusion, RGS2 expression is induced by Ang II to terminate the intracellular signaling cascade generated by Ang II. RGS2 alterations in expression levels or functionality could be implicated in deregulations of Ang II signaling and abnormal aldosterone secretion by the adrenal gland.

Sophora subprosrate polysaccharide inhibited cytokine/chemokine secretion via suppression of histone acetylation modification and NF-κb activation in PCV2 infected swine alveolar macrophage.

PubMed

Yang, Jian; Tan, Hong-Lian; Gu, Li-Yuan; Song, Man-Ling; Wu, Yuan-Ying; Peng, Jian-Bo; Lan, Zong-Bao; Wei, Ying-Yi; Hu, Ting-Jun

2017-11-01

In the present study, effect of Sophora subprosrate polysaccharide on PCV2 infection-induced inflammation and histone acetylation modification in swine alveolar macrophage 3D4/2 cells was described for the first time. The relationship between histone acetylation modifications and inflammation response was investigated. The results showed that PCV2 infection induced inflammation by promoting the secretion of TNF-α, IL-1β, IL-6 and IL-10 in 3D4/2 cells. The production of TNF-α, IL-1β and IL-6 and their mRNA expression levels markedly decreased while the level and mRNA expression of IL-10 were elevated when the cells were treated with Sophora subprosrate polysaccharide. The SSP also decreased the activity of HATs, histone H3 acetylation (Ac-H3) and histone H4 acetylation (Ac-H4), p65 phosphorylation (P-p65) in the cells infected with PCV2 while HDACs activity was down-regulated, which involved in the inhibitory effect of SSP on histone acetylation and NF-κB signaling pathways activation. Down-regulation of HAT1 mRNA expression and up-regulation of HDAC1 mRNA expression further support the inhibitory effect of SSP on histone acetylation. In conclusion, Sophora subprosrate polysaccharide antagonized inflammatory responses induced by PCV2, via mechanisms involved in histone acetylation and NF-κB signaling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

Time-course microarrays reveal early activation of the immune transcriptome in a choline-deficient mouse model of liver injury.

PubMed

Mitsumoto, Koji; Watanabe, Rina; Nakao, Katsuki; Yonenaka, Hisaki; Hashimoto, Takao; Kato, Norihisa; Kumrungsee, Thanutchaporn; Yanaka, Noriyuki

2017-09-01

Choline-deficient diet is extensively used as a model of nonalcoholic fatty liver disease (NAFLD). In this study, we explored genes in the liver for which the expression changed in response to the choline-deficient (CD) diet. Male CD-1 mice were divided into two groups and fed a CD diet with or without 0.2% choline bitartrate for one or three weeks. Hepatic levels of choline metabolites were analyzed by using liquid chromatography mass spectrometry and hepatic gene expression profiles were examined by DNA microarray analysis. The CD diet lowered liver choline metabolites after one week and exacerbated fatty liver between one and three weeks. We identified >300 genes whose expression was significantly altered in the livers of mice after consumption of this CD diet for one week and showed that liver gene expression profiles could be classified into six distinct groups. This study showed that STAT1 and interferon-regulated genes was up-regulated after the CD diet consumption and that the Stat1 mRNA level was negatively correlated with liver phosphatidylcholine level. Stat1 mRNA expression was actually up-regulated in isolated hepatocytes from the mouse liver with the CD diet. This study provides insight into the genomic effects of the CD diet through the Stat1 expression, which might be involved in NAFLD development. Copyright © 2017 Elsevier Inc. All rights reserved.

The decreased growth performance and impaired immune function and structural integrity by dietary iron deficiency or excess are associated with TOR, NF-κB, p38MAPK, Nrf2 and MLCK signaling in head kidney, spleen and skin of grass carp (Ctenopharyngodon idella).

PubMed

Guo, Yan-Lin; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Zhou, Xiao-Qiu; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Feng, Lin

2017-06-01

This study was conducted to investigate the effects of dietary iron on the growth, and immune function and structural integrity in head kidney, spleen and skin as well as the underlying signaling of young grass carp (Ctenopharyngodon idella). Total 630 grass carp (242.32 ± 0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 mg/kg (diets 2-6 were added with ferrous fumarate) and 73.50 mg/kg (diet 7 was added with ferrous sulfate) diet for 60 days. Then, a challenge test was conducted by infection of Aeromonas hydrophila for 14 days. The results firstly showed that compared with optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated the mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) p65, IκB kinases β (IKKβ) and eIF4E-binding protein (4E-BP) in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired immune function in head kidney and spleen of fish. Secondly, iron deficiency down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), and inhibitor of apoptosis protein (IAP), and decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction complexes, and up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X protein (Bax), Fas ligand (FasL), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12 and myosin light chain kinase (MLCK), and increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired structural integrity in head kidney and spleen of fish. Thirdly, iron deficiency increased skin hemorrhage and lesion morbidity, and impaired immune function and structural integrity in skin of fish. Fourthly, iron excess decreased growth and impaired the immune function and structural integrity in head kidney, spleen and skin of fish. Besides, in young grass carp, based on PWG and ability against skin hemorrhage and lesion, the efficacy of ferrous fumarate relative to ferrous sulfate was 140.32% and 126.48%, respectively, and the iron requirements based on PWG, ability against skin hemorrhage and lesion, ACP activities and MDA contents in head kidney and spleen were estimated to be 75.65, 87.03, 79.74, 78.93, 83.17 and 82.14 mg/kg diet (based on ferrous fumarate), respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential regulation of duplicate light-dependent protochlorophyllide oxidoreductases in the diatom Phaeodactylum tricornutum

DOE PAGES

Hunsperger, Heather M.; Ford, Christopher J.; Miller, James S.; ...

2016-07-01

Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability.more » As a result, for cultures maintained on a 12h light: 12h dark photoperiod at 200μEm –2 s –1 ( 200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light ( 200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μEm –2 s –1 ( 1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μEm –2 s –1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown.« less

Differential Regulation of Duplicate Light-Dependent Protochlorophyllide Oxidoreductases in the Diatom Phaeodactylum tricornutum

PubMed Central

Hunsperger, Heather M.; Cattolico, Rose Ann

2016-01-01

Background Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability. Results For cultures maintained on a 12h light: 12h dark photoperiod at 200μE m−2 s−1 (200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light (200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μE m−2 s−1 (1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μE m−2 s−1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown. Conclusion Given the sensitivity of diatom por1/POR1 to real-time light cues and adherence of por2/POR2 regulation to the diurnal cycle, we suggest that POR1 supports photoacclimation, whereas POR2 is the workhorse for daily chlorophyll synthesis. PMID:27367227

Up-regulation of COUP-TFII gene expression in the nitrofen-induced hypoplastic lung.

PubMed

Doi, Takashi; Sugimoto, Kaoru; Puri, Prem

2009-02-01

Recent studies have suggested that the retinoid signaling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung. The exact mechanism by which nitrofen acts in the RSP remains unclear. Targeted ablation of COUP-TFII, a gene encoding a transfactor regulated by the RSP, has been shown to cause Bochdalek-type congenital diaphragmatic hernia. It has been shown that COUP-TFII has 2 main roles in the RSP, (i) repressing the RSP by directly sequestering retinoid X receptors, thereby preventing heterodimerization to retinoid acid receptors and inhibiting gene transcription, and (ii) modulating the transcriptional activity of GATA proteins. We designed this study to investigate the gene expression of COUP-TFII in the nitrofen-induced hypoplastic lung. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9 of gestation. Fetuses were harvested and lungs were dissected on day 15 (D15), D18, and D21 and divided into 2 groups: control (n = 9) and nitrofen (n = 9). Real-time reverse transcription-polymerase chain reaction was performed to evaluate the relative mRNA levels of COUP-TFII expression in the hypoplastic lung. The relative mRNA levels of COUP-TFII at D15 was significantly increased in the nitrofen group (0.76 +/- 0.53) compared to controls (0.45 +/- 0.05) (P < .01). The expression levels of COUP-TFII at D18 and D21 were not significantly different between the nitrofen group and controls. Our results provide evidence for the first time that the pulmonary gene expression of COUP-TFII is up-regulated in the early stages of lung development in the nitrofen-induced hypoplastic lung. We speculate that up-regulation of COUP-TFII gene expression during the stage of branching lung morphogenesis may cause pulmonary hypoplasia by repressing RSP.

Regulation of cytoplasmic mRNA decay

PubMed Central

Schoenberg, Daniel R.; Maquat, Lynne E.

2012-01-01

Discoveries made over the past 20 years highlight the importance of mRNA decay as a means to modulate gene expression and thereby protein production. Up until recently, studies focused largely on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay, and the ribonucleases that catalyze decay. Now, current studies have begun to elucidate how the decay process is regulated. This review examines our current understanding of how mammalian-cell mRNA decay is controlled by different signaling pathways and lays out a framework for future research. PMID:22392217

Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1{alpha}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Weiping, E-mail: weiping.qin@mssm.edu; Department of Medicine, Mount Sinai School of Medicine, NY; Pan, Jiangping

Research highlights: {yields} In rat gastrocnemius muscle, dexamethasone reduced PGC-1{alpha} cellular and nuclear levels without altering mRNA levels for this factor. {yields} Dexamethasone reduced phosphorylating of p38 MAPK, which stabilizes PGC-1{alpha} and promotes its nuclear entry. {yields} Co-administration of testosterone with dexamethasone increased cellular and nuclear levels of PGC-1{alpha} protein without changing its mRNA levels. {yields} Co-administration of testosterone restored p38 MAPK levels to those of controls. -- Abstract: Glucocorticoid-induced muscle atrophy results from muscle protein catabolism and reduced protein synthesis, associated with increased expression of two muscle-specific ubiquitin ligases (MAFbx and MuRF1), and of two inhibitors of protein synthesis,more » REDD1 and 4EBP1. MAFbx, MuRF1, REDD1 and 4EBP1 are up-regulated by the transcription factors FOXO1 and FOXO3A. The transcriptional co-activator PGC-1{alpha} has been shown to attenuate many forms of muscle atrophy and to repress FOXO3A-mediated transcription of atrophy-specific genes. Dexamethasone-induced muscle atrophy can be prevented by testosterone, which blocks up-regulation by dexamethasone of FOXO1. Here, an animal model of dexamethasone-induced muscle atrophy was used to further characterize effects of testosterone to abrogate adverse actions of dexamethasone on FOXO1 levels and nuclear localization, and to determine how these agents affect PGC-1{alpha}, and its upstream activators, p38 MAPK and AMPK. In rat gastrocnemius muscle, testosterone blunted the dexamethasone-mediated increase in levels of FOXO1 mRNA, and FOXO1 total and nuclear protein. Dexamethasone reduced total and nuclear PGC-1{alpha} protein levels in the gastrocnemius; co-administration of testosterone with dexamethasone increased total and nuclear PGC-1{alpha} levels above those present in untreated controls. Testosterone blocked dexamethasone-induced decreases in activity of p38 MAPK in the gastrocnemius muscle. Regulation of FOXO1, PGC-1{alpha} and p38 MAPK by testosterone may represent a novel mechanism by which this agent protects against dexamethasone-induced muscle atrophy.« less

Expression of the genes for insulin-like growth factors and their receptors in bone during skeletal growth

NASA Technical Reports Server (NTRS)

Bikle, D. D.; Harris, J.; Halloran, B. P.; Roberts, C. T.; Leroith, D.; Morey-Holton, E.

1994-01-01

Insulin-like growth factors (IGF) are important regulators of skeletal growth. To determine whether the capacity to produce and respond to these growth factors changes during skeletal development, we measured the protein and mRNA levels for IGF-I, IGF-II, and their receptors (IGF-IR and IGF-IIR, respectively) in the tibia and femur of rats before and up to 28 mo after birth. The mRNA levels remained high during fetal development but fell after birth, reaching a nadir by 3-6 wk. This fall was most pronounced for IGF-II and IGF-IIR mRNA and least pronounced for IGF-I mRNA. However, after 6 wk, both IGF-I and IGF-IR mRNA levels recovered toward the levels observed at birth. In the prenatal bones, the signals for the mRNAs of IGF-II and IGF-IIR were stronger than the signals for the mRNAs of IGF-I and IGF-IR, although the content of IGF-I was three- to fivefold greater than that of IGF-II. IGF-II levels fell postnatally, whereas the IGF-I content rose after birth such that the ratio IGF-I/IGF-II continued to increase with age. We conclude that, during development, rat bone changes its capacity to produce and respond to IGFs with a progressive trend toward the dominance of IGF-I.

Responses of Murine and Human Macrophages to Leptospiral Infection: A Study Using Comparative Array Analysis

PubMed Central

Yang, Yingchao; Zhao, Jinping; Yang, Yutao; Cao, Yongguo; Hong, Cailing; Liu, Yuan; Sun, Lan; Huang, Minjun; Gu, Junchao

2013-01-01

Leptospirosis is a re-emerging tropical infectious disease caused by pathogenic Leptospira spp. The different host innate immune responses are partially related to the different severities of leptospirosis. In this study, we employed transcriptomics and cytokine arrays to comparatively calculate the responses of murine peritoneal macrophages (MPMs) and human peripheral blood monocytes (HBMs) to leptospiral infection. We uncovered a series of different expression profiles of these two immune cells. The percentages of regulated genes in several biological processes of MPMs, such as antigen processing and presentation, membrane potential regulation, and the innate immune response, etc., were much greater than those of HBMs (>2-fold). In MPMs and HBMs, the caspase-8 and Fas-associated protein with death domain (FADD)-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8 and caspase-3 pathways play an important role in macrophage apoptosis during leptospiral infection. In addition, the key component of the complement pathway, C3, was only up-regulated in MPMs. Furthermore, several cytokines, e.g. interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-alpha), were differentially expressed at both mRNA and protein levels in MPMs and HBMs. Some of the differential expressions were proved to be pathogenic Leptospira-specific regulations at mRNA level or protein level. Though it is still unclear why some animals are resistant and others are susceptible to leptospiral infection, this comparative study based on transcriptomics and cytokine arrays partially uncovered the differences of murine resistance and human susceptibility to leptospirosis. Taken together, these findings will facilitate further molecular studies on the innate immune response to leptospiral infection. PMID:24130911

Quercetin promotes proliferation and differentiation of oligodendrocyte precursor cells after oxygen/glucose deprivation-induced injury.

PubMed

Wu, Xiuxiang; Qu, Xuebin; Zhang, Qiang; Dong, Fuxing; Yu, Hongli; Yan, Chen; Qi, Dashi; Wang, Meng; Liu, Xuan; Yao, Ruiqin

2014-04-01

The aim of this study was to investigate quercetin's (Qu) ability to promote proliferation and differentiation of oligodendrocyte precursor cells (OPCs) under oxygen/glucose deprivation (OGD)-induced injury in vitro. The results showed that after OGD, OPCs survival rate was significantly increased by Qu as measured by Cell Counting Kit-8. Furthermore, Qu treatment reduced apoptosis of OPCs surveyed by Hoechst 33258 nuclear staining. Qu at 9 and 27 μM promoted the proliferation of OPCs the most by Brdu and Olig2 immunocytochemical staining after OGD 3 days. Also, Qu treatment for 8 days after OGD, the differentiation of OPCs to oligodendrocyte was detected by immunofluorescence staining showing that O4, Olig2, and myelin basic protein (MBP) positive cells were significantly increased compared to control group. Additionally, the protein levels of Olig2 and MBP of OPCs were quantified using western blot and mRNA levels of Olig2 and Inhibitor of DNA binding 2 (Id2) were measured by RT-PCR. Western blot showed a significant increase in Olig2 and MBP expression levels compared with controls after OGD and Qu treatment with a linear does-response curve from 3 to 81 μM. After treatment with Qu compared to its control group, Olig2 mRNA level was significantly up-regulated, whereas Id2 mRNA level was down-regulated. In conclusion, Qu at 3-27 μM can promote the proliferation and differentiation of OPCs after OGD injury and may regulate the activity of Olig2 and Id2.

[Effects of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin-resistant adipocytes].

PubMed

Tu, Jun; Luo, Xin-Xin; Li, Bing-Tao; Li, Yu; Xu, Guo-Liang

2016-06-01

Adipocytokines are closely associated with insulin resistance (IR) in adipose tissues, and they are more and more seriously taken in the study of the development of diabetes. This experiment was mainly to study the effect of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin resistant adipocytes, and investigate the molecular mechanism of berberine in enhancing insulin sensitization and application advantages of droplet digital PCR (ddPCR). ddPCR absolute quantification analysis was taken in this experiment to simply and intuitively determine the appropriate reference genes. ddPCR and quantitative Real-time PCR (qPCR) were used to compare the effect of different doses of berberine (10, 20, 50, 100 μmol•L⁻¹) on mRNA expression levels of PPARγ, adiponectin, resistin and leptin in IR 3T3-L1adipocytes. Antagonist GW9662 was added to study the inherent correlation between PPARγ and adiponectin mRNA expression levels. ddPCR results showed that the expression level of β-actin in adipocytes was stable, and suitable as reference gene for normalization of quantitative PCR data. Both of ddPCR and qPCR results showed that, as compared with IR models, the mRNA expression levels of adiponectin were decreased in the treatment with berberine (10, 20, 50, 100 μmol•L⁻¹) in a dose-dependent manner (P<0.01); the expression of PPARγ was decreased by 20, 50, 100 μmol•L⁻¹ berberine in a dose-dependent manner in qPCR assay (P<0.01) and decreased only by 50 and 100 μmol•L⁻¹ berberine in ddPCR assay (P<0.05). PPARγ specific antagonist GW9662 intervention experiment showed that adiponectin gene expression was directly relevant with PPARγ (P<0.05). ddPCR probe assay showed that various doses of berberine could significantly reduce mRNA expression levels of resistin and leptin (P<0.01) in a dose-dependent manner. In conclusion, berberine enhanced insulin sensitization effect not by up-regulating adiponect in expression of transcriptional level in PPARγ-dependent manner, but may by the elevated multimerization of adiponectin in the posttranslational regulation level. Berberine down-regulated the resistin and leptin expression levels, which could alleviate lipolysis and improve IR in adipocytes. ddPCR provided better sensitivity and linear range than qPCR, with obvious technical advantages for the detection of low abundance expression of target genes. Copyright© by the Chinese Pharmaceutical Association.

Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsui, Takanori; Yamagishi, Sho-ichi, E-mail: shoichi@med.kurume-u.ac.jp; Takeuchi, Masayoshi

2010-07-23

Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenicmore » reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression via PPAR{gamma} activation.« less

Up-regulation of Cav3.1 expression in SH-SY5Y cells induced by lidocaine hydrochloride.

PubMed

Gong, Qin; Wen, Xianjie; Li, Heng; He, Jian; Wang, Yunhua; Wu, Huiping; Wang, Hanbing; Wang, Xiaoping

2018-01-12

Neurotoxicity induced by the local anaesthetics has aroused concern. A previous study has shown that an overload of intracellular calcium was involved in the neurotoxic effect. Cav3.1 is one of the low-voltage-activated (LVA) calcium channels which play a key point to regulate the intracellular calcium ion level. This study aimed to investigate the changes of the Cav3.1 expression in the SH-SY5Y cells treated with lidocaine hydrochloride. The SH-SY5Y cells were treated with different concentrations of lidocaine hydrochloride(1 mM, 5 mM and 10 mM, namely L1 group, L5 group and L10 group) and different exposure times (1 h,12 h and 24 h), respectively. Cell viability, Cav3.1 protein and mRNA expression were detected. The results showed that cell viability decreased and Cav3.1 mRNA and protein expression increased with the concentration (from 1 mM to 10 mM) of the lidocaine hydrochloride and exposure time (from 1 h to 24 h) to the SH-SY5Y cell line increased. Those data showed that lidocaine hydrochloride induced SH-SY5Y cell toxicity and up-regulated Cav3.1mRNA and protein expression.

Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 and lymphoid CCRF-CEM leukaemic cells.

PubMed Central

Ul-Haq, R; Chitambar, C R

1993-01-01

Gallium binds to the iron transport protein transferrin (Tf), is incorporated into cells through transferrin receptors (TfR) and inhibits iron-dependent DNA synthesis. Since cellular TfR expression is tightly regulated by the availability of iron, we investigated the effects of transferrin-gallium (Tf-Ga) on TfR mRNA levels in myeloid HL60 and lymphoid CCRF-CEM cells. In HL60 cells, Tf-Ga increased TfR mRNA levels in a dose-dependent fashion. This increase in TfR mRNA was blocked by Tf-Fe and by cycloheximide. Analysis of the rate of mRNA decay in the presence of actinomycin D revealed that the half-life of TfR mRNA was increased in HL60 cells incubated with Tf-Ga. The rate of transcription of TfR mRNA was not increased by Tf-Ga. In contrast with HL60 cells, CCRF-CEM cells displayed a decrease in the level of TfR mRNA after incubation with Tf-Ga. Tf-Ga inhibited iron uptake in both HL60 and CCRF-CEM cells but increased the level of TfR mRNA only in HL60 cells, suggesting that the Tf-Ga induction of TfR mRNA was not solely due to inhibition of cellular iron uptake. At growth-inhibitory concentrations, Tf-Ga increased the TfR mRNA level in HL60 cells but decreased it in CCRF-CEM cells. Our studies suggest that in HL60 cells, gallium regulates TfR expression at the post-transcriptional level by mechanisms which require de novo protein synthesis and involve interaction with iron. The divergent effects of Tf-Ga on TfR mRNA in myeloid HL60 and lymphoid CCRF-CEM cells suggest that differences exist in the regulation of TfR expression between these two cell types. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8379943

The response of carbon metabolism and antioxidant defenses of alfalfa nodules to drought stress and to the subsequent recovery of plants.

PubMed

Naya, Loreto; Ladrera, Ruben; Ramos, Javier; González, Esther M; Arrese-Igor, Cesar; Minchin, Frank R; Becana, Manuel

2007-06-01

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N(2)ase) activity. Exposure of plants to a moderate drought (leaf water potential of -1.3 MPa) had no effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N(2)ase activity (-43%), accumulation of succinate (+36%) and Suc (+58%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (-2.1 MPa) decreased N(2)ase (-82%) and SS (-30%) activities and increased malate (+40%), succinate (+68%), and Suc (+435%). There was also up-regulation (mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe- and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (>64%) of N(2)ase, a complete recovery of Suc, and a decrease of malate (-48%) relative to control. The increase in O(2) diffusion resistance, the decrease in N(2)ase-linked respiration and N(2)ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N(2)ase activity in alfalfa nodules.

Effects of copper on growth, metamorphosis and endocrine disruption of Bufo gargarizans larvae.

PubMed

Wang, Chao; Liang, Gang; Chai, Lihong; Wang, Hongyuan

2016-01-01

Chinese toad (Bufo gargarizans) tadpoles were exposed to copper (1, 6.4, 32 and 64μgL(-1) copper) from the beginning of larval period through completion of metamorphosis. We examined the effects of chronic copper exposure on mortality, growth, time to metamorphosis, tail resorption time, body size at the metamorphic climax (Gs 42) and completion of metamorphosis (Gs 46) and thyroid gland histology. In addition, type 2 and 3 iodothyronine deiodinase (Dio2 and Dio3), thyroid hormone receptors (TRα and TRβ) mRNA levels were also measured to assess disruption of TH synthesis. Our result showed that 6.4-64μgL(-1) copper concentration increased the mortality and inhibited the growth of B. gargarizans tadpoles. In addition, significant reduction in size at Gs 42 and a time delay to Gs 42 were observed at 6.4-64μgL(-1) copper treatments. Moreover, histological examinations have clearly revealed that 64μgL(-1) copper caused follicular cell hyperplasia in thyroid gland. According to real-time PCR results, exposure to 32 and 64μgL(-1) copper significantly up-regulated mRNA expression of Dio3, but down-regulated mRNA expression of TRα and TRβ mRNA level. We concluded that copper delayed amphibian metamorphosis through changing mRNA expression of Dio3, TRα and TRβ, which suggests that copper might have the endocrine-disrupting effect. Copyright © 2015 Elsevier B.V. All rights reserved.

Nitric oxide signaling pathway regulates potassium chloride cotransporter-1 mRNA expression in vascular smooth muscle cells.

PubMed

Di Fulvio, M; Lauf, P K; Adragna, N C

2001-11-30

Rat vascular smooth muscle cells (VSMCs) express at least two mRNAs for K-Cl cotransporters (KCC): KCC1 and KCC3. cGMP-dependent protein kinase I regulates KCC3 mRNA expression in these cells. Here, we show evidence implicating the nitric oxide (NO)/cGMP signaling pathway in the expression of KCC1 mRNA, considered to be the major cell volume regulator. VSMCs, expressing soluble guanylyl cyclase (sGC) and PKG-I isoforms showed a time- and concentration-dependent increase in KCC1 mRNA levels after treatment with sodium nitroprusside as demonstrated by semiquantitative RT-PCR. sGC-dependent regulation of KCC1 mRNA expression was confirmed using YC-1, a NO-independent sGC stimulator. The sGC inhibitor LY83583 blocked the effects of sodium nitroprusside and YC-1. Moreover, 8-Br-cGMP increased KCC1 mRNA expression in a concentration- and time-dependent fashion. The 8-Br-cGMP effect was partially blocked by KT5823 but not by actinomycin D. However, actinomycin D and cycloheximide increased basal KCC1 mRNA in an additive manner, suggesting different mechanisms of action for both drugs. These findings suggest that in VSMCs, the NO/cGMP-signaling pathway participates in KCC1 mRNA regulation at the post-transcriptional level.

Expression and regulation of CNTF receptor-alpha in the in situ and in oculo grafted adult rat adrenal medulla.

PubMed

Förander, P; Brené, S; Strömberg, I

2000-02-28

Cultured and transplanted adrenal medullary cells respond to ciliary neurotrophic factor (CNTF) with neurite formation and improved cell survival although the presence of the CNTF receptor-alpha (CNTFRalpha) has been unclear. This study show that CNTFRalpha mRNA was expressed in the postnatal day 1 as well as in the adult rat adrenal medulla. The highest CNTFRalpha mRNA signal was found in the ganglion cells of the adrenal medulla. After transplantation of adrenal medullary tissue the CNTFRalpha mRNA levels were down-regulated in the chromaffin cells. CNTF treatment of grafts did not normalize the receptor levels, but treatment with nerve growth factor (NGF) did. Thus, we demonstrate that CNTFRalpha mRNA is expressed in adrenal medulla, the levels becomes down-regulated after transplantation, but normalized after treatment with NGF.

X-linked lymphocyte regulated gene 5c-like (Xlr5c-like) Is a Novel Target of Progesterone Action in Granulosa Cells of Periovulatory Rat Ovaries

PubMed Central

Mishra, Birendra; Park, Ji Yeon; Wilson, Kalin; Jo, Misung

2015-01-01

Progesterone (P4), acting through its nuclear receptor (PGR), plays an essential role in ovulation by mediating the expression of genes involved in ovulation and/or luteal formation. To identify ovulatory specific PGR-regulated genes, a preliminary microarray analysis was performed using rat granulosa cells treated with hCG ± RU486 (PGR antagonist). The transcript most highly down-regulated by RU486 was an EST (Expressed Sequence Tag) sequence (gb: BI289578.1) that matches with predicted sequence for Xlr5c-like mRNA. Since nothing is known about Xlr5c-like, we first characterized the expression pattern of Xlr5c-like mRNA in the rat ovary. The level of mRNA for Xlr5c-like is transiently up-regulated in granulosa cells of periovulatory follicles after hCG stimulation in PMSG-primed rat ovaries. The transient induction of Xlr5c-like mRNA was mimicked by hCG treatment in cultured granulosa cells from preovulatory ovaries. We further demonstrated that the LH-activated PKA, MEK, PI3K, and p38 signaling is involved in the increase in Xlr5c-like mRNA. The increase in Xlr5c-like mRNA was abolished by RU486. The inhibitory effect of RU486 was reversed by MPA (synthetic progestin), but not by dexamethasone (synthetic glucocorticoid). Furthermore, mutation of SP1/SP3 and PGR response element sites in the promoter region of Xlr5c-like decreased Xlr5c-like reporter activity. RU486 also inhibited Xlr5c-like reporter activity. ChIP assay verified the binding of PGR and SP3 to the Xlr5c-like promoter in periovulatory granulosa cells. Functionally, siRNA-mediated Xlr5c-like knockdown in granulosa cell cultures resulted in reduced levels of mRNA for Snap25, Cxcr4, and Adamts1. Recombinant Xlr5c-like protein expressed using an adenoviral approach was localized predominantly to the nucleus and to a lesser extent to the cytoplasm of rat granulosa cells. In conclusion, this is the first report showing the spatiotemporally regulated expression of Xlr5c-like mRNA by hCG in rat periovulatory ovaries. P4/PGR mediates the LH-induced increase in Xlr5c-like mRNA. In turn, Xlr5c-like is involved in regulating the expression of specific ovulatory genes such as Snap25, Cxcr4, and Adamts1, possibly acting in the nucleus of periovulatory granulosa cells. PMID:26004213

[The effect of butylphthalide on expression of NGF and BDNF in ischemia stroke tissue of rat cerebrum].

PubMed

Kong, Shuang-yan; Li, Qi-fu; Yang, Jie; He, Li

2007-06-01

To study the expressions of BDNF, BDNF mRNA, NGF and NGF mRNA in the permanent focal cerebral ischemia tissues of rats. METHHODS: Healthy male Sprague-Dawley rats were taken for this study project. According to the procedure of Zea-Longa, the rat model with permanent cerebral ischemia was established by rat middle cerebral artery obstructed (MCAO) with a nylon thread, and the model rats of neurobehavioral evaluation as 1-3 grade were randomly divided into two groups: butylphthalide group (A group) and control group (B group). A group was given with 25 mg/kg butylphthalide, B group was given with edible oil, two times every day. 3 days after occlusion, all rats were sacrificed after evaluated the neurobehavioral scores, and the samples of cerebrum were obtained after in situ perfusion and fixation with 40 g/L paraformaldehyde. 5 rats in each group were taken to tetrazolium chloride (TTC) staining for macroscopic observation of cerebral infarction area, the rest samples were processed by immunohistochemistry to evaluate effects of butylphthalide on BDNF and NGF expression, hybridization in situ to evaluate effects of butylphthalide on BDNF mRNA and NGF mRNA expression. SPSS12. 0 for statistical analysis, it was P<0. 05 as having statistical significance. Comparing to control group (B group), butylphthalide group (A group) did not have significantly pathological difference, but the grade of behavior and infarction area were apparently reduced (P<0. 05). In butylphthalide group, there was a significant expression up-regulation to BDNF, NGF, BDNF mRNA and NGF mRNA in the peripheral around infarction and cornu ammonis or hippocampus area (P<0. 05). However in the infarction area, the expressions of BDNF, NGF, BDNF mRNA and NGF mRNA had no significantly statistical difference (P> 0. 05). Comparing to control group, butylphthalide can significantly up-regulate the expressions of BDNF and NGF in genetic transcription level, and protect from the ischemia injury.

Deregulation of versican and elastin binding protein in solar elastosis.

PubMed

Knott, Anja; Reuschlein, Katja; Lucius, Ralph; Stäb, Franz; Wenck, Horst; Gallinat, Stefan

2009-04-01

Several changes in skin appearance including loss of elasticity and wrinkle formation are associated with alterations in the composition of the dermal extracellular matrix. They are induced by intrinsic aging or by environmental factors such as UV light referred to as photoaging. A general characteristic in the histology of photoaged skin is the accumulation of elastotic material suggesting impaired formation and/or massive breakdown of elastic fibres. In order to shed light on some of the underlying mechanisms we tracked two of the major players in elastic fibre formation in different skin conditions: EBP (elastin binding protein), a regulator of elastic fibre assembly and VER (versican), a component of functional elastic fibres as well as non-functional elastotic material. Using quantitative RT-PCR on skin biopsies we found that the expression levels of VER and EBP were unaltered during intrinsic skin aging. Upon acute UV stress however, VER and EBP showed different regulation patterns: VER mRNA increased after 6 h and was further up-regulated until 24 h. The EBP mRNA by contrast was reduced after 6 h but showed massive induction at 24 h after acute UV stress. In chronically sun-exposed skin, VER protein was accumulated similar to elastotic material in the extracellular space, whereas its mRNA level was consistently reduced compared to sun-protected skin. The EBP mRNA by contrast showed slightly increased expression levels in the sun-exposed area compared to its sun-protected counterpart. Based on these data we propose a model which may help to explain parts of the mechanisms leading to the formation of elastotic masses. We further hypothesize that the presence of elastotic material triggers some yet unknown feedback mechanism(s) resulting in altered expression patterns of VER and EBP in chronically sun-exposed skin.

Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

PubMed Central

Yang, Xu; Vezeridis, Peter S; Nicholas, Brian; Crisco, Joseph J; Moore, Douglas C; Chen, Qian

2006-01-01

Objective Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. Methods Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA. Results Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. Conclusion These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression. PMID:17150098

Modulation by geraniol of gene expression involved in lipid metabolism leading to a reduction of serum-cholesterol and triglyceride levels.

PubMed

Galle, Marianela; Kladniew, Boris Rodenak; Castro, María Agustina; Villegas, Sandra Montero; Lacunza, Ezequiel; Polo, Mónica; de Bravo, Margarita García; Crespo, Rosana

2015-07-15

Geraniol (G) is a natural isoprenoid present in the essential oils of several aromatic plants, with various biochemical and pharmacologic properties. Nevertheless, the mechanisms of action of G on cellular metabolism are largely unknown. We propose that G could be a potential agent for the treatment of hyperlipidemia that could contribute to the prevention of cardiovascular disease. The aim of the present study was to advance our understanding of its mechanism of action on cholesterol and TG metabolism. NIH mice received supplemented diets containing 25, 50, and 75 mmol G/kg chow. After a 3-week treatment, serum total-cholesterol and triglyceride levels were measured by commercial kits and lipid biosynthesis determined by the [(14)C] acetate incorporated into fatty acids plus nonsaponifiable and total hepatic lipids of the mice. The activity of the mRNA encoding HMGCR-the rate-limiting step in cholesterol biosynthesis-along with the enzyme levels and catalysis were assessed by real-time RT-PCR, Western blotting, and HMG-CoA-conversion assays, respectively. In-silico analysis of several genes involved in lipid metabolism and regulated by G in cultured cells was also performed. Finally, the mRNA levels encoded by the genes for the low-density-lipoprotein receptor (LDLR), the sterol-regulatory-element-binding transcription factor (SREBF2), the very-low-density-lipoprotein receptor (VLDLR), and the acetyl-CoA carboxylase (ACACA) were determined by real-time RT-PCR. Plasma total-cholesterol and triglyceride levels plus hepatic fatty-acid, total-lipid, and nonsaponifiable-lipid biosynthesis were significantly reduced by feeding with G. Even though an up-regulation of the mRNA encoding HMGCR occurred in the G treated mouse livers, the protein levels and specific activity of the enzyme were both inhibited. G also enhanced the mRNAs encoding the LDL and VLDL receptors and reduced ACACA mRNA, without altering the transcription of the mRNA encoding the SREBF2. The following mechanisms may have mediated the decrease in plasma lipids levels in mice: a down-regulation of hepatocyte-cholesterol synthesis occurred as a result of decreased HMGCR protein levels and catalytic activity; the levels of LDLR mRNA became elevated, thus suggesting an increase in the uptake of serum LDL, especially by the liver; and TG synthesis became reduced very likely because of a decrease in fatty-acid synthesis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Regulation of the corticosteroid signalling system in rainbow trout HPI axis during confinement stress.

PubMed

Kiilerich, Pia; Servili, Arianna; Péron, Sandrine; Valotaire, Claudiane; Goardon, Lionel; Leguen, Isabelle; Prunet, Patrick

2018-03-01

This study aims to shed light on corticosteroid regulation of stress in teleost fish with focus on the corticosteroid signalling system. The role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) in fish is still enigmatic, as is the function of the mineralocorticoid receptor, MR. Low plasma DOC levels and ubiquitous tissue distribution of MR question the physiological relevance of the mineralocorticoid-axis. Furthermore, the particular purpose of each of the three corticosteroid receptors in fish, the glucocorticoid receptors, GR1 and GR2, and the MR, is still largely unknown. Therefore we investigate the regulation of cortisol and DOC in plasma and mRNA levels of MR, GR1 and GR2 in the HPI-axis tissues (hypothalamus, pituitary and interrenal gland) during a detailed confinement stress time-course. Here we show a sustained up-regulation of plasma DOC levels during a confinement stress time-course. However, the low DOC levels compared to cortisol measured in the plasma do not favour an activity of DOC through MR receptors. Furthermore, we show differential contribution of the CRs in regulation and control of HPI axis activity following confinement stress. Judged by the variation of mRNA levels negative feedback regulation of cortisol release occurs on the level of the pituitary via MR and on the level of the interrenal gland via GR2. Finally, asa significant effect of confinement stress on CR expressions was observed in the pituitary gland, we completed this experiment by demonstrating that corticosteroid receptors (GR1, GR2 and MR) are co-expressed in the ACTH cells located in the adenohypophysis. Overall, these data suggest the involvement of these receptors in the regulation of the HPI axis activity by cortisol. Copyright © 2017 Elsevier Inc. All rights reserved.

Glucose Regulates the Expression of the Apolipoprotein A5 Gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fruchart, Jamila; Nowak, Maxime; Helleboid-Chapman, Audrey

2008-04-07

The apolipoprotein A5 gene (APOA5) is a key player in determining triglyceride concentrations in humans and mice. Since diabetes is often associated with hypertriglyceridemia, this study explores whether APOA5 gene expression is regulated by alteration in glucose homeostasis and the related pathways. D-glucose activates APOA5 gene expression in a time- and dose-dependent manner in hepatocytes, and the glycolytic pathway involved was determined using D-glucose analogs and metabolites. Together, transient transfections, electrophoretic mobility shift assays and chromatin immunoprecipitation assays show that this regulation occurs at the transcriptional level through an increase of USF1/2 binding to an E-box in the APOA5 promoter.more » We show that this phenomenon is not due to an increase of mRNA or protein expression levels of USF. Using protein phosphatases 1 and 2A inhibitor, we demonstrate that D-glucose regulates APOA5 gene via a dephosphorylation mechanism, thereby resulting in an enhanced USF1/2-promoter binding. Last, subsequent suppressions of USF1/2 and phosphatases mRNA through siRNA gene silencing abolished the regulation. We demonstrate that APOA5 gene is up regulated by D-glucose and USF through phosphatase activation. These findings may provide a new cross talk between glucose and lipid metabolism.« less

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers.

PubMed

Domínguez-Sánchez, María S; Sáez, Carmen; Japón, Miguel A; Aguilera, Andrés; Luna, Rosa

2011-02-17

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development.

Eukaryotic initiation factor 3 (eIF3) and 5’ mRNA leader sequences as agents of translational regulation in Arabidopsis. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

von Arnim, Albrecht G.

2015-02-04

Protein synthesis, or translation, consumes a sizable fraction of the cell’s energy budget, estimated at 5% and up to 50% in differentiated and growing cells, respectively. Plants also invest significant energy and biomass to construct and maintain the translation apparatus. Translation is regulated by a variety of external stimuli. Compared to transcriptional control, attributes of translational control include reduced sensitivity to stochastic fluctuation, a finer gauge of control, and more rapid responsiveness to environmental stimuli. Yet, our murky understanding of translational control allows few generalizations. Consequently, translational regulation is underutilized in the context of transgene regulation, although synthetic biologists aremore » now beginning to appropriate RNA-level gene regulation into their regulatory circuits. We also know little about how translational control contributes to the diversity of plant form and function. This project explored how an emerging regulatory mRNA sequence element, upstream open reading frames (uORFs), is integrated with the general translation initiation machinery to permit translational regulation on specific mRNAs.« less

Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces expression of the chemokine genes Cxcl4 and Cxcl7 in the perinatal mouse brain.

PubMed

Mitsui, Tetsuo; Taniguchi, Naofumi; Kawasaki, Nobuchika; Kagami, Yoshihiro; Arita, Jun

2011-04-01

Fetal exposure to dioxins affects brain development and influences behaviors in human and laboratory animals. However, the cellular target and mechanisms of the neurotoxic action of dioxins are largely unknown. To investigate the molecular basis for the neurotoxicity of dioxins, pregnant C57BL/6 mice were exposed to 5 µg kg(-1) body weight of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by a single gavage on gestational day 12.5 (GD 12.5), and gene expression of the whole fetal brain at GD 18.5 was profiled by DNA microarray analysis. The analysis revealed that the expression of two chemokine genes, Cxcl4 and Cxcl7, was up-regulated by TCDD exposure. Real-time PCR analysis verified that they were up-regulated by TCDD in both male and female brains, while the mRNA levels of a majority of other chemokines and their receptor genes were not affected. The up-regulation was TCDD dose-dependent and peaked at GD 15.5-18.5. In situ hybridization analysis showed that the Cxcl4 mRNA expression was localized in part of the surface of cerebral cortex and that the level was increased by TCDD treatment. These results suggest that Cxcl4 and Cxcl7 play a role in the development of neurobehavioral alterations that are triggered by in utero TCDD exposure and later surface in adults. Copyright © 2011 John Wiley & Sons, Ltd.

Effects of Reticuloendotheliosis Virus Infection on Cytokine Production in SPF Chickens

PubMed Central

Xue, Mei; Shi, Xingming; Zhao, Yan; Cui, Hongyu; Hu, Shunlei; Cui, Xianlan; Wang, Yunfeng

2013-01-01

Infection with reticuloendotheliosis virus (REV), a gammaretrovirus in the Retroviridae family, can result in immunosuppression and subsequent increased susceptibility to secondary infections. The effects of REV infection on expression of mRNA for cytokine genes in chickens have not been completely elucidated. In this study, using multiplex branched DNA (bDNA) technology, we identified molecular mediators that participated in the regulation of the immune response during REV infection in chickens. Cytokine and chemokine mRNA expression levels were evaluated in the peripheral blood mononuclear cells (PBMCs). Expression levels of interleukin (IL)-4, IL-10, IL-13 and tumor necrosis factor (TNF)-α were significantly up-regulated while interferon (IFN)-α, IFN-β, IFN-γ, IL-1β，IL-2, IL-3, IL-15, IL-17F, IL-18 and colony-stimulating factor (CSF)-1 were markedly decreased in PBMCs at all stages of infection. Compared with controls, REV infected chickens showed greater expression levels of IL-8 in PBMCs 21 and 28 days post infection. In addition, REV regulates host immunity as a suppressor of T cell proliferative responses. The results in this study will help us to understand the host immune response to virus pathogens. PMID:24358317

Effect of Dietary Purified Xanthohumol from Hop (Humulus lupulus L.) Pomace on Adipose Tissue Mass, Fasting Blood Glucose Level, and Lipid Metabolism in KK-Ay Mice.

PubMed

Takahashi, Koki; Osada, Kyoichi

2017-05-01

We previously showed that xanthohumol-rich hop extract (XRHE, ~18% xanthohumol) exerts anti-obesity effects in rats fed a high-fat diet through regulation of fatty acid metabolism. In this study, we examined the effects of dietary purified xanthohumol from XRHE (PX, ~91.9% xanthohumol) in KK-Ay mice in order to understand the anti-obesity effects of xanthohumol alone because XRHE contains 82% unknown compounds. Dietary consumption of PX significantly inhibited an increase in the visceral fat weight of mice compared to those fed control diet without PX. Plasma leptin level was significantly lower in the PX-fed group than in the control group. Dietary PX lowered hepatic fatty acid synthesis by down-regulation of SREBP1c mRNA expression in the liver. On the other hand, fatty acid β-oxidation in the liver was promoted by dietary PX through the up-regulation of PPARα mRNA expression. Moreover, the fecal levels of fatty acids and carbohydrates increased by dietary PX. PX inhibited lipase or α-amylase activity in vitro. Thus, we found that PX may exert anti-obesity effects through the regulation of lipid metabolism and inhibition of intestinal fat and carbohydrate absorption, and that xanthohumol alone may exert anti-obesity effects.

Identification of copper/zinc superoxide dismutase as a novel nitric oxide-regulated gene in rat glomerular mesangial cells and kidneys of endotoxemic rats.

PubMed

Frank, S; Zacharowski, K; Wray, G M; Thiemermann, C; Pfeilschifter, J

1999-05-01

To define the mechanism of nitric oxide (NO) action in the glomerulus, we attempted to identify genes that are regulated by NO in rat glomerular mesangial cells. We identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced in these cells by treatment with S-nitroso-glutathione as a NO-donating agent. Bacterial lipopolysaccharide (LPS) acutely decreased Cu/Zn SOD mRNA levels. The LPS-mediated decrease in Cu/Zn SOD is reversed by endogenously produced NO, as LPS also induced a delayed strong iNOS expression in these cells in vitro, which is accompanied by increased Cu/Zn SOD expression. NO dependency of Cu/Zn SOD mRNA recovery could be demonstrated by inhibition of this process by L-NG-monomethylarginine, an inhibitor of NOS enzymatic activity. To demonstrate the in vivo relevance of our observations, we have chosen LPS-treated rats as a model for induction of a systemic inflammatory response. In these animals, we demonstrate a direct coupling of Cu/Zn SOD expression levels to the presence of NO, as Cu/Zn SOD mRNA levels declined during acute inflammation in the presence of a selective inhibitor of iNOS. We propose that the up-regulation of Cu/Zn SOD by endogenous NO may serve as an adaptive, protective mechanism to prevent the formation of toxic quantities of peroxynitrite in conditions associated with iNOS induction during endotoxic shock.

Human Monocytes Accelerate Proliferation and Blunt Differentiation of Preadipocytes in Association With Suppression of C/Ebpα mRNA

PubMed Central

Couturier, Jacob; Patel, Sanjeet G.; Iyer, Dinakar; Balasubramanyam, Ashok; Lewis, Dorothy E.

2015-01-01

Obesity, type 2 diabetes, and HIV-associated lipodystrophy are associated with abnormalities in adipocyte growth and differentiation. In persons with these conditions, adipose depots contain increased numbers of macrophages, but the origins of these cells and their specific effects are uncertain. Peripheral blood mononuclear cells (PBMC)-derived monocytes, but not T cells, cocultured via transwells with primary subcutaneous preadipocytes, increased proliferation (approximately twofold) and reduced differentiation (~50%) of preadipocytes. Gene expression analyses in proliferating preadipocytes (i.e., prior to hormonal induction of terminal differentiation) revealed that monocytes down-regulated mRNA levels of CCAAT/enhancer binding protein, alpha (C/EBPα) and up-regulated mRNA levels of G0/G1 switch 2 (G0S2) message, genes important for the regulation of adipogenesis and the cell cycle. These data indicate that circulating peripheral blood monocytes can disrupt adipogenesis by interfering with a critical step in C/EBPα and G0S2 transcription required for preadipocytes to make the transition from proliferation to differentiation. Interactions between preadipocytes and monocytes also increased the inflammatory cytokines IL-6 and IL-8, as well as a novel chemotactic cytokine, CXCL1. Additionally, the levels of both IL-6 and CXCL1 were highest when preadipocytes and monocytes were cultured together, compared to each cell in culture alone. Such cross-talk amplifies the production of mediators of tissue inflammation. PMID:21869759

Low-intensity pulsed ultrasound stimulation facilitates in vitro osteogenic differentiation of human adipose-derived stem cells via up-regulation of heat shock protein (HSP)70, HSP90, and bone morphogenetic protein (BMP) signaling pathway.

PubMed

Zhang, Zhonglei; Ma, Yalin; Guo, Shaowen; He, Yi; Bai, Gang; Zhang, Wenjun

2018-05-29

Low-intensity pulsed ultrasound (LIPUS) has positive effects on osteogenic differentiation. However, the effect of LIPUS on osteogenic differentiation of human adipose-derived stem cells (hASCs) is unclear. In the present study, we investigated whether LIPUS could promote the proliferation and osteogenic differentiation of hASCs. hASCs were isolated and osteogenically induced with LIPUS stimulation at 20 and 30 mW cm -2 for 30 min day -1 Cell proliferation and osteogenic differentiation potential of hASCs were respectively analyzed by cell counting kit-8 assay, Alizarin Red S staining, real-time polymerase chain reaction, and Western blotting. The results indicated that LIPUS stimulation did not significantly affect the proliferation of hASCs, but significantly increased their alkaline phosphatase activity on day 6 of culture and markedly promoted the formation of mineralized nodules on day 21 of culture. The mRNA expression levels of runt-related transcription factor, osteopontin, and osteocalcin were significantly up-regulated by LIPUS stimulation. LIPUS stimulation did not affect the expression of heat shock protein (HSP) 27, HSP40, bone morphogenetic protein (BMP)-6 and BMP-9, but significantly up-regulated the protein levels of HSP70, HSP90, BMP-2, and BMP-7 in the hASCs. Further studies found that LIPUS increased the mRNA levels of Smad 1 and Smad 5, elevated the phosphorylation of Smad 1/5, and suppressed the expression of BMP antagonist Noggin. These findings indicated that LIPUS stimulation enhanced osteogenic differentiation of hASCs possibly through the up-regulation of HSP70 and HSP90 expression and activation of BMP signaling pathway. Therefore, LIPUS might have the potential to promote the repair of bone defect. © 2018 The Author(s).

Gene expression levels of elastin and fibulin-5 according to differences between carotid plaque regions.

PubMed

Sivrikoz, Emre; Timirci-Kahraman, Özlem; Ergen, Arzu; Zeybek, Ümit; Aksoy, Murat; Yanar, Fatih; İsbir, Turgay; Kurtoğlu, Mehmet

2015-01-01

The purpose of this study was to investigate the gene expression levels of elastin and fibulin-5 according to differences between carotid plaque regions and to correlate it with clinical features of plaque destabilization. The study included 44 endarterectomy specimens available from operated symptomatic carotid artery stenoses. The specimens were separated according to anatomic location: internal carotid artery (ICA), external carotid artery (ECA) and common carotid artery (CCA), and then stored in liquid nitrogen. The amounts of cDNA for elastin and fibulin-5 were determined by Quantitative real-time PCR (Q-RT-PCR). Target gene copy numbers were normalized using hypoxanthine-guanine phosphoribosyltransferase (HPRT1) gene. The delta-delta CT method was applied for relative quantification. Q-RT-PCR data showed that relative fibulin-5 gene expression was increased in ICA plaque regions when compared to CCA regions but not reaching significance (p=0.061). At the same time, no differences were observed in elastin mRNA level between different anatomic plaque regions (p>0.05). Moreover, elastin and fibulin-5 mRNA expression and clinical parameters were compared in ICA plaques versus CCA and ECA regions, respectively. Up-regulation of elastin and fibulin-5 mRNA levels in ICA were strongly correlated with family history of cardiovascular disease when compared to CCA (p<0.05). Up-regulation of fibulin-5 in ICA was significantly associated with diabetes, and elevated triglycerides and very low density lipoprotein (VLDL) when compared to ECA (p<0.05). The clinical significance is the differences between the proximal and distal regions of the lesion, associated with the ICA, CCA and ECA respectively, with increased fibulin-5 in the ICA region. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

A Comparative study for striatal-direct and -indirect pathway neurons to DA depletion-induced lesion in a PD rat model.

PubMed

Zheng, Xuefeng; Wu, Jiajia; Zhu, Yaofeng; Chen, Si; Chen, Zhi; Chen, Tao; Huang, Ziyun; Wei, Jiayou; Li, Yanmei; Lei, Wanlong

2018-04-16

Striatal-direct and -indirect Pathway Neurons showed different vulnerability in basal ganglia disorders. Therefore, present study aimed to examine and compare characteristic changes of densities, protein and mRNA levels of soma, dendrites, and spines between striatal-direct and -indirect pathway neurons after DA depletion by using immunohistochemistry, Western blotting, real-time PCR and immunoelectron microscopy techniques. Experimental results showed that: 1) 6OHDA-induced DA depletion decreased the soma density of striatal-direct pathway neurons (SP+), but no significant changes for striatal-indirect pathway neurons (ENK+). 2) DA depletion resulted in a decline of dendrite density for both striatal-direct (D1+) and -indirect (D2+) pathway neurons, and D2+ dendritic density declined more obviously. At the ultrastructure level, the densities of D1+ and D2+ dendritic spines reduced in the 6OHDA groups compared with their control groups, but the density of D2+ dendritic spines reduced more significant than that of D1. 3) Striatal DA depletion down-regulated protein and mRNA expression levels of SP and D1, on the contrary, ENK and D2 protein and mRNA levels of indirect pathway neurons were up-regulated significantly. Present results suggested that indirect pathway neurons be more sensitive to 6OHDA-induced DA depletion. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effects of imidacloprid on detoxifying enzyme glutathione S-transferase on Folsomia candida (Collembola).

PubMed

Sillapawattana, Panwad; Schäffer, Andreas

2017-04-01

Chemical analyses of the environment can document contamination by various xenobiotics, but it is also important to understand the effect of pollutants on living organisms. Thus, in the present work, we investigated the effect of the pesticide imidacloprid on the detoxifying enzyme glutathione S-transferase (GST) from Folsomia candida (Collembola), a standard test organism for estimating the effects of pesticides and environmental pollutants on non-target soil arthropods. Test animals were treated with different concentrations of imidacloprid for 48 h. Changes in steady-state levels of GST messenger RNA (mRNA) and GST enzyme activity were investigated. Extracted proteins were separated according to their sizes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved protein bands were detected by silver staining. The size of the glutathione (GSH) pool in Collembola was also determined. A predicted protein sequence of putative GSTs was identified with animals from control group. A 3-fold up-regulation of GST steady-state mRNA levels was detected in the samples treated with 5.0 mg L -1 imidacloprid compared to the control, while a 2.5- and 2.0- fold up-regulation was found in organisms treated with 2.5 and 7.5 mg L -1 imidacloprid, respectively. GST activity increased with increasing imidacloprid amounts from an initial activity of 0.11 μmol min -1  mg -1 protein in the control group up to 0.25 μmol min -1  mg -1 protein in the sample treated with the 5.0 mg L -1 of pesticide. By contrast, the total amount of GSH decreased with increasing imidacloprid concentration. The results suggest that the alteration of GST activity, steady-state level of GST mRNA, and GSH level may be involved in the response of F. candida to the exposure of imidacloprid and can be used as biomarkers to monitor the toxic effects of imidacloprid and other environmental pollutants on Collembola.

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

NASA Astrophysics Data System (ADS)

Park, Kiyun; Kwak, Inn-Sil

2012-12-01

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

Thiorphan-induced survival and proliferation of rat thymocytes by activation of Akt/survivin pathway and inhibition of caspase-3 activity.

PubMed

Amantini, Consuelo; Mosca, Michela; Lucciarini, Roberta; Perfumi, Marina Cecilia; Santoni, Giorgio

2008-10-01

The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK(1)R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5(+) thymocytes, namely on the CD4(+)CD8(+) (double positive; DP) and CD4(+) subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK(1)R mRNA expression as well as SP and NK(1)R protein levels in an NK(1)R-dependent manner. Thiorphan increased CD10(+)CD4(+) and CD10(+)DP thymocyte numbers, and an NK(1)R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor alpha chain expression, and concanavalin A-induced proliferation of CD5(+) thymocytes, and it inhibited spontaneous and NK(1)R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.

Gene and genon concept: coding versus regulation

PubMed Central

2007-01-01

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon. PMID:18087760

Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland

PubMed Central

Seon, Sojeong; Jeon, Daun; Kim, Heejeong; Chung, Yiwa; Choi, Narae; Yang, Hyunwon

2017-01-01

ABSTRACT Nesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17β-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulation mechanism in the pituitary of male mouse. Therefore, we examined whether nesfatin-1/NUCB2 is expressed in the male mouse pituitary and if its expression is regulated by testosterone. As a result of PCR and western blotting, we found that a large amount of nesfatin-1/NUCB2 was expressed in the pituitary and hypothalamus. The NUCB2 mRNA expression level in the pituitary was decreased after castration, but not in the hypothalamus. In addition, its mRNA expression level in the pituitary was increased after testosterone treatment in the castrated mice, whereas, the expression level in the hypothalamus was significantly decreased after the treatment with testosterone. The in vitro experiment to elucidate the direct effect of testosterone on NUCB2 mRNA expression showed that NUCB2 mRNA expression was significantly decreased with testosterone in cultured hypothalamus tissue, but increased with testosterone in cultured pituitary gland. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the male mouse pituitary and was regulated by testosterone. This data suggests that reproductive-endocrine regulation through hypothalamus-pituitary-testis axis may contribute to NUCB2 mRNA expression in the mouse hypothalamus and pituitary gland. PMID:28484746

Post-transcriptional inducible gene regulation by natural antisense RNA.

PubMed

Nishizawa, Mikio; Ikeya, Yukinobu; Okumura, Tadayoshi; Kimura, Tominori

2015-01-01

Accumulating data indicate the existence of natural antisense transcripts (asRNAs), frequently transcribed from eukaryotic genes and do not encode proteins in many cases. However, their importance has been overlooked due to their heterogeneity, low expression level, and unknown function. Genes induced in responses to various stimuli are transcriptionally regulated by the activation of a gene promoter and post-transcriptionally regulated by controlling mRNA stability and translatability. A low-copy-number asRNA may post-transcriptionally regulate gene expression with cis-controlling elements on the mRNA. The asRNA itself may act as regulatory RNA in concert with trans-acting factors, including various RNA-binding proteins that bind to cis-controlling elements, microRNAs, and drugs. A novel mechanism that regulates mRNA stability includes the interaction of asRNA with mRNA by hybridization to loops in secondary structures. Furthermore, recent studies have shown that the functional network of mRNAs, asRNAs, and microRNAs finely tunes the levels of mRNA expression. The post-transcriptional mechanisms via these RNA-RNA interactions may play pivotal roles to regulate inducible gene expression and present the possibility of the involvement of asRNAs in various diseases.

Early transcriptional changes of retinal and choroidal TGFbeta-2, RALDH-2, and ZENK following imposed positive and negative defocus in chickens.

PubMed

Simon, Perikles; Feldkaemper, Marita; Bitzer, Michaela; Ohngemach, Sibylle; Schaeffel, Frank

2004-08-24

Imposing defocus to the retina results in compensatory changes of axial eye growth. It is not clear which factors initially contribute to this process and whether they act on the post-translational, translational, or transcriptional level. We have measured early changes in mRNA levels, in response to imposed negative and positive defocus, of the transcription factor ZENK, the retinoic acid synthesis enzyme RALDH-2, and the growth factor TGFbeta-2. Chickens 11 days of age were unilaterally treated with positive or negative spectacle lenses of 7 D power. After 0, 15, 30, and 120 min, mRNA was extracted from retina and choroid, and the concentration of the mRNAs of the three candidates was measured by quantitative real time PCR in both eyes. ZENK in the retina and RALDH-2 in the choroid displayed parallel signs of defocus dependent changes in mRNA levels after 15 or 30 min, respectively. ZENK mRNA levels were reduced in the retina after 15 min with both types of lenses but were then up regulated at 30 min with positive lenses and down regulated with negative lenses, similar to the previously observed changes in ZENK protein levels. Changes of RALDH-2 and TGFbeta-2 mRNA levels were confined to the choroid. Treatment with negative lenses resulted in a rapid (15 min) and persistent decrease in TGFbeta-2 mRNA concentration in the choroid. Negative lenses provoked parallel but less pronounced alterations in the open fellow eyes. Imposed defocus triggers extensive transcriptional changes of ZENK in the retina, and of TGFbeta-2 and RALHD-2 in the choroid. Changes in retina and choroid are rapid, show no phase delay with respect to each other, and can be considered, in the case of RALDH-2 and ZENK, as specific for the sign of imposed defocus. They occur prior to any morphological changes. This is consistent with a role in causing or controlling later changes in eye growth.

Expression and regulation of the chemokine CXCL16 in Crohn’s disease and models of intestinal inflammation

PubMed Central

Diegelmann, Julia; Seiderer, Julia; Niess, Jan-Hendrik; Haller, Dirk; Göke, Burkhard; Reinecker, Hans-Christian; Brand, Stephan

2010-01-01

Background/Aims CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria and is a strong chemoattractant for CXCR6+ T cells. In this study, we determined the so far unknown expression and signal transduction of the novel CXCL16-CXCR6 chemokine-ligand receptor system in intestinal inflammation in vivo and in vitro. Methods CXCL16 mRNA was measured by quantitative PCR in human colonic biopsies of patients with Crohn’s disease (CD) as well as in the TNFΔARE mouse model of ileitis and in murine cytomegalovirus (MCMV)-induced colitis. CXCL16 serum levels were analyzed by ELISA. CXCL16-induced signal transduction was analyzed in IEC with phospho-specific antibodies for MAP kinases and Akt. Results We found an inverse expression pattern of CXCL16 and CXCR6 with highest CXCL16 mRNA levels in the proximal murine small intestine and highest CXCR6 mRNA expression in the distal colon. CXCL16 and CXCR6 mRNA were expressed in colorectal cancer (CRC)-derived IEC lines. CRC-expressed CXCR6 was functional as demonstrated by CXCL16-induced MAP kinase and Akt activation. Intestinal CXCL16 expression was elevated in the TNFΔARE mouse model of ileitis and in MCMV-induced colitis (p<0.05) and in the sera and colons of patients with CD (p<0.05), where its expression correlated highly with CXCR6 and IL-8 levels (r=0.85 and 0.89, respectively). Conclusion CRC-derived IEC express the functional CXCL16 receptor CXCR6. CXCL16 mRNA and protein expression is up-regulated in intestinal inflammation in vitro and in CD patients, suggesting an important role for this chemokine in intestinal inflammation. PMID:20848509

Hyperresponsive febrile reactions to interleukin (IL) 1α and IL-1β, and altered brain cytokine mRNA and serum cytokine levels, in IL-1β-deficient mice

PubMed Central

Alheim, Katarina; Chai, Zhen; Fantuzzi, Giamila; Hasanvan, Homa; Malinowsky, David; Di Santo, Elena; Ghezzi, Pietro; Dinarello, Charles A.; Bartfai, Tamas

1997-01-01

IL-1β is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1α and IL-1β, and compared these with response to LPS (i.p.) in wild-type and IL-1β-deficient mice. The IL-1β deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1β, IL-1α, or LPS induced hyperresponsive fevers in the IL-1β-deficient mice. We also observed phenotypic differences between wild-type and IL-1β-deficient mice in hypothalamic basal mRNA levels for IL-1α and IL-6, but not for IL-1β-converting enzyme or IL-1 receptor type I or type II. The IL-1α mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1β-deficient mice as compared with wild-type mice. The IL-1β-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type α levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1β plays an important but not obligatory role in fever induction by LPS or IL-1α, as well as in the induction of serum tumor necrosis factor type α and corticosterone responses either by LPS or by IL-1α or IL-1β. PMID:9122256

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

PubMed

Goppelt-Struebe, M; Reiser, C O; Schneider, N; Grell, M

1996-10-01

Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

PubMed Central

Ferrero, Rut; Torres, Magdalena

2002-01-01

Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

Molecular cloning and expression profiles of IGFBP-1a in common carp (Cyprinus carpio) and its expression regulation by growth hormone in hepatocytes.

PubMed

Chen, Wenbo; Lin, Haoran; Li, Wensheng

2018-04-23

In this study, we cloned and determined IGFBP-1a cDNA from common carp (Cyprinus carpio) liver. The 1655 bp full-length cDNA consisted of a 96 bp 5-untranslated region (UTR), a 789 bp open reading frame encoding 262 amino acid residues and a 770 bp 3-UTR containing seven mRNA instability motifs. Northern blot revealed a 1.8 kb IGFBP-1a transcript. IGFBP-1a mRNA was widely distributed in all tissues examined and predominantly expressed in the liver. During embryogenesis, IGFBP-1a mRNA was firstly observed in blastula stage, and significant increases were observed in body segment stage, lens formation stage and blood cycling stage. After hatching, its expression increased more than twenty times. Furthermore, hypoxia could significantly up-regulate IGFBP-1a expression in the liver and brain. IGFBP-1a expression increased with ovarian maturation and decreased at regressed stage. In testis, IGFBP-1a mRNA maintained relatively higher levels at recrudescing and matured stages, while it sharply declined at regressed stage. In primary cultured hepatocytes, IGFBP-1a gene was greatly down-regulated by growth hormone via the MAPK and PI3 kinase signaling pathways. These results suggest that IGFBP-1a may be involved in the IGF system regulating growth, development and reproduction in common carp. Copyright © 2018. Published by Elsevier Inc.

Regulation of mouse hepatic CYP2D9 mRNA expression by growth and adrenal hormones.

PubMed

Jarukamjorn, Kanokwan; Sakuma, Tsutomu; Jaruchotikamol, Atika; Oguro, Miki; Nemoto, Nobuo

2006-02-01

The constitutive expression of CYP2D9 is sexually dimorphic, namely, strong in males, but diminutive in females. Repetition of mimic growth hormone (GH) secretion pattern impressively returned the mRNA expression level to that in intact mice: the GH secretion pattern's regulation of CYP2D9 mRNA expression has been predominantly disrupted by exogenous GH-administration. The extensive decline of CYP2D9 mRNA expression becoming a sexually non-specific P450 in 9-week-old male mice exposed as neonates to monosodium L-glutamate (MSG) suggested that the male GH secretion pattern is a key to the regulation of male-specific CYP2D9 mRNA expression in adult mice. Dexamethasone (Dex) showed possibility to induce CYP2D9 mRNA expression in adult MSG-neonatally treated mice of either sex. However, the antagonism was observed by co-administration of Dex and GH in the males. Dex-administration in adrenalectomized mice significantly elevated CYP2D9 mRNA expression levels. These findings suggest that an adrenal hormone participates in the regulatory mechanism of CYP2D9 mRNA expression in association with GH.

Regulation of fish growth hormone transcription.

PubMed

Farchi-Pisanty, O; Hackett, P B; Moav, B

1995-09-01

Regulation of endogenous fish growth hormone transcription was studied using carp pituitaries in vitro. It was demonstrated that thyroid hormone (T3) and 9-cis retinoic acid have increased the steady state levels of growth hormone messenger RNA in pituitary cells, as compared with beta-actin messenger RNA levels. In contrast, estrogen failed to increase growth hormone mRNA levels. The possible involvement of thyroid hormone receptor in pituitary gene expression was demonstrated by in situ localization of both growth hormone mRNA and thyroid hormone receptor mRNA in the pituitaries as early as 4 days after fertilization.

Characterization of a novel rice gene OsATX and modulation of its expression by components of the stress signalling pathways.

PubMed

Agrawal, Ganesh K; Rakwal, Randeep; Jwa, N-S; Agrawal, Vishwanath P

2002-09-01

In our search to identify gene(s) involved in the rice self-defense responses, we cloned a novel rice (Oryza sativa L. cv. Nipponbare) gene, OsATX, a single copy gene, from the JA treated rice seedling leaves cDNA library. This gene encodes a 69 amino acid polypeptide with a predicted molecular mass of 7649.7 and a pI of 5.6. OsATX was responsive to cutting (wounding by cutting the excised leaf), over its weak constitutive expression in the healthy leaves. The critical signalling molecules, jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and hydrogen peroxide, together with protein phosphatase inhibitors, effectively up-regulated the OsATX expression with time, over the excised leaf cut control, whereas ethylene had no affect. Furthermore, copper, a heavy metal, also up-regulated OsATX expression. Moreover, induced expression of OsATX mRNA was influenced by light signal(s), and showed a requirement for de novo synthesized protein factors. Additionally, co-application of either JA or ABA with SA drastically suppressed the induced OsATX mRNA level. Finally, the blast pathogen, Magnaporthe grisea, triggered OsATX mRNA accumulation. These results strongly suggest a function/role(s) for OsATX in defense/stress responses in rice.

Differential expression of chicken dimerization cofactor of hepatocyte nuclear factor-1 (DcoH) and its novel counterpart, DcoHalpha.

PubMed Central

Kim, H; You, S; Foster, L K; Farris, J; Choi, Y J; Foster, D N

2001-01-01

We have used differential display PCR to study altered gene expression in immortalized chicken embryo fibroblasts (CEFs) that have been established in our laboratory. This technique resulted in the cloning of a novel counterpart of the previously cloned chicken dimerization cofactor of hepatocyte nuclear factor (HNF)-1 (cDcoH), which was identified as cDcoHalpha. The steady-state mRNA levels of cDcoHalpha were up-regulated in all immortal CEFs tested compared with primary CEF cells. cDcoH and cDcoHalpha showed opposite patterns of mRNA expression due to differential regulation of transcription rates, but not mRNA half-lives, in primary and immortal CEFs. Expression of cDcoHalpha increased in the late G1 and early S phases of the cell cycle, while cDcoH mRNA increased in the late S and G2/M phases. In contrast with consistent expression of both genes in primary quiescent cells, cDcoH mRNA, but not cDcoHalpha mRNA, was dramatically decreased in primary senescent cells. The highest levels of cDcoHalpha mRNA were found in the kidney, liver, heart and ovarian follicles, while the major tissues expressing cDcoH were hypothalamus, kidney and liver. cDcoH and cDcoHalpha probes did not cross-hybridize to human hepatocyte mRNA. When transfected into human HepG2 cells, both cDcoH and cDcoHalpha showed similar functional activity as measured by increased expression of a reporter gene, as well as alpha-fetoprotein and albumin genes that both contain HNF-1 binding elements in their promoters. Our results suggest that the novel chicken DcoHalpha might function as a transcriptional cofactor for HNF-1 in specific cellular-environmental states. PMID:11237869

Plasma-membrane H(+)-ATPase gene expression is regulated by NaCl in cells of the halophyte Atriplex nummularia L.

PubMed

Niu, X; Zhu, J K; Narasimhan, M L; Bressan, R A; Hasegawa, P M

1993-01-01

An Atriplex nummularia L. cDNA probe encoding the partial sequence of an isoform of the plasma-membrane H(+)-ATPase was isolated, and used to characterize the NaCl regulation of mRNA accumulation in cultured cells of this halophyte. The peptide (477 amino acids) translated from the open reading frame has the highest sequence homology to the Nicotiana plumbaginifolia plasma-membrane H(+)-ATPase isoform pma4 (greater than 80% identity) and detected a transcript of approximately 3.7 kb on Northern blots of both total and poly(A)+ RNA. The mRNA levels were comparable in unadapted cells, adapted cells (cells adapted to and growing in 342 mM NaCl) and deadapted cells (cells previously adapted to 342 mM NaCl that are now growing without salt). Increased mRNA abundance was detected in deadapted cells within 24 h after exposure to NaCl but not in unadapted cells with similar salt treatments. The NaCl up-regulation of message abundance in deadapted cells was subject to developmental control. Analogous to those reported for glycophytes, the plasma-membrane H(+)-ATPase are encoded by a multigene family in the halophyte.

Anti-inflammatory effect of miltirone on inflammatory bowel disease via TLR4/NF-κB/IQGAP2 signaling pathway.

PubMed

Wang, Hongjian; Gu, Junfei; Hou, Xuefeng; Chen, Juan; Yang, Nan; Liu, Ying; Wang, Gang; Du, Mei; Qiu, Huihui; Luo, Yi; Jiang, Ziyu; Feng, Liang

2017-01-01

Inflammatory bowel disease (IBD) is characterized by a radical imbalance in the activation of proinflammatory and anti-inflammatory signaling pathways in the gut. This study was conducted to evaluate the anti-inflammation effect of miltirone against IBD in vitro and in vivo, and try to explore the underlying mechanisms. Miltirone could extenuate the loss of colon length and weight caused by TNBS. Additionally, macroscopic scores and DAI were reduced significantly compared with the TNBS group. The levels of TNF-α, IL-1β, IL-6 and IL-8 were increased significantly with the induction by TNBS (100mg/kg) or LPS (0.5mg/mL). Interestingly, miltirone could down-regulate the levels of these increased pro-inflammatory factors in a dose-dependent manner both in vivo and in vitro. The protein and mRNA expressions of TLR4, MyD88, NF-κB p65 were up-regulated by TNBS or LPS stimulation. CRX-526, the TLR4 inhibitor, as well as miltirone could significantly suppress the increased protein and mRNA expressions. Miltirone could up-regulate the descreased IQGAP2 expression induced by LPS. All these revealed that the anti-inflammatory effect of miltirone on IBD may be via regulating TLR4/NF-κB/IQGAP2 signaling pathway. The findings might supply beneficial hints for the drug research to cure the IBD. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

TSHB mRNA is linked to cholesterol metabolism in adipose tissue.

PubMed

Moreno-Navarrete, José María; Moreno, María; Ortega, Francisco; Xifra, Gemma; Hong, Shangyu; Asara, John M; Serrano, José C E; Jové, Mariona; Pissios, Pavlos; Blüher, Matthias; Ricart, Wifredo; Portero-Otin, Manuel; Fernández-Real, José Manuel

2017-10-01

Subclinical hypothyroidism is known to be associated with increased serum cholesterol. Since thyroid-stimulating hormone (TSH) exerts an inductor effect on cholesterol biosynthesis, we aimed to investigate the relationship between TSH mRNA and cholesterol metabolism in human adipose tissue (AT). Cross-sectionally, AT TSH-β ( TSHB ) mRNA was evaluated in 4 independent cohorts in association with serum total and LDL cholesterol, and AT lipidomics. Longitudinally, the effects of statins and of diet and exercise on AT TSHB mRNA were also examined. The bidirectional relationship between cholesterol and TSHB were studied in isolated human adipocytes. TSHB mRNA was consistently detected in AT from euthyroid subjects, and positively associated with serum total- and LDL-cholesterol, and with AT-specific cholesterol metabolism-associated lipids [arachidonoyl cholesteryl ester, C8-dihydroceramide, N -stearoyl-d-sphingosine, and GlcCer(18:0, 24:1)]. Reduction of cholesterol with statins and with diet and exercise interventions led to decreased TSHB mRNA in human AT, whereas excess cholesterol up-regulated TSHB mRNA in human adipocytes. In addition, recombinant human TSH α/β administration resulted in increased HMGCR mRNA levels in human adipocytes. In mice, subcutaneous AT Tshb expression levels correlated directly with circulating cholesterol levels. In summary, current results provide novel evidence of TSHB as a paracrine factor that is modulated in parallel with cholesterol metabolism in human AT.-Moreno-Navarrete, J. M., Moreno, M., Ortega, F., Xifra, G., Hong, S., Asara, J. M., Serrano, J. C. E., Jové, M., Pissios, P., Blüher, M., Ricart, W., Portero-Otin, M., Fernández-Real, J. M. TSHB mRNA is linked to cholesterol metabolism in adipose tissue. © FASEB.

Proteomics, bioinformatics and targeted gene expression analysis reveals up-regulation of cochlin and identifies other potential biomarkers in the mouse model for deafness in usher syndrome type 1F

PubMed Central

Chance, Mark R.; Chang, Jinsook; Liu, Shuqing; Gokulrangan, Giridharan; Chen, Daniel H.-C.; Lindsay, Aaron; Geng, Ruishuang; Zheng, Qing Y.; Alagramam, Kumar

2010-01-01

Proteins and protein networks associated with cochlear pathogenesis in the Ames waltzer (av) mouse, a model for deafness in Usher syndrome 1F (USH1F), were identified. Cochlear protein from wild-type and av mice at postnatal day 30, a time point in which cochlear pathology is well established, was analyzed by quantitative 2D gel electrophoresis followed by mass spectrometry (MS). The analytic gel resolved 2270 spots; 69 spots showed significant changes in intensity in the av cochlea compared with the control. The cochlin protein was identified in 20 peptide spots, most of which were up-regulated, while a few were down-regulated. Analysis of MS sequence data showed that, in the av cochlea, a set of full-length isoforms of cochlin was up-regulated, while isoforms missing the N-terminal FCH/LCCL domain were down-regulated. Protein interaction network analysis of all differentially expressed proteins was performed with Metacore software. That analysis revealed a number of statistically significant candidate protein networks predicted to be altered in the affected cochlea. Quantitative PCR (qPCR) analysis of select candidates from the proteomic and bioinformatic investigations showed up-regulation of Coch mRNA and those of p53, Brn3a and Nrf2, transcription factors linked to stress response and survival. Increased mRNA of Brn3a and Nrf2 has previously been associated with increased expression of cochlin in human glaucomatous trabecular meshwork. Our report strongly suggests that increased level of cochlin is an important etiologic factor leading to the degeneration of cochlear neuroepithelia in the USH1F model. PMID:20097680

Differential effects of cortisol and 11-deoxycorticosterone on ion transport protein mRNA levels in gills of two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis).

PubMed

Kiilerich, Pia; Tipsmark, Christian K; Borski, Russell J; Madsen, Steffen S

2011-04-01

The role of cortisol as the only corticosteroid in fish osmoregulation has recently been challenged with the discovery of a mineralocorticoid-like hormone, 11-deoxycorticosterone (DOC), and necessitates new studies of the endocrinology of osmoregulation in fish. Using an in vitro gill explant incubation approach, DOC-mediated regulation of selected osmoregulatory target genes in the gill was investigated and compared with that of cortisol in two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis). The effects were tested in gills from both fresh water (FW)- and seawater (SW)-acclimated fish. Both cortisol and DOC caused an up-regulation of the Na(+),K(+)-ATPase α1 subunit in SW-acclimated tilapia but had no effect in FW-acclimated fish. Cortisol conferred an increase in Na(+),K(+),2Cl(-) cotransporter (NKCC) isoform 1a transcript levels in FW- and SW-acclimated tilapia, whereas DOC had a stimulatory effect only in SW-acclimated fish. Cortisol had no effect on NKCC isoform 1b mRNA levels at both salinities, while DOC stimulated this isoform in SW-acclimated fish. In striped bass, cortisol conferred an up-regulation of Na(+),K(+)-ATPase α1 and NKCC transcript levels in FW- and SW-acclimated fish, whereas DOC resulted in down-regulation of these transcripts in FW-acclimated fish. It was also found that both corticosteroids may rapidly (30 min) alter the mitogen-activated protein kinase signalling pathway in gill, inducing phosphorylation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in a salinity-dependent manner. The study shows a disparate organisation of corticosteroid signalling mechanisms involved in ion regulation in the two species and adds new evidence to a role of DOC as a mineralocorticoid hormone in teleosts.

The expression of Apoc3 mRNA is regulated by HNF4α and COUP-TFII, but not acute retinoid treatments, in primary rat hepatocytes and hepatoma cells.

PubMed

Howell, Meredith; Li, Rui; Zhang, Rui; Li, Yang; Chen, Wei; Chen, Guoxun

2014-02-01

Vitamin A status regulates obesity development, hyperlipidemia, and hepatic lipogenic gene expression in Zucker fatty (ZF) rats. The development of hyperlipidemia in acne patients treated with retinoic acid (RA) has been attributed to the induction of apolipoprotein C-III expression. To understand the role of retinoids in the development of hyperlipidemia in ZF rats, the expression levels of several selected RA-responsive genes in the liver and isolated hepatocytes from Zucker lean (ZL) and ZF rats were compared using real-time PCR. The Rarb and Srebp-1c mRNA levels are higher in the liver and isolated hepatocytes from ZF than ZL rats. The Apoc3 mRNA level is only higher in the isolated hepatocytes from ZF than ZL rats. To determine whether dynamic RA production acutely regulates Apoc3 expression, its mRNA levels in response to retinoid treatments or adenovirus-mediated overexpression of hepatocyte nuclear factor 4 alpha (HNF4α) and chicken ovalbumin upstream-transcription factor II (COUP-TFII) were analyzed. Retinoid treatments for 2-6 h did not induce the expression of Apoc3 mRNA. The overexpression of HNF4α or COUP-TFII induced or inhibited Apoc3 expression, respectively. We conclude that short-term retinoid treatments could not induce Apoc3 mRNA expression, which is regulated by HNF4α and COUP-TFII in hepatocytes.

Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

PubMed Central

2004-01-01

IL-1F7b, a novel homologue of the IL-1 (interleukin 1) family, was discovered by computational cloning. We demonstrated that IL-1F7b shares critical amino acid residues with IL-18 and binds to the IL-18-binding protein enhancing its ability to inhibit IL-18-induced interferon-γ. We also showed that low levels of IL-1F7b are constitutively present intracellularly in human blood monocytes. In this study, we demonstrate that similar to IL-18, both mRNA and intracellular protein expression of IL-1F7b are up-regulated by LPS (lipopolysaccharide) in human monocytes. In stable transfectants of murine RAW264.7 macrophage cells, there was no IL-1F7b protein expression despite a highly active CMV promoter. We found that IL-1F7b-specific mRNA was rapidly degraded in transfected cells, via a 3′-UTR (untranslated region)-independent control of IL-1F7b transcript stability. After LPS stimulation, there was a rapid transient increase in IL-1F7b-specific mRNA and concomitant protein levels. Using sequence alignment, we found a conserved ten-nucleotide homology box within the open reading frame of IL-F7b, which is flanking the coding region instability elements of some selective genes. In-frame deletion of downstream exon 5 from the full-length IL-1F7b cDNA markedly increased the levels of IL-1F7b mRNA. A similar coding region element is located in IL-18. When transfected into RAW264.7 macrophages, IL-18 mRNA was also unstable unless treated with LPS. These results indicate that both IL-1F7b and IL-18 mRNA contain functional instability determinants within their coding region, which influence mRNA decay as a novel mechanism to regulate the expression of IL-1 family members. PMID:15046617

Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.

PubMed

Santoso, D; Thornburg, R

2000-08-01

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.

Fluoroorotic Acid-Selected Nicotiana plumbaginifolia Cell Lines with a Stable Thymine Starvation Phenotype Have Lost the Thymine-Regulated Transcriptional Program1

PubMed Central

Santoso, Djoko; Thornburg, Robert

2000-01-01

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines. PMID:10938367

DAN (NBL1) specifically antagonizes BMP2 and BMP4 and modulates the actions of GDF9, BMP2, and BMP4 in the rat ovary.

PubMed

Hung, Wei-Ting; Wu, Fang-Ju; Wang, Chun-Jen; Luo, Ching-Wei

2012-05-01

Although differential screening-selected gene aberrative in neuroblastoma (DAN, official symbol NBL1) is the founding member of the DAN subfamily of bone morphogenetic protein (BMP) antagonists, its antagonizing targets, gene regulation, and physiological functions remain unclear. Using diverse cell expression systems, we found that the generation of bioactive DAN is likely to be cell type specific. Unlike other phylogenetically close members, which are covalently linked homodimers, DAN forms a noncovalently linked homodimer during folding. Purified recombinant DAN specifically blocked signaling of BMP2 and BMP4 but not that of other ovarian-expressed transforming growth factor-beta members. Although widely distributed in many organs, DAN transcript level was periodically regulated by gonadotropins. Ovarian microdissection indicated that NBL1 (DAN) mRNA is mainly expressed in granulosa cells, where its transcript level is up-regulated by the gonadotropin-driven cAMP cascade. We further investigated the local regulation and ovarian functions of DAN. NBL1 (DAN) mRNA expression in granulosa cells was up-regulated by oocyte-derived growth differentiation factor 9 (GDF9), whereas treatment with DAN significantly reversed the inhibitory effect of BMP4 on follicle-stimulating hormone-induced progesterone production in cultured granulosa cells. Our findings suggest the DAN gradient in granulosa cells, established by oocyte-derived GDF9, may serve as an antagonist barrier that modulates the actions of theca-derived BMP4 and granulosa/theca-derived BMP2 during folliculogenesis both spatially and temporally.

Elevated expression of neuropeptide signaling genes in the eyestalk ganglia and Y-organ of Gecarcinus lateralis individuals that are refractory to molt induction.

PubMed

Pitts, Natalie L; Schulz, Hanna M; Oatman, Stephanie R; Mykles, Donald L

2017-12-01

Molting is induced in decapod crustaceans via multiple leg autotomy (MLA) or eyestalk ablation (ESA). MLA removes five or more walking legs, which are regenerated and become functional appendages at ecdysis. ESA eliminates the primary source of molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), which suppress the production of molting hormones (ecdysteroids) from the molting gland or Y-organ (YO). Both MLA and ESA are effective methods for molt induction in Gecarcinus lateralis. However, some G. lateralis individuals are refractory to MLA, as they fail to complete ecdysis by 12weeks post-MLA; these animals are in the "blocked" condition. Quantitative polymerase chain reaction was used to quantify mRNA levels of neuropeptide and mechanistic target of rapamycin (mTOR) signaling genes in YO, eyestalk ganglia (ESG), thoracic ganglion (TG), and brain of intact and blocked animals. Six of the seven neuropeptide signaling genes, three of four mTOR signaling genes, and Gl-elongation factor 2 (EF2) mRNA levels were significantly higher in the ESG of blocked animals. Gl-MIH and Gl-CHH mRNA levels were higher in the TG and brain of blocked animals and levels increased in both control and blocked animals in response to ESA. By contrast, mRNA levels of Gl-EF2 and five of the 10 MIH signaling pathway genes in the YO were two to four orders of magnitude higher in blocked animals compared to controls. These data suggest that increased MIH and CHH synthesis in the ESG contributes to the prevention of molt induction by MLA in blocked animals. The up-regulation of MIH signaling genes in the YO of blocked animals suggests that the YO is more sensitive to MIH produced in the ESG, as well as MIH produced in brain and TG of ESA animals. Both the up-regulation of MIH signaling genes in the YO and of Gl-MIH and Gl-CHH in the ESG, TG, and brain appear to contribute to some G. lateralis individuals being refractory to MLA and ESA. Copyright © 2017 Elsevier Inc. All rights reserved.

Innate Immune Responses in ALV-J Infected Chicks and Chickens with Hemangioma In Vivo.

PubMed

Feng, Min; Dai, Manman; Xie, Tingting; Li, Zhenhui; Shi, Meiqing; Zhang, Xiquan

2016-01-01

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression. Since the precise mechanism of the innate immune response induced by ALV-J is unknown, we investigated the antiviral innate immune responses induced by ALV-J in chicks and chickens that had developed tumors. Spleen levels of interleukin-6 (IL-6), IL-10, IL-1β, and interferon-β (IFN-β) were not significantly different between the infected chick groups and the control groups from 1 day post hatch to 7 days post hatch. However, IL-6, IL-1β, and IFN-β protein levels in the three clinical samples with hemangiomas were dramatically increased compared to the healthy samples. In addition, the anti-inflammatory cytokine IL-10 increased sharply in two of three clinical samples. We also found a more than 20-fold up-regulation of ISG12-1 mRNA at 1 day post infection (d.p.i.) and a twofold up-regulation of ZC3HAV1 mRNA at 4 d.p.i. However, there were no statistical differences in ISG12-1 and ZC3HAV1 mRNA expression levels in the tumorigenesis phase. ALV-J infection induced a significant increase of Toll-like receptor 7 (TLR-7) at 1 d.p.i. and dramatically increased the mRNA levels of melanoma differentiation-associated gene 5 (MDA5) in the tumorigenesis phase. Moreover, the protein levels of interferon regulatory factor 1 (IRF-1) and signal transducer and activator of transcription 1 (STAT1) were decreased in chickens with tumors. These results suggest that ALV-J was primarily recognized by chicken TLR7 and MDA5 at early and late in vivo infection stages, respectively. ALV-J strain SCAU-HN06 did not induce any significant antiviral innate immune response in 1 week old chicks. However, interferon-stimulated genes were not induced normally during the late phase of ALV-J infection due to a reduction of IRF1 and STAT1 expression.

Cold-Induced Accumulation of hsp90 Transcripts in Brassica napus.

PubMed Central

Krishna, P.; Sacco, M.; Cherutti, J. F.; Hill, S.

1995-01-01

Characterization of the expression of hsp90 genes of Brassica napus by northern blot analysis and immunoblotting showed that the hsp90 mRNA and protein are present in all B. napus tissues examined, albeit at different levels. High levels of hsp90 mRNA and protein were found in young and rapidly dividing tissues such as shoot apices and flower buds, suggesting that hsp90 may have an important role in plant growth and development. A significant increase in hsp90 mRNA levels was detected in seedlings exposed to 5[deg]C. The transcript levels reached a maximum within 1 d of cold treatment and remained elevated for the entire duration of cold treatment. The levels of hsp90 mRNA rapidly decreased to the level found in control plants upon return to 20[deg]C. The cold-induced accumulation of hsp90 mRNA closely resembles the expression of two previously identified cold-regulated genes of B. napus. We have also confirmed cold regulation of hsp90 mRNA in spinach (Spinacea oleracea). Our results suggest a role for hsp90 in adaptation to cold temperature stress. PMID:12228411

Effect of emodin on mobility signal transduction system of gallbladder smooth muscle in Guinea pig with cholelithiasis.

PubMed

Fang, Bang-Jiang; Shen, Jun-Yi; Zhang, Hua; Zhou, Shuang; Lyu, Chuan-Zhu; Xie, Yi-Qiang

2016-10-01

To study the effect of emodin on protein and gene expressions of the massagers in mobility signal transduction system of cholecyst smooth muscle cells in guinea pig with cholesterol calculus. The guinea pigs were randomly divided into 4 groups, such as control group, gall-stone (GS) group, emodin group and ursodeoxycholic acid (UA) group. Cholesterol calculus models were induced in guinea pigs of GS, emodin and UA groups by lithogenic diet, while emodin or UA were given to the corresponding group for 7 weeks. The histomorphological and ultrastructure change of gallbladder were detected by microscope and electron microscope, the content of plasma cholecystokinin (CCK) and [Ca 2+ ] i were analyzed successively by radioimmunoassay and flow cytometry. The protein and mRNA of Gsα, Giα and Cap in cholecyst cells were determined by western blotting and real time polymerase chain reaction (RT-PCR). Emodin or UA can relieve pathogenic changes in epithelial cells and muscle cells in gallbladder of guinea pig with cholesterol calculus by microscope and transmission electron microscope. In the cholecyst cells of GS group, CCK levels in plasma and [Ca 2+ ] i decreased, the protein and mRNA of GS were down-regulated, the protein and mRNA of Gi and Cap were up-regulated. Emodin significantly decreased the formative rate of gallstone, improved the pathogenic change in epithelial cells and muscle cells, increased CCK levels in plasma and [Ca 2+ ] i in cholecyst cells, enhanced the protein and mRNA of Gs in cholecyst cells, reduced the protein and mRNA of Gi and Cap in cholecyst cells in guinea pig with cholesterol calculus. The dysfunction of gallbladder contraction gives rise to the disorders of mobility signal transduction system in cholecyst smooth muscle cells, including low content of plasma CCK and [Ca 2+ ] i in cholecyst cells, abnormal protein and mRNA of Gs, Gi and Cap. Emodin can enhance the contractibility of gallbladder and alleviate cholestasis by regulating plasma CCK levels, [Ca 2+ ] i in cholecyst cells and the protein and mRNA of Gs, Gi and Cap. Copyright © 2016 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.

Evidences for involvement of growth hormone and insulin-like growth factor in ovarian development of starry flounder (Platichthys stellatus).

PubMed

Xu, Yongjiang; Wang, Bin; Liu, Xuezhou; Shi, Bao; Zang, Kun

2017-04-01

Although gonadotrophins are major regulators of ovarian function in teleosts and other vertebrates, accumulating evidence indicates that the growth hormone (GH)-insulin-like growth factor (IGF) axis also plays an important role in fish reproduction. As a first step to understand the physiological role of the GH-IGF system in the ovarian development of starry flounder (Platichthys stellatus), the expression profiles of GH and IGF messenger RNAs (mRNAs) and plasma GH, IGF-I, estradiol-17β (E2), and testosterone (T) levels during the ovarian development were investigated. The developmental stages of ovaries were divided into five stages (II, III, IV, V, and VI) by histological analysis. The hepatosomatic index (HSI) and gonadosomatic index (GSI) values increased and peaked at stage IV and stage V, respectively, and then declined at stage VI. Pituitary GH mRNA levels decreased sharply at stage III and raised to top level at stage VI. The hepatic IGF-I mRNA levels ascended to maximum value at stage V and then declined significantly at stage VI. However, the hepatic IGF-II mRNA levels remained stable and increased significantly at stage VI. In contrast, the ovarian IGF-I mRNA levels increased gradually and peaked at stage VI. The ovarian IGF-II mRNA levels were initially stable and increased significantly at stage V until the top level at stage VI. Consistent with the pituitary GH mRNA levels, plasma GH levels reduced sharply at stage III and remained depressed until stage V and then raised remarkably at stage VI. Plasma IGF-I level peaked at stage V and then declined to initial level. Plasma E2 level peaked at stage IV and then dramatically descended to the basal level. Plasma T level peaked at stage V and then declined significantly back to the basal level. Based on statistical analysis, significant positive correlations between hepatic IGF-I mRNA and GSI, ovarian IGF-II mRNA and hepatic IGF-II mRNA, ovarian IGF-I mRNA and ovarian IGF-II mRNA, and plasma IGF-I and plasma T were observed, respectively. These results suggest that the GH-IGF system may be involved in the ovarian development of starry flounder; GH and IGFs appear to play distinct roles in the regulation of the ovarian development in paracrine/autocrine manners. These findings extend our knowledge of the roles of the GH-IGF axis on reproduction regulation in fish.

MicroRNAs May Mediate the Down-Regulation of Neurokinin-1 Receptor in Chronic Bladder Pain Syndrome

PubMed Central

Sanchez Freire, Veronica; Burkhard, Fiona C.; Kessler, Thomas M.; Kuhn, Annette; Draeger, Annette; Monastyrskaya, Katia

2010-01-01

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B1 receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor. PMID:20008142

Chronic restraint stress up-regulates GLT-1 mRNA and protein expression in the rat hippocampus: Reversal by tianeptine

PubMed Central

Reagan, Lawrence P.; Rosell, Daniel R.; Wood, Gwendolyn E.; Spedding, Michael; Muñoz, Carmen; Rothstein, Jeffrey; McEwen, Bruce S.

2004-01-01

Excitatory amino acids play a key role in stress-induced remodeling of dendrites in the hippocampus as well as in suppression of neurogenesis in the dentate gyrus. The regulation of extracellular glutamate levels has been suggested as a potential mechanism through which repeated stress causes dendritic remodeling of CA3 pyramidal neurons. Accordingly, the current study examined the distribution and regulation of the glia glutamate transporter GLT-1 and the recently identified GLT isoform, GLT-1b, in the hippocampus of rats subjected to chronic restraint stress (CRS). We also examined the ability of the antidepressant tianeptine, which blocks CRS-induced dendritic remodeling, to modulate CRS-mediated changes in GLT-1 and GLT-1b expression. CRS increased GLT-1 mRNA expression in the dentate gyrus and CA3 region of Ammon's horn, increases that were inhibited by tianeptine. CRS more selectively increased GLT-1 protein levels in the subregion where dendritic remodeling is most prominent, namely the CA3 region, increases that were also inhibited by tianeptine administration. In contrast, GLT-1b mRNA expression was not modulated in the hippocampus in any of these groups, but CRS increased GLT-1b protein levels in all hippocampal subfields examined, increases that were unaffected by tianeptine treatment. These results point to the importance of understanding the mechanism for the differential and subregional regulation of GLT-1 isoforms in neuronal and glial compartments in the hippocampus as a basis for understanding the effects of chronic stress on structural plasticity as well as the neuroprotective properties of agents such as tianeptine. PMID:14766991

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum.

PubMed

Venkataramanan, Keerthi P; Min, Lie; Hou, Shuyu; Jones, Shawn W; Ralston, Matthew T; Lee, Kelvin H; Papoutsakis, E Terry

2015-01-01

Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

The Role of Inducible Hsp70, and Other Heat Shock Proteins, in Adaptive Complex of Cold Tolerance of the Fruit Fly (Drosophila melanogaster)

PubMed Central

Štětina, Tomáš; Koštál, Vladimír; Korbelová, Jaroslava

2015-01-01

Background The ubiquitous occurrence of inducible Heat Shock Proteins (Hsps) up-regulation in response to cold-acclimation and/or to cold shock, including massive increase of Hsp70 mRNA levels, often led to hasty interpretations of its role in the repair of cold injury expressed as protein denaturation or misfolding. So far, direct functional analyses in Drosophila melanogaster and other insects brought either limited or no support for such interpretations. In this paper, we analyze the cold tolerance and the expression levels of 24 different mRNA transcripts of the Hsps complex and related genes in response to cold in two strains of D. melanogaster: the wild-type and the Hsp70- null mutant lacking all six copies of Hsp70 gene. Principal Findings We found that larvae of both strains show similar patterns of Hsps complex gene expression in response to long-term cold-acclimation and during recovery from chronic cold exposures or acute cold shocks. No transcriptional compensation for missing Hsp70 gene was seen in Hsp70- strain. The cold-induced Hsps gene expression is most probably regulated by alternative splice variants C and D of the Heat Shock Factor. The cold tolerance in Hsp70- null mutants was clearly impaired only when the larvae were exposed to severe acute cold shock. No differences in mortality were found between two strains when the larvae were exposed to relatively mild doses of cold, either chronic exposures to 0°C or acute cold shocks at temperatures down to -4°C. Conclusions The up-regulated expression of a complex of inducible Hsps genes, and Hsp70 mRNA in particular, is tightly associated with cold-acclimation and cold exposure in D. melanogaster. Genetic elimination of Hsp70 up-regulation response has no effect on survival of chronic exposures to 0°C or mild acute cold shocks, while it negatively affects survival after severe acute cold shocks at temperaures below -8°C. PMID:26034990

The Role of Inducible Hsp70, and Other Heat Shock Proteins, in Adaptive Complex of Cold Tolerance of the Fruit Fly (Drosophila melanogaster).

PubMed

Štětina, Tomáš; Koštál, Vladimír; Korbelová, Jaroslava

2015-01-01

The ubiquitous occurrence of inducible Heat Shock Proteins (Hsps) up-regulation in response to cold-acclimation and/or to cold shock, including massive increase of Hsp70 mRNA levels, often led to hasty interpretations of its role in the repair of cold injury expressed as protein denaturation or misfolding. So far, direct functional analyses in Drosophila melanogaster and other insects brought either limited or no support for such interpretations. In this paper, we analyze the cold tolerance and the expression levels of 24 different mRNA transcripts of the Hsps complex and related genes in response to cold in two strains of D. melanogaster: the wild-type and the Hsp70- null mutant lacking all six copies of Hsp70 gene. We found that larvae of both strains show similar patterns of Hsps complex gene expression in response to long-term cold-acclimation and during recovery from chronic cold exposures or acute cold shocks. No transcriptional compensation for missing Hsp70 gene was seen in Hsp70- strain. The cold-induced Hsps gene expression is most probably regulated by alternative splice variants C and D of the Heat Shock Factor. The cold tolerance in Hsp70- null mutants was clearly impaired only when the larvae were exposed to severe acute cold shock. No differences in mortality were found between two strains when the larvae were exposed to relatively mild doses of cold, either chronic exposures to 0°C or acute cold shocks at temperatures down to -4°C. The up-regulated expression of a complex of inducible Hsps genes, and Hsp70 mRNA in particular, is tightly associated with cold-acclimation and cold exposure in D. melanogaster. Genetic elimination of Hsp70 up-regulation response has no effect on survival of chronic exposures to 0°C or mild acute cold shocks, while it negatively affects survival after severe acute cold shocks at temperatures below -8°C.

Neurotrophins and their receptors in the rat pituitary gland: regulation of BDNF and trkB mRNA levels by adrenal hormones.

PubMed

Kononen, J; Soinila, S; Persson, H; Honkaniemi, J; Hökfelt, T; Pelto-Huikko, M

1994-12-01

We studied the expression of messenger ribonucleic acids (mRNAs) for neurotrophins and neurotrophin receptors in the rat pituitary gland and examined the influence of adrenal hormones on their mRNA levels, using in situ hybridization and Northern blot analysis. The only neurotrophin present at detectable levels in the pituitary was brain-derived neurotrophic factor (BDNF), which was observed in the anterior and intermediate lobes. Several transcripts of the putative receptor for BDNF, trkB, were present in the anterior and posterior lobes of the pituitary. A low amount of trkC mRNA was found in both the anterior and the intermediate lobe. Dexamethasone treatment decreased both BDNF and trkB mRNA levels in the anterior lobe of the pituitary. Adrenalectomy had no effect on trkB expression, but it decreased BDNF mRNA levels in comparison to the control animals. This effect could not be reversed by dexamethasone substitution, suggesting that BDNF, mRNA levels may be regulated not only by glucocorticoids but also by other adrenal hormones. These results demonstrate that BDNF, trkB and trkC are expressed in the pituitary gland and that glucocorticoids and possibly other adrenal hormones may modulate pituitary functions by regulating the expression of neurotrophic factors and their receptors. Whether BDNF acts as a secreted hormone, a trophic factor, or has autocrine/paracrine functions within the pituitary through its receptor, trkB, remains to be studied.

Molecular mechanism for the operation of nitrogen control in cyanobacteria.

PubMed Central

Luque, I; Flores, E; Herrero, A

1994-01-01

In cyanobacteria, ammonium exerts a negative regulation of the expression of proteins involved in the assimilation of nitrogen sources alternative to ammonium. In Synechococcus, mRNA levels of genes encoding proteins for nitrate and ammonium assimilation were observed to be negatively regulated by ammonium, and ammonium-regulated transcription start points were identified for those genes. The NtcA protein is a positive regulator of genes subjected to nitrogen control by ammonium. Mutants lacking NtcA exhibited only basal mRNA levels of the regulated genes, even in the absence of ammonium, indicating that NtcA exerts its regulatory action by positively influencing mRNA levels of the nitrogen-regulated genes. NtcA was observed to bind directly to the promoters of nitrogen-regulated genes, and the palindromic DNA sequence GTAN8TAC was identified as a sequence signature for NtcA-target sites. The structure of the nitrogen-, NtcA-regulated promoters of Synechococcus was determined to be constituted by a -10, Pribnow-like box in the form TAN3T, and an NtcA-binding site that substituted for the canonical -35 box. Images PMID:8026471

Role of Endogenous Cholecystokinin on Growth of Human Pancreatic Cancer

PubMed Central

Matters, Gail L.; McGovern, Christopher; Harms, John F.; Markovic, Kevin; Anson, Krystal; Jayakumar, Calpurnia; Martenis, Melissa; Awad, Christina; Smith, Jill P.

2012-01-01

Cholecystokinin (CCK) and gastrin stimulate growth of pancreatic cancer. Although down regulation of gastrin inhibits growth of pancreatic cancer, the contribution of endogenous CCK to tumor growth is unknown. The purpose of this study was to evaluate the role of endogenous CCK on autocrine growth of pancreatic cancer. Pancreatic cancer cell lines were analyzed for CCK mRNA and peptide expression by real time RT-PCR and radioimmunoassay, respectively. The effect of endogenous CCK on growth was evaluated by treating cancer cells with CCK neutralizing antibodies and by down regulating CCK mRNA by RNAi. Wild type pancreatic cancer cells expressed significantly lower CCK mRNA and peptide levels than gastrin. Neither treatment of pancreatic cancer cells with CCK antibodies nor the down regulation of CCK mRNA and peptide by shRNAs altered growth in vitro or in vivo. Conversely, when gastrin mRNA expression was down regulated, the same cells failed to produce tumors in spite of having sustained levels of endogenous CCK. Pancreatic cancer cells produce CCK and gastrin; however, the autocrine production of gastrin is more important for stimulating tumor growth. PMID:21186400

Adipose tissue CIDEA is associated, independently of weight variation, to change in insulin resistance during a longitudinal weight control dietary program in obese individuals.

PubMed

Montastier, Emilie; Déjean, Sébastien; Le Gall, Caroline; Saris, Wim H M; Langin, Dominique; Viguerie, Nathalie

2014-01-01

Weight loss reduces risk factors associated with obesity. However, long-term metabolic improvement remains a challenge. We investigated quantitative gene expression of subcutaneous adipose tissue in obese individuals and its relationship with low calorie diet and long term weight maintenance induced changes in insulin resistance. Three hundred eleven overweight and obese individuals followed a dietary protocol consisting of an 8-week low calorie diet followed by a 6-month ad libitum weight-maintenance diet. Individuals were clustered according to insulin resistance trajectories assessed using homeostasis model assessment of insulin resistance (HOMA-IR) index. Adipose tissue mRNA levels of 267 genes selected for regulation according to obesity, metabolic status and response to dieting was assessed using high throughput RT-qPCR. A combination of discriminant analyses was used to identify genes with regulation according to insulin resistance trajectories. Partial correlation was used to control for change in body mass index. Three different HOMA-IR profile groups were determined. HOMA-IR improved during low calorie diet in the 3 groups. At the end of the 6-month follow-up, groups A and B had reduced HOMA-IR by 50%. In group C, HOMA-IR had returned to baseline values. Genes were differentially expressed in the adipose tissue of individuals according to groups but a single gene, CIDEA, was common to all phases of the dietary intervention. Changes in adipose tissue CIDEA mRNA levels paralleled variations in insulin sensitivity independently of change in body mass index. Overall, CIDEA was up-regulated in adipose tissue of individuals with successful long term insulin resistance relapse and not in adipose tissue of unsuccessful individuals. The concomitant change in adipose tissue CIDEA mRNA levels and insulin sensitivity suggests a beneficial role of adipose tissue CIDEA in long term glucose homeostasis, independently of weight variation. ClinicalTrials.gov NCT00390637.

Induction of hypoxia-inducible factor-1alpha and activation of caspase-3 in hypoxia-reoxygenated bone marrow stroma is negatively regulated by the delayed production of substance P.

PubMed

Qian, J; Ramroop, K; McLeod, A; Bandari, P; Livingston, D H; Harrison, J S; Rameshwar, P

2001-10-15

The bone marrow (BM), which is the major site of immune cell development in the adult, responds to different stimuli such as inflammation and hemorrhagic shock. Substance P (SP) is the major peptide encoded by the immune/hemopoietic modulator gene, preprotachykinin-1 (PPT-I). Differential gene expression using a microarray showed that SP reduced hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA levels in BM stroma. Because long-term hypoxia induced the expression of PPT-I in BM mononuclear cells, we used timeline studies to determine whether PPT-I is central to the biologic responses of BM stroma subjected to 30-min hypoxia (pO(2) = 35 mm Hg) followed by reoxygenation. HIF-1alpha mRNA and protein levels were increased up to 12 h. At this time, beta-PPT-I mRNA was detected with the release of SP at 16 h. SP release correlated with down-regulation of HIF-1alpha to baseline. A direct role for SP in HIF-1alpha expression was demonstrated as follows: 1) transient knockout of beta-PPT-I showed an increase in HIF-1alpha expression up to 48 h of reoxygenation; and 2) HIF-1alpha expression remained baseline during reoxygenation when stroma was subjected to hypoxia in the presence of SP. Reoxygenation activated the PPT-I promoter with concomitant nuclear translocation of HIF-1alpha that can bind to the respective consensus sequences within the PPT-I promoter. SP reversed active caspase-3, an indicator of apoptosis and erythropoiesis, to homeostasis level after reoxygenation of hypoxic stroma. The results show that during reoxgenation the PPT-I gene acts as a negative regulator on the expression of HIF-1alpha and active caspase-3 in BM stroma subjected to reoxygenation.

Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

PubMed

Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

2015-03-01

Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation. © 2015 International Society for Neurochemistry.

RNA splicing and splicing regulator changes in prostate cancer pathology.

PubMed

Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

2017-09-01

Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

Possible Involvement of Photoperiodic Regulation in Reproductive Endocrine System of Female Olive Flounder Paralichthys olivaceus.

PubMed

Kim, Hyun Chul; Lee, Chi Hoon; Hur, Sung Pyu; Kim, Byeong Hoon; Park, Jun Young; Lee, Young Don

2015-03-01

This study investigated possible involvement of photoperiodic regulation in reproductive endocrine system of female olive flounder. To investigate the influence on brain-pituitary axis in endocrine system by regulating photoperiod, compared expression level of Kisspeptin and sbGnRH mRNA in brain and FSH-β, LH-β and GH mRNA in pituitary before and after spawning. Photoperiod was treated natural photoperiod and long photoperiod (15L:9D) conditions from Aug. 2013 to Jun. 2014. Continuous long photoperiod treatment from Aug. (post-spawning phase) was inhibited gonadal development of female olive flounder. In natural photoperiod group, the Kiss2 expression level a significant declined in Mar. (spawning period). And also, FSH-β, LH-β and GH mRNA expression levels were increasing at this period. However, in long photoperiod group, hypothalamic Kiss2, FSH-β, LH-β and GH mRNA expression levels did not show any significant fluctuation. These results suggest that expression of hypothalamic Kiss2, GtH and GH in the pituitary would change in response to photoperiod and their possible involvement of photoperiodic regulation in reproductive endocrine system of the BPG axis.

[Electroacupuncture Intervention Enhances Splenic Natural Killer Cell Activity via Inhibiting Phosphorylation of ERK 5 in the Hypothalamus of Surgically Traumatized Rats].

PubMed

Chen, Yan; Li, Jing; Zhu, Ke-ying; Xiao, Sheng; Wang, Yan-qing; Wu, Gen-cheng; Wang, Jun

2015-06-01

To observe the effect of electroacupuncture (EA) on cytotoxic activity of splenic natural killer (NK) cells after surgical trauma via extracellular signal-regulated kinase (ERK) 5 pathway in the rats' hypothalamus, so as to explore its mechanism underlying improving immune disorders after surgery. Sprague-Dawley rats were randomly divided into the following 6 groups: control, trauma model, EA, sham EA, 4 nmol-BIX 02188 (an inhibitor for ERK 5 catalytic activity) and 20 nmol-BIX 02188 (n = 6 rats per group). The surgical trauma model was established by making a longitudinal incision (6 cm in length) along the median line of the back to expose the spinal column and another longitudinal incision along the abdominal median line. EA (2 Hz/15 Hz, 1 - 2 mA) was applied to bilateral "Zusanli" (ST 36) for 30 min immediately after surgery. For rats of the BIX groups, intra-lateral ventricular microinjection of BIX 02188 (10 µL, 4 nmol or 20 nmol, or saline for control rats) was conducted 30 min before the surgery. The expression level and protein of phosphorylated ERK 5 (p-ERK 5) and corticotropin-releasing factor (CRF) protein were measured by immunohistochemistry and Western blot, respectively. The cytotoxicity of splenic NK cells and the expression of splenic Perforin and Granzyme-B genes were measured by lactate dehydrogenase (LDH) release assay and real-time PCR, respectively. In comparison with the control group, hypothalamic p-ERK 5 immunoactivity, p-ERK 5 protein and CRF protein expression levels were significantly up-regulated in the model group (P<0. 01, P<0. 05), while splenic NK cell cytotoxicity and Perforin mRNA and Granzyme-B mRNA expression levels were notably down-regulated in the model group (P <0. 05, P < 0. 01). Following EA and administration of ERK 5 antagonist, the increased expression levels of p-ERK 5 immunoactivity in the EA group, and p-ERK 5 and CRF proteins in both EA and 20 nmol-BIX 02188 groups were obviously down-regulated (P<0. 05, P<0. 01), without changes in the sham EA and 4 nmol-BIX 02188 groups (P>0. 05) except the increased p-ERK 5 protein in the 4 nmol-BIX 02188 group. In addition, the down-regulated NK cell activity, Perforin mRNA and Granzyme-B mRNA expression levels were significantly reversed in the EA and 20 nmol-BIX 02188 groups (P<0. 05, P<0. 01). No significant differences were found between the EA group and 20 nmol-BIX 02188 group in down-regulating hypothalamic p-ERK 5 and CRF protein expression and up-regulating splenic NK cytotoxicity and Perforin and Granzyme-B gene expression (P>0. 05). EA can promote the cytotoxicity of splenic NK cells in surgical trauma rats, which may be closely associated with its functions in down-regulating trauma-induced activation of ERK 5 pathway and production of CRF in the hypothalamus.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

PubMed Central

2011-01-01

Background One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. Methods The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. Results We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. Conclusions These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development. PMID:21329510

Thyroid hormone deiodinase type 2 mRNA levels in sea lamprey (Petromyzon marinus) are regulated during metamorphosis and in response to a thyroid challenge.

PubMed

Stilborn, S Salina M; Manzon, Lori A; Schauenberg, Jennifer D; Manzon, Richard G

2013-03-01

Thyroid hormones (THs) are crucial for normal vertebrate development and are the one obligate morphogen that drives amphibian metamorphosis. However, contrary to other metamorphosing vertebrates, lampreys exhibit a sharp drop in serum TH early in metamorphosis, and anti-thyroid agents such as potassium perchlorate (KClO(4)) induce metamorphosis. The type 2 deiodinase (D2) enzyme is a key regulator of TH availability during amphibian metamorphosis. We set out to determine how D2 may be involved in the regulation of lamprey metamorphosis and thyroid homeostasis. We cloned a 1.8Kb Petromyzon marinus D2 cDNA that includes the entire protein coding region and a selenocysteine (Sec) codon. Northern blotting indicated that the lamprey D2 mRNA is the longest reported to date (>9Kb). Using real-time PCR, we showed that intestinal and hepatic D2 mRNA levels were elevated prior to and during the early stages of metamorphosis and then declined dramatically to low levels that were sustained for the remainder of metamorphosis. These data are consistent with previously reported changes in serum TH levels and deiodinase activity. Treatment of larvae with either TH or KClO(4) significantly affected D2 mRNA levels in the intestine and liver. These D2 mRNA levels during metamorphosis and in response to thyroid challenges suggest that D2 may function in the regulation of TH levels during lamprey metamorphosis and the maintenance of TH homeostasis. Copyright © 2013 Elsevier Inc. All rights reserved.

Transcriptional Regulation of Seprase in Invasive Melanoma Cells by Transforming Growth Factor-β Signaling*

PubMed Central

Tulley, Shaun; Chen, Wen-Tien

2014-01-01

The tumor invasive phenotype driven by seprase expression/activity has been widely examined in an array of malignant tumor cell types; however, very little is known about the transcriptional regulation of this critical protease. Seprase (also named fibroblast activation protein-α, antiplasmin-cleaving enzyme, and dipeptidyl prolyl peptidase 5) is expressed at high levels by stromal fibroblast, endothelial, and tumor cells in a variety of invasive tumors but is undetectable in the majority of normal adult tissues. To examine the transcriptional regulation of the gene, we cloned the human seprase promoter and demonstrated that endogenous seprase expression and exogenous seprase promoter activity are high in invasive melanoma cells but not in non-invasive melanoma cells/primary melanocytes. In addition, we identified a crucial TGF-β-responsive cis-regulatory element in the proximal seprase promoter region that enabled robust transcriptional activation of the gene. Treatment of metastatic but not normal/non-invasive cells with TGF-β1 caused a rapid and profound up-regulation of endogenous seprase mRNA, which coincided with an abolishment of the negative regulator c-Ski, and an increase in binding of Smad3/4 to the seprase promoter in vivo. Blocking TGF-β signaling in invasive melanoma cells through overexpression of c-Ski, chemically using SB-431542, or with a neutralizing antibody against TGF-β significantly reduced seprase mRNA levels. Strikingly, RNAi of seprase in invasive cells greatly diminished their invasive potential in vitro as did blocking TGF-β signaling using SB-431542. Altogether, we found that seprase is transcriptionally up-regulated in invasive melanoma cells via the canonical TGF-β signaling pathway, supporting the roles of both TGF-β and seprase in tumor invasion and metastasis. PMID:24727589

Molecular cloning, characterization, and expression analysis of a heat shock protein (HSP) 70 gene from Paphia undulata.

PubMed

Wu, Xiangwei; Tan, Jing; Cai, Mingyi; Liu, Xiande

2014-06-15

In this study, a full-length HSP70 cDNA from Paphia undulata was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length cDNA is 2,351 bp, consisting of a 5'-untranslated region (UTR) of 83 bp, a 3'-UTR of 315 bp, and an open reading frame (ORF) of 1,953 bp. This cDNA encodes 650 amino acids with an estimated molecular weight of 71.3 kDa and an isoelectric point of 5.51. Based on the amino acid sequence analysis and phylogenetic analysis, this HSP70 gene was identified as a member of the cytoplasmic HSP70 family, being the constitutive expression, and it was designated as PuHSC70. The distribution of PuHSC70 mRNA in the mantle, digestive gland, adductor muscle, gonad, gill, heart, and hemocytes suggested that PuHSC70 is ubiquitously expressed. The mRNA levels of PuHSC70 under high temperature and high salinity stresses were analyzed by real-time PCR. Under high temperature stress of 32°C, PuHSC70 mRNA in the mantle, digestive gland, gill, and heart was significantly up-regulated at 1h and 2h, and it was then progressively down-regulated. In the adductor muscle, the level of PuHSC70 mRNA gradually increased throughout the study period; the mRNA levels in the gonad and hemocytes increased significantly at 4h and 8h (P<0.05) and then decreased at 8h and 14 h, respectively, however they increased again afterwards, reaching the highest levels at 50h. Under high salinity (32 ‰) stress, the mRNA levels of PuHSC70 in the mantle and gonad were increased significantly only at 24h and 48 h (P<0.05), and at the rest of the study period they were slightly elevated. Compared with the pretreatment level, the levels of expression in the digestive gland and gill were unchanged or reduced throughout the study period. The levels of PuHSC70 mRNA in the adductor muscle, hemocytes, and heart were significantly increased, reaching a maximum at 24h, and then they gradually decreased; moreover, in the heart, the mRNA expression recovered to the pretreatment level at 50h; while in the adductor muscle and hemocytes, the expression level remained higher than that of the control. The cloning and expression analyses of PuHSC70 provide theoretical basis to further study the mechanism of physiological response to thermal and high salinity stresses. Copyright © 2014 Elsevier B.V. All rights reserved.

Impact of co-exposure with butachlor and triadimefon on thyroid endocrine system in larval zebrafish.

PubMed

Cao, Chuyan; Wang, Qiangwei; Jiao, Fang; Zhu, Guonian

2016-09-01

Butachlor (BTL) and triadimefon (TDF), the widely used herbicide and fungicide, are unavoidable enter into the aquatic environment. However, there were limited study regarding to the joint toxicity of these two pesticides on fish at present. To evaluate the potential thyroid-disrupting toxicity and exposed to different concentrations of BTL mixed with TDF. Zebrafish embryo (n=3) were exposed to 0.01 and 0.05 fold of LC50 from the acute joint toxicity test, of which 0.32mg/L (BTL) and 9.41mg/L (TDF) for single or mixture agents (BTL: 0.0064mg/L, 0.032mg/L; TDF: 0.1882mg/L, 0.9410mg/L; co-exposure: 0.0032mg/L BTL+0.0941mg/L TDF, 0.016mg/l BTL+0.4705mg/L TDF) after 10-day post-fertilization. Hatching, malformation, survival rates and thyroid hormones (THs), genes expression involved in HPT-axis of embryos were measured and detected in control and separately/co-exposure treatments. THs contents were evaluated by ELISA kit and the expression levels of genes were determined by RT-PCR. Hatching, malformation and survival rates of embryos exposed to single BTL exhibited no statistically significant difference from the control besides decreased of high concentration in survival rates. Exposure to TDF reduced hatching, survival rate and increased malformation. The combined exposure to BTL and TDF resulted in greater adverse effects on embryonic development. BTL exposure significantly increased free T3 and T4 contents. Elevated free T3 content was also observed in the larvae exposed with single BTL. Co-exposure of the two pesticides caused greater enhanced of T3 and T4 levels. Furthermore, gene data showed BTL up-regulated the mRNA expression of tpo, tshβ, tg, ttr, dio2, TDF up-regulated the mRNA expression of tpo, trα, ttr, dio2 and down-regulated trβ gene. The mixture of the two pesticides caused up-regulation mRNA expression of trα, trβ, tg, ttr, dio2. BTL and TDF resulted in adverse effects on zebrafish embryonic development and caused thyroid endocrine disruption, BTL and TDF have a synergistic effect on development and thyroid endocrine by enhanced level of thyroid hormone. Copyright © 2016 Elsevier GmbH. All rights reserved.

Uncovering Hidden Layers of Cell Cycle Regulation through Integrative Multi-omic Analysis

PubMed Central

Aviner, Ranen; Shenoy, Anjana; Elroy-Stein, Orna; Geiger, Tamar

2015-01-01

Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression. PMID:26439921

Regulation of tyrosine hydroxylase gene expression during differentiation of neuroblastoma cells.

PubMed

Summerhill, E M; Wood, K; Fishman, M C

1987-07-01

Differentiation of N1E-115 neuroblastoma cells into neuron-like cells, with extension of neurites and acquisition of excitable membranes, can be induced by dimethyl sulfoxide (DMSO). We have found this differentiation to be accompanied by an increase in tyrosine hydroxylase (TH) mRNA, an increase disproportionate to changes in mRNAs for other measured, non-neuron-specific genes. The mRNA increases slowly over several days and falls gradually after removal of DMSO. Nuclear run-on studies suggest that a change in the rate of transcription cannot explain the increase in steady-state mRNA levels. TH mRNA half-life does, however, increase. This suggests that regulation is exerted in this case not at the level of transcription but rather at that of mRNA stability.

E. coli infection modulates the pharmacokinetics of oral enrofloxacin by targeting P-glycoprotein in small intestine and CYP450 3A in liver and kidney of broilers.

PubMed

Guo, Mengjie; Sun, Yong; Zhang, Yu; Bughio, Shamsuddin; Dai, Xiaohua; Ren, Weilong; Wang, Liping

2014-01-01

P-glycoprotein (P-gp) expression determines the absorption, distribution, metabolism and excretion of many drugs in the body. Also, up-regulation of P-gp acts as a defense mechanism against acute inflammation. This study examined expression levels of abcb1 mRNA and localization of P-gp protein in the liver, kidney, duodenum, jejunum and ileum in healthy and E. coli infected broilers by real time RT-PCR and immunohistochemistry. Meanwhile, pharmacokinetics of orally administered enrofloxacin was also investigated in healthy and infected broilers by HPLC. The results indicated that E. coli infection up-regulated expression of abcb1 mRNA levels significantly in the kidney, jejunum and ileum (P<0.05), but not significantly in the liver and duodenum (P>0.05). However, the expression level of CYP 3A37 mRNA were observed significantly decreased only in liver and kidney of E. coli infected broilers (P<0.05) compared with healthy birds. Furthermore, the infection reduced absorption of orally administered enrofloxacin, significantly decreased Cmax (0.34 vs 0.98 µg mL(-1), P = 0.000) and AUC0-12h (4.37 vs 8.88 µg mL(-1) h, P = 0.042) of enrofloxacin, but increased Tmax (8.32 vs 3.28 h, P = 0.040), T1/2a(2.66 vs 1.64 h(-1), P = 0.050) and V/F (26.7 vs 5.2 L, P = 0.040). Treatment with verapamil, an inhibitor of P-gp, significantly improved the absorption of enrofloxacin in both healthy and infected broilers. The results suggest that the E. coli infection induces intestine P-gp expression, altering the absorption of orally administered enrofloxacin in broilers.

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

PubMed

Easty, D J; Guthrie, B A; Maung, K; Farr, C J; Lindberg, R A; Toso, R J; Herlyn, M; Bennett, D C

1995-06-15

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

Impaired intestinal immune barrier and physical barrier function by phosphorus deficiency: Regulation of TOR, NF-κB, MLCK, JNK and Nrf2 signalling in grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila.

PubMed

Chen, Kang; Zhou, Xiao-Qiu; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Feng, Lin

2018-03-01

In aquaculture, the occurrence of enteritis has increased and dietary nutrition is considered as one of the major strategies to solve this problem. In the present study, we assume that dietary phosphorus might enhance intestinal immune barrier and physical barrier function to reduce the occurrence of enteritis in fish. To test this assumption, a total of 540 grass carp (Ctenopharyngodon idella) were investigated by feeding graded levels of available phosphorus (0.95-8.75 g/kg diet) and then infection with Aeromonas hydrophila. The results firstly showed that phosphorus deficiency decreased the ability to combat enteritis, which might be related to the impairment of intestinal immune barrier and physical barrier function. Compared with optimal phosphorus level, phosphorus deficiency decreased fish intestinal antimicrobial substances activities or contents and down-regulated antimicrobial peptides mRNA levels leading to the impairment of intestinal immune response. Phosphorus deficiency down-regulated fish intestinal anti-inflammatory cytokines mRNA levels and up-regulated the mRNA levels of pro-inflammatory cytokines [except IL-1β and IL-12p35 in distal intestine (DI) and IL-12p40] causing aggravated of intestinal inflammatory responses, which might be related to the signalling molecules target of rapamycin and nuclear factor kappa B. In addition, phosphorus deficiency disturbed fish intestinal tight junction function and induced cell apoptosis as well as oxidative damage leading to impaired of fish intestinal physical barrier function, which might be partially associated with the signalling molecules myosin light chain kinase, c-Jun N-terminal protein kinase and NF-E2-related factor 2, respectively. Finally, based on the ability to combat enteritis, dietary available phosphorus requirement for grass carp (254.56-898.23 g) was estimated to be 4.68 g/kg diet. Copyright © 2017. Published by Elsevier Ltd.

Hamp1 mRNA and plasma hepcidin levels are influenced by sex and strain but do not predict tissue iron levels in inbred mice.

PubMed

McLachlan, Stela; Page, Kathryn E; Lee, Seung-Min; Loguinov, Alex; Valore, Erika; Hui, Simon T; Jung, Grace; Zhou, Jie; Lusis, Aldons J; Fuqua, Brie; Ganz, Tomas; Nemeth, Elizabeta; Vulpe, Chris D

2017-11-01

Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin ( Hamp1 ) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels. NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. Copyright © 2017 the American Physiological Society.

MiR-138 promotes smooth muscle cells proliferation and migration in db/db mice through down-regulation of SIRT1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Juan; Li, Li; Yun, Hui-fang

2015-08-07

Background: Diabetic vascular smooth muscle cells (VSMCs) exhibit significantly increased rates of proliferation and migration, which was the most common pathological change in atherosclerosis. In addition, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified. Methods: The expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and in SMC lines C-12511 exposed to high glucose with qRT-PCR and western blot. The regulation of miR-138 on the expression of SMCs was detected with luciferase report assay. VSMCsmore » proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. Results: We discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 3′-UTR was highly increased by miR-138 inhibitor and reduced by miR-138 mimic. In addition, we examined that the up-regulation of NF-κB induced by high glucose in SMCs was reversed by resveratrol and miR-138 inhibitor. MTT and migration assays showed that miR-138 inhibitor attenuated the proliferation and migration of smooth muscle cells. Conclusion: In this study, we revealed that miR-138 might promote proliferation and migration of SMC in db/db mice through suppressing the expression of SIRT1. - Highlights: • Higher mRNA level of miR-138 was observed in SMCs from db/db mice. • The mRNA and protein level of SIRT1 in SMCs from db/db mice were greatly reduced. • miR-138 could regulate the expression of SIRT1 in SMCs. • SIRT1 overexpression reversed the up-regulation of acetylized p65 and NF-κB induced by high glucose. • MiR-138 inhibitor reversed VSMCs proliferation and migration induced by high glucose.« less

Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

PubMed Central

Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

2014-01-01

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

Posttranscriptional mRNA processing as a mechanism for regulation of human A1 adenosine receptor expression.

PubMed Central

Ren, H; Stiles, G L

1994-01-01

The human A1 adenosine receptor gene contains six exons with exons 1, 2, 3, 4, and part of 5 representing 5' untranslated regions. Reverse transcription-PCR with exon-specific primers showed two distinct transcripts containing either exons 3, 5, and 6 or exons 4, 5, and 6, with exons 3 and 4 being mutually exclusive. No mature mRNAs containing exons 1 and 2 have been detected. All human tissues that express any A1 receptors contain mRNA with exons 4, 5, and 6. Tissues which express high levels of A1 receptors contain mRNA with exons 3, 5, and 6. Exon 4 contains two upstream ATG codons whereas exon 3 contains none. COS cells transfected with expression vectors containing exon 4 (exons 1-6, 3-6, or Ex4-6) express much lower levels of A1 receptors than vectors without exon 4 (exons 3, 5, and 6). Mutation of upstream ATG codons in exon 4 leads to 3- to 7-fold increased A1 receptor expression, up to the level seen with the construct containing exons 3, 5, and 6. Thus, in human tissues "basal" levels of A1 receptors can be expressed by use of mRNA containing exons 4, 5, and 6, but when high levels are needed, alternative transcripts with exons 3, 5, and 6 are produced. Images PMID:8197148

The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1, Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

PubMed Central

Liu, Chang; Wu, Zhe; Sun, Hong-chen

2009-01-01

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups (n=24). Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-β1 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket. PMID:20687301

Long non-coding RNA metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) interacts with estrogen receptor and predicted poor survival in breast cancer.

PubMed

Huang, Nai-Si; Chi, Ya-Yun; Xue, Jing-Yan; Liu, Meng-Ying; Huang, Sheng; Mo, Miao; Zhou, Shu-Ling; Wu, Jiong

2016-06-21

Metastasis associated in lung adenocarcinoma transcript 1 (MALAT1), a lncRNA that was first recognized as a prognostic parameter for patient survival of stage I lung cancer, is up-regulated in multiple human malignancies, including breast cancer. However, the mechanism of its function remained elusive. In the current study, by examining MALAT1 expression on mRNA level, we demonstrated that compared with MCF10A, MALAT1 expression was up-regulated in the majority of breast cancer cell lines (9/12). In 26 pairs of estrogen receptor (ER)-positive breast cancer samples, MALAT1 expression was significantly up-regulated compared with adjacent normal tissues (P = 0.012). Furthermore, of 204 breast cancer patients, high MALAT1 expression was associated with positive ER (P = 0.023) and progesterone receptor (PR) (P = 0.024) status. Further analysis using TCGA database revealed that ER and its target genes PGR and CCND1, were overexpressed in MALAT1 altered group compared with unaltered group, both on the mRNA and protein level. Lastly, we verified MALAT1's prognostic value in breast cancer. At the cut-off value of 75%, MALAT1 was the only independent prognostic factor of recurrence-free survival (RFS) in ER-negative patients in a multivariate Cox regression model (hazard ratio [HR] = 2.83, 95% confidence interval [CI] 1.02-7.83). MALAT1 overexpression was also associated with poor RFS in tamoxifen treated ER-positive breast cancer patients, which might serve as a potential biomarker to predict endocrine treatment sensitivity.

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides.

PubMed

Nicolás, Francisco E; Vila, Ana; Moxon, Simon; Cascales, María D; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M; Garre, Victoriano

2015-03-25

RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants. Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway. This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.

Regulation of neuropeptide Y gene expression in rat brain.

PubMed

Lindefors, N; Brené, S; Herrera-Marschitz, M; Persson, H

1990-01-01

NPY mRNA expression was studied in rat brain using in situ hybridization and RNA blot analysis. Transsynaptic regulation of NPY gene expression was specifically studied in caudate-putamen and frontoparietal (somatosensory) cortex of rats with unilateral lesion of midbrain dopamine neurons and in sham-injected animals. NPY mRNA expression in these two brain regions and the regulation of midbrain dopamine neurons were compared with that of SOM, PPT, CCK and GAD mRNA expression. Neurons expressing NPY and SOM mRNA showed a similar distribution and the expression of both NPY and SOM appears to be regulated by dopamine in a similar fashion. Following a unilateral dopamine deafferentation, the numerical density of both NPY and SOM mRNA expressing neurons almost doubled in the lesioned rat caudate-putamen with no change in the average grain density over positive neurons. Hence, in the intact caudate-putamen dopamine appears to normally suppress expression of these two neuropeptide genes. An activation of both NPY and SOM mRNA expression in many non- or low-expressing neurons is seen when the level of dopamine is decreased. In the frontoparietal cortex, on the other hand, dopamine appears to stimulate NPY and SOM gene expression. RNA blot analysis shows clear-cut changes of NPY mRNA levels in both caudate-putamen and frontoparietal cortex consistent with the changes observed using in situ hybridization. No evidence was found for a change in CCK mRNA expression by the dopamine deafferentation, while PPT mRNA expression decreased in the deafferented caudate-putamen. Consequently, dopamine exerts dissimilar effects on the expression of different neuropeptide genes, that in turn do not respond in the same way in different brain regions. Indirect evidence is also presented indicating that dopamine regulates NPY mRNA expression in a subpopulation of neurons that possibly also express GAD mRNA, both in caudate-putamen and in frontoparietal cortex.

Dietary zinc deficiency reduced growth performance, intestinal immune and physical barrier functions related to NF-κB, TOR, Nrf2, JNK and MLCK signaling pathway of young grass carp (Ctenopharyngodon idella).

PubMed

Song, Zheng-Xing; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Zhou, Xiao-Qiu; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Feng, Lin

2017-07-01

Our study investigated the effects of dietary zinc (Zn) deficiency on growth performance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (244.14 ± 0.40 g) were fed graded levels of zinc lactate (10.71, 30.21, 49.84, 72.31, 92.56, 110.78 mg Zn/kg diet) and one zinc sulfate group (56.9 mg Zn/kg diet) for 60 days. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila for 14 days. These results indicated that compared with optimal dietary Zn level, dietary Zn deficiency (10.71 mg/kg diet) decreased the production of antibacterial compounds, up-regulated pro-inflammatory cytokines related to nuclear factor kappa B (NF-κB) and down-regulated anti-inflammatory cytokines related to target of rapamycin (TOR) in three intestinal segments of young grass carp (P < 0.05), suggesting that dietary Zn deficiency could impair intestinal immune barrier of fish; decreased the activities and mRNA levels of antioxidant enzymes related to NF-E2-related factor 2 (Nrf2), up-regulated the mRNA levels of caspase-3, -7, -8, -9 related to p38 mitogen activated protein (p38 MAPK) and c-Jun N-terminal protein kinase (JNK), down-regulated the mRNA levels of tight junction complexes (TJs) related to myosin light chain kinase (MLCK) in three intestinal segments of young grass carp (P < 0.05), demonstrating that dietary Zn deficiency could injury intestinal physical barrier of fish. Besides, the Zn requirements (zinc lactate as Zn source) based on percent weight gain (PWG), against enteritis morbidity, acid phosphatase (ACP) activity in the proximal intestine (PI) and malondialdehyde (MDA) content in the PI of young grass carp was estimated to be 61.2, 61.4, 69.2 and 69.5 mg/kg diet, respectively. Finally, based on specific growth rate (SGR), feed efficiency (FE) and against enteritis morbidity of young grass carp, the efficacy of zinc lactate relative to zinc sulfate were 132.59%, 135.27% and 154.04%, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Down-regulation of Decapping Protein 2 mediates chronic nicotine exposure-induced locomotor hyperactivity in Drosophila.

PubMed

Ren, Jing; Sun, Jinghan; Zhang, Yunpeng; Liu, Tong; Ren, Qingzhong; Li, Yan; Guo, Aike

2012-01-01

Long-term tobacco use causes nicotine dependence via the regulation of a wide range of genes and is accompanied by various health problems. Studies in mammalian systems have revealed some key factors involved in the effects of nicotine, including nicotinic acetylcholine receptors (nAChRs), dopamine and other neurotransmitters. Nevertheless, the signaling pathways that link nicotine-induced molecular and behavioral modifications remain elusive. Utilizing a chronic nicotine administration paradigm, we found that adult male fruit flies exhibited locomotor hyperactivity after three consecutive days of nicotine exposure, while nicotine-naive flies did not. Strikingly, this chronic nicotine-induced locomotor hyperactivity (cNILH) was abolished in Decapping Protein 2 or 1 (Dcp2 or Dcp1) -deficient flies, while only Dcp2-deficient flies exhibited higher basal levels of locomotor activity than controls. These results indicate that Dcp2 plays a critical role in the response to chronic nicotine exposure. Moreover, the messenger RNA (mRNA) level of Dcp2 in the fly head was suppressed by chronic nicotine treatment, and up-regulation of Dcp2 expression in the nervous system blocked cNILH. These results indicate that down-regulation of Dcp2 mediates chronic nicotine-exposure-induced locomotor hyperactivity in Drosophila. The decapping proteins play a major role in mRNA degradation; however, their function in the nervous system has rarely been investigated. Our findings reveal a significant role for the mRNA decapping pathway in developing locomotor hyperactivity in response to chronic nicotine exposure and identify Dcp2 as a potential candidate for future research on nicotine dependence.

Down-Regulation of Decapping Protein 2 Mediates Chronic Nicotine Exposure-Induced Locomotor Hyperactivity in Drosophila

PubMed Central

Ren, Jing; Sun, Jinghan; Zhang, Yunpeng; Liu, Tong; Ren, Qingzhong; Li, Yan; Guo, Aike

2012-01-01

Long-term tobacco use causes nicotine dependence via the regulation of a wide range of genes and is accompanied by various health problems. Studies in mammalian systems have revealed some key factors involved in the effects of nicotine, including nicotinic acetylcholine receptors (nAChRs), dopamine and other neurotransmitters. Nevertheless, the signaling pathways that link nicotine-induced molecular and behavioral modifications remain elusive. Utilizing a chronic nicotine administration paradigm, we found that adult male fruit flies exhibited locomotor hyperactivity after three consecutive days of nicotine exposure, while nicotine-naive flies did not. Strikingly, this chronic nicotine-induced locomotor hyperactivity (cNILH) was abolished in Decapping Protein 2 or 1 (Dcp2 or Dcp1) -deficient flies, while only Dcp2-deficient flies exhibited higher basal levels of locomotor activity than controls. These results indicate that Dcp2 plays a critical role in the response to chronic nicotine exposure. Moreover, the messenger RNA (mRNA) level of Dcp2 in the fly head was suppressed by chronic nicotine treatment, and up-regulation of Dcp2 expression in the nervous system blocked cNILH. These results indicate that down-regulation of Dcp2 mediates chronic nicotine-exposure-induced locomotor hyperactivity in Drosophila. The decapping proteins play a major role in mRNA degradation; however, their function in the nervous system has rarely been investigated. Our findings reveal a significant role for the mRNA decapping pathway in developing locomotor hyperactivity in response to chronic nicotine exposure and identify Dcp2 as a potential candidate for future research on nicotine dependence. PMID:23300696

Particulate matter triggers depressive-like response associated with modulation of inflammatory cytokine homeostasis and brain-derived neurotrophic factor signaling pathway in mice.

PubMed

Liu, Xuemei; Qian, Xin; Xing, Jing; Wang, Jinhua; Sun, Yixuan; Wang, Qin'geng; Li, Huiming

2018-04-23

Particulate matter (PM) exposure may contribute to depressive-like response in mice. However, few studies have evaluated the adaptive impacts of long-term PM exposure on depressive-like response associated with systemic inflammation and brain-derived neurotrophic factor (BDNF) signaling pathway. We studied the association among depressive-like behaviors, mRNA levels of pro- and anti-inflammatory cytokines, and the expression of BDNF signaling pathway in mice by long-term PM exposure. C57BL/6 male mice were exposed to ambient air alongside control mice breathing air filtered through a high-efficiency air PM (HEPA) filter. Depressive-like behaviors were assessed together with pro-inflammatory, anti-inflammatory cytokine mRNA levels and the modulation of BDNF pathway in hippocampus and olfactory-bulb of mice exposed to PM for 4, 8, and 12 weeks. Exposure to HEPA filtered air for 4 weeks may exert antidepressant like effects in mice. Pro-inflammatory cytokines were up-regulated while the expression of BDNF, its high-affinity receptor tropomyosin-related kinase B (TrkB), and the transcription factor cAMP-response-element binding protein (CREB) were down-regulated in ambient air mice. However, after 8 weeks, there was no significant difference in the rate of depressive-like behaviors between the two groups. After 12 weeks, mice exposed to ambient air again had a higher rate of depressive-like behaviors, significant up-regulation of pro-inflammatory cytokines, down-regulation of interleukin-10 (IL-10), BDNF, TrkB, and CREB than HEPA mice. Ultrafine PM in brain tissues of mice exposed to ambient air was observed. Our results suggest continuous high-level PM exposure alters the depressive-like response in mice and induces a damage-repair-imbalance reaction.

Natural antisense transcript-targeted regulation of inducible nitric oxide synthase mRNA levels.

PubMed

Yoshigai, Emi; Hara, Takafumi; Araki, Yoshiro; Tanaka, Yoshito; Oishi, Masaharu; Tokuhara, Katsuji; Kaibori, Masaki; Okumura, Tadayoshi; Kwon, A-Hon; Nishizawa, Mikio

2013-04-01

Natural antisense transcripts (asRNAs) are frequently transcribed from mammalian genes. Recently, we found that non-coding asRNAs are transcribed from the 3' untranslated region (3'UTR) of the rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide. The iNOS asRNA stabilizes iNOS mRNA by interacting with the mRNA 3'UTR. Furthermore, single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence were found to reduce iNOS mRNA levels by interfering with mRNA-asRNA interactions in rat hepatocytes. This method was named natural antisense transcript-targeted regulation (NATRE) technology. In this study, we detected human iNOS asRNA expressed in hepatocarcinoma and colon carcinoma tissues. The human iNOS asRNA harbored a sequence complementary to an evolutionarily conserved region of the iNOS mRNA 3'UTR. When introduced into hepatocytes, iNOS sense oligonucleotides that were modified by substitution with partial phosphorothioate bonds and locked nucleic acids or 2'-O-methyl nucleic acids greatly reduced levels of iNOS mRNA and iNOS protein. Moreover, sense oligonucleotides and short interfering RNAs decreased iNOS mRNA to comparable levels. These results suggest that NATRE technology using iNOS sense oligonucleotides could potentially be used to treat human inflammatory diseases and cancers by reducing iNOS mRNA levels. Copyright © 2013 Elsevier Inc. All rights reserved.

[Autophagy modulates the levels of inflammatory cytokines in macrophages induced by lipopolysaccharide].

PubMed

Ren, Caiyuan; Zhang, Xiangying; Shi, Hongbo; Chen, Dexi; Duan, Zhongping; Zhang, Huanhu; Ren, Feng

2017-05-01

Objective To analyze the effect of autophagy on inflammatory response in macrophages induced by lipopolysaccharide (LPS) and investigate its molecular mechanism. Methods Bone marrow mesenchymal stem cells, which were separated from the femora of mice, were cultured and induced to differentiate into primary macrophages in vitro. The inflammatory cell model was established by stimulating the primary macrophages with LPS. Autophagy was inhibited by 3-methyladenine (3-MA) or promoted by rapamycin. Green fluorescent protein-microtubule associated protein 1 light chain 3 (GFP-LC3) plasmid was used to transfect primary macrophages and the percentage of cells with GFP-LC3 puncta were counted in the different groups. The mRNA levels of LC3B, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), IL-6 and IL-12p40 were detected by real-time quantitative PCR, and the protein levels of LC3B, nuclear factor κB (NF-κB) and IκBα were determined by Western blotting. Results LC3B mRNA and protein expression levels were gradually up-regulated and the autophagosomes increased in the macrophages 2, 4 and 6 hours after treated by LPS. Compared with only LPS treatment group, autophagy inhibition by 3-MA pretreatment promoted the mRNA expressions of inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-12p40, and the autophagy induction by rapamycin pretreatment suppressed TNF-α, IL-1β, IL-6 and IL-12p40. Meanwhile, 3-MA or rapamycin pretreatment further regulated the protein expressions of IκBα and p-NF-kBp65 induced by LPS in macrophages. Conclusion Autophagy can suppress the LPS-induced inflammatory response in macrophages by regulating NF-κB signaling pathway.

Mincle in the innate immune response of mice fungal keratitis.

PubMed

Yu, Guo-Rong; Lin, Jing; Zhang, Jie; Che, Cheng-Ye; Peng, Xu-Dong; Li, Cui; Hu, Li-Ting; Zhu, Guo-Qiang; He, Kun; Zhao, Gui-Qiu

2018-01-01

To investigate how macrophage inducible C-type lectin (Mincle) influences inflammation in mice fungal keratitis induced by Aspergillus fumigatus ( A. fumigatus ). C57BL/6 mice were infected with A. fumigatus after pretreated with Mincle agonist TDB or Mincle neutralizing antibody (MincleAb), taking DMSO or IgG as control group respectively. The cornea lesions were monitored with slit-lamp microscope and evaluated by clinical score. Mincle expression was assessed using reverse transcription-ploymerase chain reaction (RT-PCR) and immunostaining. The expression of cytokines (IL-1β, TNF-α and IL-6) chemokines (CXCL-1 and MIP-2) was determined by RT-PCR and ELISA. Neutrophil infiltration was observed by immunostaining. The levels of nitric oxide (NO) generated by corneas were tested by Griess reaction. Mincle mRNA and protein levels were higher in infected corneas than normal corneas of C57BL/6 mice, saving clinical scores revealed differences. When pretreated with Mincle agonist TDB, the mRNA and protein levels of IL-1β, TNF-α and IL-6 in infected corneas were significantly increased compared with the control group ( P <0.01). Results of the counterpart in corneas pretreated with Mincle neutralizing antibody was decreased consistently ( P <0.01). Expression of CXCL1 and MIP-2 mRNA levels were up-regulated in TDB group and down-regulated in MincleAb group ( P <0.01), coincide with neutrophil aggregation degree in corneas showed by immunostaining. As for the concentration of NO, it was promoted in TDB group compared with DMSO control group, and decreased in MincleAb group compared with IgG control group. Mincle plays a dual role in mice fungal keratitis. It participates in the innate immune system by enhancing inflammation. What's more, Mincle can mediate cytotoxic effects by regulating the formation of NO.

Vitamin D3 supplementation alleviates rotavirus infection in pigs and IPEC-J2 cells via regulating the autophagy signaling pathway.

PubMed

Tian, Gang; Liang, Xiaofang; Chen, Daiwen; Mao, Xiangbing; Yu, Jie; Zheng, Ping; He, Jun; Huang, Zhiqing; Yu, Bing

2016-10-01

Vitamin D had an anti-infection effect and benefited to the intestinal health. Autophagy signaling pathway was regulated by vitamin D3 to inhibit the infection of human immunodeficiency virus type-1. Rotavirus (RV) was a major cause of the severe diarrheal disease in young children and young animals. Although evidence suggested that vitamin D3 attenuates the negative effects of RV infection via the retinoic acid-inducible gene I signaling pathway, little is known of its antiviral effect whether through the regulation of autophagy. The present study was performed to investigate whether vitamin D3 alleviates RV infection in pig and porcine small intestinal epithelial cell line (IPEC-J2) models via regulating the autophagy signaling pathway. RV administration increased the Beclin 1 mRNA abundance in porcine jejunum and ileum. 5000 IU/kg dietary vitamin D3 supplementation greatly up-regulated LC3-II/LC3-I ratios and PR-39 mRNA expression under the condition of RV challenged. The viability of IPEC-J2 was significantly inhibited by RV infection. Incubation with 25-hydroxyvitamin D3 significantly decreased the concentrations of RV antigen and non-structural protein 4 (NSP4), and up-regulated the mRNA expression of Beclin 1 and PR-39 in the RV-infected IPEC-J2 cells. And then, based on the 25-hydroxyvitamin D3 treatment and RV infection, LC3-II mRNA expression in cells was inhibited by an autophagy inhibitor 3-methyladenine (3-MA). Bafilomycin A1 (Baf A1, a class of inhibitors of membrane ATPases, inhibits maturation of autophagic vacuoles) treatment numerically enhanced the LC3-II mRNA abundance, but had no effect on NSP4 concentration. Furthermore, 25-hydroxyvitamin D3 decreased the p62 mRNA expression and increased porcine cathelicidins (PMAP23, PG1-5 and PR-39) mRNA expression in the RV-infected cells. Taken together, these results indicated that vitamin D3 attenuates RV infection through regulating autophagic maturation and porcine cathelicidin genes expression. Copyright © 2016 Elsevier Ltd. All rights reserved.

Competition between RNA-binding proteins CELF1 and HuR modulates MYC translation and intestinal epithelium renewal

PubMed Central

Liu, Lan; Ouyang, Miao; Rao, Jaladanki N.; Zou, Tongtong; Xiao, Lan; Chung, Hee Kyoung; Wu, Jing; Donahue, James M.; Gorospe, Myriam; Wang, Jian-Ying

2015-01-01

The mammalian intestinal epithelium is one of the most rapidly self-renewing tissues in the body, and its integrity is preserved through strict regulation. The RNA-binding protein (RBP) ELAV-like family member 1 (CELF1), also referred to as CUG-binding protein 1 (CUGBP1), regulates the stability and translation of target mRNAs and is implicated in many aspects of cellular physiology. We show that CELF1 competes with the RBP HuR to modulate MYC translation and regulates intestinal epithelial homeostasis. Growth inhibition of the small intestinal mucosa by fasting in mice was associated with increased CELF1/Myc mRNA association and decreased MYC expression. At the molecular level, CELF1 was found to bind the 3′-untranslated region (UTR) of Myc mRNA and repressed MYC translation without affecting total Myc mRNA levels. HuR interacted with the same Myc 3′-UTR element, and increasing the levels of HuR decreased CELF1 binding to Myc mRNA. In contrast, increasing the concentrations of CELF1 inhibited formation of the [HuR/Myc mRNA] complex. Depletion of cellular polyamines also increased CELF1 and enhanced CELF1 association with Myc mRNA, thus suppressing MYC translation. Moreover, ectopic CELF1 overexpression caused G1-phase growth arrest, whereas CELF1 silencing promoted cell proliferation. These results indicate that CELF1 represses MYC translation by decreasing Myc mRNA association with HuR and provide new insight into the molecular functions of RBPs in the regulation of intestinal mucosal growth. PMID:25808495

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Effects of pectin pentaoligosaccharide from Hawthorn ( Crataegus pinnatifida Bunge. var. Major) on the activity and mRNA levels of enzymes involved in fatty acid oxidation in the liver of mice fed a high-fat diet.

PubMed

Li, Tuo-Ping; Zhu, Ru-Gang; Dong, Yin-Ping; Liu, Yong-Hui; Li, Su-Hong; Chen, Gang

2013-08-07

The regulatory effects of haw pectin pentaoligosaccharide (HPPS) on fatty acid oxidation-related enzyme activities and mRNA levels were investigated in the liver of high fat diet induced hyperlipidemic mice. Results showed that HPPS (150 mg/kg for 10 weeks) significantly suppresses weight gain (32.3 ± 0.26 and 21.1 ± 0.14 g for high-fat diet and HPPS groups, respectively), decreases serum triacylglycerol levels (1.64 ± 0.09 and 0.91 ± 0.02 mmol/L, respectively), and increases lipid excretion in feces (55.7 ± 0.38 and 106.4 ± 0.57 mg/g for total lipid, respectively), compared to high-fat diet as control. HPPS significantly increased the hepatic fatty acid oxidation-related enzyme activities of acyl-CoA oxidase, carnitine palmitoyltransferase I, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase by 53.8, 74.2, 47.1, and 24.2%, respectively. Meanwhile, the corresponding mRNAs were up-regulated by 89.6, 85.8, 82.9, and 30.9%, respectively. Moreover, HPPS was able to up-regulate the gene and protein expressions of peroxisome proliferator-activated receptor α. Results suggest that continuous HPPS ingestion may be used as dietary therapy to prevent obesity and cardiovascular diseases.

PKA- and PKC-dependent regulation of angiopoietin 2 mRNA in human granulosa lutein cells.

PubMed

Witt, P S; Pietrowski, D; Keck, C

2004-02-01

New blood vessels develop from preexisting vessels in response to growth factors or hypoxic conditions. Recent studies have shown that angiopoietin 2 (ANGPT-2) plays an important role in the modulation of angiogenesis and vasculogenesis in humans and mice. The signaling pathways that lead to the regulation of ANGPT-2 are largely unclear. Here, we report that protein kinase C and protein kinase A activators (ADMB, 8-Cl-cAMP) increased the mRNA levels of ANGPT-2 in human Granulosa cells, whereas PKC and PKA Inhibitors (Rp-cAMP, GO 6983) decreased markedly the level of ANGPT-2 mRNA. Due to varying specificity of the modulators for certain protein kinases subunits, we conclude that the conventional PKCs, but not PKC alpha and beta1, the atypical PKCs and the PKA I, are involved in the regulation of ANGPT-2. These findings may help to explain the role of both PKA and PKC dependent signaling cascades in the regulation of ANGPT-2 mRNA.

Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

NASA Astrophysics Data System (ADS)

Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

2017-06-01

MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

PROTEASOME INHIBITOR TREATMENT REDUCED FATTY ACID, TRIACYLGLYCEROL AND CHOLESTEROL SYNTHESIS

PubMed Central

Oliva, Joan; French, Samuel W.; Li, Jun; Bardag-Gorce, Fawzia

2014-01-01

In the present study, the beneficial effects of proteasome inhibitor treatment in reducing ethanol-induced steatosis were investigated. A microarray analysis was performed on the liver of rats injected with PS-341 (Bortezomib, Velcade®), and the results showed that proteasome inhibitor treatment significantly reduced the mRNA expression of SREBP-1c, and the downstream lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. ELOVL6, which is responsible for fatty acids long chain elongation, was also significantly down regulated by proteasome inhibitor treatment. Moreover, PS-341 administration significantly reduced the expression of acyl-glycerol-3-phosphate acyltransferase (AGPAT), and diacylglycerol acyltransferase (DGAT), enzyme involved in triacylglycerol (TAG) synthesis. Finally, PS-341 was found to down regulate the enzymes 3-hydroxy-3-methylglutaryl-CoenzymeA synthase (HMG-CoA synthase) that is responsible for cholesterol synthesis. Proteasome inhibitor was also found to play a role in intestinal lipid adsorption because apolipoproteins A (apoA-I, apoAII, apoA-IV and ApoCIII) were down regulated by proteasome inhibitor treatment, especially ApoA-II that is known to be a marker of alcohol consumption. Proteasome inhibitor treatment also decreased apobec-1 complementation factor (ACF) leading to lower level of editing and production of ApoB protein. Moreover apolipoprotein C-III, a major component of chylomicrons was significantly down regulated. However, lipoprotein lipase (Lpl) and High density lipoprotein binding protein (Hdlbp) mRNA levels were increased by proteasome inhibitor treatment. These results suggested that proteasome inhibitor treatment could be used to reduce the alcohol-enhanced lipogenesis and alcohol-induced liver steatosis. A morphologic analysis, performed on the liver of rats fed ethanol for one month and treated with PS-341, showed that proteasome inhibitor treatment significantly decreased ethanol-induced liver steatosis. SREBP-1c, FAS and ACC were increased by ethanol feeding alone, but were significantly decreased when proteasome inhibitor was administered to rats fed ethanol. Our results also show that both mRNA and protein levels of these lipogenic enzymes, up regulated by ethanol, were then down regulated when proteasome inhibitor was administered to rats fed ethanol. It was also confirmed that alcohol feeding caused an increase in AGPAT and DGAT, which was prevented by proteasome inhibitor treatment of the animal fed ethanol. Chronic alcohol feeding did not affect the gene expression of HMG-CoA synthase. However, PS341 administration significantly reduced the HMG-CoA synthase mRNA levels, confirming the results obtained with the microarray analysis. C/EBP transcription factors alpha (CCAAT/enhancer-binding protein alpha) has been shown to positively regulate SREBP-1c mRNA expression, thus regulating lipogenesis. Proteasome inhibition caused a decrease in C/EBP alpha mRNA expression, indicating that C/EBP down regulation may be the mechanism by which proteasome inhibitor treatment reduced lipogenesis. In conclusion, our results indicate that proteasome activity is not only involved in down regulating fatty acid synthesis and triacylglycerol synthesis, but also cholesterol synthesis and intestinal lipid adsorption. Proteasome inhibitor, administrated at a non-toxic low dose, played a beneficial role in reducing lipogenesis caused by chronic ethanol feeding and these beneficial effects are obtained because of the specificity and reversibility of the proteasome inhibitor used. PMID:22445925

Coordinated Expression of Cyclin-dependent Kinase-4 and its Regulators in Human Oral Tumors

PubMed Central

POI, MING J.; KNOBLOCH, THOMAS J.; SEARS, MARTA T.; UHRIG, LANA K.; WARNER, BLAKE M.; WEGHORST, CHRISTOPHER M.; LI, JUNAN

2014-01-01

Background/Aim While aberrant expression of cyclin-dependent kinase-4 (CDK4) has been found in squamous cell carcinoma of the head and neck (SCCHN), the associations between CDK4 and its regulators, namely, cyclin D1, cyclin E, gankyrin, SEI1, and BMI1 in gene expression remain to be explored. Herein we investigated the mRNA profiles of these oncogenes and their interrelations in different oral lesion tissues. Materials and Methods Thirty SCCHN specimens and patient-matched high at-risk mucosa (HARM) and 16 healthy control specimens were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. Results The mRNA levels of CDK4, cyclin D1, gankyrin, SEI1, BMI1 were significantly elevated in both HARM and SCCHN (in comparison with control specimens), and statistically significant correlations were found among these markers in gene expression. Conclusion Up-regulation of CDK4 and its regulators takes place in oral cancer progression in a coordinate manner, and HARM and SCCHN share a similar molecular signature within the CDK4-pRB pathway. PMID:24982332

Hedgehog signaling pathway is active in GBM with GLI1 mRNA expression showing a single continuous distribution rather than discrete high/low clusters.

PubMed

Chandra, Vikas; Das, Tapojyoti; Gulati, Puneet; Biswas, Nidhan K; Rote, Sarang; Chatterjee, Uttara; Ghosh, Samarendra N; Deb, Sumit; Saha, Suniti K; Chowdhury, Anup K; Ghosh, Subhashish; Rudin, Charles M; Mukherjee, Ankur; Basu, Analabha; Dhara, Surajit

2015-01-01

Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression--as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution-unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the "high-Hh" cluster of MB but 5.6 fold higher than that of the "low-Hh" cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

Alterations of overused supraspinatus tendon: a possible role of glycosaminoglycans and HARP/pleiotrophin in early tendon pathology.

PubMed

Attia, Mohamed; Scott, Alexander; Duchesnay, Arlette; Carpentier, Gilles; Soslowsky, Louis J; Huynh, Minh Bao; Van Kuppevelt, Toin H; Gossard, Camille; Courty, José; Tassoni, Marie-Claude; Martelly, Isabelle

2012-01-01

Supraspinatus tendon overuse injuries lead to significant pain and disability in athletes and workers. Despite the prevalence and high social cost of these injuries, the early pathological events are not well known. We analyzed the potential relation between glycosaminoglycan (GAG) composition and phenotypic cellular alteration using a rat model of rotator cuff overuse. Total sulfated GAGs increased after 4 weeks of overuse and remained elevated up to 16 weeks. GAG accumulation was preceded by up-regulation of decorin, versican, and aggrecan proteoglycans (PGs) mRNAs and proteins and biglycan PG mRNA after 2 weeks. At 2 weeks, collagen 1 transcript decreased whereas mRNAs for collagen 2, collagen 3, collagen 6, and the transcription factor Sox9 were increased. Protein levels of heparin affine regulatory peptide (HARP)/pleiotrophin, a cytokine known to regulate developmental chondrocyte formation, were enhanced especially at 4 weeks, without up-regulation of HARP/pleiotrophin mRNA. Further results suggest that the increased GAGs present in early lesions may sequester HARP/pleiotrophin, which could contribute to a loss of tenocyte's phenotype. All these modifications are characteristic of a shift towards the chondrocyte phenotype. Identification of these early changes in the extra-cellular matrix may help to prevent the progression of the pathology to more disabling, degenerative alterations. Copyright © 2011 Orthopaedic Research Society.

Upregulation of endothelin receptors A and B in the nitrofen induced hypoplastic lung occurs early in gestation.

PubMed

Dingemann, Jens; Doi, Takashi; Ruttenstock, Elke; Puri, Prem

2010-01-01

Pulmonary hypoplasia and persistent pulmonary hypertension (PPH) aggravate clinical courses in congenital diaphragmatic hernia (CDH). Endothelin 1 enhances PPH by vasoconstriction and proliferation of vessel walls. Up-regulation of pulmonary Endothelin Receptors A and B (EDNRA, EDNRB) has been reported in human CDH and animal models, but the onset of those alterations during lung development remains unclear. We hypothesized that pulmonary expression of EDNRA and EDNRB is up-regulated at early gestational stages in the nitrofen model. Pregnant rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Embryos were sacrificed on D15, D18 and D21 and divided into nitrofen- and control group. Pulmonary RNA was extracted and mRNA levels of EDNRA and EDNRB were determined by real-time PCR. Immunohistochemistry for protein expression of both receptors was performed. mRNA levels of EDNRA and EDNRB were significantly increased in the nitrofen group on D15, D18 and D21. Immunohistochemistry revealed increased pulmonary vascular expression of EDNRA and EDNRB compared to controls. Altered expression of EDNRA and EDNRB is an early event in lung morphogenesis in the nitrofen model. We speculate that pulmonary arteries in CDH become excessively muscularised in early fetal life, becoming unable to adapt normally at birth.

Simulated microgravity reduces mRNA levels of multidrug resistance genes 4 and 5 in non-metastatic human melanoma cells

NASA Astrophysics Data System (ADS)

Eiermann, Peter; Tsiockas, Wasiliki; Hauslage, Jens; Hemmersbach, Ruth; Gerzer, Rupert; Ivanova, Krassimira

Multidrug resistance proteins (MRP) are members of the ATP-binding cassette transporter superfamily that are able to export a large variety of substances into the extracellular space in-cluding nucleoside and nucleotide base analogs used in antiviral and anticancer therapy. MRP4 and 5 (MRP4/5) particularly transport cyclic nucleotides, e.g. guanosine 3',5'-cyclic monophos-phate (cGMP). The second messenger cGMP, which is synthesized by the catalytic activity of the guanylyl cyclase (GC), plays an import role in vasodilatation, smooth muscle relaxation, and nitric oxide (NO)-induced perturbation of melanocyte-extracellular matrix interactions. In previous studies we have reported that different GC isoforms are responsible for cGMP synthe-sis in melanocytic cells. Normal human melanocytes and non-metastatic melanoma cell lines predominantly express the NO-sensitive soluble GC isoform (sGC), a heterodimeric protein consisting of α and β subunits. Metastatic melanoma cells lack the expression of the β sub-unit and show up-regulated activities of the particulate isoforms. We have further found that long-term exposure to hypergravity (5 g for 24 h) induced an increased cGMP export in normal human melanocytes, and non-metastatic, but not in metastatic human melanoma cells as a re-sult of up-regulated MRP4/5 expression. The aim of the present study is to investigate whether simulated microgravity may also alter the expression of MRP4/5 in non-metastatic melanoma cells. Experiments were performed using a fast-rotating clinostat (60 rpm) with one rotation axis. The non-metastatic 1F6 melanoma cells were exposed to simulated microgravity (up to 1.21x10-2 g) for 24 h. The mRNA analyses were performed by a relative calibrator-normalized and efficiency corrected quantitative polymerase chain reaction (Light Cycler R , Roche). Our data show a reduced expression of approximately 35% for MRP4 and of 50% for MRP5 in simulated microgravity in comparison to 1 g controls. Also, the mRNA levels of sGC α and β were down-regulated by about 31% and 22%, respectively. Thus, the reduced expression of MRP4/5 could be related to the decrease in mRNA levels for the sGC subunits. In addition, the long-term exposure to simulated microgravity did not alter cellular morphology. Taken together, the results of our studies indicate that the expression of MRP4/5 in non-metastatic melanoma cells is inversely regulated by hypergravity and simulated microgravity. Finally, a reduced expression of MRP4 and MRP5 may increase the availability of drugs in cells and influence astronaut medication.

Microarray profiling analysis uncovers common molecular mechanisms of rubella virus, human cytomegalovirus, and herpes simplex virus type 2 infections in ECV304 cells.

PubMed

Mo, X; Xu, L; Yang, Q; Feng, H; Peng, J; Zhang, Y; Yuan, W; Wang, Y; Li, Y; Deng, Y; Wan, Y; Chen, Z; Li, F; Wu, X

2011-08-01

To study the common molecular mechanisms of various viruses infections that might result in congential cardiovascular diseases in perinatal period, changes in mRNA expression levels of ECV304 cells infected by rubella virus (RUBV), human cytomegalovirus (HCMV), and herpes simplex virus type 2 (HSV-2) were analyzed using a microarray system representing 18,716 human genes. 99 genes were found to exhibit differential expression (80 up-regulated and 19 down-regulated). Biological process analysis showed that 33 signaling pathways including 22 genes were relevant significantly to RV, HCMV and HSV-II infections. Of these 33 biological processes, 28 belong to one-gene biological processes and 5 belong to multiple-gene biological processes. Gene annotation indicated that the 5 multiple-gene biological processes including regulation of cell growth, collagen fibril organization, mRNA transport, cell adhesion and regulation of cell shape, and seven down- or up-regulated genes [CRIM1 (cysteine rich transmembrane BMP regulator 1), WISP2 (WNT1 inducible signaling pathway protein 2), COL12A1 (collagen, type XII, alpha 1), COL11A2 (collagen, type XI, alpha 2), CNTN5 (contactin 5), DDR1 (discoidin domain receptor tyrosine kinase 1), VEGF (vascular endothelial growth factor precursor)], are significantly correlated to RUBV, HCMV and HSV-2 infections in ECV304 cells. The results obtained in this study suggested the common molecular mechanisms of viruses infections that might result in congential cardiovascular diseases.

Pre-acclimation to low ammonia improves ammonia handling in common carp (Cyprinus carpio) when exposed subsequently to high environmental ammonia.

PubMed

Shrivastava, Jyotsna; Sinha, Amit Kumar; Datta, Surjya Narayan; Blust, Ronny; De Boeck, Gudrun

2016-11-01

We tested whether exposing fish to low ammonia concentrations induced acclimation processes and helped fish to tolerate subsequent (sub)lethal ammonia exposure by activating ammonia excretory pathways. Common carp (Cyprinus carpio) were pre-exposed to 0.27mM ammonia (∼10% 96h LC 50 ) for 3, 7 and 14days. Thereafter, each of these pre-exposed and parallel naïve groups were exposed to 1.35mM high environmental ammonia (HEA, ∼50% 96h LC 50 ) for 12h and 48h to assess the occurrence of ammonia acclimation based on sub-lethal end-points, and to lethal ammonia concentrations (2.7mM, 96h LC 50 ) in order to assess improved survival time. Results show that fish pre-exposed to ammonia for 3 and 7days had a longer survival time than the ammonia naïve fish. However, this effect disappeared after prolonged (14days) pre-exposure. Ammonia excretion rate (J amm ) was strongly inhibited (or even reversed) in the unacclimated groups during HEA. On the contrary, after 3days the pre-exposure fish maintained J amm while after 7days these pre-acclimated fish were able to increase J amm efficiently. Again, this effect disappeared after 14days of pre-acclimation. The efficient ammonia efflux in pre-acclimated fish was associated with the up-regulation of branchial mRNA expression of ammonia transporters and exchangers. Pre-exposure with ammonia for 3-7days stimulated an increment in the transcript level of gill Rhcg-a and Rhcg-b mRNA relative to the naïve control group and the up-regulation of these two Rhcg homologs was reinforced during subsequent HEA exposure. No effect of pre-exposure was noted for Rhbg. Relative to unacclimated fish, the transcript level of Na + /H + exchangers (NHE-3) was raised in 3-7days pre-acclimated fish and remained higher during the subsequent HEA exposure while gill H + -ATPase activities and mRNA levels were not affected by pre-acclimation episodes. Likewise, ammonia pre-acclimated fish with or without HEA exposure displayed pronounced up-regulation in Na + /K + -ATPase activity and mRNA expression relative to the corresponding ammonia naïve groups. Overall, these data suggest that ammonia acclimation was evident for both lethal and the sub-lethal endpoints through priming mechanisms in ammonia excretory transcriptional processes, but these acclimation effects were transient and disappeared after prolonged pre-exposure. Copyright © 2016 Elsevier B.V. All rights reserved.

Tucum-Do-Cerrado (Bactris setosa Mart.) Consumption Modulates Iron Homeostasis and Prevents Iron-Induced Oxidative Stress in the Rat Liver

PubMed Central

Fustinoni-Reis, Adriana M.; Arruda, Sandra F.; Dourado, Lívia P. S.; da Cunha, Marcela S. B.; Siqueira, Egle M. A.

2016-01-01

This study investigated the effect of tucum-do-cerrado consumption in the oxidative status of iron-supplemented rats. Four groups of rats were treated: Control (AIN-93G), Tuc (AIN-93G added of tucum-do-cerrado), Fe (AIN-93G iron-enriched), or TucFe (AIN-93G with tucum-do-cerrado and iron-enriched) diet, for 30 days. Iron-enriched diet increased serum, liver, spleen, and intestine iron levels; transferrin saturation; liver lipid oxidation; mRNA levels of hepatic Hamp and Bmp6, and Nrf2 in the intestine. Tucum-do-cerrado consumption reduced spleen lipid and protein oxidation; mRNA levels of hepatic Hamp and Ftl, and increased serum antioxidant capacity and hepatic mRNA levels of Bmp6, Hmox1, Nqo1, and Nrf2. TucFe diet consumption abrogated the liver Hamp iron-induced up-regulation, prevented intestinal iron accumulation; hepatic lipid peroxidation; splenic protein damage, and the increase of catalase, glutathione reductase, and glutathione peroxidase activity in some tissues. These results suggest that tucum-do-cerrado protects tissues against oxidative damage, by reducing iron availability in liver and consequently inhibiting liver Hamp expression. PMID:26901220

Absolute gene expression patterns of thioredoxin and glutaredoxin redox systems in mouse.

PubMed

Jurado, Juan; Prieto-Alamo, María-José; Madrid-Rísquez, José; Pueyo, Carmen

2003-11-14

This work provides the first absolute expression patterns of genes coding for all known components of both thioredoxin (Trx) and glutaredoxin (Grx) systems in mouse: Trx1, Trx2, Grx1, Grx2, TrxR1, TrxR2, thioredoxin/glutathione reductase, and glutathione reductase. We devised a novel assay that, combining the advantages of multiplex and real-time PCR, streamlines the quantitation of the actual mRNA copy numbers in whole-animal experiments. Quantitations reported establish differences among adult organs and embryonic stages, compare mRNA decay rates, explore the significance of alternative mRNA isoforms derived from TrxR1 and Grx2 genes, and examine the time-course expression upon superoxide stress promoted by paraquat. Collectively, these quantitations show: i) unique expression profiles for each transcript and mouse organ examined, yet with some general trends like the higher amounts of mRNA species coding for thioredoxins than those coding for the reductases that control their redox states and activities; ii) continuous expression during embryogenesis with outstanding up-regulations of Trx1 and TrxR1 mRNAs in specific temporal sequences; iii) drastic differences in mRNA stability, liver decay rates range from 2.8 h (thioredoxin/glutathione reductase) to >/= 35 h (Trx1 and Trx2), and directly correlate with mRNA steady-state values; iv) testis-specific differences in the amounts (relative to total isoforms) of transcripts yielding the mitochondrial Grx2a and 67-kDa TrxR1 variants; and v) coordinated up-regulation of TrxR1 and glutathione reductase mRNAs in response to superoxide stress in an organ-specific manner. Further insights into in vivo roles of these redox systems should be gained from more focused studies of the mechanisms underlying the vast differences reported here at the transcript level.

Effects of the non-steroidal anti-inflammatory drug(NSAID) naproxen on gene expression of antioxidant enzymes in zebrafish (Danio rerio).

PubMed

Stancová, V; Ziková, A; Svobodová, Z; Kloas, W

2015-09-01

The aim of this study was to investigate the effects of naproxen on the gene expression of antioxidant enzymes in adult zebrafish. Surprisingly, after 2 weeks exposure no significant effect on the mRNA expression of the target genes was found in the liver. However, mRNA levels of three genes were altered significantly in the intestine. The expression of Ucp-2 decreased at the environmental concentration of 1μg/L while mRNA expression of GST p2 increased at the concentration of 100μg/L. The mRNA level for the antioxidant enzyme CAT was up-regulated significantly at both the concentrations used. Exposure to naproxen caused only moderate effects on the expression of antioxidant genes in the intestine rather than in the liver, which demonstrates that the intestine is more sensitive to waterborne naproxen exposure than the liver. Interestingly, the adverse side effects of NSAIDs occur in the gastrointestinal tract of humans. To our knowledge, this is the first study that has focused on transcriptional effects of naproxen on zebrafish. Copyright © 2015 Elsevier B.V. All rights reserved.

Up-regulation of Hsp27 by ERα/Sp1 facilitates proliferation and confers resistance to apoptosis in human papillary thyroid cancer cells.

PubMed

Mo, Xiao-Mei; Li, Li; Zhu, Ping; Dai, Yu-Jie; Zhao, Ting-Ting; Liao, Ling-Yao; Chen, George G; Liu, Zhi-Min

2016-08-15

17β-estradiol (E2) has been suggested to play a role in the development and progression of papillary thyroid cancer. Heat shock protein 27 (Hsp27) is a member of the Hsp family that is responsible for cell survival under stressful conditions. Previous studies have shown that the 5'-promoter region of Hsp27 gene contains a specificity protein-1 (Spl) and estrogen response element half-site (ERE-half), which contributes to Hsp27 induction by E2 in breast cancer cells. However, it is unclear whether Hsp27 can be up-regulated by E2 and which estrogen receptor (ER) isoform and tethered transcription factor are involved in this regulation in papillary thyroid cancer cells. In the present study, we demonstrated that Hsp27 can be effectively up-regulated by E2 at mRNA and protein levels in human K1 and BCPAP papillary thyroid cancer cells which have more than two times higher level of ERα than that of ERβ. The up-regulation of Hsp27 by E2 is mediated by ERα/Sp1 and ERβ has repressive effect on this ERα/Sp1-mediated up-regulation of Hsp27. Moreover, we showed that the up-regulation of Hsp27 by ERα/Sp1 facilitates proliferation and confers resistance to apoptosis through interaction with procaspase-3. Targeting this pathway may be a potential strategy for therapy of papillary thyroid cancer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effects of recombinant human leptin administration on hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco: in vivo and in vitro studies.

PubMed

Song, Yu-Feng; Wu, Kun; Tan, Xiao-Ying; Zhang, Li-Han; Zhuo, Mei-Qin; Pan, Ya-Xiong; Chen, Qi-Liang

2015-02-01

The present study was conducted to investigate the effects and mechanism of leptin influencing lipid metabolism in yellow catfish Pelteobagrus fulvidraco. To this end, hepatic lipid (in vivo experiment) and intracellular triglyceride (TG) (in vitro experiment) content, the activities and/or expression level of several enzymes (CPT-1, 6PGD, G6PD, FAS, ME and ICDH) as well as the mRNA expression of transcription factors (PPARα, PPARγ and SREBP-1) involved in lipid metabolism were determined. Using the primary hepatocytes of yellow catfish, specific inhibitors AG490 (JAK-STAT inhibitor) and wortmannin (IRS-PI3K inhibitor) were used to explore the signaling pathways of leptin effects on lipid metabolism. Intraperitoneal injection of recombinant human leptin (rt-hLEP) significantly reduced hepatic lipid content, activities of lipogenic enzymes (6PGD, G6PD, ME, ICDH and FAS) as well as mRNA levels of 6PGD, G6PD, FAS, PPARγ and SREBP-1 genes, but up-regulated activity and mRNA level of CPT-1 and PPARα. Using primary hepatocytes, rt-hLEP incubation also reduced intracellular TG content, mRNA levels of G6PD and PPARγ genes, but enhanced mRNA levels of PPARα, CPT-1 and SREBP-1. Leptin-induced effects could partially be reversed by specific inhibitors AG490, suggesting that JAK-STAT signaling pathways played important roles in the process of leptin-induced changes in lipid metabolism. Wortmannin significantly suppressed the decrease of TG content induced by leptin, reflecting that IRS-PI3K was involved in the leptin-mediate changes as well. To our knowledge, the present study provides, for the first time, evidence that rt-hLEP can increase lipolysis and reduce lipogenesis at the both enzymatic and molecular levels in fish with the combination of in vivo with in vitro studies, which serves to increase our understanding into the roles and mechanisms of leptin regulating lipid metabolism in fish. Copyright © 2015 Elsevier Inc. All rights reserved.

Vitamin E deficiency depressed fish growth, disease resistance, and the immunity and structural integrity of immune organs in grass carp (Ctenopharyngodon idella): Referring to NF-κB, TOR and Nrf2 signaling.

PubMed

Pan, Jia-Hong; Feng, Lin; Jiang, Wei-Dan; Wu, Pei; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu; Liu, Yang

2017-01-01

This study investigated the effects of dietary vitamin E on growth, disease resistance and the immunity and structural integrity of head kidney, spleen and skin in grass carp (Ctenopharyngodon idella). The fish were fed six diets containing graded levels of vitamin E (0, 45, 90, 135, 180 and 225 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila. The results showed that compared with optimal vitamin E supplementation, vitamin E deficiency caused depressed growth, poor survival rates and increased skin lesion morbidity in grass carp. Meanwhile, vitamin E deficiency decreased lysozyme and acid phosphatase activities, complement component 3 and complement component 4 contents in the head kidney, spleen and skin of grass carp (P < 0.05). Moreover, vitamin E deficiency down-regulated antimicrobial peptides (Hepcidin, liver-expressed antimicrobial peptide-2A, -2B, β-defensin), IL-10, TGFβ1, IκBα, TOR and S6K1 mRNA levels (P < 0.05) and up-regulated IL-1β, IL-6, IL-8, IFN-γ2 and TNFα, NF-κB p65, IKKα, IKKβ and 4EBP1 (not in the head kidney) mRNA levels (P < 0.05). In addition, vitamin E deficiency caused oxidative damage, decreased superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) activities, and down-regulated the mRNA levels of antioxidant enzymes and signaling molecules Nrf2 (P < 0.05). Vitamin E deficiency also induced apoptosis by up-regulating capase-2, -3, -7, and -8 mRNA levels in the head kidney, spleen and skin of grass carp. In conclusion, this study indicated that dietary vitamin E deficiency depressed fish growth, impaired the immune function and disturbed the structural integrity of the head kidney, spleen and skin in grass carp, but optimal vitamin E supplementation can reverse those negative effects in fish. The optimal vitamin E requirements for young grass carp (266.39-1026.63 g) to achieve optimal growth performance and disease resistance based on the percent weight gain (PWG) and skin lesion morbidity were estimated to be 116.2 and 130.9 mg/kg diet, respectively. Meanwhile, based on immune indicator (LA activity in the head kidney) and antioxidant indicator (protection of spleen against MDA), the optimal vitamin E requirements for young grass carp were estimated to be 123.8 and 136.4 mg/kg diet, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of GHRP-2 and Cysteamine Administration on Growth Performance, Somatotropic Axis Hormone and Muscle Protein Deposition in Yaks (Bos grunniens) with Growth Retardation

PubMed Central

Hu, Rui; Wang, Zhisheng; Peng, Quanhui; Zou, Huawei; Wang, Hongze; Yu, Xiaoqiang; Jing, Xiaoping; Wang, Yixin; Cao, Binghai; Bao, Shanke; Zhang, Wenhua; Zhao, Suonan; Ji, Hanzhong; Kong, Xiangying; Niu, Quanxi

2016-01-01

The objective of this study was to investigate the effects of growth hormone-releasing peptide-2 (GHRP-2) and cysteamine (CS) administration on growth performance in yaks with growth retardation and try to elucidate its regulatory mechanisms. Trial 1, thirty-six 1-year-old Qinghai high plateau yaks (body weight 38–83.2 kg) were randomly chosen for body weight and jugular blood samples collection. The relationship between body weight and serum GHRH (P < 0.05, R = 0.45), GH (P < 0.05, R = 0.47), IGF-1 (P < 0.05, R = 0.62) was significantly correlated in yaks colonies with lighter body weights. Trial 2, fifteen 1-year-old Qinghai high plateau yaks with growth retardation (average body weight 54.8 ± 8.24 kg) were randomly selected and assigned to negative control group (NG), GHRP-2 injection group (GG) and cysteamine feeding group (CG), with 5 yaks per group. Another five 1-year-old Qinghai high plateau yaks with normal growth performance (average body weight 75.3 ± 2.43 kg) were selected as positive control group (PG). The average daily gain (ADG) of the GG and CG were significantly higher than those in the PG and NG (P < 0.05). Both GHRP-2 and CS administration significantly enhanced the myofiber diameter and area of skeletal muscle (P<0.05). GHRP-2 significantly enhanced the serum GH and IGF-1 levels (P < 0.05), and up-regulated GHR, IGF-1 and IGF-1R mRNA expression in the liver and skeletal muscle (P < 0.05), enhanced the mRNA expression of PI3K, AKt and mTOR in the skeletal muscle (P<0.05). CS significantly reduced the serum SS levels and the hypothalamus SS mRNA expression (P < 0.05), and enhanced GHR and IGF-1 mRNA expression in the liver (P < 0.05), decreased the mRNA expression of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) mRNA (P < 0.05). Conclusions: Growth retardation in yaks was primarily due to somatotropic axis hormones secretion deficiency. Both GHRP-2 and CS administration can accelerate growth performance and GH, IGF-1 secretion in yaks with growth retardation. GHRP-2 enhanced muscle protein deposition mainly by up-regulated the protein synthesis pathways, whereas CS worked mainly by down-regulated the ubiquitin-proteasome pathway. PMID:26894743

Effects of GHRP-2 and Cysteamine Administration on Growth Performance, Somatotropic Axis Hormone and Muscle Protein Deposition in Yaks (Bos grunniens) with Growth Retardation.

PubMed

Hu, Rui; Wang, Zhisheng; Peng, Quanhui; Zou, Huawei; Wang, Hongze; Yu, Xiaoqiang; Jing, Xiaoping; Wang, Yixin; Cao, Binghai; Bao, Shanke; Zhang, Wenhua; Zhao, Suonan; Ji, Hanzhong; Kong, Xiangying; Niu, Quanxi

2016-01-01

The objective of this study was to investigate the effects of growth hormone-releasing peptide-2 (GHRP-2) and cysteamine (CS) administration on growth performance in yaks with growth retardation and try to elucidate its regulatory mechanisms. Trial 1, thirty-six 1-year-old Qinghai high plateau yaks (body weight 38-83.2 kg) were randomly chosen for body weight and jugular blood samples collection. The relationship between body weight and serum GHRH (P < 0.05, R = 0.45), GH (P < 0.05, R = 0.47), IGF-1 (P < 0.05, R = 0.62) was significantly correlated in yaks colonies with lighter body weights. Trial 2, fifteen 1-year-old Qinghai high plateau yaks with growth retardation (average body weight 54.8 ± 8.24 kg) were randomly selected and assigned to negative control group (NG), GHRP-2 injection group (GG) and cysteamine feeding group (CG), with 5 yaks per group. Another five 1-year-old Qinghai high plateau yaks with normal growth performance (average body weight 75.3 ± 2.43 kg) were selected as positive control group (PG). The average daily gain (ADG) of the GG and CG were significantly higher than those in the PG and NG (P < 0.05). Both GHRP-2 and CS administration significantly enhanced the myofiber diameter and area of skeletal muscle (P<0.05). GHRP-2 significantly enhanced the serum GH and IGF-1 levels (P < 0.05), and up-regulated GHR, IGF-1 and IGF-1R mRNA expression in the liver and skeletal muscle (P < 0.05), enhanced the mRNA expression of PI3K, AKt and mTOR in the skeletal muscle (P<0.05). CS significantly reduced the serum SS levels and the hypothalamus SS mRNA expression (P < 0.05), and enhanced GHR and IGF-1 mRNA expression in the liver (P < 0.05), decreased the mRNA expression of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) mRNA (P < 0.05). Growth retardation in yaks was primarily due to somatotropic axis hormones secretion deficiency. Both GHRP-2 and CS administration can accelerate growth performance and GH, IGF-1 secretion in yaks with growth retardation. GHRP-2 enhanced muscle protein deposition mainly by up-regulated the protein synthesis pathways, whereas CS worked mainly by down-regulated the ubiquitin-proteasome pathway.

Effects of rhynchophylline on GluN1 and GluN2B expressions in primary cultured hippocampal neurons.

PubMed

He, Yan; Zeng, Sheng-Ya; Zhou, Shi-Wen; Qian, Gui-Sheng; Peng, Kang; Mo, Zhi-Xian; Zhou, Ji-Yin

2014-10-01

N-methyl-d-aspartate (NMDA) receptor subunits GluN1 and GluN2B in hippocampal neurons play key roles in anxiety. Our previous studies show that rhynchophylline, an active component of the Uncaria species, down-regulates GluN2B expression in the hippocampal CA1 area of amphetamine-induced rat. The effects of rhynchophylline on expressions of GluN1 and GluN2B in primary hippocampal neurons in neonatal rats in vitro were investigated. Neonatal hippocampal neurons were cultured with neurobasal-A medium. After incubation for 6h or 48 h with rhynchophylline (non-competitive NMDAR antagonist) and MK-801 (non-competitive NMDAR antagonist with anxiolytic effect, as the control drug) from day 6, neuron toxicity, mRNA and protein expressions of GluN1 and GluN2B were analyzed. GluN1 is mainly distributed on neuronal axons and dendritic trunks, cytoplasm and cell membrane near axons and dendrites. GluN2B is mainly distributed on the membrane, dendrites, and axon membranes. GluN1 and GluN2B are codistributed on dendritic trunks and dendritic spines. After 48 h incubation, a lower concentration of rhynchophylline (lower than 400 μmol/L) and MK-801 (lower than 200 μmol/L) have no toxicity on neonatal hippocampal neurons. Rhynchophylline up-regulated GluN1 mRNA expression at 6h and mRNA and protein expressions at 48h, but down-regulated GluN2B mRNA and protein expressions at 48 h. However, GluN1 and GluN2B mRNA expressions were down-regulated at 6h, and mRNA and protein expressions were both up-regulated by MK-801 at 48h. These findings show that rhynchophylline reciprocally regulates GluN1 and GluN2B expressions in hippocampal neurons, indicating a potential anxiolytic property for rhynchophylline. Copyright © 2014 Elsevier B.V. All rights reserved.

[Regulatory effect and mechanism of RNA binding motif protein 38 on the expression of progesterone receptor in human breast cancer ZR-75-1 cells].

PubMed

Lou, P P; Li, C L; Xia, T S; Shi, L; Wu, J; Zhou, X J; Wang, Y; Ding, Q

2016-06-23

To investigate the regulatory mechanism of RNA binding motif protein 38 (RNPC1) on the expression of progesterone receptor (PR) in breast cancer cell line ZR-75-1. Lentiviral vector was used to induce overexpression of RNPC1 in ZR-75-1 cells. qRT-PCR and Western blot were used to assess the regulatory effect of RNPC1 on PR expression. Actinomycin was used to detect the regulatory mechanism involved. Immunohistochemical (IHC) staining was used to determine the protein expression of RNPC1 and PR in 80 breast cancer tissues. IHC staining showed that the expression of RNPC1 was significantly higher in the PR positive breast cancer tissues than that in the PR negative breast cancer tissues (P<0.05). The qRT-PCR results showed that overexpression of RNPC1 in ZR-75-1 cells significantly upregulated the mRNA level of PR (1.764±0.028 vs. 1.001±0.037, P<0.01), whereas knockdown of RNPC1 did the opposite (0.579± 0.007 vs. 1.000±0.002, P<0.01). The Western blot results also showed that overexpression of RNPC1 up-regulated PR levels, while knockdown of RNPC1 resulted in down-regulation of PR levels in the ZR-75-1 cells.The actinomycin assay showed that overexpression of RNPC1 increased the mRNA stability of PR. The half-life of PR mRNA was increased from 4.0 h to 6.5 h. Knockdown of RNPC1 decreased the mRNA stability of PR and the half-life of PR transcript was decreased from 4.1 h to 3.0 h. RNPC1 plays a crucial role in regulating the expression of PR in breast cancer ZR-75-1 cells.

mRNA Transcript Abundance during Plant Growth and the Influence of Li + Exposure

DOE PAGES

Duff, M. C.; Kuhne, W. W.; Halverson, N. V.; ...

2014-10-23

Lithium (Li) toxicity in plants is, at a minimum, a function of Li + concentration, exposure time, species and growth conditions. Most plant studies with Li + focus on short-term acute exposures. This study examines short- and long-term effects of Li + exposure in Arabidopsis with Li + uptake studies and measured shoot mRNA transcript abundance levels in treated and control plants. Stress, pathogen-response and arabinogalactan protein genes were typically more up-regulated in older (chronic, low level) Li +-treatment plants and in the much younger plants from acute high-level exposures. The gene regulation behavior of high-level Li + resembled priormore » studies due to its influence on: inositol synthesis, 1-aminocyclopropane-1-carboxylate synthases and membrane ion transport. In contrast, chronically-exposed plants had gene regulation responses that were indicative of pathogen, cold, and heavy-metal stress, cell wall degradation, ethylene production, signal transduction, and calcium-release modulation. Acute Li + exposure phenocopies magnesium-deficiency symptoms and is associated with elevated expression of stress response genes that could lead to consumption of metabolic and transcriptional energy reserves and the dedication of more resources to cell development. In contrast, chronic Li + exposure increases expression signal transduction genes. The identification of new Li +-sensitive genes and a gene-based “response plan” for acute and chronic Li + exposure are delineated.« less

mRNA Transcript abundance during plant growth and the influence of Li(+) exposure.

PubMed

Duff, M C; Kuhne, W W; Halverson, N V; Chang, C-S; Kitamura, E; Hawthorn, L; Martinez, N E; Stafford, C; Milliken, C E; Caldwell, E F; Stieve-Caldwell, E

2014-12-01

Lithium (Li) toxicity in plants is, at a minimum, a function of Li(+) concentration, exposure time, species and growth conditions. Most plant studies with Li(+) focus on short-term acute exposures. This study examines short- and long-term effects of Li(+) exposure in Arabidopsis with Li(+) uptake studies and measured shoot mRNA transcript abundance levels in treated and control plants. Stress, pathogen-response and arabinogalactan protein genes were typically more up-regulated in older (chronic, low level) Li(+)-treatment plants and in the much younger plants from acute high-level exposures. The gene regulation behavior of high-level Li(+) resembled prior studies due to its influence on: inositol synthesis, 1-aminocyclopropane-1-carboxylate synthases and membrane ion transport. In contrast, chronically-exposed plants had gene regulation responses that were indicative of pathogen, cold, and heavy-metal stress, cell wall degradation, ethylene production, signal transduction, and calcium-release modulation. Acute Li(+) exposure phenocopies magnesium-deficiency symptoms and is associated with elevated expression of stress response genes that could lead to consumption of metabolic and transcriptional energy reserves and the dedication of more resources to cell development. In contrast, chronic Li(+) exposure increases expression signal transduction genes. The identification of new Li(+)-sensitive genes and a gene-based "response plan" for acute and chronic Li(+) exposure are delineated. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

PubMed Central

Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

2013-01-01

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

Transcriptome responses in the rectal gland of fed and fasted spiny dogfish shark (Squalus acanthias) determined by suppression subtractive hybridization.

PubMed

Deck, Courtney A; McKay, Sheldon J; Fiedler, Tristan J; LeMoine, Christophe M R; Kajimura, Makiko; Nawata, C Michele; Wood, Chris M; Walsh, Patrick J

2013-12-01

Prior studies of the elasmobranch rectal gland have demonstrated that feeding induces profound and rapid up regulation of the gland's ability to secrete concentrated NaCl solutions and the metabolic capacity to support this highly ATP consuming process. We undertook the current study to attempt to determine the degree to which up regulation of mRNA transcription was involved in the gland's activation. cDNA libraries were created from mRNA isolated from rectal glands of fasted (7days post-feeding) and fed (6h and 22h post-feeding) spiny dogfish sharks (Squalus acanthias), and the libraries were subjected to suppression subtractive hybridization (SSH) analysis. Quantitative real time PCR (qPCR) was also used to ascertain the mRNA expression of several genes revealed by the SSH analysis. In total the treatments changed the abundance of 170 transcripts, with 103 up regulated by feeding, and 67 up regulated by fasting. While many of the changes took place in 'expected' Gene Ontology (GO) categories (e.g., metabolism, transport, structural proteins, DNA and RNA turnover, etc.), KEGG analysis revealed a number of categories which identify oxidative stress as a topic of interest for the gland. GO analysis also revealed that branched chain essential amino acids (e.g., valine, leucine, isoleucine) are potential metabolic fuels for the rectal gland. In addition, up regulation of transcripts for many genes in the anticipated GO categories did not agree (i.e., fasting down regulated in feeding treatments) with previously observed increases in their respective proteins/enzyme activities. These results suggest an 'anticipatory' storage of selected mRNAs which presumably supports the rapid translation of proteins upon feeding activation of the gland. © 2013 Elsevier Inc. All rights reserved.

Multiple Transduction Pathways Mediate Thyrotropin Receptor Signaling in Preosteoblast-Like Cells

PubMed Central

Boutin, Alisa; Neumann, Susanne

2016-01-01

It has been shown that the TSH receptor (TSHR) couples to a number of different signaling pathways, although the Gs-cAMP pathway has been considered primary. Here, we measured the effects of TSH on bone marker mRNA and protein expression in preosteoblast-like U2OS cells stably expressing TSHRs. We determined which signaling cascades are involved in the regulation of IL-11, osteopontin (OPN), and alkaline phosphatase (ALPL). We demonstrated that TSH-induced up-regulation of IL-11 is primarily mediated via the Gs pathway as IL-11 was up-regulated by forskolin (FSK), an adenylyl cyclase activator, and inhibited by protein kinase A inhibitor H-89 and by silencing of Gαs by small interfering RNA. OPN levels were not affected by FSK, but its up-regulation was inhibited by TSHR/Gi-uncoupling by pertussis toxin. Pertussis toxin decreased p38 MAPK kinase phosphorylation, and a p38 inhibitor and small interfering RNA knockdown of p38α inhibited OPN induction by TSH. Up-regulation of ALPL expression required high doses of TSH (EC50 = 395nM), whereas low doses (EC50 = 19nM) were inhibitory. FSK-stimulated cAMP production decreased basal ALPL expression, whereas protein kinase A inhibition by H-89 and silencing of Gαs increased basal levels of ALPL. Knockdown of Gαq/11 and a protein kinase C inhibitor decreased TSH-stimulated up-regulation of ALPL, whereas a protein kinase C activator increased ALPL levels. A MAPK inhibitor and silencing of ERK1/2 inhibited TSH-stimulated ALPL expression. We conclude that TSH regulates expression of different bone markers via distinct signaling pathways. PMID:26950201

MicroRNA-494-3p targets CXCR4 to suppress the proliferation, invasion, and migration of prostate cancer.

PubMed

Shen, Peng-fei; Chen, Xue-qin; Liao, Yong-chuan; Chen, Ni; Zhou, Qiao; Wei, Qiang; Li, Xiang; Wang, Jia; Zeng, Hao

2014-05-01

Although SDF-1/CXCR4 pathway is a potential mechanism of tumor proliferation and progression, the mechanism of controlling CXCR4 expression is not fully understood. This study was to confirm that miR-494-3p might be a potentially post-transcriptional regulator of CXCR4 and over-expression of miR-494 might suppress prostate cancer progression and metastasis. We firstly postulated the post-transcriptional regulation of CXCR4 by miR-494-3p through bioinformatics analysis, and then it was demonstrated that miR-494-3p could regulate the CXCR4 mRNA post-transcriptionally by binding to the predicted site by dual reporter gene assays. The biological effect of miR-494-3p on prostate cancer cells proliferation, apoptosis, migration, and invasion was measured by MTT, TUNEL, flow cytometry, migration, and invasion assays. It was shown that the mRNA and protein expression levels of CXCR4 were significantly up-regulated in PC-3 and DU145, whereas barely detected in LNCaP and RWPE-1. However, the CXCR4 protein levels were inversely related to the mature miR-494-3p expression levels in RWPE-1 and prostate cancer cells. The constitutive over-expression of miR-494-3p could down-regulate the protein level of CXCR4 in PC-3 and DU145. MiR-494-3p also could bind to the seed sequences in the 3'-UTR of the CXCR4 gene. Artificial over-expression of miR-494-3p could inhibit the growth, promote the apoptosis, and inhibit the migration and invasion of PC-3 and DU145 cells in vivo. Our results suggested that miR-494-3p might play crucial role in prostate cancer by post-transcriptional regulation to CXCR4 mRNA. MiR-494-3p/CXCR4 pathway may be a potential therapeutic target to prevent prostate cancer progression and metastasis. © 2014 Wiley Periodicals, Inc.

Amphetamine regulation of acetylcholine and gamma-aminobutyric acid in nucleus accumbens.

PubMed

Lindefors, N; Hurd, Y L; O'Connor, W T; Brené, S; Persson, H; Ungerstedt, U

1992-01-01

In situ hybridization histochemistry and in vivo microdialysis were combined to study the effect of amphetamine on the expression of choline acetyltransferase and glutamate decarboxylase67 mRNA and in vivo release of acetylcholine and GABA in rat medial nucleus accumbens. Differential effects on acetylcholine and GABA neurons by a single challenge injection of amphetamine (1.5 mg/kg, s.c.) were apparent in saline-pretreated and amphetamine-pretreated (same dose, twice daily for the previous seven days) rats. Extracellular acetylcholine levels were increased up to 50% over a prolonged period following both single and repeated amphetamine. In contrast, extracellular concentrations of GABA were gradually decreased to half the control values, but only in rats receiving repeated amphetamine. Although the increase of acetylcholine release was not associated with any change in choline acetyltransferase mRNA levels, the number of neurons expressing high levels of glutamate decarboxylase67 mRNA was decreased (28%) following repeated injections. Thus we suggest that amphetamine decreases extracellular GABA levels by a slow mechanism, associated with the decreased expression of glutamate decarboxylase67 mRNA in a subpopulation of densely labeled neurons in the medial nucleus accumbens. The delayed response by GABA to amphetamine may reflect supersensitivity in the activity of postsynaptic gamma-aminobutyric acid-containing neurons in nucleus accumbens as a consequence of the repeated amphetamine treatment.

Single nucleotide polymorphisms at miR-146a/196a2 and their primary ovarian insufficiency-related target gene regulation in granulosa cells.

PubMed

Cho, Sung Hwan; An, Hui Jeong; Kim, Kyung Ah; Ko, Jung Jae; Kim, Ji Hyang; Kim, Young Ran; Ahn, Eun Hee; Rah, HyungChul; Lee, Woo Sik; Kim, Nam Keun

2017-01-01

MicroRNAs post-transcriptionally regulate gene expression in animals and plants. The aim of this study was to identify new target genes for microRNA polymorphisms (miR-146aC>G and miR-196a2T>C) in primary ovarian insufficiency (POI). We cloned and transfected miR-146aC>G and miR-196a2T>C into human granulosa cells and used microarrays and qPCR-arrays to examine the changes in the messenger RNA expression profile. We show miR-146aC>G and miR-196a2T>C change the mRNA expression patterns in granulosa cell. In each case, mRNAs were up or down-regulated after treatments with miR-146a C or G and miR-196a2 T or C. We found that miR-146a led to a significantly altered regulation of the mRNA levels of FOXO3, FOXL2 and CCND2 compared to controls. We also found that the polymorphisms of miR-146a led to a significantly altered regulation of CCND2 and FOXO3. Our results suggest that miR-146aC>G and miR-196a2T>C can regulate the levels of many of their target transcripts. In addition, specific target genes of miR-146aC>G polymorphisms may be involved in granulosa cell regulation.

Single nucleotide polymorphisms at miR-146a/196a2 and their primary ovarian insufficiency-related target gene regulation in granulosa cells

PubMed Central

Cho, Sung Hwan; An, Hui Jeong; Kim, Kyung Ah; Ko, Jung Jae; Kim, Ji Hyang; Kim, Young Ran; Ahn, Eun Hee; Rah, HyungChul; Lee, Woo Sik

2017-01-01

MicroRNAs post-transcriptionally regulate gene expression in animals and plants. The aim of this study was to identify new target genes for microRNA polymorphisms (miR-146aC>G and miR-196a2T>C) in primary ovarian insufficiency (POI). We cloned and transfected miR-146aC>G and miR-196a2T>C into human granulosa cells and used microarrays and qPCR-arrays to examine the changes in the messenger RNA expression profile. We show miR-146aC>G and miR-196a2T>C change the mRNA expression patterns in granulosa cell. In each case, mRNAs were up or down-regulated after treatments with miR-146a C or G and miR-196a2 T or C. We found that miR-146a led to a significantly altered regulation of the mRNA levels of FOXO3, FOXL2 and CCND2 compared to controls. We also found that the polymorphisms of miR-146a led to a significantly altered regulation of CCND2 and FOXO3. Our results suggest that miR-146aC>G and miR-196a2T>C can regulate the levels of many of their target transcripts. In addition, specific target genes of miR-146aC>G polymorphisms may be involved in granulosa cell regulation. PMID:28841705

A High Serum Iron Level Causes Mouse Retinal Iron Accumulation Despite an Intact Blood-Retinal Barrier

PubMed Central

Zhao, Liangliang; Li, Yafeng; Song, Delu; Song, Ying; Theurl, Milan; Wang, Chenguang; Cwanger, Alyssa; Su, Guanfang; Dunaief, Joshua L.

2015-01-01

The retina can be shielded by the blood-retinal barrier. Because photoreceptors are damaged by excess iron, it is important to understand whether the blood-retinal barrier protects against high serum iron levels. Bone morphogenic protein 6 (Bmp6) knockout mice have serum iron overload. Herein, we tested whether the previously documented retinal iron accumulation in Bmp6 knockout mice might result from the high serum iron levels or, alternatively, low levels of retinal hepcidin, an iron regulatory hormone whose transcription can be up-regulated by Bmp6. Furthermore, to determine whether increases in serum iron can elevate retinal iron levels, we i.v. injected iron into wild-type mice. Retinas were analyzed by real-time quantitative PCR and immunofluorescence to assess the levels of iron-regulated genes/proteins and oxidative stress. Retinal hepcidin mRNA levels in Bmp6 knockout retinas were the same as, or greater than, those in age-matched wild-type retinas, indicating that Bmp6 knockout does not cause retinal hepcidin deficiency. Changes in mRNA levels of L ferritin and transferrin receptor indicated increased retinal iron levels in i.v. iron-injected wild-type mice. Oxidative stress markers were elevated in photoreceptors of mice receiving i.v. iron. These findings suggest that elevated serum iron levels can overwhelm local retinal iron regulatory mechanisms. PMID:25174877

Low Medium pH Value Enhances Anthocyanin Accumulation in Malus Crabapple Leaves

PubMed Central

Tian, Ji; Jin, Kaina; Yao, Yuncong

2014-01-01

Anthocyanin is a critical factor involved in coloration of plant tissues, but the mechanism how medium pH values affect anthocyanin accumulation in woody plants is unknown. We analyzed anthocyanin composition and the expression of elements encoding anthocyanin and flavonols biosynthesis underlying different medium pH values by using three different leave color type cultivars. HPLC analysis demonstrated that high medium pH values treatment induced a dramatic decrease in the concentration of cyaniding in crabapple leaves. Conversely, the high medium pH values induced up-regulation of the content of flavones and flavonols, suggesting that low pH treatment-induced anthocyanin accumulation. Quantitative real time PCR experiment showed the expression level of anthocyanidin synthase (McANS) and uridine diphosphate glucose flavonoid 3-O-glucosyltransferase (McUFGT) was up-regulated by low pH values treatment, and high medium pH value treatment up-regulate the transcription level of flavonol synthase (McFLS). Meanwhile, several MYB TFs have been suggested in the regulation of pH responses. These results strongly indicate that the low pH treatment-induced anthocyanin accumulation is mediated by the variation of mRNA transcription of the anthocyanin biosynthetic genes. PMID:24914811

Effects of nitrate on metamorphosis, thyroid and iodothyronine deiodinases expression in Bufo gargarizans larvae.

PubMed

Wang, Ming; Chai, Lihong; Zhao, Hongfeng; Wu, Minyao; Wang, Hongyuan

2015-11-01

Chinese toad (Bufo gargarizans) tadpoles were exposed to nitrate (10, 50 and 100mg/L NO3-N) from the beginning of the larval period through metamorphic climax. We examined the effects of chronic nitrate exposure on metamorphosis, mortality, body size and thyroid gland. In addition, thyroid hormone (TH) levels, type II iodothyronine deiodinase (Dio2) and type III iodothyronine deiodinase (Dio3) mRNA levels were also measured to assess disruption of TH synthesis. Results showed that significant metamorphic delay and mortality increased were caused in larvae exposed to 100mg/L NO3-N. The larvae exposed to 100mg/L NO3-N clearly exhibited a greater reduction in thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels. Moreover, treatment with NO3-N induced down-regulation of Dio2 mRNA levels and up-regulation of Dio3 mRNA levels, reflecting the disruption of thyroid endocrine. It seems that increased mass and body size may be correlated with prolonged metamorphosis. Interestingly, we observed an exception that exposure to 100mg/L NO3-N did not exhibit remarkable alterations of thyroid gland size. Compared with control groups, 100mg/L NO3-N caused partial colloid depletion in the thyroid gland follicles. These results suggest that nitrate can act as a chemical stressor inducing retardation in development and metamorphosis. Therefore, we concluded that the presence of high concentrations nitrate can influence the growth, decline the survival, impair TH synthesis and induce metamorphosis retardation of B. gargarizans larvae. Copyright © 2015 Elsevier Ltd. All rights reserved.

JBP485 improves gentamicin-induced acute renal failure by regulating the expression and function of Oat1 and Oat3 in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Xinjin; Meng, Qiang; Liu, Qi

2013-09-01

We investigated the effects of JBP485 (an anti-inflammatory dipeptide and a substrate of OAT) on regulation of the expression and function of renal Oat1 and Oat3, which can accelerate the excretion of accumulated uremic toxins (e.g. indoxyl sulfate) in the kidney to improve gentamicin-induced ARF in rats. JBP485 caused a significant decrease in the accumulation of endogenous substances (creatinine, blood urea nitrogen and indoxyl sulfate) in vivo, an increase in the excretion of exogenous compounds (lisinopril and inulin) into urine, and up-regulation of the expressions of renal Oat1 and Oat3 in the kidney tissues and slices via substrate induction. Tomore » determine the effect of JBP485 on the accelerated excretion of uremic toxins mediated by Oat1 and Oat3, the mRNA and protein expression levels of renal basolateral Oats were assessed by quantitative real-time PCR, western blot, immunohistochemical analysis and an immunofluorescence method. Gentamicin down-regulated the expression of Oats mRNA and protein in rat kidney, and these effects were reversed after administration of JBP485. In addition, JBP485 caused a significant decrease in MPO and MDA levels in the kidney, and improved the pathological condition of rat kidney. These results indicated that JBP485 improved acute renal failure by increasing the expression and function of Oat1 and Oat3, and by decreasing overoxidation of the kidney in gentamicin-induced ARF rats. - Highlights: • JBP485 could up-regulate function and expression of Oat1 and Oat3 in kidney. • Effects of JBP485 on ARF are mediated by stimulating excretion of uremic toxins. • JBP485 protected against gentamicin-induced ARF by decreasing MPO and MDA.« less

Early social deprivation impairs pair bonding and alters serum corticosterone and the NAcc dopamine system in mandarin voles.

PubMed

Yu, Peng; An, Shucheng; Tai, Fadao; Wang, Jianli; Wu, Ruiyong; Wang, Bo

2013-12-01

Early life stress has a long-term negative impact on emotion, learning, memory and adult sexual behavior, and these deficits most likely impair pair bonding. Here, we investigated whether early social deprivation (ED) affects the formation of pair bonds in socially monogamous mandarin voles (Microtus mandarinus). In a partner preference test (PPT), ED-reared adult females and males did not show a preference for their partner, spent more time exploring the cage of an unfamiliar animal and directed high levels of aggression toward unfamiliar animals. In social interaction test, ED increased exploring behavior only in females, but increased movement around the partner and reduced inactivity in both males and females. Three days of cohabitation did not alter serum corticosterone levels in ED-reared males, but increased corticosterone levels in males that received bi-parental care (PC). Interestingly, serum corticosterone levels in ED- and PC-reared females declined after cohabitation. ED significantly increased basal serum corticosterone levels in males, but had no effect on females. ED significantly up-regulated the levels of dopamine and the mRNA expression of dopamine 1-type receptor (D1R) in the nucleus accumbens (NAcc) in females and males. ED suppressed dopamine 2-type receptor mRNA (D2R) expression in females, but increased this in males. After three days of cohabitation, levels of D1R mRNA and D2R mRNA expression changed in opposite directions in PC-reared voles, but in the same direction in ED-reared males, and only the expression of D2R mRNA increased in ED-reared females. Our results indicate that early social deprivation inhibits pair bonding at adulthood. This inhibition is possibly associated with sex-specific alterations in serum corticosterone, levels of dopamine and mRNA expression of two types of dopamine receptors in the NAcc. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

PubMed

Komninou, Eliza Rossi; Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Basso, Andrea Cristina; Jornada, Denise Soledade; Deschamps, João Carlos; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Bordignon, Vilceu; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

2016-01-01

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

Acute and long-term transcriptional responses in sulfur mustard-exposed SKH-1 hairless mouse skin.

PubMed

Vallet, V; Poyot, T; Cléry-Barraud, C; Coulon, D; Sentenac, C; Peinnequin, A; Boudry, I

2012-03-01

Sulfur mustard (HD) ranks among the alkylating chemical warfare agents. Skin contact with HD produces an inflammatory response that evolves into separation at the epidermal-dermal junction conducting to blistering and epidermis necrosis. Up to now, current treatment strategies of HD burns have solely consisted in symptomatic management of skin damage. Therapeutic efficacy studies are still being conducted; classically using appropriate animal skin toxicity models. In order to substantiate the use of SKH-1 hairless mouse as an appropriate model for HD-induced skin lesions, we investigate the time-dependent quantitative gene expression of various selected transcripts associated to the dorsal skin exposure to HD saturated vapors. Using quantitative real time polymerase chain reaction (RT-qPCR), the expression of interleukins (IL-1β and IL-6), tumor necrosis factor (TNF)-α, macrophage inflammatory proteins (MIP)-2α (also called Cxcl2) and MIP-1αR (also called Ccr1), matrix metalloproteases (MMP-9 and MMP-2), laminin γ2 monomer (Lamc2) and keratin (K)1 was determined up to 21 days after HD challenge in order to allow enough time for wound repair to begin. Specific transcript RT-qPCR analysis demonstrated that IL-6, IL-1β, Ccr1, Cxcl2 mRNA levels increased as early as 6 h in HD-exposed skins and remained up-regulated over a 14-day period. Topical application of HD also significantly up-regulated MMP-9, TNF-α, and Lamc2 expression at specific time points. In contrast, MMP-2 mRNA levels remained unaffected by HD over the time-period considered, whereas that long-term study revealed that K1 mRNA level significantly increased only 21 days after HD challenge. Our study hereby provides first-hand evidence to substantiate a long period variation expression in the inflammatory cytokine, MMPs and structural components following cutaneous HD exposure in hairless mouse SKH-1. Our data credit the use of SKH-1 for investigating mechanisms of HD-induced skin toxicity and for the development of pharmacological countermeasures.

Acute heat stress up-regulates neuropeptide Y precursor mRNA expression and alters brain and plasma concentrations of free amino acids in chicks.

PubMed

Ito, Kentaro; Bahry, Mohammad A; Hui, Yang; Furuse, Mitsuhiro; Chowdhury, Vishwajit S

2015-09-01

Heat stress causes an increase in body temperature and reduced food intake in chickens. Several neuropeptides and amino acids play a vital role in the regulation of food intake. However, the responses of neuropeptides and amino acids to heat-stress-induced food-intake regulation are poorly understood. In the current study, the hypothalamic mRNA expression of some neuropeptides related to food intake and the content of free amino acids in the brain and plasma was examined in 14-day-old chicks exposed to a high ambient temperature (HT; 40±1 °C for 2 or 5 h) or to a control thermoneutral temperature (CT; 30±1 °C). HT significantly increased rectal temperature and plasma corticosterone level and suppressed food intake. HT also increased the expression of neuropeptide Y (NPY) and agouti-signaling protein (ASIP) precursor mRNA, while no change was observed in pro-opiomelanocortin, cholecystokinin, ghrelin, or corticotropin-releasing hormone precursor mRNA. It was further found that the diencephalic content of free amino acids - namely, tryptophan, leucine, isoleucine, valine and serine - was significantly higher in HT chicks with some alterations in their plasma amino acids in comparison with CT chicks. The induction of NPY and ASIP expression and the alteration of some free amino acids during HT suggest that these changes can be the results or causes the suppression of food intake. Copyright © 2015. Published by Elsevier Inc.

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

PubMed Central

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-01-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA. PMID:8943327

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

PubMed

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-12-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

Endoplasmic Reticulum Stress in Mice Increases Hepatic Expression of Genes Carrying a Premature Termination Codon via a Nutritional Status-Independent GRP78-Dependent Mechanism.

PubMed

Harada, Nagakatsu; Okuyama, Maiko; Yoshikatsu, Aya; Yamamoto, Hironori; Ishiwata, Saori; Hamada, Chikako; Hirose, Tomoyo; Shono, Masayuki; Kuroda, Masashi; Tsutsumi, Rie; Takeo, Jiro; Taketani, Yutaka; Nakaya, Yutaka; Sakaue, Hiroshi

2017-11-01

Nonsense-mediated mRNA decay (NMD) degrades mRNAs carrying a premature termination codon (PTC) in eukaryotes. Cellular stresses, including endoplasmic reticulum (ER) stress, inhibit NMD, and up-regulate PTC-containing mRNA (PTC-mRNA) levels in several cell lines. However, whether similar effects exist under in vivo conditions that involve systemic nutritional status is unclear. Here, we compared the effects of pharmacological induction of ER stress with those of nutritional interventions on hepatic PTC-mRNA levels in mice. In mouse livers, the ER stress inducer tunicamycin increased PTC-mRNA levels of endogenous marker genes. Tunicamycin decreased body weight and perturbed nutrient metabolism in mice. Food restriction or deprivation mimicked the effect of tunicamycin on weight loss and metabolism, but did not increase PTC-mRNA levels. Hyperphagia-induced obesity also had little effect on hepatic PTC-mRNA levels. Meanwhile, in mouse liver phosphorylation of eIF2α, a factor that regulates NMD, was increased by both tunicamycin and nutritional interventions. Hepatic expression of GRP78, a central chaperone in ER stress responses, was increased by tunicamycin but not by the nutritional interventions. In cultured liver cells (Hepa), exogenous overexpression of a phosphomimetic eIF2α failed to increase PTC-mRNA levels. However, GRP78 overexpression in Hepa cells increased PTC-mRNA and PTC-mRNA-derived protein levels. ER stress promoted localization of GRP78 to mitochondria, and exogenous expression of a GRP78 fusion protein targeted to mitochondria mimicked the effect of wild type GRP78. These results indicate that GRP78, but not nutritional status, is a potent up-regulator of hepatic PTC-mRNA levels during induction of ER stress in vivo. J. Cell. Biochem. 118: 3810-3824, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

MAT2B promotes adipogenesis by modulating SAMe levels and activating AKT/ERK pathway during porcine intramuscular preadipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Cunzhen; Chen, Xiaochang; Wu, Wenjing

Intramuscular fat (IMF) has been demonstrated as one of the crucial factors of livestock meat quality. The MAT2B protein with MAT2α catalyzes the formation of methyl donor S- adenosylmethionine (SAMe) to mediate cell metabolism including proliferation and apoptosis. However, the regulatory effect of MAT2B on IMF deposition is still unclear. In this study, the effect of MAT2B on adipogenesis and its potential mechanism during porcine intramuscular preadipocyte differentiation was studied. The results showed that overexpression of MAT2B promoted adipogenesis and significantly up-regulated the mRNA and protein levels of adipogenic marker genes including FASN, PPARγ and aP2, consistently, knockdown of MAT2Bmore » inhibited lipid accumulation and down-regulated the mRNA and protein levels of the above genes. Furthermore, flow cytometry and EdU-labeling assay indicated that MAT2B regulate adipogenesis was partly due to influence intracellular SAMe levels and further affect cell clonal expansion. Also, increased expression of MAT2B activated the phosphorylations of AKT and ERK1/2, whereas knockdown of MAT2B blocked AKT signaling and repressed the phosphorylation of ERK1/2. Moreover, the inhibitory effect of LY294002 (a specific PI3K inhibitor) on the activities of AKT and ERK1/2 was partially recovered by overexpression of MAT2B in porcine intramuscular adipocytes. Finally, Co-IP experiments showed that MAT2B can directly interact with AKT. Taken together, our findings suggested that MAT2B acted as a positive regulator through modifying SAMe levels as well as activating AKT/ERK signaling pathway to promote porcine intramuscular adipocyte differentiation. - Highlights: • MAT2B up-regulates the expression of adipogenic marker genes and promotes porcine intramuscular preadipocyte differentiation. • MAT2B influences intracellular SAMe levels and further affects cell clonal expansion. • MAT2B interacts with AKT and activates AKT/ERK signaling pathway.« less

Sex steroid hormone metabolism takes place in human ocular cells.

PubMed

Coca-Prados, Miguel; Ghosh, Sikha; Wang, Yugang; Escribano, Julio; Herrala, Annakaisa; Vihko, Pirkko

2003-08-01

Steroids are potentially important mediators in the pathophysiology of ocular diseases. In this study, we report on the gene expression in the human eye of a group of enzymes, the 17beta-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation of sex steroid hormones. In the eye, the ciliary epithelium, a neuroendocrine secretory epithelium, co-expresses the highest levels of 17HSD2 and 5 mRNAs, and in lesser level 17HSD7 mRNA. The regulation of gene expression of these enzymes was investigated in vitro in cell lines, ODM-C4 and chronic open glaucoma (GCE), used as cell models of the human ciliary epithelium. The estrogen, 17beta-estradiol (10(-7) M) and androgen agonist, R1881 (10(-8) M) elicited in ODM-C4 and GCE cells over a 24 h time course a robust up-regulation of 17HSD7 mRNA expression. 17HSD2 was up-regulated by estradiol in ODM-C4 cells, but not in GCE cells. Under steady-state conditions, ODM-C4 cells exhibited a predominant 17HSD2 oxidative enzymatic activity. In contrast, 17HSD2 activity was low or absent in GCE cells. Our collective data suggest that cultured human ciliary epithelial cells are able to metabolize estrogen, androgen and progesterone, and that 17HSD2 and 7 in these cells are sex steroid hormone-responsive genes and 17HSD7 is responsible to keep on intra/paracrine estrogenic milieu.

Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation and mGluR signaling

PubMed Central

Muddashetty, Ravi S.; Nalavadi, Vijayalaxmi C.; Gross, Christina; Yao, Xiaodi; Xing, Lei; Laur, Oskar; Warren, Stephen T.; Bassell, Gary J.

2011-01-01

Summary The molecular mechanism how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 is reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a novel mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation. PMID:21658607

Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.

We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNAmore » staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.« less

Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

NASA Technical Reports Server (NTRS)

Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

1997-01-01

Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

Molecular cloning, characterization, and expression analysis of ghrelin and cholecystokinin in the pigeon (Columba livia).

PubMed

Xie, P; Wan, X P; Bu, Z; Zou, X T

2016-11-01

Ghrelin and cholecystokinin (CCK) are multifunctional peptides. In the current study, complete sequences of ghrelin (800 bp) and CCK (739 bp) were firstly cloned in Columba livia by using rapid amplification of cDNA ends (RACE) method. The open reading frames of ghrelin (351bp) and CCK (393bp) encoded 116 amino acids and 130 amino acids, respectively. Sequence comparison indicated that pigeon ghrelin and CCK shared high identity with those reported in other avian species. Quantitative real-time PCR analysis found that ghrelin and CCK mRNAs expressed in three intestinal segments of pigeon during development. Both ghrelin and CCK showed generally higher expressions at days posthatch than embryonic periods regardless of intestinal segments. In duodenum and ileum, the expressions of ghrelin and CCK mRNA reached the peak values at 8 d posthatch. Jejunum CCK mRNA level increased linearly after hatching, and reached the highest point at posthatch 28 d. Based on documented effects of long chain fatty acids (LCFAs) on pigeon ghrelin and CCK expression were also investigated in vitro. Higher concentrations (50 μM or 250 μM) of linoleic acid, α-linolenic acid or arachidonic acid can significantly increase ghrelin mRNA level in pigeon jejunum. However, for oleic acid, the induction of ghrelin gene expressions needed a lower concentration (5 μM). 5 μM of linoleic acid, α-linolenic acid or arachidonic acid and 250 μM palmitic acid repressed CCK expression significantly. A higher concentration (250 μM) of oleic acid or α-linolenic acid can up-regulate CCK mRNA level significantly. Our results indicated that ghrelin and CCK may act key functions in pigeon intestine development and their expressions could be regulated by LCFAs. © 2016 Poultry Science Association Inc.

Dietary isohumulones, the bitter components of beer, raise plasma HDL-cholesterol levels and reduce liver cholesterol and triacylglycerol contents similar to PPARalpha activations in C57BL/6 mice.

PubMed

Miura, Yutaka; Hosono, Mayu; Oyamada, Chiaki; Odai, Hideharu; Oikawa, Shinichi; Kondo, Keiji

2005-04-01

The effects of dietary isohumulones, the main components accounting for the bitter taste of beer, on lipid metabolism were examined. Young female C57BL/6N mice were fed diets containing isomerized hop extract (IHE), which consists mainly of isohumulones. Administration of IHE with an atherogenic (high-fat and high-cholesterol) diet for 2 weeks resulted in a significant increase in plasma HDL-cholesterol (P<0.01), along with a concomitant reduction in the atherosclerosis index, an increase in liver weight and a decrease in body weight gain in a dose-dependent manner. When animals received IHE with either a cholesterol or a basal diet for 1 week, significant decreases in the liver content of cholesterol (P<0.01) and triacylglycerol (cholesterol diet, P<0.01) were observed. Quantitative analyses of hepatic mRNA levels revealed that IHE administration resulted in up-regulation of mRNA for acyl-CoA oxidase, acyl-CoA synthetase, hydroxymethylglutaryl-CoA synthetase, lipoprotein lipase and fatty acid transport protein, and down-regulation of mRNA for Apo CIII and Apo AI. Administration of purified isohumulones effectively resulted in the same changes as IHE. Administration of fenofibrate, an agonist for PPARalpha, with a cholesterol diet caused marked hepatomegaly, an increase in plasma HDL-cholesterol, a decrease in hepatic cholesterol content, and alterations in hepatic mRNA levels similar to those observed in mice given IHE. Taken together, these results suggest that the modulation of lipid metabolism observed in mice fed diets containing isohumulones is, at least in part, mediated by activation of PPARalpha.

[The Impact of Electroacupuncture Intervention on Expression of 5-HTR 1 B/2 C Genes in Mice under Radiation Stimulation from Mobile Phone].

PubMed

Dai, Jian-yu; Chen, Yi-guo; Zhang, Xiao-qing

2015-08-01

To observe the effect of electroacupuncture (EA) stimulation of "Yifen" (TE 17), "Shenshu" (BL 23) on the expression of 5-hydroxytryptamine receptor 1 B (5-HTR 1 B) mRNA and 5-hydroxytryptamine receptor 2 C (5-HTR 2 C) mRNA in the cochlear nucleus tissue in mice experiencing radiation from mobile phone, so as to explore its mechanisms underlying improvement of tinnitus. Thirty Kunming mice were randomly divided into control group (n = 6) and modeling group (n = 24). The tinnitus model was established by giving the mice with mobile phone-radiation for 1 h in the morning and 1 h in the afternoon, continuously for 40 days. EA stimulation was applied to "Yifeng" (TE 17) group (n = 6) and "Shenshu" (BL 23) group (n = 6) for 20 min, once a day for 7 days. The expression of 5-THR 1 B/2 C mRNA in the cochlear nucleus was assayed by fluorescence quantitative polymerase chain reaction (real time-PCR). The expression level of 5-HTR 1 B was significantly lower in the model group than in the control group (P < 0.05), while that of 5-HTR 2 C mRNA significantly increased (P < 0.01). TE 17 group received a significant acupoint intervention effect (P < 0.01). Compared with TE 17 group, BL 23 group received a weaker effect (P < 0.05). EA of TE 17 can up-regulate expression level of 5-HTR 1 B and down-regulate expression level of 5-HTR 2 C in the cochlear nucleus in mice experiencing mobile-phone radiation.

[Effect of sodium phenylbutyrate on the apoptosis of human tongue squamous cancer cell line and expression of p21 and survivin genes].

PubMed

Chen, Wei-qiang; Feng, Feng-lan; Gu, Hong-biao; Pan, De-shun

2010-07-01

To examine the effects of sodium phenylbutyrate on the apoptosis of human tongue squamous cancer cell line and expression of p21 and survivin genes. The inhibition effects of sodium phenylbutyrate on Tca8113 and human tongue squamous cell carcinoma (TCSSA) cell lines were detected by methyl thiazoly terazolium (MTT) and the apoptosis of the cancer cells after being induced by sodium phenylbutyrate examined by flow cytometry (FCM). The expression of p21 and survivin genes were observed with Western blotting and RT-PCR. Compared with control group, the level of p21 mRNA and protein of Tca8113 cellline increased to 0.09 ± 0.08 and increased 0.72 ± 0.10, that of TCSSA cellline increased 1.34 ± 0.12 and 1.56 ± 0.09 (P < 0.05). Compared with control group, the level of surrive mRNA and protein of Tca8113 cellline decreased to 1.10 ± 0.05 and 1.14 ± 1.10, that of TCSSA cellline decreased to 0.12 ± 0.08 and 0.94 ± 0.09 (P < 0.05). Sodium phenylbutyrate inhibited the cell proliferation, promoted cell apoptosis and arrested the cells in G₁/G₀ phase. The amount of p21 mRNA and protein were increased, and the expression of survivin gene was decreased. Sodium phenylbutyrate exhibited remarkable inhibitory effects on human tongue squamous cancer cell proliferation and induced cancer cell apoptosis. The mechanism may be due to up-regulation of p21 gene and down-regulation of survivin gene. The mRNA level of p21 gene and survivin gene showed a strong correlation.

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells.

PubMed

Di Fulvio, Mauricio; Lauf, Peter K; Shah, Shalin; Adragna, Norma C

2003-05-01

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.

[Effect of Moxibustion on Learning-memory Ability and Hippocampal Amyloid beta Protein Overexpression in Mild Cognitive Impairment Rats].

PubMed

Zhu, Cai-feng; Sun, Jian-jian; Han, Wei; Yang, Jun

2016-04-01

To observe the effect of moxibustion of "Baihui" (GV 20), etc. on learning-memory ability, hip- pocampal amyloid beta (AP) protein expression and immune activity in mild cognitive impairment (MCI) rats, so as to reveal its mechanism underlying improving cognitive impairment. A total of 48 SD rats were randomly divided into normal, model, moxibustion, and medication groups (n = 12 in each group). The MCI model was established by intraperitoneal injection of 2 mL mixture solution containing D-galactose (120 mg - kg- - d-) and Sodium Nitrite (90 mg x kg(-1) x d(-1)), once daily for 40 days. Moxibustion (separated by Radix Aconiti Praeparata cake) was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min, once daily for 2 weeks, with one day's rest between two weeks. The rats of the medication group were given with Nimodipine (2 mg x kg(-1) x d(-1), t.i.d.) by lavage for 2 weeks (except Sundays). The learning-memory ability was detected by Morris maze water swimming tasks. The expression level of hippocampal AP protein was detected by immunohistochemistry, and those of hippocampal presenilin-1 (PS-1) mRNA and cleaving enzyme (BACE-1) mRNA were detected by real time-PCR, and serum IL-6 level was assayed by ELISA. Following modeling, the average escape latency of location navigation tests of Morris maze water swimming tests, the expression levels of hippocampal Abeta protein, PS-1 mRNA and BACE-1 mRNA, and serum IL-6 content were significantly increased in the model group( P<0.01) , while the target-platform crossing times and the percentage of target-quadrant swimming duration of spacial probe trials were remarkably decreased in the model group (P<0.01). After moxibustion, the increased escape latency, hippocampal AP protein, PS-i mHNA and BACE-1 mRNA ex- pression and serum IL-6 content, and the decreased target-platform crossing times and the percentage of target-quadrant swim- ming duration were reversed in both moxibustion and medication groups (P<0.01). The effects of the moxibustion group were obviously superior to those of the medication group in decreasing the escape latency, and in up-regulating the target-platform crossing times, the percentage of target-quadrant swimming duration, and down-regulating hippocampal Abeta protein, PS-1 mHNA and BACE-1 mRNA expression levels and serum IL-6 content (P<0.05). Moxibustion is effective in improving MCI rats' learning-memory ability, which may be associated with its functions in down-regulating the levels of hippocampal Abeta protein, PS-1 mRNA and BACE-1 mRNA expression and serum IL-6 content, possibly by blocking Abeta overexpression-induced inflammation cascade.

Protein Biochemistry and Expression Regulation of Cadmium/Zinc Pumping ATPases in the Hyperaccumulator Plants Arabidopsis halleri and Noccaea caerulescens

PubMed Central

Mishra, Seema; Mishra, Archana; Küpper, Hendrik

2017-01-01

P1B-ATPases are decisive for metal accumulation phenotypes, but mechanisms of their regulation are only partially understood. Here, we studied the Cd/Zn transporting ATPases NcHMA3 and NcHMA4 from Noccaea caerulescens as well as AhHMA3 and AhHMA4 from Arabidopsis halleri. Protein biochemistry was analyzed on HMA4 purified from roots of N. caerulescens in active state. Metal titration of NcHMA4 protein with an electrochromic dye as charge indicator suggested that HMA4 reaches maximal ATPase activity when all internal high-affinity Cd2+ binding sites are occupied. Although HMA4 was reported to be mainly responsible for xylem loading of heavy metals for root to shoot transport, the current study revealed high expression of NcHMA4 in shoots as well. Further, there were additional 20 and 40 kD fragments at replete Zn2+ and toxic Cd2+, but not at deficient Zn2+ concentrations. Altogether, the protein level expression analysis suggested a more multifunctional role of NcHMA4 than previously assumed. Organ-level transcription analysis through quantitative PCR of mRNA in N. caerulescens and A. halleri confirmed the strong shoot expression of both NcHMA4 and AhHMA4. Further, in shoots NcHMA4 was more abundant in 10 μM Zn2+ and AhHMA4 in Zn2+ deficiency. In roots, NcHMA4 was up-regulated in response to deficient Zn2+ when compared to replete Zn2+ and toxic Cd2+ treatment. In both species, HMA3 was much more expressed in shoots than in roots, and HMA3 transcript levels remained rather constant regardless of Zn2+ supply, but were up-regulated by 10 μM Cd2+. Analysis of cellular expression by quantitative mRNA in situ hybridisation showed that in A. halleri, both HMA3 and HMA4 mRNA levels were highest in the mesophyll, while in N. caerulescens they were highest in the bundle sheath of the vein. This is likely related to the different final storage sites for hyperaccumulated metals in both species: epidermis in N. caerulescens, mesophyll in A. halleri. PMID:28588597

Protein Biochemistry and Expression Regulation of Cadmium/Zinc Pumping ATPases in the Hyperaccumulator Plants Arabidopsis halleri and Noccaea caerulescens.

PubMed

Mishra, Seema; Mishra, Archana; Küpper, Hendrik

2017-01-01

P 1B -ATPases are decisive for metal accumulation phenotypes, but mechanisms of their regulation are only partially understood. Here, we studied the Cd/Zn transporting ATPases NcHMA3 and NcHMA4 from Noccaea caerulescens as well as AhHMA3 and AhHMA4 from Arabidopsis halleri . Protein biochemistry was analyzed on HMA4 purified from roots of N. caerulescens in active state. Metal titration of NcHMA4 protein with an electrochromic dye as charge indicator suggested that HMA4 reaches maximal ATPase activity when all internal high-affinity Cd 2+ binding sites are occupied. Although HMA4 was reported to be mainly responsible for xylem loading of heavy metals for root to shoot transport, the current study revealed high expression of NcHMA4 in shoots as well. Further, there were additional 20 and 40 kD fragments at replete Zn 2+ and toxic Cd 2+ , but not at deficient Zn 2+ concentrations. Altogether, the protein level expression analysis suggested a more multifunctional role of NcHMA4 than previously assumed. Organ-level transcription analysis through quantitative PCR of mRNA in N. caerulescens and A. halleri confirmed the strong shoot expression of both NcHMA4 and AhHMA4 . Further, in shoots NcHMA4 was more abundant in 10 μM Zn 2+ and AhHMA4 in Zn 2+ deficiency. In roots, NcHMA4 was up-regulated in response to deficient Zn 2+ when compared to replete Zn 2+ and toxic Cd 2+ treatment. In both species, HMA3 was much more expressed in shoots than in roots, and HMA3 transcript levels remained rather constant regardless of Zn 2+ supply, but were up-regulated by 10 μM Cd 2+ . Analysis of cellular expression by quantitative mRNA in situ hybridisation showed that in A. halleri , both HMA3 and HMA4 mRNA levels were highest in the mesophyll, while in N. caerulescens they were highest in the bundle sheath of the vein. This is likely related to the different final storage sites for hyperaccumulated metals in both species: epidermis in N. caerulescens , mesophyll in A. halleri .

Differential regulation of ATP binding cassette protein A1 expression and ApoA-I lipidation by Niemann-Pick type C1 in murine hepatocytes and macrophages.

PubMed

Wang, Ming-Dong; Franklin, Vivian; Sundaram, Meenakshi; Kiss, Robert S; Ho, Kenneth; Gallant, Michel; Marcel, Yves L

2007-08-03

Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr alpha mRNA is not changed, and Lxr alpha target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [(35)S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate.

β1- and β2-adrenergic receptor stimulation differ in their effects on PGC-1α and atrogin-1/MAFbx gene expression in chick skeletal muscle.

PubMed

Shimamoto, Saki; Ijiri, Daichi; Kawaguchi, Mana; Nakashima, Kazuki; Tada, Osamu; Inoue, Hiroki; Ohtsuka, Akira

2017-09-01

Adrenaline changes expression of the genes encoding peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), which is known as a regulator of muscle size, and atrogin-1/muscle atrophy F-box (MAFbx), which is a muscle-specific ubiquitin ligase. However, the subtype of β-adrenergic receptor (β-AR) involved in regulating these genes in skeletal muscle is not yet well defined. In this study, the effects of intraperitoneal injection of adrenaline and three β 1-3 -AR selective agonists on chick skeletal muscle metabolism were examined, to evaluate the functions of β-AR subtypes. Adrenaline decreased atrogin-1/MAFbx mRNA levels accompanied by an increase in PGC-1α mRNA and protein levels. However, among the three selective agonists, only the β 1 -AR agonist, dobutamine, increased PGC-1α mRNA and protein levels, while the β 2 -AR agonist, clenbuterol, suppressed atrogin-1/MAFbx mRNA levels. In addition, preinjection of the β 1 -AR antagonist, acebutolol, and the β 2 -AR antagonist, butoxamine, inhibited the adrenaline-induced increase in PGC-1α mRNA levels and the decrease in atrogin-1/MAFbx mRNA levels, respectively. Compared with adrenaline administration, the β 3 -AR agonist, BRL37344, decreased PGC-1α mRNA levels and increased atrogin-1/MAFbx mRNA levels. These results suggest that, in chick skeletal muscle, PGC-1α is induced via the β 1 -AR, while atrogin-1/MAFbx is suppressed via the β 2 -AR. Copyright © 2017. Published by Elsevier Inc.

Zearalenone and 17 β-estradiol induced damages in male rats reproduction potential; evidence for ERα and ERβ receptors expression and steroidogenesis.

PubMed

Adibnia, Elmira; Razi, Mazdak; Malekinejad, Hassan

2016-09-15

The estrogen receptors (ERs)-dependent effects of Zearalenone (ZEA) on structure and function of the testis as well as sperm parameters were compared with 17-β estradiol as endogenous substance. For this purpose, 30 mature male rats were assigned into five groups as; control (appropriate volume of normal saline, i. p.), ZEA-received (1, 2 and 4 mg/kg, b. w., i. p.) and 17 β-estradiol (E2)-received (appropriate dose of 0.1 mg/kg, i. p.). Following 28 days, the mRNA levels of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) in the testis and sperms and the expression of them at protein levels in testicles were estimated. Mitochondrial content of germinal epithelium, Leydig cells steroid foci, sperm quality parameters and serum level of testosterone were assessed. Fluorescent techniques were used for analyzing apoptosis and mRNA damage in necrotic cells. ZEA reduced the mRNA and protein levels of ERα in testicles while up-regulated the ERβ expression. The mRNA level of ERα decreased in sperms of ZEA and E2-received animals. No remarkable changes were found for ERβ expression in sperms from ZEA and E2-received animals. ZEA reduced the Leydig cells steroidogenesis, mitochondrial content of germinal cells and elevated cellular apoptosis and necrosis dose-dependently. E2 reduced the testosterone concentration, enhanced the apoptosis and reduced sperm quality. Our data suggest that ZEA-induced detrimental effects in the structure and function of testis, may attribute to changing the ERs expression at mRNA and translational level. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transcriptomic analysis reveals the flooding tolerant mechanism in flooding tolerant line and abscisic acid treated soybean.

PubMed

Yin, Xiaojian; Hiraga, Susumu; Hajika, Makita; Nishimura, Minoru; Komatsu, Setsuko

2017-03-01

Soybean is highly sensitive to flooding stress and exhibits markedly reduced plant growth and grain yield under flooding conditions. To explore the mechanisms underlying initial flooding tolerance in soybean, RNA sequencing-based transcriptomic analysis was performed using a flooding-tolerant line and ABA-treated soybean. A total of 31 genes included 12 genes that exhibited similar temporal patterns were commonly changed in these plant groups in response to flooding and they were mainly involved in RNA regulation and protein metabolism. The mRNA expression of matrix metalloproteinase, glucose-6-phosphate isomerase, ATPase family AAA domain-containing protein 1, and cytochrome P450 77A1 was up-regulated in wild-type soybean under flooding conditions; however, no changes were detected in the flooding-tolerant line or ABA-treated soybean. The mRNA expression of cytochrome P450 77A1 was specifically up-regulated in root tips by flooding stress, but returned to the level found in control plants following treatment with the P450 inhibitor uniconazole. The survival ratio and root fresh weight of plants were markedly improved by 3-h uniconazole treatment under flooding stress. Taken together, these results suggest that cytochrome P450 77A1 is suppressed by uniconazole treatment and that this inhibition may enhance soybean tolerance to flooding stress.

Regulation of atrial natriuretic peptide clearance receptors in mesangial cells by growth factors.

PubMed

Paul, R V; Wackym, P S; Budisavljevic, M; Everett, E; Norris, J S

1993-08-25

Rat mesangial cells can express both 130-kDa guanylyl cyclase-coupled and 66-kDa non-coupled atrial natriuretic peptide (ANP) receptors (ANPR-A and ANPR-C, respectively). Exposure of mesangial cells, grown in 20% fetal calf serum, to 0.1% serum for 24 h increased total ANP receptor density more than 2-fold (Bmax = 87 versus 37 fmol/mg of cell protein) without changing binding affinity (Kd = 94 versus 88 pM). Radioligand binding and cross-linking studies demonstrated that up-regulation of ANP binding after serum deprivation was entirely due to an increase in ANPR-C, with little or no change in ANPR-A. Inhibition of protein synthesis with cycloheximide blocked up-regulation after serum deprivation. Steady-state ANPR-C mRNA level was increased 15-fold by serum deprivation, as judged by Northern blotting. There was no change in ANPR-A mRNA. Platelet-derived growth factor and phorbol myristate acetate, when added to low serum medium, blocked or reversed the effect of serum deprivation on ANPR-C. We conclude that synthesis and expression of ANPR-C but not ANPR-A is suppressed by serum, platelet-derived growth factor, and phorbol myristate acetate. Suppression of ANPR-C in vivo could contribute to mesangial cell proliferative responses to growth factors.

Cell type-specific regulation of beta2-adrenoceptor mRNA by agonists.

PubMed

Danner, S; Lohse, M J

1997-07-16

Prolonged agonist stimulation of beta2-adrenoceptors results in receptor down-regulation which is often paralleled by a reduction of the corresponding mRNA. In this study, we investigated the agonist-dependent regulation of beta2-adrenoceptor mRNA in DDT1-MF2 smooth muscle cells and C6 glioma cells. In DDT1-MF2 cells the half-life of the mRNA was 12 h in monolayer compared to 2 h in suspension cultures. Under both conditions, the agonist isoproterenol reduced this half-life by a factor of 2. In contrast, in C6 glioma cells isoproterenol had no effect on the mRNA stability, even though it reduced mRNA levels by approximately 50%. Isoproterenol-induced downregulation of beta2-adrenoceptor mRNA was completely blocked in C6 cells by the presence of a protein synthesis inhibitor, while this was not so in DDT1-MF2-cells. These data show that beta2-adrenoceptor downregulation occurs via cell-type specific mechanisms.

Cloning of the chaperonin t-complex polypeptide 1 gene from Schistosoma mansoni and studies of its expression levels under heat shock and oxidative stress.

PubMed

Campos, E G; Hamdan, F F

2000-03-01

The protein TCP-1 (t-complex polypeptide 1) is a subunit of the hetero-oligomeric complex CCT (chaperonin containing TCP- 1) present in the eukaryotic cytosol. Chaperone function may be critical for the development and survival of the different life stages of Schistosoma mansoni, a parasite that is exposed to drastic environmental changes during its development. We isolated a full-length S. mansoni TCP-1 cDNA (SmTCP-1A) encoding a protein highly homologous with TCP-1. The deduced SmTCP-1A amino-acid sequence shows up to 65% identity with other eukaryotic CCT family members. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the mRNA expression levels of SmTCP-1A in adult S. mansoni were down-regulated in worms subjected to heat shock and oxidative stress conditions. This down-regulation of SmTCP-1A mRNA may reflect a switch in CCT subunits as an adaptive response to heat shock and oxidative stress conditions.

Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1β gene expression in pancreatic islet β-cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakabayashi, Hiroko; Ohta, Yasuharu, E-mail: yohta@yamaguchi-u.ac.jp; Yamamoto, Masayoshi

2013-05-03

Highlights: •Arnt mRNA expressed in a circadian manner in mouse pancreatic islets. •Expressions of Dbp and Arnt damped in the islets of a diabetic model mouse. •DBP and E4BP4 regulate Arnt promoter activity by direct binding. •Arnt may have a role in connecting circadian rhythm and metabolism. -- Abstract: Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1β (HIF-1β) has emerged as a potential determinant of pancreatic β-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expressionmore » have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4). By conducting gene expression studies using the islets of Wfs1{sup −/−} A{sup y}/a mice that develop severe diabetes due to β-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the Arnt promoter in MIN6 cells. These results suggest that in mouse pancreatic islets mRNA expression of Arnt fluctuates significantly in a circadian manner and that the down-regulation of Dbp and up-regulation E4bp4 contribute to direct suppression of Arnt expression in diabetes.« less

Regulation of the steady state level of Fc gamma RI mRNA by IFN-gamma and dexamethasone in human monocytes, neutrophils, and U-937 cells.

PubMed

Pan, L Y; Mendel, D B; Zurlo, J; Guyre, P M

1990-07-01

The high affinity IgG FcR Fc gamma RI, CD64, plays important roles in the immune response. Fc gamma RI is predominantly expressed on monocytes and macrophages, and barely detectable on neutrophils. rIFN-gamma markedly increases the expression of Fc gamma RI on neutrophils, monocytes, macrophages and myeloid cell lines such as U-937, HL-60, and THP-1. Glucocorticoids inhibit the augmentation of Fc gamma RI expression by rIFN-gamma on neutrophils and myeloid cell lines, but enhance the augmentation of Fc gamma RI expression by rIFN-gamma on monocytes. In this study, we examined the effect of rIFN-gamma and dexamethasone (Dex) on the steady state level of Fc gamma RI mRNA in U-937 cells, neutrophils, and monocytes by hybridizing total RNA with the Fc gamma RI cDNA probe, p135. We found that the amount of Fc gamma RI mRNA increased within 1 h of treatment with rIFN-gamma in all three cell types. This initial induction of Fc gamma RI mRNA by rIFN-gamma was completely blocked by an inhibitor of RNA synthesis, actinomycin D, suggesting that the rIFN-gamma-mediated induction of Fc gamma RI mRNA is dependent on gene transcription. Dex, used in combination with rIFN-gamma, partially blocked the induction of Fc gamma RI mRNA by rIFN-gamma in U-937 cells and neutrophils, but caused a synergistic increase in Fc gamma RI mRNA levels in monocytes. The inhibitory effect of Dex on the steady state level of Fc gamma RI mRNA in U-937 cells was blocked by an inhibitor of protein synthesis, cycloheximide, suggesting that Dex-induced proteins were involved in the regulation of Fc gamma RI expression. This study indicates that the regulation of Fc gamma RI expression on U-937 cells, neutrophils, and monocytes by rIFN-gamma and Dex occurs, at least in part, at the mRNA level. rIFN-gamma increases the steady state level of Fc gamma RI mRNA through a common pathway among U-937 cells, neutrophils, and monocytes, whereas the effect of Dex on rIFN-gamma-induced Fc gamma RI mRNA is cell-type specific.

Leptin controls ketone body utilization in hypothalamic neuron.

PubMed

Narishima, Ryota; Yamasaki, Masahiro; Hasegawa, Shinya; Yoshida, Saki; Tanaka, Shinya; Fukui, Tetsuya

2011-03-03

Leptin is an appetite-controlling peptide secreted from adipose tissue. Previously, we showed that the gene expression of acetoacetyl-CoA synthetase (AACS), the ketone body-utilizing enzyme for lipid synthesis, was suppressed by leptin deficiency-induced obesity in white adipose tissue. In this study, to clarify the effects of leptin on ketone body utilization in the central nervous system, we examined the effects of leptin signaling on AACS expression. In situ hybridization analysis of ob/ob and db/db mice revealed that AACS mRNA level was reduced by leptin deficiency in the arcuate nucleus (Arc) and ventromedial hypothalamic nucleus (VMH) in hypothalamus but not in other brain regions. Moreover, AACS mRNA level was increased by leptin treatment both in primary cultured neural cells and in N41 neural-like cells. In N41 cells, AACS level was decreased by AMPK inducer but increased by AMPK inhibitor. These results suggest that the up-regulation of AACS expression by leptin is due to the suppression of AMPK activity via neural leptin signaling and that the deficiency of this regulation may be responsible for neurological disorders in central appetite control. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

PubMed Central

Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

2013-01-01

CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of nucleolin's actions on CSF-1 mRNA and describe the dependence of miR-130a- and miR-301a-directed CSF-1 mRNA decay and inhibition of ovarian cancer cell motility on nucleolin. PMID:23471483

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

PubMed

Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

2003-09-01

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

CXCL1 and CXCR2 as potential markers for vital reactions in skin contusions.

PubMed

He, Jie-Tao; Huang, Hong-Yan; Qu, Dong; Xue, Ye; Zhang, Kai-Kai; Xie, Xiao-Li; Wang, Qi

2018-06-01

Detection of the vitality of wounds is one of the most important issues in forensic practice. This study investigated mRNA and protein levels of CXCL1 and CXCR2 in skin wounds in mice and humans. Western blot analysis of CXCL1 and CXCR2 protein levels showed no difference between wounded and intact skin. However, mRNA levels demonstrated higher expression of CXCL1 and CXCR2 in contused mouse and human skin, compared with intact skin. At postmortem there were no remarkable changes in CXCL1 and CXCR2 mRNA levels in contused mouse skin. Increased mRNA expression was observed in contused mouse skin up to 96 h and 72 h after death for CXCL1 and CXCR2 respectively. In human samples of wounded skin, increased CXCL1 mRNA levels were detected up to 48 h after autopsy in all 5 cases, while increased CXCR2 mRNA levels were observed 48 h after autopsy in 4 of 5 cases. These findings suggest that the levels of CXCL1 and CXCR2 mRNA present in contused skin can be used as potential markers for a vital reaction in forensic practice.

Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella).

PubMed

Xu, Hui-Jun; Jiang, Wei-Dan; Feng, Lin; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2016-05-01

This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4 (C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, B-cell lymphoma-2, inhibitor of apoptosis protein, NF-E2-related factor 2 mRNA levels (P < 0.05), increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl contents (P < 0.05), and up-regulated Claudin-12, 15a, -15b, Fas ligand, mitogen-activated protein kinase kinase 6, p38 mitogen-activated protein kinase, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1, caspase-3, -7, -8, -9, Kelch-like ECH-associating protein (Keap) 1a and Keap 1b mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen structural integrity through depression of antioxidative ability, induction of apoptosis and disruption of tight junctional complexes. In addition, except the activities of ACP and MnSOD, and mRNA expression levels of TGF-β1, Occludin and MnSOD, the effect of vitamin C on fish head kidney, spleen and skin immunity and structural integrity other indicators model are similar under infection of A. hydrophila. Finally, the vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8 mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively. Copyright © 2016. Published by Elsevier Ltd.

Emodin opposes chronic unpredictable mild stress induced depressive-like behavior in mice by upregulating the levels of hippocampal glucocorticoid receptor and brain-derived neurotrophic factor.

PubMed

Li, Meng; Fu, Qiang; Li, Ying; Li, Shanshan; Xue, Jinsong; Ma, Shiping

2014-10-01

Emodin, the major active component of Rhubarb, has shown neuroprotective activity. This study is attempted to investigate whether emodin possesses beneficial effects on chronic unpredictable mild stress (CUMS)-induced behavioral deficits (depression-like behaviors) and explore the possible mechanisms. ICR mice were subjected to chronic unpredictable mild stress for 42 consecutive days. Then, emodin and fluoxetine (positive control drug) were administered for 21 consecutive days at the last three weeks of CUMS procedure. The classical behavioral tests: open field test (OFT), sucrose preference test (SPT), tail suspension test (TST) and forced swimming test (FST) were applied to evaluate the antidepressant effects of emodin. Then plasma corticosterone concentration, hippocampal glucocorticoid receptor (GR) and brain-derived neurotrophic factor (BDNF) levels were tested to probe the mechanisms. Our results indicated that 6 weeks of CUMS exposure induced significant depression-like behavior, with high, plasma corticosterone concentration and low hippocampal GR and BDNF expression levels. Whereas, chronic emodin (20, 40 and 80 mg/kg) treatments reversed the behavioral deficiency induced by CUMS exposure. Treatment with emodin normalized the change of plasma corticosterone level, which demonstrated that emodin could partially restore CUMS-induced HPA axis impairments. Besides, hippocampal GR (mRNA and protein) and BDNF (mRNA) expressions were also up-regulated after emodin treatments. In conclusion, emodin remarkably improved depression-like behavior in CUMS mice and its antidepressant activity is mediated, at least in part, by the up-regulating GR and BDNF levels in hippocampus. Copyright © 2014 Elsevier B.V. All rights reserved.

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Konnai, Satoru; Usui, Tatsufumi; Ikeda, Manabu

2005-09-01

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-{alpha} and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-{alpha}-induced responses, in this study we examined the TNF-{alpha}-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-{alpha} (rTNF-{alpha}) was significantly higher than those from ALmore » cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5{sup +} or sIgM{sup +} cells and these cells showed resistance to TNF-{alpha}-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-{alpha}-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection.« less

Ras-dva Is a Novel Pit-1- and Glucocorticoid-Regulated Gene in the Embryonic Anterior Pituitary Gland

PubMed Central

Ellestad, Laura E.

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5′-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5′-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland. PMID:23161868

Ras-dva is a novel Pit-1- and glucocorticoid-regulated gene in the embryonic anterior pituitary gland.

PubMed

Ellestad, Laura E; Porter, Tom E

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

Visually induced changes in components of the retinoic acid system in fundal layers of the chick.

PubMed

Bitzer, M; Feldkaemper, M; Schaeffel, F

2000-01-01

Eye growth is visually regulated via messengers that are released from the retina. The retina involves a yet unknown algorithm to analyse the projected image so that the appropriate growth rates for the back of the eye are ensured. One biochemical candidate that could act as a growth controller, is retinoic acid (RA). Previous work (Seko, Shimokawa and Tokoro, 1996; Mertz et al., 1999) has shown that retinal and choroidal RA levels are indeed predictably changed by visual conditions that cause myopia or hyperopia, respectively. We have studied in which fundal tissues aldehyde dehydrogenase-2 (AHD2) and retinaldehyde dehydrogenase-2 (RALDH2), enzymes involved in RA synthesis, are expressed and at which levels the effects of vision on RA levels may be controlled. Using Northern blot analysis, we have found that the retinal mRNA level of the AHD2 is up-regulated after 3 days of treatment with negative lenses (negative lenses place the image behind the retina). The abundance of the retinal mRNA of a RA receptor, RAR-beta, was up-regulated already after 6 hr of treatment with positive lenses (positive lenses place the image in front of the retina). The up-regulation persisted for at least 1 week. Finally, we have studied the effects of an inhibitor of RA synthesis, disulfiram, on the visual control of eye growth. We found inhibition of myopia as induced by frosted goggles ('deprivation myopia') but no significant inhibitory effects on refractive errors induced by +7D or -7D lenses. Our results are in line with the hypothesis that RA may play a role in the visual control of eye growth. The RA system differs from a number of other candidates (dopamine, cholinergic agents, opiates) in that it distinguishes between positive and negative defocus, similar to the immediate early gene ZENK (Stell et al., 1999). The exact time kinetics of the changes have still to be worked out since it is possible that the changes in RA relate to already occurring changes in growth rather than to initial steps of the signaling cascade. Copyright 2000 Academic Press.

Up-regulated ephrinB3/EphB3 expression in intractable temporal lobe epilepsy patients and pilocarpine induced experimental epilepsy rat model.

PubMed

Huang, Hao; Li, Ruohan; Yuan, Jinxian; Zhou, Xin; Liu, Xi; Ou, Shu; Xu, Tao; Chen, Yangmei

2016-05-15

EphB family receptor tyrosine kinases, in cooperation with cell surface-bound ephrinB ligands, play a critical role in maintenance of dendritic spine morphogenesis, axons guidance, synaptogenesis, synaptic reorganization and plasticity in the central nervous system (CNS). However, the expression pattern of ephrinB/EphB in intractable temporal lobe epilepsy (TLE) and the underlying molecular mechanisms during epileptogenesis remain poorly understood. Here we investigated the expression pattern and cellular distribution of ephrinB/EphB in intractable TLE patients and lithium chloride-pilocarpine induced TLE rats using real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry, double-labeled immunofluorescence and Western blot analysis. Compared to control groups, ephrinB3 and EphB3 mRNA expression were significantly up-regulated in intractable TLE patients and TLE rats, while the mRNA expression trend of ephrinB1/2 and EphB1/2/4/6 in intractable TLE patients and TLE rats were inconsistent. Western blot analysis and semi-quantitative immunohistochemistry confirmed that ephrinB3 and EphB3 protein level were up-regulated in intractable TLE patients and TLE rats. At the same time, double-labeled immunofluorescence indicate that ephrinB3 was expressed mainly in the cytoplasm and protrusions of glia and neurons, while EphB3 was expressed mainly in the cytoplasm of neurons. Taken together, up-regulated expression of ephrinB3/EphB3 in intractable TLE patients and experimental TLE rats suggested that ephrinB3/EphB3 might be involved in the pathogenesis of TLE. Copyright © 2016 Elsevier B.V. All rights reserved.

Cichorium intybus L. promotes intestinal uric acid excretion by modulating ABCG2 in experimental hyperuricemia.

PubMed

Wang, Yu; Lin, Zhijian; Zhang, Bing; Nie, Anzheng; Bian, Meng

2017-01-01

Excessive production and/or reduced excretion of uric acid could lead to hyperuricemia, which could be a major cause of disability. Hyperuricemia has received increasing attention in the last few decades due to its global prevalence. Cichorium intybus L., commonly known as chicory, is a perennial herb of the asteraceae family. It was previously shown to exert potent hypouricemic effects linked with decreasing uric acid formation in the liver by down-regulating the activity of xanthine oxidase, and increasing uric acid excretion by up-regulating the renal OAT3 mRNA expression. The present study aimed to evaluate its extra-renal excretion and possible molecular mechanism underlying the transporter responsible for intestinal uric acid excretion in vivo. Chicory was administered intragastrically to hyperuricemic rats induced by drinking 10% fructose water. The uricosuric effect was evaluated by determining the serum uric acid level as well as the intestinal uric acid excretion by HPLC. The location and expression levels of ATP-binding cassette transporter, sub-family G, member 2 (ABCG2) in jejunum and ileum were analyzed. The administration of chicory decreased the serum uric acid level significantly and increased the intestinal uric acid excretion obviously in hyperuricemic rats induced by 10% fructose drinking. Staining showed that ABCG2 was expressed in the apical membrane of the epithelium and glands of the jejunum and ileum in rats. Further examination showed that chicory enhanced the mRNA and protein expressions of ABCG2 markedly in a dose-dependent manner in jejunum and ileum. These findings indicate that chicory increases uric acid excretion by intestines, which may be related to the stimulation of intestinal uric acid excretion via down-regulating the mRNA and protein expressions of ABCG2.

The expression of transforming growth factor beta in pregnant rat myometrium is hormone and stretch dependent.

PubMed

Shynlova, Oksana; Tsui, Prudence; Dorogin, Anna; Langille, B Lowell; Lye, Stephen J

2007-09-01

From a quiescent state in early pregnancy to a highly contractile state in labor, the myometrium displays tremendous growth and remodeling. We hypothesize that the transforming growth factor beta (TGFbeta) system is involved in the differentiation of pregnant myometrium throughout gestation and labor. Furthermore, we propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial TGFbetas. The expression of TGFbeta1-3 mRNAs and proteins was examined by real-time PCR, Western immunoblot, and localized with immunohistochemistry in the rat uterus throughout pregnancy and labor. Tgfbeta1-3 genes were expressed differentially in pregnant myometrium. Tgfbeta2 gene was not affected by pregnancy, whereas the Tgfbeta1 gene showed a threefold increase during the second half of gestation. In contrast, we observed a dramatic bimodal change in Tgfbeta3 gene expression throughout pregnancy. Tgfbeta3 mRNA levels first transiently increased at mid-gestation (11-fold on day 14) and later at term (45-fold at labor, day 23). Protein expression levels paralleled the changes in mRNA. Treatment of pregnant rats with the progesterone (P4) receptor antagonist RU486 induced premature labor on day 19 and increased Tgfbeta3 mRNA, whereas artificial maintenance of elevated P4 levels at late gestation (days 20-23) caused a significant decrease in the expression of Tgfbeta3 gene. In addition, Tgfbeta3 was up-regulated specifically in the gravid horn of unilaterally pregnant rats subjected to a passive biological stretch imposed by the growing fetuses, but not in the empty horn. Collectively, these data indicate that the TGFbeta family contributes in the regulation of myometrial activation at term integrating mechanical and endocrine signals for successful labor contraction.

Up-regulation of aldolase A and methylglyoxal production in adipocytes.

PubMed

Liu, Jianghai; Desai, Kaushik; Wang, Rui; Wu, Lingyun

2013-04-01

We previously reported that up-regulation of aldolase B, a key enzyme in fructose metabolism, was mainly responsible for vascular methylglyoxal (MG) overproduction under different pathological conditions. Here we investigated whether aldolase A, an enzyme of the glycolytic pathway, also caused MG overproduction in insulin-sensitive adipocytes. The relative contributions of different metabolic pathways or enzymes to MG generation were evaluated in cultured 3T3-L1 adipocytes. Glucose (25 mM) had no effect on aldolase A gene expression, but insulin (100 nM) up-regulated aldolase A mRNA and protein levels in the absence or presence of 25 mM glucose in adipocytes. Treatment with insulin increased levels of basal or glucose (25 mM)-induced MG and glucose 6-phosphate. However, insulin, glucose (25 mM) or their combination had no effect on cellular levels of sorbitol and fructose, but down-regulated gene expression of aldolase B to a similar extent, when compared with the control group. Incubation of 3T3-L1 adipocytes with fructose, acetone, acetol, threonine or glycine (25 mM), with or without insulin did not alter cellular MG levels. The elevated MG levels induced by insulin, glucose (25 mM) or their combination in adipocytes was completely reduced by siRNA knock down of aldolase A or application of 2-deoxy-D-glucose (a non-specific inhibitor of glucose uptake and glycolysis), but not by knock down of aldolase B. Insulin enhanced MG overproduction in insulin-sensitive adipocytes by up-regulating aldolase A, a mechanism that could be involved in the development of insulin resistance and obesity. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Silver nanoparticles administered to chicken affect VEGFA and FGF2 gene expression in breast muscle and heart

NASA Astrophysics Data System (ADS)

Hotowy, Anna; Sawosz, Ewa; Pineda, Lane; Sawosz, Filip; Grodzik, Marta; Chwalibog, André

2012-07-01

Nanoparticles of colloidal silver (AgNano) can influence gene expression. Concerning trials of AgNano application in poultry nutrition, it is useful to reveal whether they affect the expression of genes crucial for bird development. AgNano were administered to broiler chickens as a water solution in two concentrations (10 and 20 ppm). After dissection of the birds, breast muscles and hearts were collected. Gene expression of FGF2 and VEGFA on the mRNA and protein levels were evaluated using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay methods. The results for gene expression in the breast muscle revealed changes on the mRNA level ( FGF2 was up-regulated, P < 0.05) but not on the protein level. In the heart, 20 ppm of silver nanoparticles in drinking water increased the expression of VEGFA ( P < 0.05), at the same time decreasing FGF2 expression both on the transcriptional and translational levels. Changes in the expression of these genes may lead to histological changes, but this needs to be proven using histological and immunohistochemical examination of tissues. In general, we showed that AgNano application in poultry feeding influences the expression of FGF2 and VEGFA genes on the mRNA and protein levels in growing chicken.

Distinct mechanisms underlie adaptation of proximal tubule Na+/H+ exchanger isoform 3 in response to chronic metabolic and respiratory acidosis.

PubMed

Silva, Pedro Henrique Imenez; Girardi, Adriana Castello Costa; Neri, Elida Adalgisa; Rebouças, Nancy Amaral

2012-04-01

The Na(+/)H(+) exchanger isoform 3 (NHE3) is essential for HCO(3)(-) reabsorption in renal proximal tubules. The expression and function of NHE3 must adapt to acid-base conditions. The goal of this study was to elucidate the mechanisms responsible for higher proton secretion in proximal tubules during acidosis and to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and, if so, to identify the relevant parameters that may trigger these distinct adaptive responses. We achieved metabolic acidosis by lowering HCO(3)(-) concentration in the cell culture medium and respiratory acidosis by increasing CO(2) tension in the incubator chamber. We found that cell-surface NHE3 expression was increased in response to both forms of acidosis. Mild (pH 7.21 ± 0.02) and severe (6.95 ± 0.07) metabolic acidosis increased mRNA levels, at least in part due to up-regulation of transcription, whilst mild (7.11 ± 0.03) and severe (6.86 ± 0.01) respiratory acidosis did not up-regulate NHE3 expression. Analyses of the Nhe3 promoter region suggested that the regulatory elements sensitive to metabolic acidosis are located between -466 and -153 bp, where two consensus binding sites for SP1, a transcription factor up-regulated in metabolic acidosis, were localised. We conclude that metabolic acidosis induces Nhe3 promoter activation, which results in higher mRNA and total protein level. At the plasma membrane surface, NHE3 expression was increased in metabolic and respiratory acidosis alike, suggesting that low pH is responsible for NHE3 displacement to the cell surface.

Intramuscular nerve damage in lacerated skeletal muscles may direct the inflammatory cytokine response during recovery.

PubMed

Pereira, Barry P; Tan, Bee Leng; Han, Hwan Chour; Zou, Yu; Aung, Khin Zarchi; Leong, David T

2012-07-01

The expression of inflammatory cytokines and growth factors in surgically repaired lacerated muscles over a 12-week recovery phase was investigated. We hypothesized that these expression levels are influenced by both neural and muscular damage within lacerated muscles. Microarrays were confirmed with reverse transcription-polymerase chain reaction assays and histology of biopsies at the lesion of three simulated lacerated muscle models in 130 adult rats. The lacerated medial gastrocnemius with the main intramuscular nerve branch either cut (DN), crushed but leaving an intact nerve sheath (RN); or preserved intact (PN) were compared. At 4 weeks, DN had a higher number of interleukins up-regulated. DN and RN also had a set of Bmp genes significantly expressed between 2 and 8 weeks (P ≤ 0.05). By 12 weeks, DN had a poorer and slower myogenic recovery and greater fibrosis formation correlating with an up-regulation of the Tgf-β gene family. DN also showed poorer re-innervation with higher mRNA expression levels of nerve growth factor (Ngf) and brain-derived neurotrophin growth factor (Bdnf) over RN and PN. This study demonstrates that the inflammatory response over 12 weeks in lacerated muscles may be directed by the type of intramuscular nerve damage, which can influence the recovery at the lesion site. Inflammatory-related genes associated to the type of intramuscular nerve damage include Gas-6, Artemin, Fgf10, Gdf8, Cntf, Lif, and Igf-2. qPCR also found up-regulation of Bdnf (1-week), neurotrophin-3 (2w), Lif (4w), and Ngf (4w, 8w) mRNA expressions in DN, making them possible candidates for therapeutic treatment to arrest the poor recovery in muscle lacerations (250). Copyright © 2012 Wiley Periodicals, Inc.

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Zidong Donna; Klaassen, Curtis D., E-mail: cklaasse@kumc.edu

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors.more » In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs.« less

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis.

PubMed

Spangler, Jacob B; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

PubMed Central

Spangler, Jacob B.; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cunha, Elizabeth S.; Kawahara, Rebeca; Kadowaki, Marina K.

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cellmore » cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.« less

Opposite effects on regulation of urea synthesis by early and late uraemia in rats.

PubMed

Nielsen, Susanne Schouw; Grøfte, Thorbjørn; Grønbaek, Henning; Tygstrup, Niels; Vilstrup, Hendrik

2007-04-01

Acute and chronic kidney failure lead to catabolism with loss of lean body mass. Up-regulation of hepatic urea synthesis may play a role for the loss of body nitrogen and for the level of uraemia. The aims were to investigate the effects of early and late experimental renal failure on the regulation of hepatic urea synthesis and the expression of urea cycle enzyme genes in the liver. We examined the in vivo capacity of urea nitrogen synthesis, mRNA levels of urea cycle enzyme genes, and N-balances 6 days and 21 days after 5/6th partial nephrectomy in rats, and compared these data with pair- and free-fed control animals. Compared with pair-fed animals, early uraemia halved the in vivo urea synthesis capacity and decreased urea gene expressions (P<0.05). In contrast, late uraemia up-regulated in vivo urea synthesis and expression of all urea genes (P<0.05), save that of the flux-generating enzyme carbamoyl phosphate synthetase. The N-balance in rats with early uraemia was markedly negative (P<0.05) and near zero in late uraemia. Early uraemia down-regulated urea synthesis, so hepatic ureagenesis was not in itself involved in the negative N-balance. In contrast, late uraemia up-regulated urea synthesis, which probably contributed towards the reduced N-balance of this condition. These time-dependent, opposite effects on the uraemia-induced regulation of urea synthesis in vivo were not related to food restriction and probably mostly reflected regulation on gene level.

Fasting increases the phosphorylation of AMPK and expression of sirtuin1 in muscle of adult male northern elephant seals (Mirounga angustirostris).

PubMed

Lee, Debby; Martinez, Bridget; Crocker, Daniel E; Ortiz, Rudy M

2017-02-01

Fasting typically suppresses thyroid hormone (TH)-mediated cellular events and increases sirtuin 1 (SIRT1) activity. THs may regulate metabolism through nongenomic pathways and directly through activation of adenosine monophosphate-activated protein kinase (AMPK). Adult male elephant seals ( Mirounga angustirostris ) are active, hypermetabolic, and normothermic during their annual breeding fast, which is characterized by stable TH levels. However, the contribution of TH to maintenance of their fasting metabolism is unknown. To investigate the fasting effects on cellular TH-mediated events and its potential association with SIRT1 and AMPK, we quantified plasma TH levels, mRNA expressions of muscle SIRT1 and TH-associated genes as well as the phosphorylation of AMPK in adult, male northern elephant seals ( n  = 10/fasting period) over 8 weeks of fasting (early vs. late). Deiodinase type I (DI1) expression increased twofold with fasting duration suggesting that the potential for TH-mediated cellular signaling is increased. AMPK phosphorylation increased 61 ± 21% with fasting suggesting that cellular metabolism is increased. The mRNA expression of the TH transporter, monocarboxylate transporter 10 (MCT10), increased 2.4-fold and the TH receptor (THr β -1) decreased 30-fold suggesting that cellular uptake of T 4 is increased, but its subsequent cellular effects such as activation of AMPK are likely nongenomic. The up-regulation of SIRT1 mRNA expression (2.6-fold) likely contributes to the nongenomic activation of AMPK by TH, which may be necessary to maintain the expression of PGC-1 α These coordinated changes likely contribute to the up-regulation of mitochondrial metabolism to support the energetic demands associated with prolonged fasting in adult seals. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

High expression of GPER1, EGFR and CXCR1 is associated with lymph node metastasis in papillary thyroid carcinoma.

PubMed

Tang, Cui; Yang, Lei; Wang, Ni; Li, Li; Xu, Man; Chen, George G; Liu, Zhi-Min

2014-01-01

Clinical and epidemiological studies have shown that estrogen may be involved in the development and progression of papillary thyroid carcinoma (PTC). G protein-coupled estrogen receptor 1 (GPER1) is a novel seven-transmembrane estrogen receptor that functions alongside traditional nuclear estrogen receptors (ERs) to regulate the cellular responses to estrogen. The purpose of this study was to examine GPER1, EGFR and CXCR1 expression in PTC and to assess the association of their expression with clinicopathological indicators. GPER1, EGFR and CXCR1 protein expression in 129 PTCs, 61 nodular hyperplasia and 118 normal thyroid tissue specimens were analyzed using immunohistochemistry. The protein expression levels of these three molecules were up-regulated in PTCs. High protein expression of GPER1, EGFR and CXCR1 was significantly correlated with lymph node metastasis (LNM) (P ≤ 0.001). Furthermore, GPER1, EGFR and CXCR1 protein expression were correlated with one another. Concomitant high expression of these molecules had stronger correlation with LNM than did each alone (P = 0.002 for GPER1/EGFR, P = 0.013 for GPER1/CXCR1, P = 0.018 for EGFR/CXCR1 and P < 0.001 for GPER1/EGFR/CXCR1). Additionally, GPER1, EGFR and CXCR1 mRNA expression was assessed in 30 PTCs, 10 nodular hyperplasia and 10 normal thyroid tissue specimens using real-time RT-PCR. GPER1, EGFR and CXCR1 mRNA expression levels were up-regulated in PTCs, and high mRNA expression of GPER1, EGFR and CXCR1 was significantly correlated with LNM (P < 0.001 for all these three molecules). These results demonstrated that the evaluation of GPER1, EGFR and CXCR1 expression in PTC may be useful in predicting the risk of LNM.

Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels.

PubMed Central

Horton, J D; Cuthbert, J A; Spady, D K

1993-01-01

The concentration of LDL in plasma is strongly influenced by the amount and the type of lipid in the diet. Recent studies in the hamster have shown that dietary fatty acids differentially affect circulating LDL levels primarily by altering receptor-dependent LDL uptake in the liver. To investigate the mechanistic basis of this effect, rates of receptor-dependent LDL transport in the liver were correlated with LDL receptor protein and mRNA levels in hamsters fed safflower oil or coconut oil and varying amounts of cholesterol. Hepatic LDL receptor activity was significantly lower in animals fed coconut oil than in animals fed safflower oil at all levels of cholesterol intake (26, 53, and 61% lower at cholesterol intakes of 0, 0.06, and 0.12%, respectively). These fatty acid-induced changes in hepatic LDL receptor activity were accompanied by parallel changes in hepatic LDL receptor protein and mRNA levels, suggesting that dietary fatty acids regulate the LDL receptor pathway largely at the mRNA level. Images PMID:8349814

Substance P activates ADAM9 mRNA expression and induces α-secretase-mediated amyloid precursor protein cleavage.

PubMed

Marolda, R; Ciotti, M T; Matrone, C; Possenti, R; Calissano, P; Cavallaro, S; Severini, C

2012-04-01

Altered levels of Substance P (SP), a neuropeptide endowed with neuroprotective and anti-apoptotic properties, were found in brain areas and spinal fluid of Alzheimer's disease (AD) patients. One of the hallmarks of AD is the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). In the present study, we confirmed, the neurotrophic action of SP in cultured rat cerebellar granule cells (CGCs) and investigated its effects on APP metabolism. Incubation with low (5 mM) potassium induced apoptotic cell death of CGCs and amyloidogenic processing of APP, whereas treatment with SP (200 nM) reverted these effects via NK1 receptors. The non-amyloidogenic effect of SP consisted of reduction of Aβ(1-42), increase of sAPPα and enhanced α-secretase activity, without a significant change in steady-state levels of cellular APP. The intracellular mechanisms whereby SP alters APP metabolism were further investigated by measuring mRNA and/or steady-state protein levels of key enzymes involved with α-, β- and γ-secretase activity. Among them, Adam9, both at the mRNA and protein level, was the only enzyme to be significantly down-regulated following the induction of apoptosis (K5) and up-regulated after SP treatment. In addition to its neuroprotective properties, this study shows that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

Epigallocatechin-3-gallate (EGCG) up-regulates miR-15b expression thus attenuating store operated calcium entry (SOCE) into murine CD4+ T cells and human leukaemic T cell lymphoblasts.

PubMed

Zhang, Shaqiu; Al-Maghout, Tamer; Bissinger, Rosi; Zeng, Ni; Pelzl, Lisann; Salker, Madhuri S; Cheng, Anchun; Singh, Yogesh; Lang, Florian

2017-10-27

CD4 + T cells are key elements in immune responses and inflammation. Activation of T cell receptors in CD4 + T cells triggers cytosolic Ca 2+ release with subsequent store operated Ca 2+ entry (SOCE), which is accomplished by the pore forming Ca 2+ release activated Ca 2+ (CRAC) channel Orai1 and its regulator stromal cell-interaction molecule 2 (STIM2). Green tea polyphenol epigallocatechin-3-gallate (EGCG) acts as a potent anti-inflammatory and anti-oxidant agent for various types of cells including immune cells. However, how post-transcriptional gene regulators such as miRNAs are involved in the regulation of Ca 2+ influx into murine CD4 + T cells and human Jurkat T cells through EGCG is not defined. EGCG treatment of murine CD4 + T cells significantly down-regulated the expression of STIM2 and Orai1 both at mRNA and protein levels. Furthermore, EGCG significantly decreased SOCE in both murine and human T cells. EGCG treatment increased miRNA-15b (miR-15b) abundance in both murine and human T cells. Bioinformatics analysis reveals that miR-15b, which has a STIM2 binding site, is involved in the down-regulation of SOCE. Overexpression of miR-15b significantly decreased the mRNA and protein expression of STIM2 and Orai1 in murine T cells. Treatment of Jurkat T cells with 10 μM EGCG further decreased mTOR and PTEN protein levels. EGCG decreased mitochondrial membrane potential (MMP) in both human and murine T cells. In conclusion, the observations suggest that EGCG inhibits the Ca 2+ entry into murine and human T cells, an effect accomplished at least in part by up-regulation of miR-15b.

Changes in barrier health status of the gill for grass carp (Ctenopharyngodon idella) during valine deficiency: Regulation of tight junction protein transcript, antioxidant status and apoptosis-related gene expression.

PubMed

Feng, Lin; Luo, Jian-Bo; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

2015-08-01

This study investigated the effects of dietary valine on tight junction protein transcription, antioxidant status and apoptosis on grass carp gills (Ctenopharyngodon idella). Fish were fed six different experimental diets containing graded levels of valine (4.3, 8.0, 10.6, 13.1, 16.7, 19.1 g/kg). The results indicated that valine deficiency decreased Claudin b, Claudin 3, Occludin and ZO-1 transcription and increased Claudin 15 expression in the fish gill (P < 0.05). These effects were partly due to the down-regulation of interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and IκB α and the up-regulation of relative mRNA expression of interleukin 1β (IL-1β), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α) and nuclear factor κB P65 (NF-κB P65) (P < 0.05). However, valine deficiency and valine supplementation did not have a significant effect on Claudin c and Claudin 12 expression in grass carp gills (P > 0.05). Valine deficiency also disrupted antioxidant status in the gill by decreasing anti-superoxide radicals and hydroxyl radical capacity, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) (P < 0.05). These results may be ascribed to the down-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the up-regulation of Kelch-like-ECH-associated protein 1 (Keap1) (P < 0.05). Additionally, valine deficiency induced DNA fragmentation via the up-regulation of Caspase 3, Caspase 8 and Caspase 9 expressions (P < 0.05). These results may be ascribed to the improvement in ROS levels in the fish gill (P < 0.05). Taken together, the results showed that valine deficiency impaired the structural integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, antioxidant enzymes, NF-κB p65, IκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gill. Copyright © 2015 Elsevier Ltd. All rights reserved.

Leptin Regulation of Gonadotrope Gonadotropin-Releasing Hormone Receptors As a Metabolic Checkpoint and Gateway to Reproductive Competence

PubMed Central

Odle, Angela K.; Akhter, Noor; Syed, Mohsin M.; Allensworth-James, Melody L.; Beneš, Helen; Melgar Castillo, Andrea I.; MacNicol, Melanie C.; MacNicol, Angus M.; Childs, Gwen V.

2018-01-01

The adipokine leptin signals the body’s nutritional status to the brain, and particularly, the hypothalamus. However, leptin receptors (LEPRs) can be found all throughout the body and brain, including the pituitary. It is known that leptin is permissive for reproduction, and mice that cannot produce leptin (Lep/Lep) are infertile. Many studies have pinpointed leptin’s regulation of reproduction to the hypothalamus. However, LEPRs exist at all levels of the hypothalamic–pituitary–gonadal axis. We have previously shown that deleting the signaling portion of the LEPR specifically in gonadotropes impairs fertility in female mice. Our recent studies have targeted this regulation to the control of gonadotropin releasing hormone receptor (GnRHR) expression. The hypotheses presented here are twofold: (1) cyclic regulation of pituitary GnRHR levels sets up a target metabolic checkpoint for control of the reproductive axis and (2) multiple checkpoints are required for the metabolic signaling that regulates the reproductive axis. Here, we emphasize and explore the relationship between the hypothalamus and the pituitary with regard to the regulation of GnRHR. The original data we present strengthen these hypotheses and build on our previous studies. We show that we can cause infertility in 70% of female mice by deleting all isoforms of LEPR specifically in gonadotropes. Our findings implicate activin subunit (InhBa) mRNA as a potential leptin target in gonadotropes. We further show gonadotrope-specific upregulation of GnRHR protein (but not mRNA levels) following leptin stimulation. In order to try and understand this post-transcriptional regulation, we tested candidate miRNAs (identified with in silico analysis) that may be binding the Gnrhr mRNA. We show significant upregulation of one of these miRNAs in our gonadotrope-Lepr-null females. The evidence provided here, combined with our previous work, lay the foundation for metabolically regulated post-transcriptional control of the gonadotrope. We discuss possible mechanisms, including miRNA regulation and the involvement of the RNA binding protein, Musashi. We also demonstrate how this regulation may be vital for the dynamic remodeling of gonadotropes in the cycling female. Finally, we propose that the leptin receptivity of both the hypothalamus and the pituitary are vital for the body’s ability to delay or slow reproduction during periods of low nutrition. PMID:29354094

Locally Applied Leptin Induces Regional Aortic Wall Degeneration Preceding Aneurysm Formation in ApoE Deficient Mice

PubMed Central

Tao, Ming; Yu, Peng; Nguyen, Binh T.; Mizrahi, Boaz; Savion, Naphtali; Kolodgie, Frank D.; Virmani, Renu; Hao, Shuai; Ozaki, C. Keith; Schneiderman, Jacob

2013-01-01

Objective Leptin promotes atherosclerosis and vessel wall remodeling. As abdominal aorta aneurysm (AAA) formation involves tissue remodeling, we hypothesized that local leptin synthesis initiates and promotes this process. Methods and Results Human surgical AAA walls were analyzed for antigen and mRNA levels of leptin and leptin receptor (ObR), as well as mRNA for matrix metalloproteinases (MMP)-9, and MMP-12. Leptin and ObR antigen were evident in all AAAs, and, leptin, MMP-9, and MMP-12 mRNA was increased relative to age-matched non-dilated controls. To simulate in vivo local leptin synthesis, ApoE-/- mice were subjected to a para-visceral peri-aortic application of low-dose leptin. Leptin-treated aortas exhibited decreased TGFβ and increased MMP-9 mRNA levels 5 days after surgery, and ObR mRNA was up-regulated by day 28. Serial ultrasonography demonstrated accelerated regional aortic diameter growth after 28 days, correlating with local medial degeneration, increased MMP-9, MMP-12 and peri-adventitial macrophage clustering. Furthermore, the combination of local peri-aortic leptin and systemic angiotensin II administration augmented medial MMP-9 synthesis and aortic aneurysm size. Conclusions Leptin is locally synthesized in human AAA wall. Para-visceral aortic leptin in ApoE-/- mice induces local medial degeneration, and augments angiotensin II-induced AAA, thus suggesting novel mechanistic links between leptin and AAA formation. PMID:23220275

Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells.

PubMed Central

Riser, B. L.; Cortes, P.; Heilig, C.; Grondin, J.; Ladson-Wofford, S.; Patterson, D.; Narins, R. G.

1996-01-01

Glomerular distention from increased intraglomerular pressure stretches mesangial cells (MCs). Stretching MCs in culture stimulates extracellular matrix accumulation, suggesting that this may be a mechanism for glomerular hypertension-associated glomerulosclerosis. We examined whether mechanical stretching serves as a stimulus for the synthesis and activation of the prosclerotic molecule transforming growth factor (TGF)-beta, thus providing a potential system for auto-induction of extracellular matrix. Rat MCs cultured on flexible-bottom plates were subjected to cyclic stretching for up to 3 days and then assayed for TGF-beta mRNA, secretion of TGF-beta, and localization of active TGF-beta by immunostaining. MCs contained mRNA for all three mammalian isoforms of TGF-beta. Cyclic stretching for 36 hours increased TGF-beta1 and TGF-beta3 mRNA levels approximately twofold, without altering the levels of TGF-beta2 mRNA. This was followed at 48 to 72 hours by the increased secretion of both latent and active TGF-beta1. Latent, but not active, TGF-beta3 secretion also increased whereas the levels of TGF-beta2 were unaffected by mechanical force. The stretching force in this system is unequally distributed over the culture membrane. Localization of active TGF-beta by immunostaining demonstrated that the quantity of cell-associated cytokine across the culture was directly proportional to the zonal amplitude of the stretching force. These results demonstrate that stretching force stimulates MCs to selectively release and activate TGF-beta1. This mechanical induction of TGF-beta1 may help explain the increased extracellular matrix associated with intraglomerular hypertension. Images Figure 1 Figure 3 PMID:8669477

Mechanically induced c-fos expression is mediated by cAMP in MC3T3-E1 osteoblasts

NASA Technical Reports Server (NTRS)

Fitzgerald, J.; Hughes-Fulford, M.

1999-01-01

In serum-deprived MC3T3-E1 osteoblasts, mechanical stimulation caused by mild (287 x g) centrifugation induced a 10-fold increase in mRNA levels of the proto-oncogene, c-fos. Induction of c-fos was abolished by the cAMP-dependent protein kinase inhibitor H-89, suggesting that the transient c-fos mRNA increase is mediated by cAMP. Down-regulation of protein kinase C (PKC) activity by chronic TPA treatment failed to significantly reduce c-fos induction, suggesting that TPA-sensitive isoforms of PKC are not responsible for c-fos up-regulation. In addition, 287 x g centrifugation increased intracellular prostaglandin E2 (PGE2) levels 2.8-fold (P<0. 005). Since we have previously shown that prostaglandin E2 (PGE2) can induce c-fos expression via a cAMP-mediated mechanism, we asked whether the increase in c-fos mRNA was due to centrifugation-induced PGE2 release. Pretreatment with the cyclooxygenase inhibitors indomethacin and flurbiprofen did not hinder the early induction of c-fos by mechanical stimulation. We conclude that c-fos expression induced by mild mechanical loading is dependent primarily on cAMP, not PKC, and initial induction of c-fos is not necessarily dependent on the action of newly synthesized PGE2.

[Expression of cation-chloride cotransporters KCC2 and NKCC1 in brainstem of para- chlorophenylalanine-induced acute insomnia rats].

PubMed

Lin, Fang-ju; Yang, Xiao-su; Yang, De; Zou, Yan-qun

2013-05-21

To explore the possible roles of KCC2 and NKCC1 in the pathological mechanism of acute insomnia in rats. A total of 18 Sprague-Dawley rats were randomly selected into model, interference and normal control groups.The expressions of KCC2 and NKCC1 in brainstem were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.The concentration of intracellular Cl(-) ([Cl(-)]i) in brainstem was detected by fluorescence probe MQAE with laser confocal microscopy. (1) Comparing with the control group, both KCC2 mRNA and protein expression were down-regulated in the model and interference groups (mRNA:0.196 ± 0.021 vs 0.939 ± 0.109, P < 0.05; 0.485 ± 0.026 vs 0.939 ± 0.109, P < 0.05; protein expression:0.363 ± 0.058 vs 0.967 ± 0.155, P < 0.05; 0.663 ± 0.106 vs 0.967 ± 0.155, P < 0.05).However they became up-regulated in the interference group versus the model group (mRNA: 0.485 ± 0.026 vs 0.196 ± 0.021, P < 0.05; protein expression:0.663 ± 0.106 vs 0.363 ± 0.058, P < 0.05). (2) Comparing with the control group, both NKCC1 mRNA and protein expression in the model group were slightly up-regulated.But statistical difference was insignificant (mRNA: 0.344 ± 0.026 vs 0.320 ± 0.019, P > 0.05; protein expression:0.244 ± 0.010 vs 0.230 ± 0.021, P > 0.05).There was down-regulation in the interference group versus the model and control groups (mRNA: 0.066 ± 0.031 vs 0.320 ± 0.019, P < 0.05; 0.066 ± 0.031 vs 0.344 ± 0.026, P < 0.05; protein expression:0.131 ± 0.012 vs 0.230 ± 0.021, P < 0.05; 0.131 ± 0.012 vs 0.244 ± 0.010, P < 0.05). (3) Comparing with the control group, [Cl(-)]i became up-regulated in the model group (0.0315 ± 0.0039 vs 0.0164 ± 0.0019, P < 0.05).It was down-regulated in the interference group versus the model group (0.0182 ± 0.0013 vs 0.0315 ± 0.0039, P < 0.05), but higher than control group without statistical difference (0.0182 ± 0.0013 vs 0.0164 ± 0.0019, P > 0.05). The down-regulation of KCC2 and rise of [Cl(-)]i in brainstem may participate in the pathological mechanism of acute insomnia in rats. And the mechanism of sedative-hypnotic diazepam may be operate through an up-regulation of KCC2, a down-regulation of NKCC1 and decreased [Cl(-)]i.

Ethylene-Regulated Floral Volatile Synthesis in Petunia Corollas1[w

PubMed Central

Underwood, Beverly A.; Tieman, Denise M.; Shibuya, Kenichi; Dexter, Richard J.; Loucas, Holly M.; Simkin, Andrew J.; Sims, Charles A.; Schmelz, Eric A.; Klee, Harry J.; Clark, David G.

2005-01-01

In many flowering plants, such as petunia (Petunia × hybrida), ethylene produced in floral organs after pollination elicits a series of physiological and biochemical events, ultimately leading to senescence of petals and successful fertilization. Here, we demonstrate, using transgenic ethylene insensitive (44568) and Mitchell Diploid petunias, that multiple components of emission of volatile organic compounds (VOCs) are regulated by ethylene. Expression of benzoic acid/salicylic acid carboxyl methyltransferase (PhBSMT1 and 2) mRNA is temporally and spatially down-regulated in floral organs in a manner consistent with current models for postpollination ethylene synthesis in petunia corollas. Emission of methylbenzoate and other VOCs after pollination and exogenous ethylene treatment parallels a reduction in PhBSMT1 and 2 mRNA levels. Under cyclic light conditions (day/night), PhBSMT mRNA levels are rhythmic and precede emission of methylbenzoate by approximately 6 h. When shifted into constant dark or light conditions, PhBSMT mRNA levels and subsequent methylbenzoate emission correspondingly decrease or increase to minimum or maximum levels observed during normal conditions, thus suggesting that light may be a more critical influence on cyclic emission of methylbenzoate than a circadian clock. Transgenic PhBSMT RNAi flowers with reduced PhBSMT mRNA levels show a 75% to 99% decrease in methylbenzoate emission, with minimal changes in other petunia VOCs. These results implicate PhBSMT1 and 2 as genes responsible for synthesis of methylbenzoate in petunia. PMID:15849311

Lower glutamic acid decarboxylase 65kD mRNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia

PubMed Central

Glausier, JR; Kimoto, S; Fish, KN; Lewis, DA

2014-01-01

Background Altered GABA signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in schizophrenia and schizoaffective disorder. PFC levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67kD (GAD67) has been consistently reported to be lower in these disorders, but the status of the second GABA-synthesizing enzyme, GAD65, remains unclear. Methods GAD65 mRNA levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. GAD65 relative protein levels were quantified in a subset of subject pairs by confocal immunofluorescence microscopy. Results Mean GAD65 mRNA levels were 13.6% lower in schizoaffective disorder subjects, but did not differ in schizophrenia subjects, relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein measures within schizoaffective disorder subjects was not attributable to factors commonly comorbid with the diagnosis. Conclusions In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in subjects with schizoaffective disorder relative to subjects with schizophrenia, these findings may support an interpretation that GAD65 down-regulation provides a homeostatic response complementary to GAD67 down-regulation expression that serves to reduce inhibition in the face of lower PFC network activity. PMID:24993056

Identification and expression analysis of the sting gene, a sensor of viral DNA, in common carp Cyprinus carpio.

PubMed

Cao, X L; Chen, J J; Cao, Y; Nie, G X; Su, J G

2016-05-01

Stimulator of interferon gene (sting) was identified and characterized from common carp Cyprinus carpio. The sting messenger (m)RNA encoded a polypeptide of 402 amino acids with a calculated molecular mass of 46·184 kDa and an isoelectronic point of 6·08. The deduced protein of sting contained a signal peptide, three transmembrane motifs in the N-terminal region and four putative motifs (RXR) found in resident endoplasmic reticulum proteins. mRNA expression of sting was present in twelve investigated tissues, and was up-regulated by koi herpesvirus (KHV) in vivo and in vitro. The transcription of sting was altered by poly(I:C) and poly(dT:dA) stimulation in vitro. The findings suggested that sting is an inducible gene involved in innate immunity against DNA- and RNA-derived pathogens. To investigate defence mechanisms in C. carpio development, sting level in embryos, larvae and juvenile fish was monitored following KHV challenge. The sting message was negligible in embryos prior to hatching, but observed at higher transcriptional levels throughout larval and juvenile stages. Investigation showed the mRNA expression profiles of genes encoding for proteins promoting various functions in the interferon pathway, from pattern recognition receptors to antiviral genes, to be significantly induced in all examined organs by in vivo infection with KHV. Following KHV infection, the ifn message was significantly downregulated in spleen, head kidney, brain and hepatopancreas but notably up-regulated in gill, intestine and skin, suggesting that ifn induction might be related to the mucosal immune system and virus anti-ifn mechanisms. These results provided the basis for further research into the role and mechanisms of sting in fishes. © 2016 The Fisheries Society of the British Isles.

Development of fetal and placental innate immune responses during establishment of persistent infection with bovine viral diarrhea virus.

PubMed

Smirnova, Natalia P; Webb, Brett T; Bielefeldt-Ohmann, Helle; Van Campen, Hana; Antoniazzi, Alfredo Q; Morarie, Susan E; Hansen, Thomas R

2012-08-01

Transplacental viral infections are dependent upon complex interactions between feto-placental and maternal immune responses and the stage of fetal development at which the infection occurs. Bovine viral diarrhea virus (BVDV) has the ability to cross the placenta and infect the fetus. Infection early in gestation with non-cytopathic (ncp) BVDV leads to persistent infection. Establishment of fetal persistent infection results in life-long viremia, virus-specific immunotolerance, and may have detrimental developmental consequences. We have previously shown that heifers infected experimentally with ncp BVDV type 2 on d. 75 of gestation had transient robust up-regulation of the type I interferon (IFN) stimulated genes (ISGs) 3-15 days after viral inoculation. Blood from persistently infected (PI) fetuses, collected 115 days post maternal infection, demonstrated moderate chronic up-regulation of ISGs. This infection model was used to delineate timing of the development of innate immune responses in the fetus and placenta during establishment of persistent infection. It was hypothesized that: (i) chronic stimulation of innate immune responses occurs following infection of the fetus and (ii) placental production of the type I IFN contributes to up-regulation of ISGs in PI fetuses. PI fetuses, generated by intranasal inoculation of pregnant heifers with ncp BVDV, and control fetuses from uninfected heifers, were collected via Cesarean sections on d. 82, 89, 97, 192, and 245 of gestation. Fetal viremia was confirmed starting on d. 89. Significant up-regulation of mRNA encoding cytosolic dsRNA sensors -RIG-I and MDA5 - was detected on d. 82-192. Detection of viral dsRNA by cytosolic sensors leads to the stimulation of ISGs, which was reflected in significant up-regulation of ISG15 mRNA in fetal blood on d. 89, 97, and 192. No difference in IFN-α and IFN-β mRNA concentration was found in fetal blood or caruncular tissue, while a significant increase in both IFN-α and IFN-β mRNA was seen in cotyledons from PI fetuses on d. 192. It is concluded that fetuses respond to early gestational ncp BVDV infection by induction of the type I IFN pathway, resulting in chronic up-regulation of ISGs. Cotyledonary tissue contributes to up-regulation of ISGs by increased production of IFNs. The innate immune response might partially curtail viral replication in PI fetuses, but is not able to eliminate the virus in the absence of a virus-specific adaptive immune response. Copyright © 2012 Elsevier B.V. All rights reserved.

Effects of added fermentable carbohydrates in the diet on intestinal proinflammatory cytokine-specific mRNA content in weaning piglets.

PubMed

Pié, S; Awati, A; Vida, S; Falluel, I; Williams, B A; Oswald, I P

2007-03-01

There is increasing evidence showing that dietary supplementation with prebiotics can be effective in the treatment of intestinal inflammation. Because weaning time is characterized by rapid intestinal inflammation, this study investigated the effect of a diet supplemented with a combination of 4 fermentable carbohydrates (lactulose, inulin, sugarbeet pulp, and wheat starch) on the mRNA content of proinflammatory cytokines in newly weaned piglets. Cytokines (IL-1beta, IL-6, IL-8, IL-12p40, IL-18, and tumor necrosis factor-alpha) were analyzed using a semiquantitative reverse-transcription PCR technique on d 1, 4, and 10 in the ileum and colon of piglets fed either a test diet (CHO) or a control diet. In addition to the diet, the effect of enforced fasting on cytokine mRNA content was also evaluated. No effect of fasting was observed on the pro-inflammatory cytokine mRNA content. Our results showed that the CHO diet induced an up-regulation of IL-6 mRNA content in the colon of piglets 4 d postweaning. This up-regulation was specific for the animals fed the CHO diet and was not observed in animals fed the control diet. An increase in IL-1beta mRNA content was also observed on d 4 postweaning in all of the piglets. Correlations between proinflammatory cytokines and the end-products of fermentation indicated that the regulation of cytokines may be linked with some of the fermentation end-products such as branched-chain fatty acids, which are in turn end-products of protein fermentation.

Enrofloxacin and Probiotic Lactobacilli Influence PepT1 and LEAP-2 mRNA Expression in Poultry.

PubMed

Pavlova, Ivelina; Milanova, Aneliya; Danova, Svetla; Fink-Gremmels, Johanna

2016-12-01

Expression of peptide transporter 1 (PepT1) and liver-expressed antimicrobial peptide 2 (LEAP-2) in chickens can be influenced by food deprivation, pathological conditions and drug administration. Effect of three putative probiotic Lactobacillus strains and enrofloxacin on the expression of PepT1 and LEAP-2 mRNA was investigated in Ross 308 chickens. One-day-old chicks (n = 24) were allocated to following groups: control (without treatment); group treated with probiotics via feed; group treated with a combination of probiotics and enrofloxacin; and a group given enrofloxacin only. The drug was administered at a dose of 10 mg kg -1 , via drinking water for 5 days. Samples from liver, duodenum and jejunum were collected 126 h after the start of the treatment. Expression levels of PepT1 and LEAP-2 were determined by real-time polymerase chain reaction and were statistically evaluated by Mann-Whitney test. Enrofloxacin administered alone or in combination with probiotics provoked a statistically significant up-regulation of PepT1 mRNA levels in the measured organ sites. These changes can be attributed to a tendency of improvement in utilization of dietary peptide and in body weight gain. LEAP-2 mRNA expression levels did not change significantly in enrofloxacin-treated chickens in comparison with control group.

Rac1 GTPase regulates 11β hydroxysteroid dehydrogenase type 2 and fibrotic remodeling.

PubMed

Lavall, Daniel; Schuster, Pia; Jacobs, Nadine; Kazakov, Andrey; Böhm, Michael; Laufs, Ulrich

2017-05-05

The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression ( r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression ( r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Group I mGluR-Regulated Translation of the Neuronal Glutamate Transporter, Excitatory Amino Acid Carrier 1 (EAAC1)

PubMed Central

Ross, John R.; Ramakrishnan, Hariharasubramanian; Porter, Brenda E.; Robinson, Michael B.

2011-01-01

Recently, we demonstrated that mRNA for the neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), is found in dendrites of hippocampal neurons in culture and in dendrites of hippocampal pyramidal cells after pilocarpine-induced status epilepticus (SE). We also showed that SE increased the levels of EAAC1 mRNA ~15-fold in synaptoneurosomes. In the present study, the effects of SE on the distribution EAAC1 protein in hippocampus were examined. In addition, the effects of Group 1 mGluR receptor activation on the levels of EAAC1 protein were examined in synaptoneurosomes prepared from sham control animals and from animals that experience pilocarpine-induced SE. We find that EAAC1 immunoreactivity increases in pyramidal cells of the hippocampus after 3 h of SE. In addition, the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), caused an increase in EAAC1 protein levels in hippocampal synaptoneurosomes; this effect of DHPG was much larger (~3- to 5-fold) after 3 h of SE. The DHPG-induced increases in EAAC1 protein were blocked by two different inhibitors of translation but not by inhibitors of transcription. mGluR1 or mGluR5 antagonists completely blocked the DHPG-induced increases in EAAC1 protein. DHPG also increased the levels of GluR2/3 protein, but this effect was not altered by SE. The DHPG-induced increase in EAAC1 protein was blocked by an inhibitor of the mammalian target of rapamycin (mTOR) or an inhibitor of extracellular signal-regulated kinase (ERK). These studies provide the first evidence EAAC1 translation can be regulated, and they show that regulated translation of EAAC1 is up-regulated after SE. PMID:21371038

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture

PubMed Central

Meißner, Joachim D; Gros, Gerolf; Scheibe, Renate J; Scholz, Michael; Kubis, Hans-Peter

2001-01-01

The addition of cyclosporin A (500 ng ml−1) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 × 10−7m) was added to the medium, a partial fast-to-slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation. PMID:11351029

ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders

PubMed Central

Nam, Hyeyoung; Wang, Chia-Yu; Zhang, Lin; Zhang, Wei; Hojyo, Shintaro; Fukada, Toshiyuki; Knutson, Mitchell D.

2013-01-01

The liver, pancreas, and heart are particularly susceptible to iron-related disorders. These tissues take up plasma iron from transferrin or non-transferrin-bound iron, which appears during iron overload. Here, we assessed the effect of iron status on the levels of the transmembrane transporters, ZRT/IRT-like protein 14 and divalent metal-ion transporter-1, which have both been implicated in transferrin- and non-transferrin-bound iron uptake. Weanling male rats (n=6/group) were fed an iron-deficient, iron-adequate, or iron-overloaded diet for 3 weeks. ZRT/IRT-like protein 14, divalent metal-ion transporter-1 protein and mRNA levels in liver, pancreas, and heart were determined by using immunoblotting and quantitative reverse transcriptase polymerase chain reaction analysis. Confocal immunofluorescence microscopy was used to localize ZRT/IRT-like protein 14 in the liver and pancreas. ZRT/IRT-like protein 14 and divalent metal-ion transporter-1 protein levels were also determined in hypotransferrinemic mice with genetic iron overload. Hepatic ZRT/IRT-like protein 14 levels were found to be 100% higher in iron-loaded rats than in iron-adequate controls. By contrast, hepatic divalent metal-ion transporter-1 protein levels were 70% lower in iron-overloaded animals and nearly 3-fold higher in iron-deficient ones. In the pancreas, ZRT/IRT-like protein 14 levels were 50% higher in iron-overloaded rats, and in the heart, divalent metal-ion transporter-1 protein levels were 4-fold higher in iron-deficient animals. At the mRNA level, ZRT/IRT-like protein 14 expression did not vary with iron status, whereas divalent metal-ion transporter-1 expression was found to be elevated in iron-deficient livers. Immunofluorescence staining localized ZRT/IRT-like protein 14 to the basolateral membrane of hepatocytes and to acinar cells of the pancreas. Hepatic ZRT/IRT-like protein 14, but not divalent metal-ion transporter-1, protein levels were elevated in iron-loaded hypotransferrinemic mice. In conclusion, ZRT/IRT-like protein 14 protein levels are up-regulated in iron-loaded rat liver and pancreas and in hypotransferrinemic mouse liver. Divalent metal-ion transporter-1 protein levels are down-regulated in iron-loaded rat liver, and up-regulated in iron-deficient liver and heart. Our results provide insight into the potential contributions of these transporters to tissue iron uptake during iron deficiency and overload. PMID:23349308

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nicolas, Francisco E.; Vila, Ana; Moxon, Simon

Here, RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which theymore » derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway. In conclusion, this work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.« less

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

DOE PAGES

Nicolas, Francisco E.; Vila, Ana; Moxon, Simon; ...

2015-03-25

Here, RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which theymore » derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway. In conclusion, this work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.« less

Growth-inducing effects of argon plasma on soybean sprouts via the regulation of demethylation levels of energy metabolism-related genes.

PubMed

Zhang, Jiao Jiao; Jo, Jin Oh; Huynh, Do Luong; Mongre, Raj Kumar; Ghosh, Mrinmoy; Singh, Amit Kumar; Lee, Sang Baek; Mok, Young Sun; Hyuk, Park; Jeong, Dong Kee

2017-02-07

This study was conducted to determine the effects of argon plasma on the growth of soybean [Glycine max (L.) Merr.] sprouts and investigate the regulation mechanism of energy metabolism. The germination and growth characteristics were modified by argon plasma at different potentials and exposure durations. Upon investigation, plasma treatment at 22.1 kV for 12 s maximized the germination and seedling growth of soybean, increasing the concentrations of soluble protein, antioxidant enzymes, and adenosine triphosphate (ATP) as well as up-regulating ATP a1, ATP a2, ATP b1, ATP b2, ATP b3, target of rapamycin (TOR), growth-regulating factor (GRF) 1-6, down-regulating ATP MI25 mRNA expression, and increasing the demethylation levels of the sequenced region of ATP a1, ATP b1, TOR, GRF 5, and GRF 6 of 6-day-old soybean sprouts. These observations indicate that argon plasma promotes soybean seed germination and sprout growth by regulating the demethylation levels of ATP, TOR, and GRF.

Growth-inducing effects of argon plasma on soybean sprouts via the regulation of demethylation levels of energy metabolism-related genes

NASA Astrophysics Data System (ADS)

Zhang, Jiao Jiao; Jo, Jin Oh; Huynh, Do Luong; Mongre, Raj Kumar; Ghosh, Mrinmoy; Singh, Amit Kumar; Lee, Sang Baek; Mok, Young Sun; Hyuk, Park; Jeong, Dong Kee

2017-02-01

This study was conducted to determine the effects of argon plasma on the growth of soybean [Glycine max (L.) Merr.] sprouts and investigate the regulation mechanism of energy metabolism. The germination and growth characteristics were modified by argon plasma at different potentials and exposure durations. Upon investigation, plasma treatment at 22.1 kV for 12 s maximized the germination and seedling growth of soybean, increasing the concentrations of soluble protein, antioxidant enzymes, and adenosine triphosphate (ATP) as well as up-regulating ATP a1, ATP a2, ATP b1, ATP b2, ATP b3, target of rapamycin (TOR), growth-regulating factor (GRF) 1-6, down-regulating ATP MI25 mRNA expression, and increasing the demethylation levels of the sequenced region of ATP a1, ATP b1, TOR, GRF 5, and GRF 6 of 6-day-old soybean sprouts. These observations indicate that argon plasma promotes soybean seed germination and sprout growth by regulating the demethylation levels of ATP, TOR, and GRF.

Autocrine pathways involving S100A8 and/or S100A9 that are postulated to regulate the immunological functions of macrophages in rats.

PubMed

Okada, Kohki; Arai, Satoshi; Nakase, Hiroshi; Kohno, Hisashi; Nakamura, Fumihiko; Takeda, Mayu; Toda, Yoshinobu; Itoh, Hiroshi; Adachi, Souichi; Ikemoto, Masaki

2015-01-02

The development of ulcerative colitis (UC) is closely associated with abnormally functioning macrophages. Rat S100A8 (r-S100A8) and r-S100A9 (S100 proteins) is abundantly expressed in immune cells of myeloid origin, macrophages; however, it remains unclear why r-S100A9 is dominantly expressed in the macrophages of UC rats (UCR). The purpose of this study was to verify the immunological roles of S100 proteins in UCR. We observed the distribution of S100 protein-positive macrophages in the large colons of UCR using a fluorescent immunological staining method, so that S100 protein-positive macrophages were restricted to the rectal tissues of the UCR, and that the mRNA levels of r-S100A8 and r-S100A9 were up-regulated by stimulation with recombinant rat S100A8 (rr-S100A8) alone and rr-S100A9 alone, respectively. When the changes in the mRNA levels of r-S100A8 and r-S100A9 in macrophages were examined in in vitro study by PCR and real-time PCR, the mRNA levels of anti-inflammatory and inflammatory cytokines increased selectively after stimulation with rr-S100A8 alone and rr-S100A9 alone, respectively. These results suggest that autocrine signal transduction pathways involving S100 proteins regulate the immunological functions of macrophages to maintain homeostasis in the gastrointestinal tract. This may be depended on expression balance of S100 proteins in macrophages. It is strongly suggested that in UCR the immune functions of macrophages are regulated in a complex manner by r-S100A8 and/or r-S100A9 through undefined autocrine pathways on the cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Up-regulation of peroxidase proliferator-activated receptor gamma in cholesteatoma.

PubMed

Hwang, Soon Jae; Kang, Hee Joon; Song, Jae-Jun; Kang, Jae Seong; Woo, Jeong Soo; Chae, Sung Won; Lee, Heung-Man

2006-01-01

To evaluate the localization and expression of peroxidase proliferator-activated receptor (PPAR)gamma in cholesteatoma epithelium. Experimental study. Reverse-transcription polymerase chain reaction was performed on cholesteatoma tissues from 10 adult patients undergoing tympanomastoid surgery for middle ear cholesteatoma and on 10 samples of normal external auditory canal skin tissue. The expression levels of PPARgamma to glyceraldehyde-3-phosphate dehydrogenase transcripts were semiquantified by densitometry. We also characterized the cellular localization of the PPARgamma protein immunohistochemically. Ki-67 was also localized to compare the proliferative activity of cells in cholesteatoma epithelium and in normal external auditory canal skin. PPARgamma mRNA and protein were detected in normal external auditory canal skin and in cholesteatoma epithelium. The expression level of PPARgamma mRNA in cholesteatoma was significantly increased compared with that in normal external auditory canal skin. PPARgamma protein was expressed in cells mainly in the granular and prickle cell layers. However, the intensity of its expression was generally decreased in the parabasal layer of the cholesteatoma epithelium. Ki-67 was expressed in the nuclei of cells in the basal and parabasal layers, and a greater number of cells were Ki-67 immunopositive in cholesteatoma epithelium. PPARgamma is up-regulated in the cholesteatoma epithelium compared with normal external auditory canal skin. These results suggest that PPARgamma may play an important role in the pathogenesis of cholesteatoma.

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

PubMed

Joo, Jin Woo; Kim, Hyun-Soo; Do, Sung-Im; Sung, Ji-Youn

2018-05-01

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trempe, J.P.; Carter, B.J.

1988-01-01

The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/submore » 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.« less

Dietary capsanthin, the main carotenoid in paprika (Capsicum annuum), alters plasma high-density lipoprotein-cholesterol levels and hepatic gene expression in rats.

PubMed

Aizawa, Koichi; Inakuma, Takahiro

2009-12-01

The effects of dietary capsanthin, the main carotenoid in paprika (Capsicum annuum), on lipid metabolism were examined. Young male Wistar rats were fed diets containing paprika powder, paprika organic solvent extract, residue of paprika extract, and purified capsanthin. Administration of purified capsanthin for 2 weeks resulted in a significant increase in plasma HDL-cholesterol (P < 0.05) without detectable differences in plasma total cholesterol and TAG concentrations. A statistically significant correlation (r 0.567; P < 0.001) was found between dietary capsanthin concentrations and plasma HDL-cholesterol concentrations. Animals receiving diets containing two different capsanthin concentrations exhibited dose-dependent increases in plasma HDL-cholesterol (r 0.597; P < 0.005). While capsanthin was absent in the liver of animals fed the basal diet, it increased markedly in capsanthin-fed animals (P < 0.001). Quantitative analyses of hepatic mRNA levels revealed that capsanthin administration resulted in up-regulation of mRNA for apoA5 and lecithin cholesterol acyltransferase (LCAT), without significant differences in other mRNA levels related to HDL-cholesterol metabolism. These results suggest that capsanthin had an HDL-cholesterol-raising effect on plasma, and the potential to increase cholesterol efflux to HDL particles by increasing apoA5 levels and/or enhancement of LCAT activity.

Acute exposure to waterborne cadmium induced oxidative stress and immunotoxicity in the brain, ovary and liver of zebrafish (Danio rerio).

PubMed

Zheng, Jia-Lang; Yuan, Shuang-Shuang; Wu, Chang-Wen; Lv, Zhen-Ming

2016-11-01

Cadmium (Cd) is an environmental contaminant that poses serious risks to aquatic organisms and their associated ecosystem. The mechanisms underlying Cd-induced oxidative stress and immunotoxicity in fish remain largely unknown. In this study, adult female zebrafish were exposed to 0 (control), 1mgL -1 Cd for 24h and 96h, and the oxidative stress and inflammatory responses induced by Cd were evaluated in the brain, liver and ovary. Reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) increased in a time-dependent manner after treatment with Cd in the brain and liver. The increase may result from the disturbance of genes including copper and zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), inducible nitric oxide synthase (iNOS), and ciclooxigenase-2 (COX-2) at mRNA, protein and activity levels. Although ROS, NO and MDA were not significantly affected by Cd in the ovary, the up-regulation of Cu/Zn-SOD, CAT, iNOS, and COX-2 was observed. Exposure to Cd induced a sharp increase in the protein levels of tumor necrosis factor alpha (TNF-α) in the brain, liver and ovary, possibly contributing to activate inflammatory responses. Furthermore, we also found a dramatic increase in mRNA levels of NF-E2-related factor 2 (Nrf2) and nuclear transcription factor κB (NF-κB) at 24h in the liver and ovary. The corresponding changes in the mRNA levels of Kelch-like-ECH-associated protein 1 (Keap1a and Keap1b) and the inhibitor of κBα (IκBαa and IκBαb) may contribute to regulate the transcriptional activity of Nrf2 and NF-κB, respectively. Contrarily, mRNA levels of Nrf2, NF-κB, Keap1, Keap1b, IκBαa and IκBαb remained stable at 24 and 96h in the brain. Taken together, we demonstrated Cd-induced oxidative stress and immunotoxicity in fish, possibly through transcriptional regulation of Nrf2 and NF-κB and gene modifications at transcriptional, translational, post-translational levels, which would greatly extend our understanding on the Cd toxicity. Copyright © 2016. Published by Elsevier B.V.

New operant model of reinstatement of food-seeking behavior in mice.

PubMed

Martín-García, Elena; Burokas, Aurelijus; Kostrzewa, Elzbieta; Gieryk, Agnieszka; Korostynski, Michal; Ziolkowska, Barbara; Przewlocka, Barbara; Przewlocki, Ryszard; Maldonado, Rafael

2011-05-01

A major problem in treating obesity is the high rate of relapse to abnormal food-taking behavior when maintaining diet. The present study evaluates the reinstatement of extinguished palatable food-seeking behavior induced by cues previously associated with the palatable food, re-exposure to this food, or stress. The participation of the opioid and dopamine mechanisms in the acquisition, extinction, and cue-induced reinstatement was also investigated. C57BL/6 mice were first trained on a fixed-ratio-1 schedule of reinforcement to obtain chocolate-flavored pellets during 20 days, which was associated to a stimulus light. Operant behavior was then extinguished during 20 daily sessions. mRNA levels of opioid peptide precursors and dopamine receptors were evaluated in the brain by in situ hybridization and RT-PCR techniques. A reinstatement of food-seeking behavior was only obtained after exposure to the food-associated cue. A down-regulation of prodynorphin mRNA was found in the dorsal striatum and nucleus accumbens after the acquisition, extinction, and reinstatement of the operant behavior. Extinction and reinstatement of this operant response enhanced proenkephalin mRNA in the dorsal striatum and/or the nucleus accumbens core. Down-regulation of D2 receptor expression was observed in the dorsal striatum and nucleus accumbens after reinstatement. An up-regulation of PDYN mRNA expression was found in the hypothalamus after extinction and reinstatement. This study provides a new operant model in mice for the evaluation of food-taking behavior and reveals specific changes in the dopamine and opioid system associated to the behavioral responses directed to obtain a natural reward.

Molecular mechanisms underlying the effects of acupuncture on neuropathic pain

PubMed Central

Ju, Ziyong; Cui, Huashun; Guo, Xiaohui; Yang, Huayuan; He, Jinsen; Wang, Ke

2013-01-01

Acupuncture has been used to treat neuropathic pain for a long time, but its mechanisms of action remain unknown. In this study, we observed the effects of electroacupuncture and manual acu-puncture on neuropathic pain and on ephrin-B/EphB signaling in rats models of chronic constriction injury-induced neuropathic pain. The results showed that manual acupuncture and elec-puncture significantly reduced mechanical hypersensitivity following chronic constriction injury, es-pecially electroacupuncture treatment. Real-time PCR results revealed that ephrin-B1/B3 and EphB1/B2 mRNA expression levels were significantly increased in the spinal dorsal horns of chronic constriction injury rats. Electroacupuncture and manual acupuncture suppressed the high sion of ephrin-B1 mRNA, and elevated EphB3/B4 mRNA expression. Electroacupuncture signifi-cantly enhanced the mRNA expression of ephrin-B3 and EphB3/B6 in the dorsal horns of neuro-pathic pain rats. Western blot results revealed that electroacupuncture in particular, and manual acupuncture, significantly up-regulated ephrin-B3 protein levels in rat spinal dorsal horns. The re-sults of this study suggest that acupuncture could activate ephrin-B/EphB signaling in neuropathic pain rats and improve neurological function. PMID:25206545

Molecular mechanisms underlying the effects of acupuncture on neuropathic pain.

PubMed

Ju, Ziyong; Cui, Huashun; Guo, Xiaohui; Yang, Huayuan; He, Jinsen; Wang, Ke

2013-09-05

Acupuncture has been used to treat neuropathic pain for a long time, but its mechanisms of action remain unknown. In this study, we observed the effects of electroacupuncture and manual acu-puncture on neuropathic pain and on ephrin-B/EphB signaling in rats models of chronic constriction injury-induced neuropathic pain. The results showed that manual acupuncture and elec-puncture significantly reduced mechanical hypersensitivity following chronic constriction injury, es-pecially electroacupuncture treatment. Real-time PCR results revealed that ephrin-B1/B3 and EphB1/B2 mRNA expression levels were significantly increased in the spinal dorsal horns of chronic constriction injury rats. Electroacupuncture and manual acupuncture suppressed the high sion of ephrin-B1 mRNA, and elevated EphB3/B4 mRNA expression. Electroacupuncture signifi-cantly enhanced the mRNA expression of ephrin-B3 and EphB3/B6 in the dorsal horns of neuro-pathic pain rats. Western blot results revealed that electroacupuncture in particular, and manual acupuncture, significantly up-regulated ephrin-B3 protein levels in rat spinal dorsal horns. The re-sults of this study suggest that acupuncture could activate ephrin-B/EphB signaling in neuropathic pain rats and improve neurological function.

Expression of the cytokeratin endo A gene during early mouse embryogenesis.

PubMed Central

Duprey, P; Morello, D; Vasseur, M; Babinet, C; Condamine, H; Brûlet, P; Jacob, F

1985-01-01

Expression of cytokeratin endo A has been analyzed during mouse blastocyst formation and embryonal carcinoma cell differentiation. To study the regulation of endo A expression, nuclease S1 mapping experiments have been performed on RNA extracted from two-cell to 7.5-day embryos. Low levels of endo A mRNA begin to be detectable in eight-cell embryos. The amount of this mRNA increases at the blastocyst stage, suggesting that endo A expression is regulated at the mRNA level during blastocyst formation. At this stage, in situ hybridization studies show that endo A mRNA is present in the trophectoderm but not in the inner cell mass. In 7.5-day embryos, endo A mRNAs are also detectable in the endoderm layer and in the amnion. Images PMID:2417224

SOX15 regulates proliferation and migration of endometrial cancer cells.

PubMed

Rui, Xiaohui; Xu, Yun; Jiang, Xiping; Guo, Caixia; Jiang, Jingting

2017-10-31

The study aimed to investigate the effects of Sry-like high mobility group box 15 ( SOX15 ) on proliferation and migration of endometrial cancer (EC) cells. Immunohistochemistry (IHC) was applied to determine the expression of SOX15 in EC tissues and adjacent tissues. We used cell transfection method to construct the HEC-1-A and Ishikawa cell lines with stable overexpression and low expression SOX15 Reverse-transcription quantitative real-time PCR (RT-qPCR) and Western blot were performed to examine expression of SOX15 mRNA and SOX15 protein, respectively. By conducting a series of cell proliferation assay and migration assay, we analyzed the influence of SOX15 overexpression or low expression on EC cell proliferation and migration. The expression of SOX15 mRNA and protein in EC tissues was significantly lower than that in adjacent tissues. After lentivirus-transfecting SOX15 , the expression level of SOX15 mRNA and protein was significantly increased in cells of SOX15 group, and decreased in sh- SOX15 group. Overexpression of SOX15 could suppress cell proliferation, while down-regulation of SOX15 increased cell proliferation. Flow cytometry results indicated that overexpression of SOX15 induced the ratio of cell-cycle arrest in G 1 stage. In addition, Transwell migration assay results showed that SOX15 overexpression significantly inhibited cell migration, and also down-regulation of SOX15 promoted the migration. As a whole, SOX15 could regulate the proliferation and migration of EC cells and up- regulation of SOX15 could be valuable for EC treatment. © 2017 The Author(s).

Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation

NASA Technical Reports Server (NTRS)

Reichler, S. A.; Balk, J.; Brown, M. E.; Woodruff, K.; Clark, G. B.; Roux, S. J.

2001-01-01

The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiss, Daniel L.; Hou, Dezhi; Gross, Robert H.

Highlights: Black-Right-Pointing-Pointer Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. Black-Right-Pointing-Pointer Identified novel 3 Prime UTR cis-acting element that destabilizes a reporter mRNA. Black-Right-Pointing-Pointer Show exosome subunits are required for cis-acting element-mediated mRNA instability. Black-Right-Pointing-Pointer Define precise sequence requirements of novel cis-acting element. Black-Right-Pointing-Pointer Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3 Prime -5 Prime exoribonuclease and endoribonuclease. Although itmore » is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3 Prime untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of mRNA turnover that involves direct Dis3 and other exosome subunit recruitment to and/or regulation on mRNA substrates.« less

Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

PubMed Central

Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

2016-01-01

The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries. PMID:27886048

Tissue Factor Inflammatory Response Regulated by Promoter Genotype and p38 MAPK in Neonatal vs. Adult Microvascular Endothelial Cells

PubMed Central

Buzby, Jeffrey S.; Williams, Shirley A.; Imfeld, Karen L.; Kunicki, Thomas J.; Nugent, Diane J.

2014-01-01

Objective and design Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18 base pair deletion (D) or insertion (I) at -1208. We sought to determine the relationship between these haplotypes and interleukin-1 (IL-1α)-induced TF expression in neonatal versus adult HMVEC. Results IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed 3-fold higher levels of TF mRNA, 2-fold higher TF protein, and 4-fold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC. Conclusions Up-regulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants. PMID:24385191

Zinc-finger protein 418 overexpression protects against cardiac hypertrophy and fibrosis

PubMed Central

Huang, Zirui; Zhu, Zhilin; Xu, Chunli; Teng, Lin; He, Ling; Gu, Chen; Yi, Cai

2017-01-01

Background This study aimed to investigated the effect and mechanism of zinc-finger protein 418 (ZNF418) on cardiac hypertrophy caused by aortic banding (AB), phenylephrine (PE) or angiotensin II (Ang II) in vivo and in vitro. Methods The expression of ZNF418 in hearts of patients with dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) and AB-induced cardiac hypertrophy mice, as well as in Ang II- or PE-induced hypertrophic primary cardiomyocytes was detected by western blotting. Then, the expression of ZNF418 was up-regulated or down-regulated in AB-induced cardiac hypertrophy mice and Ang II -induced hypertrophic primary cardiomyocytes. The hypertrophic responses and fibrosis were evaluated by echocardiography and histological analysis. The mRNA levels of hypertrophy markers and fibrotic markers were detected by RT-qPCR. Furthermore, the phosphorylation and total levels of c-Jun were measured by western blotting. Results ZNF418 was markedly down-regulated in hearts of cardiac hypertrophy and hypertrophic primary cardiomyocytes. Down-regulated ZNF418 exacerbated the myocyte size and fibrosis, moreover increased the mRNA levels of ANP, BNP, β-MHC, MCIP1.4, collagen 1a, collagen III, MMP-2 and fibronection in hearts of AB-treated ZNF418 knockout mice or Ang II-treated cardiomyocytes with AdshZNF418. Conversely, these hypertrophic responses were reduced in the ZNF418 transgenic (TG) mice treated by AB and the AdZNF418-transfected primary cardiomyocytes treated by Ang II. Additionally, the deficiency of ZNF418 enhanced the phosphorylation level of c-jun, and overexpression of ZNF418 suppressed the phosphorylation level of c-jun in vivo and in vitro. Conclusion ZNF418 maybe attenuate hypertrophic responses by inhibiting the activity of c-jun/AP-1. PMID:29065170

Resveratrol Reduces Steroidogenesis in Rat Ovarian Theca-Interstitial Cells: The Role of Inhibition of Akt/PKB Signaling Pathway

PubMed Central

Ortega, Israel; Villanueva, Jesus A.; Wong, Donna H.; Cress, Amanda B.; Sokalska, Anna; Stanley, Scott D.

2012-01-01

Polycystic ovary syndrome is characterized by theca-interstitial hyperplasia and increased expression of steroidogenic genes, leading to excessive androgen production. Resveratrol, a natural polyphenol, promotes apoptosis and reduces rat theca-interstitial cell growth, in part by inhibiting the mevalonate pathway and decreasing the availability of substrates of isoprenylation [farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP)]. This study evaluated the effect of resveratrol on rat theca-interstitial cell steroidogenesis. Because resveratrol may activate sirtuins, this study also investigated whether steroidogenesis was affected by sirtuin inhibitors (nicotinamide, sirtinol). Theca-interstitial cells were cultured with or without resveratrol (1–10 μm), GGPP (30 μm), FPP (30 μm), nicotinamide (1 mm), and/or sirtinol (10 μm). Resveratrol did not affect progesterone levels but reduced androgen production in a concentration-dependent fashion (androstenedione by up to 78% and androsterone by up to 76%). This inhibitory effect correlated with a decrease in mRNA expression of genes regulating androgen production, especially Cyp17a1 (by up to 73%). GGPP and FPP had no effect on androgen levels and Cyp17a1 mRNA levels and did not alter the effects induced by resveratrol. Similarly, sirtuin inhibitors did not reverse resveratrol-induced inhibition of steroidogenesis. However, resveratrol decreased activity of serine-threonine kinase/protein kinase B pathway, a cell-signaling pathway involved in ovarian steroidogenesis. The present findings indicate that resveratrol reduces androgen production primarily by inhibiting Cyp17a1 mRNA expression, and this inhibition may be mediated, in part, by blocking the activity of the serine-threonine kinase/protein kinase B pathway. These findings may be of clinical relevance to conditions associated with excessive production of androgens by theca cells, such as polycystic ovary syndrome. PMID:22719052

Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

PubMed Central

Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

2003-01-01

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

Neuronal ELAV proteins enhance mRNA stability by a PKCα-dependent pathway

PubMed Central

Pascale, Alessia; Amadio, Marialaura; Scapagnini, Giovanni; Lanni, Cristina; Racchi, Marco; Provenzani, Alessandro; Govoni, Stefano; Alkon, Daniel L.; Quattrone, Alessandro

2005-01-01

More than 1 in 20 human genes bear in the mRNA 3′ UTR a specific motif called the adenine- and uridine-rich element (ARE), which posttranscriptionally determines its expression in response to cell environmental signals. ELAV (embryonic lethal abnormal vision) proteins are the only known ARE-binding factors that are able to stabilize the bound mRNAs, thereby positively controlling gene expression. Here, we show that in human neuroblastoma SH-SY5Y cells, neuron-specific ELAV (nELAV) proteins (HuB, HuC, and HuD) are up-regulated and redistributed by 15 min of treatment with the activators of PKC phorbol esters and bryostatin-1. PKC stimulation also induces nELAV proteins to colocalize with the translocated PKCα isozyme preferentially on the cytoskeleton, with a concomitant increase of nELAV threonine phosphorylation. The same treatment promotes stabilization of growth-associated protein 43 (GAP-43) mRNA, a well known nELAV target, and induces an early increase in GAP-43 protein concentration, again only in the cytoskeletal cell fraction. Genetic or pharmacological inactivation of PKCα abolishes nELAV protein cytoskeletal up-regulation, GAP-43 mRNA stabilization, and GAP-43 protein increase, demonstrating the primary role of this specific PKC isozyme in the cascade of nELAV recruitment. Finally, in vivo PKC activation is associated with an up-regulation of nELAV proteins in the hippocampal rat brain. These findings suggest a model for gene expression regulation by nELAV proteins through a PKCα-dependent pathway that is relevant for the cellular programs in which ARE-mediated control plays a pivotal role. PMID:16099831

NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway

PubMed Central

Bühlmann, Melanie; Walrad, Pegine; Rico, Eva; Ivens, Alasdair; Capewell, Paul; Naguleswaran, Arunasalam; Roditi, Isabel; Matthews, Keith R.

2015-01-01

Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5′UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA export. PMID:25873624

Changes in cholecystokinin and peptide Y gene expression with feeding in yellowtail (Seriola quinqueradiata): relation to pancreatic exocrine regulation.

PubMed

Murashita, Koji; Fukada, Haruhisa; Hosokawa, Hidetsuyo; Masumoto, Toshiro

2007-03-01

In fish, the regulation of digestive enzyme secretion by hormonal control such as cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptide is not well understood. To investigate the roles of fish CCK and peptide Y (PY) in digestive enzyme secretion, mRNA levels of CCK and PY, pyloric caeca enzyme activities and mRNA levels of pancreatic digestive enzymes (lipase, trypsin and amylase) were measured at pre- and post-prandial stages in yellowtail. Pyloric caeca were sampled at 0, 0.5, 1.5, 3, 6, 12, 24 and 48 h after feeding. The mRNA levels of trypsin and amylase increased after feeding, suggesting that transcription was induced by feed ingestion. Digestive enzyme activities decreased in exocrine pancreas after feeding, suggesting the stored enzyme was secreted from pancreas post-prandially. mRNA levels for CCK displayed a time-dependent increase, peaking between 1.5 and 3 h after-feeding followed by a rapid decrease 3 to 6 h after feeding. The mRNA expression pattern of PY was inverse to the pattern of CCK, decreasing until 1.5 h after feeding and then rising to initial levels by 12 h after feeding. These results suggest that CCK and PY work antagonistically in the exocrine pancreas of yellowtail.

E. coli Infection Modulates the Pharmacokinetics of Oral Enrofloxacin by Targeting P-Glycoprotein in Small Intestine and CYP450 3A in Liver and Kidney of Broilers

PubMed Central

Guo, Mengjie; Sun, Yong; Zhang, Yu; Bughio, Shamsuddin; Dai, Xiaohua; Ren, Weilong; Wang, Liping

2014-01-01

P-glycoprotein (P-gp) expression determines the absorption, distribution, metabolism and excretion of many drugs in the body. Also, up-regulation of P-gp acts as a defense mechanism against acute inflammation. This study examined expression levels of abcb1 mRNA and localization of P-gp protein in the liver, kidney, duodenum, jejunum and ileum in healthy and E. coli infected broilers by real time RT-PCR and immunohistochemistry. Meanwhile, pharmacokinetics of orally administered enrofloxacin was also investigated in healthy and infected broilers by HPLC. The results indicated that E. coli infection up-regulated expression of abcb1 mRNA levels significantly in the kidney, jejunum and ileum (P<0.05), but not significantly in the liver and duodenum (P>0.05). However, the expression level of CYP 3A37 mRNA were observed significantly decreased only in liver and kidney of E. coli infected broilers (P<0.05) compared with healthy birds. Furthermore, the infection reduced absorption of orally administered enrofloxacin, significantly decreased Cmax (0.34 vs 0.98 µg mL−1, P = 0.000) and AUC0-12h (4.37 vs 8.88 µg mL−1 h, P = 0.042) of enrofloxacin, but increased Tmax (8.32 vs 3.28 h, P = 0.040), T1/2a(2.66 vs 1.64 h−1, P = 0.050) and V/F (26.7 vs 5.2 L, P = 0.040). Treatment with verapamil, an inhibitor of P-gp, significantly improved the absorption of enrofloxacin in both healthy and infected broilers. The results suggest that the E. coli infection induces intestine P-gp expression, altering the absorption of orally administered enrofloxacin in broilers. PMID:24498193

PGC1α -1 Nucleosome Position and Splice Variant Expression and Cardiovascular Disease Risk in Overweight and Obese Individuals.

PubMed

Henagan, Tara M; Stewart, Laura K; Forney, Laura A; Sparks, Lauren M; Johannsen, Neil; Church, Timothy S

2014-01-01

PGC1α, a transcriptional coactivator, interacts with PPARs and others to regulate skeletal muscle metabolism. PGC1α undergoes splicing to produce several mRNA variants, with the NTPGC1α variant having a similar biological function to the full length PGC1α (FLPGC1α). CVD is associated with obesity and T2D and a lower percentage of type 1 oxidative fibers and impaired mitochondrial function in skeletal muscle, characteristics determined by PGC1α expression. PGC1α expression is epigenetically regulated in skeletal muscle to determine mitochondrial adaptations, and epigenetic modifications may regulate mRNA splicing. We report in this paper that skeletal muscle PGC1α  -1 nucleosome (-1N) position is associated with splice variant NTPGC1α but not FLPGC1α expression. Division of participants based on the -1N position revealed that those individuals with a -1N phased further upstream from the transcriptional start site (UP) expressed lower levels of NTPGC1α than those with the -1N more proximal to TSS (DN). UP showed an increase in body fat percentage and serum total and LDL cholesterol. These findings suggest that the -1N may be a potential epigenetic regulator of NTPGC1α splice variant expression, and -1N position and NTPGC1α variant expression in skeletal muscle are linked to CVD risk. This trial is registered with clinicaltrials.gov, identifier NCT00458133.

PPARγ and MyoD are differentially regulated by myostatin in adipose-derived stem cells and muscle satellite cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Feng; Deng, Bing; Wen, Jianghui

2015-03-06

Myostatin (MSTN) is a secreted protein belonging to the transforming growth factor-β (TGF-β) family that is primarily expressed in skeletal muscle and also functions in adipocyte maturation. Studies have shown that MSTN can inhibit adipogenesis in muscle satellite cells (MSCs) but not in adipose-derived stem cells (ADSCs). However, the mechanism by which MSTN differently regulates adipogenesis in these two cell types remains unknown. Peroxisome proliferator-activated receptor-γ (PPARγ) and myogenic differentiation factor (MyoD) are two key transcription factors in fat and muscle cell development that influence adipogenesis. To investigate whether MSTN differentially regulates PPARγ and MyoD, we analyzed PPARγ and MyoDmore » expression by assessing mRNA, protein and methylation levels in ADSCs and MSCs after treatment with 100 ng/mL MSTN for 0, 24, and 48 h. PPARγ mRNA levels were downregulated after 24 h and upregulated after 48 h of treatment in ADSCs, whereas in MSCs, PPARγ levels were downregulated at both time points. MyoD expression was significantly increased in ADSCs and decreased in MSCs. PPARγ and MyoD protein levels were upregulated in ADSCs and downregulated in MSCs. The CpG methylation levels of the PPARγ and MyoD promoters were decreased in ADSCs and increased in MSCs. Therefore, this study demonstrated that the different regulatory adipogenic roles of MSTN in ADSCs and MSCs act by differentially regulating PPARγ and MyoD expression. - Highlights: • PPARγ and MyoD mRNA and protein levels are upregulated by myostatin in ADSCs. • PPARγ and MyoD mRNA and protein levels are downregulated by myostatin in MSCs. • PPARγ exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • MyoD exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • PPARγ and MyoD are differentially regulated by myostatin in ADSCs and MSCs.« less

Androgens Up-regulate Transcription of the Notch Inhibitor Numb in C2C12 Myoblasts via Wnt/β-Catenin Signaling to T Cell Factor Elements in the Numb Promoter*

PubMed Central

Liu, Xin-Hua; Wu, Yong; Yao, Shen; Levine, Alice C.; Kirschenbaum, Alexander; Collier, Lauren; Bauman, William A.; Cardozo, Christopher P.

2013-01-01

Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3β. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which β-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling. PMID:23649620

Cytokine expression in patients with bladder pain syndrome/interstitial cystitis ESSIC type 3C.

PubMed

Logadottir, Yr; Delbro, Dick; Fall, Magnus; Gjertsson, Inger; Jirholt, Pernilla; Lindholm, Catharina; Peeker, Ralph

2014-11-01

Bladder wall nitric oxide production in patients with bladder pain syndrome type 3C is increased compared to undetectable nitric oxide in patients with nonHunner bladder pain syndrome and healthy controls. However, the underlying mechanism/s of the increased nitric oxide production is largely unknown. We compared mRNA expression of a select group of cytokines in patients with bladder pain syndrome/interstitial cystitis type 3C and in pain-free controls. Cold cup biopsies from 7 patients with bladder pain syndrome type 3C and 6 healthy subjects were analyzed. mRNA expression of IL-4, 6, 10 and 17A, iNOS, TNF-α, TGF-β and IFN-γ was estimated by real-time polymerase chain reaction. IL-17 protein expression was determined by immunohistochemistry. Mast cells were labeled with tryptase to evaluate cell appearance and count. IL-6, 10 and 17A, and iNOS mRNA levels as well as the number of mast cells infiltrating the bladder mucosa were significantly increased in patients with bladder pain syndrome type 3C compared to healthy controls. TNF-α, TGF-β and IFN-γ mRNA levels were similar in patients and controls. IL-17A expression at the protein level was up-regulated and localized to inflammatory cells and urothelium in patients with bladder pain syndrome type 3C. Patients with bladder pain syndrome/interstitial cystitis had increased mRNA levels of IL-17A, 10 and 6, and iNOS. IL-17A might be important in the inflammatory process. To our knowledge the increase in IL-17A is a novel finding that may have new treatment implications. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

The Staphylococcus aureus protein-coding gene gdpS modulates sarS expression via mRNA-mRNA interaction.

PubMed

Chen, Chuan; Zhang, Xu; Shang, Fei; Sun, Haipeng; Sun, Baolin; Xue, Ting

2015-08-01

Staphylococcus aureus is an important Gram-positive pathogen responsible for numerous diseases ranging from localized skin infections to life-threatening systemic infections. The virulence of S. aureus is essentially determined by a wide spectrum of factors, including cell wall-associated proteins and secreted toxins that are precisely controlled in response to environmental changes. GGDEF domain protein from Staphylococcus (GdpS) is the only conserved staphylococcal GGDEF domain protein that is involved not in c-di-GMP synthesis but in the virulence regulation of S. aureus NCTC8325. Our previous study showed that the inactivation of gdpS generates an extensive change of virulence factors together with, in particular, a major Spa (protein A) surface protein. As reported, sarS is a direct positive regulator of spa. The decreased transcript levels of sarS in the gdpS mutant compared with the parental NCTC8325 strain suggest that gdpS affects spa through interaction with sarS. In this study, site mutation and complementary experiments showed that the translation product of gdpS was not involved in the regulation of transcript levels of sarS. We found that gdpS functioned through direct RNA-RNA base pairing with the 5' untranslated region (5'UTR) of sarS mRNA and that a putative 18-nucleotide region played a significant role in the regulatory process. Furthermore, the mRNA half-life analysis of sarS in the gdpS mutant showed that gdpS positively regulates the mRNA levels of sarS by contributing to the stabilization of sarS mRNA, suggesting that gdpS mRNA may regulate spa expression in an RNA-dependent pathway. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

In Ovo injection of betaine affects hepatic cholesterol metabolism through epigenetic gene regulation in newly hatched chicks.

PubMed

Hu, Yun; Sun, Qinwei; Li, Xiaoliang; Wang, Min; Cai, Demin; Li, Xi; Zhao, Ruqian

2015-01-01

Betaine is reported to regulate hepatic cholesterol metabolism in mammals. Chicken eggs contain considerable amount of betaine, yet it remains unknown whether and how betaine in the egg affects hepatic cholesterol metabolism in chicks. In this study, eggs were injected with betaine at 2.5 mg/egg and the hepatic cholesterol metabolism was investigated in newly hatched chicks. Betaine did not affect body weight or liver weight, but significantly increased the serum concentration (P < 0.05) and the hepatic content (P < 0.01) of cholesterol. Accordingly, the cholesterol biosynthetic enzyme HMGCR was up-regulated (P < 0.05 for both mRNA and protein), while CYP7A1 which converts cholesterol to bile acids was down-regulated (P < 0.05 for mRNA and P = 0.07 for protein). Moreover, hepatic protein content of the sterol-regulatory element binding protein 1 which regulates cholesterol and lipid biosynthesis, and the mRNA abundance of ATP binding cassette sub-family A member 1 (ABCA1) which mediates cholesterol counter transport were significantly (P < 0.05) increased in betaine-treated chicks. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and adenosylhomocysteinase-like 1 were increased (P < 0.05), which was associated with global genomic DNA hypermethylation (P < 0.05) and diminished gene repression mark histone H3 lysine 27 trimethylation (P < 0.05). Furthermore, CpG methylation level on gene promoters was found to be increased (P < 0.05) for CYP7A1 yet decreased (P < 0.05) for ABCA1. These results indicate that in ovo betaine injection regulates hepatic cholesterol metabolism in chicks through epigenetic mechanisms including DNA and histone methylations.

In Ovo Injection of Betaine Affects Hepatic Cholesterol Metabolism through Epigenetic Gene Regulation in Newly Hatched Chicks

PubMed Central

Hu, Yun; Sun, Qinwei; Li, Xiaoliang; Wang, Min; Cai, Demin; Li, Xi; Zhao, Ruqian

2015-01-01

Betaine is reported to regulate hepatic cholesterol metabolism in mammals. Chicken eggs contain considerable amount of betaine, yet it remains unknown whether and how betaine in the egg affects hepatic cholesterol metabolism in chicks. In this study, eggs were injected with betaine at 2.5 mg/egg and the hepatic cholesterol metabolism was investigated in newly hatched chicks. Betaine did not affect body weight or liver weight, but significantly increased the serum concentration (P < 0.05) and the hepatic content (P < 0.01) of cholesterol. Accordingly, the cholesterol biosynthetic enzyme HMGCR was up-regulated (P < 0.05 for both mRNA and protein), while CYP7A1 which converts cholesterol to bile acids was down-regulated (P < 0.05 for mRNA and P = 0.07 for protein). Moreover, hepatic protein content of the sterol-regulatory element binding protein 1 which regulates cholesterol and lipid biosynthesis, and the mRNA abundance of ATP binding cassette sub-family A member 1 (ABCA1) which mediates cholesterol counter transport were significantly (P < 0.05) increased in betaine-treated chicks. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and adenosylhomocysteinase-like 1 were increased (P < 0.05), which was associated with global genomic DNA hypermethylation (P < 0.05) and diminished gene repression mark histone H3 lysine 27 trimethylation (P < 0.05). Furthermore, CpG methylation level on gene promoters was found to be increased (P < 0.05) for CYP7A1 yet decreased (P < 0.05) for ABCA1. These results indicate that in ovo betaine injection regulates hepatic cholesterol metabolism in chicks through epigenetic mechanisms including DNA and histone methylations. PMID:25860502

Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c.

PubMed

Costet, Philippe; Cariou, Bertrand; Lambert, Gilles; Lalanne, Florent; Lardeux, Bernard; Jarnoux, Anne-Laure; Grefhorst, Aldo; Staels, Bart; Krempf, Michel

2006-03-10

Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory element-binding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4-5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulin-mediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 microm T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia.

Effects of nitrite stress on mRNA expression of antioxidant enzymes, immune-related genes and apoptosis-related proteins in Marsupenaeus japonicus.

PubMed

Zheng, Jinbin; Mao, Yong; Su, Yongquan; Wang, Jun

2016-11-01

Nitrite accumulation in aquaculture systems is a potential risk factor that may trigger stress responses in aquatic organisms. However, the mechanisms regulating the responses of shrimp to nitrite stress remain unclear. In this study, full-length cDNA sequences of two apoptosis-related genes, caspase-3 and defender against apoptotic death (DAD-1), were cloned from Marsupenaeus japonicus for the first time, and their expression levels and tissue distribution were analyzed by quantitative real-time PCR (qRT-PCR). The full lengths of Mjcaspase-3 and MjDAD-1 were 1203 bp and 640 bp respectively, with deduced amino acid (AA) sequences of 321 and 114 AA. Mjcaspase-3 was predominantly expressed in haemocytes and weakly expressed in the seven other tissues tested. MjDAD-1 was mainly expressed in the defense and digestive tissues, especially in the hepatopancreas and hemocytes. To explore the influence of nitrite stress on the genetic response of antioxidant enzymes, immune-related genes and apoptosis-related proteins, the mRNA expression profiles of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 in response to nitrite stress were analyzed by qRT-PCR. The mRNA levels of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 show both time- and dose-dependent changes in response to nitrite stress. The mRNA expression levels of MjCAT and MjSOD peaked at 6 h for all nitrite concentrations tested (p < 0.05) and the up-regulated of MjCAT and MjSOD exhibited a positive correlation with the nitrite concentration. The mRNA expression levels of Mj-ilys and Mj-sty gradually decreased during the experiment period. Mjcaspase-3 mRNA level reached a maximum at 6 h (p < 0.05), and MjDAD-1 reached its peak at 12 h and 48 h in 10 mg/L and 20 mg/L nitrite, respectively. In addition, CAT and SOD activity showed changes in response to nitrite stress that mirrored the induced expression of MjCAT and MjMnSOD, and prolonged nitrite exposure reduced the activity of CAT. This study provided basic data for further elucidating the responses of shrimp to nitrite stress at the molecular level. Copyright © 2016. Published by Elsevier Ltd.

Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voelkl, Jakob; Alesutan, Ioana; Pakladok, Tatsiana

Highlights: • Cardiac Anxa7 expression was up-regulated following TAC. • The hypertrophic response following TAC was augmented in Anxa7-deficient mice. • Silencing of Anxa7 increased indicators of HL-1 cardiomyocytes hypertrophy. • Silencing of Anxa7 induced Nfatc1 nuclear translocation. • Silencing of Anxa7 enhanced NFAT-dependent transcriptional activity. - Abstract: Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca{sup 2+} signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7{sup −/−}) and wild-type mice (anxa7{supmore » +/+}) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7{sup −/−} mice than in anxa7{sup +/+} mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following β-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions.« less

Iron regulation of hepcidin despite attenuated Smad1,5,8 signaling in mice without transferrin receptor 2 or Hfe

PubMed Central

Corradini, Elena; Rozier, Molly; Meynard, Delphine; Odhiambo, Adam; Lin, Herbert Y.; Feng, Qi; Migas, Mary C.; Britton, Robert S.; Babitt, Jodie L.; Fleming, Robert E.

2011-01-01

Background & Aims HFE and transferrin receptor 2 (TFR2) are each necessary for the normal relationship between body iron status and liver hepcidin expression. In murine Hfe and Tfr2 knockout models of hereditary hemochromatosis (HH), signal transduction to hepcidin via the bone morphogenetic protein 6 (Bmp6)/Smad1,5,8 pathway is attenuated. We examined the effect of dietary iron on regulation of hepcidin expression via the Bmp6/Smad1,5,8 pathway using mice with targeted disruption of Tfr2, Hfe, or both genes. Methods Hepatic iron concentrations and mRNA expression of Bmp6 and hepcidin were compared with wild-type mice in each of the HH models on standard or iron-loading diets. Liver phospho-Smad (P-Smad)1,5,8 and Id1 mRNA levels were measured as markers of Bmp/Smad signaling. Results While Bmp6 expression was increased, liver hepcidin and Id1 expression were decreased in each of the HH models compared with wild-type mice. Each of the HH models also demonstrated attenuated P-Smad1,5,8 levels relative to liver iron status. Mice with combined Hfe/Tfr2 disruption were most affected. Dietary iron loading increased hepcidin and Id1 expression in each of the HH models. Compared with wild-type mice, HH mice demonstrated attenuated (Hfe knockout) or no increases in P-Smad1,5,8 levels in response to dietary iron loading. Conclusions These observations demonstrate that Tfr2 and Hfe are each required for normal signaling of iron status to hepcidin via Bmp6/Smad1,5,8 pathway. Mice with combined loss of Hfe and Tfr2 up-regulate hepcidin in response to dietary iron loading without increases in liver BMP6 mRNA or steady-state P-Smad1,5,8 levels. PMID:21745449

Hepatic expression of transcription factors affecting developmental regulation of UGT1A1 in the Han Chinese population.

PubMed

Nie, Ya-Li; He, Hang; Li, Jiang-Feng; Meng, Xiang-Guang; Yan, Liang; Wang, Pei; Wang, Shu-Jie; Bi, Hong-Zheng; Zhang, Li-Rong; Kan, Quan-Cheng

2017-01-01

Complete or partial inactivity of UGT1A1, the unique enzyme responsible for bilirubin glucuronidation, is commonly associated with hyperbilirubinemia. We investigated the dynamic expression of UGT1A1, and that of the transcription factors (TFs) involved in its developmental regulation, during human hepatic growth in Han Chinese individuals. Eighty-eight prenatal, pediatric, and adult liver samples were obtained from Han Chinese individuals. Quantitative real-time polymerase chain reaction was used to evaluate mRNA expression of UGT1A1 and TFs including PXR, CAR, HNF1A, HNF4A, PPARA, etc. UGT1A1 protein levels and metabolic activity were determined by western blotting and high-performance liquid chromatography. Direct sequencing was employed to genotype UGT1A1*6 (211G˃A) and UGT1A1*28 (TA6˃TA7) polymorphisms. UGT1A1 expression was minimal in prenatal samples, but significantly elevated during pediatric and adult stages. mRNA and protein levels and metabolic activity were prominently increased (120-, 20-, and 10-fold, respectively) in pediatric and adult livers compared to prenatal samples. Furthermore, expression did not differ appreciably between pediatric and adult periods. Dynamic expression of TFs, including PXR, CAR, HNF1A, HNF4A, and PPARA, was consistent with UGT1A1 levels at each developmental stage. A pronounced correlation between expression of these TFs and that of UGT1A1 (P < 0.001) was observed. Moreover, UGT1A1*6 and UGT1A1*28 polymorphisms reduced levels of UGT1A1 by up to 40-60 %. Hepatic expression of transcription factors is associated with developmental regulation of UGT1A1 in the Han Chinese population. Moreover, UGT1A1 polymorphisms are associated with reduced expression of UGT1A1 mRNA and protein, as well as enzyme activity.

The effect of low-intensity pulsed ultrasound on wound healing using scratch assay in epithelial cells.

PubMed

Iwanabe, Yujiro; Masaki, Chihiro; Tamura, Akiko; Tsuka, Shintaro; Mukaibo, Taro; Kondo, Yusuke; Hosokawa, Ryuji

2016-10-01

Low-intensity pulsed ultrasound (LIPUS) is widely used in medical fields because it shortens the time required for biologic wound healing in fracture treatment. Also, in dental fields, LIPUS should be effectively employed for implant treatment. However, most of the relevant reports have been published on its effects on bone formation around implants, and the effects of LIPUS on soft tissue healing remain unclear. In the present study, we examined the effects of LIPUS on soft tissue healing using gingival epithelial cells. Gingival epithelial cells were cultured on a dish, followed by LIPUS exposure at a frequency of 3MHz for 15min. The cells were counted with a hemocytometer, and a scratch assay was conducted by measuring the closing area of the scratch wound using a microscope. Following LIPUS exposure, total RNA was collected for microarray analysis. In addition, real-time PCR was performed to examine the mRNA expression level of integrin α6β4. Furthermore, total protein was collected to examine the protein expression level of integrin α6β4 by western blotting. The cell count and scratch assay demonstrated that LIPUS exposure promoted cell proliferation and scratch-wound closure. Microarray analysis demonstrated the increased expression levels of adhesion-related genes, including integrin. Real-time PCR analysis demonstrated that LIPUS exposure significantly up-regulated the mRNA expression level of integrin α6β4. Western blotting showed intense staining of integrin α6β4. LIPUS exposure promotes wound closure in the scratch assay and up-regulates the expression level of integrin α6β4 as compared with the control. Copyright © 2016 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

Exercise training attenuated chronic cigarette smoking-induced up-regulation of FIZZ1/RELMα in lung of rats.

PubMed

Ma, Wan-li; Cai, Peng-cheng; Xiong, Xian-zhi; Ye, Hong

2013-02-01

FIZZ/RELM is a new gene family named "found in inflammatory zone" (FIZZ) or "resistin-like molecule" (RELM). FIZZ1/RELMα is specifically expressed in lung tissue and associated with pulmonary inflammation. Chronic cigarette smoking up-regulates FIZZ1/RELMα expression in rat lung tissues, the mechanism of which is related to cigarette smoking-induced airway hyperresponsiveness. To investigate the effect of exercise training on chronic cigarette smoking-induced airway hyperresponsiveness and up-regulation of FIZZ1/RELMα, rat chronic cigarette smoking model was established. The rats were treated with regular exercise training and their airway responsiveness was measured. Hematoxylin and eosin (HE) staining, immunohistochemistry and in situ hybridization of lung tissues were performed to detect the expression of FIZZ1/RELMα. Results revealed that proper exercise training decreased airway hyperresponsiveness and pulmonary inflammation in rat chronic cigarette smoking model. Cigarette smoking increased the mRNA and protein levels of FIZZ1/RELMα, which were reversed by the proper exercise. It is concluded that proper exercise training prevents up-regulation of FIZZ1/RELMα induced by cigarette smoking, which may be involved in the mechanism of proper exercise training modulating airway hyperresponsiveness.

ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

PubMed Central

Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

2015-01-01

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

Atypical antipsychotic medications increase postprandial triglyceride and glucose levels in male rats: relationship with stearoyl-CoA desaturase activity.

PubMed

McNamara, Robert K; Jandacek, Ronald; Rider, Therese; Tso, Patrick; Cole-Strauss, Allyson; Lipton, Jack W

2011-06-01

Recent preclinical and clinical evidence suggests that the stearoyl-CoA desaturase-1 (Scd1) enzyme plays a key role in the regulation of triglyceride (TG) biosynthesis and insulin sensitivity, and in vitro studies have found that antipsychotic medications up-regulate Scd1 mRNA expression. To investigate these effects in vivo, rats were treated with risperidone (1.5, 3, and 6mg/kg/d), paliperidone (1.5, 3, and 6mg/kg/d), olanzapine (2.5, 5, and 10mg/kg/d), quetiapine (5, 10, and 20mg/kg/d), haloperidol (1, and 3mg/kg/d) or vehicle through their drinking water for 40days. Effects on liver Scd1 mRNA expression and an index of Scd1 activity (the plasma 18:1/18:0 ratio, 'desaturation index') were determined, as were postprandial plasma triglyceride (TG), glucose, insulin, and polyunsaturated fatty acid (PUFA) levels. All atypical antipsychotics increased the plasma 18:1/18:0 ratio, but not liver Scd1 mRNA expression, at doses found to also increase plasma TG levels. Among all rats (n=122), the plasma 18:1/18:0 ratio accounted for 56% of the variance in TG concentrations. The plasma 18:1/18:0 ratio was also positively associated with erythrocyte and heart membrane phospholipid 18:1n-9 composition. All antipsychotics except risperidone increased glucose levels at specific doses, and none of the antipsychotics significantly altered insulin levels. The plasma 18:1/18:0 ratio accounted for 20% of the variance in glucose levels. Plasma omega-3 and omega-6 PUFA levels were inversely correlated with the plasma 18:1/18:0 ratio and TG and glucose levels. These in vivo data demonstrate that different atypical antipsychotic medications increase the plasma 18:1/18:0 ratio in association with elevations in postprandial TG and glucose levels, and that concomitant elevations in PUFA biosynthesis oppose these effects. Copyright © 2011 Elsevier B.V. All rights reserved.

Atypical Antipsychotic Medications Increase Postprandial Triglyceride and Glucose Levels in Male Rats: Relationship with Stearoyl-CoA Desaturase Activity

PubMed Central

McNamara, Robert K.; Jandacek, Ronald; Rider, Therese; Tso, Patrick; Cole-Strauss, Allyson; Lipton, Jack W.

2011-01-01

Recent preclinical and clinical evidence suggests that the stearoyl-CoA desaturase-1 (Scd1) enzyme plays a key role in the regulation of triglyceride (TG) biosynthesis and insulin sensitivity, and in vitro studies have found that antipsychotic medications up-regulate Scd1 mRNA expression. To investigate these effects in vivo, rats were treated with risperidone (1.5, 3, 6 mg/kg/d), paliperidone (1.5, 3, 6 mg/kg/d), olanzapine (2.5, 5, 10 mg/kg/d), quetiapine (5, 10, 20 mg/kg/d), haloperidol (1, 3 mg/kg/d) or vehicle through their drinking water for 40 d. Effects on liver Scd1 mRNA expression and an index of Scd1 activity (the plasma 18:1/18:0 ratio, ‘deaturation index’) were determined, as were postprandial plasma triglyceride (TG), glucose, insulin, and polyunsaturated fatty acid (PUFA) levels. All atypical antipsychotics increased the plasma 18:1/18:0 ratio, but not liver Scd1 mRNA expression, at doses found to also increase plasma TG levels. Among all rats (n=122), the plasma 18:1/18:0 ratio accounted for 56% of the variance in TG concentrations. The plasma 18:1/18:0 ratio was also positively associated with erythrocyte and heart membrane phospholipid 18:1n-9 composition. All antipsychotics except risperidone increased glucose levels at specific doses, and none of the antipsychotics significantly altered insulin levels. The plasma 18:1/18:0 ratio accounted for 20% of the variance in glucose levels. Plasma omega-3 and omega-6 PUFA levels were inversely correlated with the plasma 18:1/18:0 ratio and TG and glucose levels. These in vivo data demonstrate that different atypical antipsychotic medications increase the plasma 18:1/18:0 ratio in association with elevations in postprandial TG levels, and that concomitant elevations in PUFA biosynthesis oppose these effects. PMID:21474290

Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets.

PubMed

Muller, Laurent; Mitsuhashi, Masato; Simms, Patricia; Gooding, William E; Whiteside, Theresa L

2016-02-04

Tumor cell-derived exosomes (TEX) suppress functions of immune cells. Here, changes in the gene profiles of primary human T lymphocytes exposed in vitro to exosomes were evaluated. CD4(+) Tconv, CD8(+) T or CD4(+) CD39(+) Treg were isolated from normal donors' peripheral blood and co-incubated with TEX or exosomes isolated from supernatants of cultured dendritic cells (DEX). Expression levels of 24-27 immune response-related genes in these T cells were quantified by qRT-PCR. In activated T cells, TEX and DEX up-regulated mRNA expression levels of multiple genes. Multifactorial data analysis of ΔCt values identified T cell activation and the immune cell type, but not exosome source, as factors regulating gene expression by exosomes. Treg were more sensitive to TEX-mediated effects than other T cell subsets. In Treg, TEX-mediated down-regulation of genes regulating the adenosine pathway translated into high expression of CD39 and increased adenosine production. TEX also induced up-regulation of inhibitory genes in CD4(+) Tconv, which translated into a loss of CD69 on their surface and a functional decline. Exosomes are not internalized by T cells, but signals they carry and deliver to cell surface receptors modulate gene expression and functions of human T lymphocytes.

Multiparametric in situ mRNA hybridization analysis to predict disease recurrence in patients with colon carcinoma.

PubMed Central

Kitadai, Y.; Ellis, L. M.; Tucker, S. L.; Greene, G. F.; Bucana, C. D.; Cleary, K. R.; Takahashi, Y.; Tahara, E.; Fidler, I. J.

1996-01-01

We examined the expression level of several genes that regulate different steps of metastasis in formalin-fixed, paraffin-embedded archival specimens of primary human colon carcinomas from patients with at least 5 years of follow-up. The expression of epidermal growth factor receptor, basic fibroblast growth factor, type IV collagenase, E-cadherin, and multidrug resistance (mdr-1) was examined by a colorimetric in situ mRNA hybridization technique concentrating on reactivity at the periphery of the neoplasms. The in situ hybridization technique revealed inter- and intratumor heterogeneity for expression of the metastasis-related genes. The expression of basic fibroblast growth factor, collagenase type IV, epidermal growth factor receptor, and mdr-1 mRNA was higher in Dukes's stage D than in Dukes' stage B tumors. Among the 22 Dukes' stage B neoplasms, 5 specimens exhibited a high expression level of epidermal growth factor receptor, basic fibroblast growth factor, and collagenase type IV. Clinical outcome data (5-year follow-up) revealed that all 5 patients with Dukes' stage B tumors developed distant metastasis (recurrent disease), whereas the other 17 patients with Dukes' stage B tumors expressing low levels of the metastasis-related genes were disease-free. Multivariate analysis identified high levels of expression of collagenase type IV and low levels of expression of E-cadherin as independent factors significantly associated with metastasis or recurrent disease. More specifically, metastatic or recurrent disease was associated with a high ratio (> 1.35) of expression of collagenase type IV to E-cadherin (specificity of 95%). Collectively, the data show that multiparametric in situ hybridization analysis for several metastasis-related genes may predict the metastatic potential, and hence the clinical outcome, of individual lymph-node-negative human colon cancers. Images Figure 1 Figure 2 PMID:8909244

Microarray analysis of potential genes in the pathogenesis of recurrent oral ulcer.

PubMed

Han, Jingying; He, Zhiwei; Li, Kun; Hou, Lu

2015-01-01

Recurrent oral ulcer seriously threatens patients' daily life and health. This study investigated potential genes and pathways that participate in the pathogenesis of recurrent oral ulcer by high throughput bioinformatic analysis. RT-PCR and Western blot were applied to further verify screened interleukins effect. Recurrent oral ulcer related genes were collected from websites and papers, and further found out from Human Genome 280 6.0 microarray data. Each pathway of recurrent oral ulcer related genes were got through chip hybridization. RT-PCR was applied to test four recurrent oral ulcer related genes to verify the microarray data. Data transformation, scatter plot, clustering analysis, and expression pattern analysis were used to analyze recurrent oral ulcer related gene expression changes. Recurrent oral ulcer gene microarray was successfully established. Microarray showed that 551 genes involved in recurrent oral ulcer activity and 196 genes were recurrent oral ulcer related genes. Of them, 76 genes up-regulated, 62 genes down-regulated, and 58 genes up-/down-regulated. Total expression level up-regulated 752 times (60%) and down-regulated 485 times (40%). IL-2 plays an important role in the occurrence, development and recurrence of recurrent oral ulcer on the mRNA and protein levels. Gene microarray can be used to analyze potential genes and pathways in recurrent oral ulcer. IL-2 may be involved in the pathogenesis of recurrent oral ulcer.

Lipopolysaccharide-regulated production of bone sialoprotein and interleukin-8 in human periodontal ligament fibroblasts: the role of toll-like receptors 2 and 4 and the MAPK pathway.

PubMed

Zhang, Y; Li, X

2015-04-01

Lipopolysaccharide (LPS) on the cell wall of periodontal pathogens is a major mediator of the inflammatory response and can enhance alveolar bone resorption in periodontitis. Bone sialoprotein is an early marker of osteoblast differentiation. The proinflammatory cytokine, interleukin-8 (IL-8), induces osteoclast differentiation, maturation and maintenance of bone resorption activity. However, the effects of LPS from periodontal pathogens on the expression of bone sialoprotein and IL-8 in human osteoblasts and the mechanism of periodontal bone metabolism regulation are rather unclear. The objectives of this study were to determine the effects of Porphyromonas gingivalis LPS on the production of bone sialoprotein and IL-8 in human periodontal ligament fibroblasts (hPDLFs), and to investigate whether toll-like receptor (TLR) 2, TLR4 and MAPKs pathways are involved in the regulation of production of bone sialoprotein and IL-8 by P. gingivalis LPS. The third-generation of hPDLFs were cultured with mineralization-inducing culture medium. After hPDLFs were treated with P. gingivalis LPS, bone sialoprotein and IL-8 mRNA expression were detected using Real time PCR. Then hPDLFs were transiently transfected with siTLR2 or siTLR4 (20 nm) or inhibited by MAPK signaling pathways inhibitors, and then bone sialoprotein and IL-8 mRNA and protein expression were also detected using Real time PCR and western blotting. Treatments with 0.01 and 0.1 mg/L of P. gingivalis LPS for 8 h up-regulated bone sialoprotein mRNA expression, whereas 10 and 100 mg/L of P. gingivalis LPS induced a significant decrease in the expression of bone sialoprotein mRNA. In contrast, IL8 mRNA levels were increased significantly by 10 mg/L of P. gingivalis LPS. Interestingly, small interfering RNA (siRNA) knock down of the TLR2 and ERK1/2 inhibitor, PD98059, abolished the effects of P. gingivalis LPS on the bone sialoprotein mRNA level, whereas siRNA knock down of the TLR2 and p38 MAPK inhibitor, SB203580, blocked the effect of P. gingivalis LPS on IL-8 in hPDLFs. This study suggests that in hPDLFs, P. gingivalis LPS suppresses bone sialoprotein and enhances IL-8 gene and protein expression via TLR2 and ERK1/2 or the p38 MAPK signaling pathway, respectively. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A transgenic approach to study argininosuccinate synthetase gene expression

PubMed Central

2014-01-01

Background Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. Results Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene. Conclusions We demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene. PMID:24884799

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122; Zhao, Zhe

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels highermore » in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.« less

Abscisic acid regulation of DC8, a carrot embryonic gene. [Daucus carota

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatzopoulos, P.; Fong, F.; Sung, Z.R.

1990-10-01

DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage embryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decreasemore » to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 {times} 10{sup {minus}7} molar ABA while 10{sup {minus}6} molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.« less

c-fms mRNA is regulated posttranscriptionally by 1,25(OH)2D3 in HL-60 cells.

PubMed

Biskobing, D M; Fan, D; Rubin, J

1997-09-01

Macrophage colony-stimulating factor (MCSF) is required for normal osteoclast and macrophage development. The receptor for MCSF (c-fms) is expressed on the pluripotent precursor and mature osteoclasts and macrophages. We have previously shown in myelomonocytic HL-60 cells that phorbol myristate acetate (PMA) upregulates c-fms mRNA expression. This induction of c-fms is inhibited by 1,25(OH)2D3. The major regulatory control of c-fms mRNA levels by PMA has been identified as posttranscriptional. However, a role of transcript elongation in controlling levels of c-fms mRNA has also been suggested. To better understand the 1,25(OH)2D3 regulation of c-fms mRNA expression we studied nuclear run on, mRNA stability, and transcript elongation in HL-60 cells treated with 10 ng/ml phorbol myristate acetate, 10 nM 1,25(OH)2D3 alone or combined. We demonstrated by nuclear run on that c-fms was constitutively transcribed in 1,25(OH)2D3 as well as control and PMA-treated cells. Transcript elongation was evaluated by RT-PCR for exon 2 or exon 3. Both exons were minimally expressed in control and 1,25(OH)2D3-treated cells, and increased in PMA-treated cells; this increased expression was inhibited by the addition of 1,25(OH)2D3. These results fail to show differential transcript elongation. Measurement of mRNA stability demonstrated decreased mRNA half-life to 5 hours in cells treated with PMA and 1,25(OH)2D3 compared with a half-life of 8 hours in cells treated with PMA alone. Our findings demonstrate that c-fms is regulated by 1,25(OH)2D3 at the posttranscriptional level by changes in mRNA stability. This gives the cell the ability to respond to local signals with rapid changes in c-fms levels altering the ability of the cell to respond to MCSF.

Nitric oxide-sensitive guanylyl cyclase is differentially regulated by nuclear and non-nuclear estrogen pathways in anterior pituitary gland.

PubMed

Cabilla, Jimena P; Nudler, Silvana I; Ronchetti, Sonia A; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

2011-01-01

17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway. © 2011 Cabilla et al.

FTO regulates the chemo-radiotherapy resistance of cervical squamous cell carcinoma (CSCC) by targeting β-catenin through mRNA demethylation.

PubMed

Zhou, Shun; Bai, Zhou-Lan; Xia, Di; Zhao, Zhi-Jun; Zhao, Ren; Wang, Yan-Yang; Zhe, Hong

2018-05-01

The role of N 6 -methyladenosine (m 6 A) demethylase fat mass and obesity-associated protein (FTO) in the regulation of chemo-radiotherapy resistance remains largely unknown. Here, we show that the mRNA level of FTO is elevated in cervical squamous cell carcinoma (CSCC) tissues when compared with respective adjacent normal tissues. FTO enhances the chemo-radiotherapy resistance both in vitro and in vivo through regulating expression of β-catenin by reducing m 6 A levels in its mRNA transcripts and in turn increases excision repair cross-complementation group 1 (ERCC1) activity. Clinically, the prognostic value of FTO for overall survival is found to be dependent on β-catenin expression in human CSCC samples. Taken together, these findings uncover a critical function for FTO and its substrate m 6 A in the regulation of chemo-radiotherapy resistance, which may bear potential clinical implications for CSCC treatment. © 2018 Wiley Periodicals, Inc.

Different effects of blue and red light-emitting diodes on antioxidant responses in the liver and ovary of zebrafish Danio rerio.

PubMed

Yuan, Shuang-Shuang; Xu, Huan-Zhi; Liu, Li-Qin; Zheng, Jia-Lang

2017-04-01

The present study assessed the effects of a white fluorescent bulb (the control) and two different light-emitting diodes (blue LEDs, LDB; red, LDR) on growth, morphology, and oxidative stress in the liver and ovary of zebrafish for 5 weeks. Growth maintained relatively constant under LDB condition, but was reduced under LDR condition. In the liver, hepatosomatic index (HSI) and protein carbonylation (PC) increased under LDR condition, whereas lipid peroxidation (LPO) declined and HSI remained unchanged under LDB condition. The decrease in oxidative damage by LDB could be attributed to the up-regulated levels of mRNA, protein, and activity of Cu/Zn-SOD and CAT. A failure to activate the activity of both enzymes may result in the enhanced PC levels under LDR condition, though both genes were up-regulated at transcriptional and translational levels. In the ovary, although gonadosomatic index sharply increased under LDR condition, LPO and PC dramatically accumulated. The increase in oxidative damage by LDR might result from the down-regulated levels of protein and activity of Cu/Zn-SOD and CAT, though both genes were up-regulated at a transcriptional level. Furthermore, a sharp increase in expression of transcription factor Nrf2 that targets antioxidant genes was observed in the liver but not in the ovary under LDB and LDR conditions. In conclusion, our data demonstrated a positive effect of LDB and negative effect of LDR on fish antioxidant defenses, emphasizing the potentials of LDB as an effective light source in fish farming.

Dietary proanthocyanidins boost hepatic NAD(+) metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats.

PubMed

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-04-22

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD(+)) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD(+) precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD(+). Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD(+) availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD(+) levels.

Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats

PubMed Central

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A.; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-01-01

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD+) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD+ precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD+. Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD+ availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD+ levels. PMID:27102823

Postnatal handling does not normalize hypothalamic corticotropin-releasing factor mRNA levels in animals prenatally exposed to ethanol.

PubMed

Gabriel, Kara I; Glavas, Maria M; Ellis, Linda; Weinberg, Joanne

2005-06-09

Postnatal handling has been shown to attenuate some of the deficits in developmental outcome observed following prenatal ethanol exposure (E) although it appears to be ineffective at ameliorating the hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness to stressors that has been observed in adult E animals. However, the effects of postnatal handling on central regulation of HPA activity in E animals, particularly with regard to alterations in steady-state hypothalamic corticotropin-releasing factor (CRF) activity, have not been examined. In the present study, offspring from E, pair-fed (PF), and ad-libitum-fed control (C) groups were exposed to daily handling during the first 2 weeks of life (H) or were left entirely undisturbed until weaning (NH). Basal CRF and arginine vasopressin (AVP) mRNA in the parvocellular portion of the paraventricular nucleus (pPVN) of the hypothalamus were assessed at 90-110 days of age. Prenatal ethanol exposure resulted in elevated basal pPVN CRF mRNA levels compared to those in ad-libitum-fed controls. Handling altered CRF mRNA levels in a sex-specific and prenatal treatment-specific manner. Females showed no significant effects of handling. In contrast, handling decreased CRF mRNA levels in PF and C but not E males compared to their NH counterparts. There were no effects of prenatal ethanol or postnatal handling on AVP mRNA levels. These findings indicate that prenatal ethanol exposure results in elevated basal CRF mRNA levels in adulthood and that handling appears to be ineffective in normalizing those elevations, supporting the suggestion that altered basal HPA regulation in E animals may, at least in part, underlie their HPA hyperresponsiveness to stressors.

Endoglin regulates renal ischaemia-reperfusion injury.

PubMed

Docherty, Neil G; López-Novoa, José M; Arevalo, Miguel; Düwel, Annette; Rodriguez-Peña, Ana; Pérez-Barriocanal, Fernando; Bernabeu, Carmelo; Eleno, Nélida

2006-08-01

Renal ischaemia-reperfusion (I-R) can cause acute tubular necrosis and chronic renal deterioration. Endoglin, an accessory receptor for Transforming Growth Factor-beta1 (TGF-beta1), is expressed on activated endothelium during macrophage maturation and implicated in the control of fibrosis, angiogenesis and inflammation. Endoglin expression was monitored over 14 days after renal I-R in rats. As endoglin-null mice are not viable, the role of endoglin in I-R was studied by comparing renal I-R injury in haploinsufficient mice (Eng(+/-)) and their wild-type littermates (Eng(+/+)). Renal function, morphology and molecular markers of acute renal injury and inflammation were compared. Endoglin mRNA up-regulation in the post-ischaemic kidneys of rats occurred at 12 h after I-R; endoglin protein levels were elevated throughout the study period. Expression was initially localized to the vascular endothelium, then extended to fibrotic and inflamed areas of the interstitium. Two days after I-R, plasma creatinine elevation and acute tubular necrosis were less marked in Eng(+/-) than in Eng(+/+) mice. Significant up-regulation of endoglin protein was found only in the post-ischaemic kidneys of Eng(+/+) mice and coincided with an increased mRNA expression of the TGF-beta1 and collagen IV (alpha1) chain genes. Significant increases in vascular cell adhesion molecule-1 (VCAM-1) and inducible nitric oxide synthase (iNOS) expression, nitrosative stress, myeloperoxidase activity and CD68 staining for macrophages were evident in post-ischaemic kidneys of Eng(+/+), but not Eng(+/-) mice, suggesting that impaired endothelial activation and macrophage maturation may account for the reduced injury in post-ischaemic kidneys of Eng(+/-) mice. Endoglin is up-regulated in the post-ischaemic kidney and endoglin-haploinsufficient mice are protected from renal I-R injury. Endoglin may play a primary role in promoting inflammatory responses following renal I-R.

Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease

PubMed Central

Yin, Xiangling; Manczak, Maria; Reddy, P. Hemachandra

2016-01-01

The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. PMID:26908605

Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease.

PubMed

Yin, Xiangling; Manczak, Maria; Reddy, P Hemachandra

2016-05-01

The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl2.

PubMed

Damiano, Fabrizio; Testini, Mariangela; Tocci, Romina; Gnoni, Gabriele V; Siculella, Luisa

2018-04-01

Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5' untranslated region (5' UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5' UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl 2 , up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5' UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

[N(omega)-nitro-L-arginine methyl ester inhibits the up-regulated expression of neuronal nitric oxide synthase/NMDA receptor in the morphine analgesia tolerance rats].

PubMed

Yu, Ling; Xue, Fu-Shan; Li, Cheng-Wen; Xu, Ya-Chao; Zhang, Guo-Hua; Liu, Kun-Peng; Liu, Yi; Sun, Hai-Tao

2006-12-25

The effect of systemic administration of nonspecific nitric oxide synthase inhibitor (N(omega)-nitro-L-arginine methyl ester, L-NAME) on morphine analgesia tolerance was observed by using the thermal tail-flick method, and the roles of NO and NMDA receptors in morphine analgesia tolerance were evaluated on the basis of the expressions of nNOS mRNA, NR1A mRNA and NR2A mRNA in spinal cord and midbrain. Thirty-six healthy adult Sprague-Dawley rats were randomly divided into six groups (6 rats per group). Group 1, control group, received a subcutaneous (s.c.) injection of normal saline (1 ml); Groups 2, 3, 4, 5 and 6, the treatment groups received s.c. injection of L-NAME 10 mg/kg, L-NAME 20 mg/kg, morphine 10 mg/kg, L-NAME 10 mg/kg + morphine 10 mg/kg, and L-NAME 20 mg/kg + morphine 10 mg/kg, respectively. All rats received s.c. injections twice per day (8:00 and 17:00). The tail-flick latency (TFL) was measured in each rat before the injection as a baseline value, and then TFL at 50 min after the 1st injection every day as the measuring values. The animals (except for groups 2 and 5) were decapitated at 80 min after the last injection on the 8th day. The spinal segments and midbrain were removed for analysis of nNOS mRNA, NR1A mRNA and NR2A mRNA expressions by the RT-PCR method. The results showed that TFL remained unchangeable in group 2 compared with baseline value during the 7-day observation, while increased significantly on the 7th day in group 3. In group 4, TFL was longest on the 1st day, then decreased gradually from the 2nd day to the 6th day, and restored to the baseline value on the 6th day. In group 5, TFL showed a decreasing tendency during the 7-day observation, but was still significantly longer than the baseline value on the 7th day. The changes of TFL obtained in group 6 were similar to those in group 5. The results of RT-PCR showed that as compared with group 1, nNOS mRNA expressions in spinal cord and midbrain were significantly down-regulated in group 3, but the expressions of the NR1A mRNA and NR2A mRNA in both groups were similar, while the nNOS mRNA, NR(1A) mRNA and NR(2A) mRNA expressions were all significantly up-regulated in group 4. As compared with group 4, the expressions of nNOS mRNA, NR(1A) mRNA and NR(2A) mRNA were significantly inhibited in group 6. These results suggest that the expressions of nNOS and NMDA receptors in spinal cord and midbrain were significantly up-regulated in the rats with morphine analgesia tolerance. Chronic co-administration of L-NAME could effectively inhibit the morphine-induced overexpressions of nNOS and NMDA receptors, and postpone the development of morphine analgesia tolerance. Based on the results of this study, it is concluded that NO/NMDA receptor in spinal cord and midbrain is closely related to the development of morphine analgesia tolerance.

Biotin deficiency up-regulates TNF-alpha production in murine macrophages.

PubMed

Kuroishi, Toshinobu; Endo, Yasuo; Muramoto, Koji; Sugawara, Shunji

2008-04-01

Biotin, a water-soluble vitamin of the B complex, functions as a cofactor of carboxylases that catalyze an indispensable cellular metabolism. Although significant decreases in serum biotin levels have been reported in patients with chronic inflammatory diseases, the biological roles of biotin in inflammatory responses are unclear. In this study, we investigated the effects of biotin deficiency on TNF-alpha production. Mice were fed a basal diet or a biotin-deficient diet for 8 weeks. Serum biotin levels were significantly lower in biotin-deficient mice than biotin-sufficient mice. After i.v. administration of LPS, serum TNF-alpha levels were significantly higher in biotin-deficient mice than biotin-sufficient mice. A murine macrophage-like cell line, J774.1, was cultured in a biotin-sufficient or -deficient medium for 4 weeks. Cell proliferation and biotinylation of intracellular proteins were decreased significantly in biotin-deficient cells compared with biotin-sufficient cells. Significantly higher production and mRNA expression of TNF-alpha were detected in biotin-deficient J774.1 cells than biotin-sufficient cells in response to LPS and even without LPS stimulation. Intracellular TNF-alpha expression was inhibited by actinomycin D, indicating that biotin deficiency up-regulates TNF-alpha production at the transcriptional level. However, the expression levels of TNF receptors, CD14, and TLR4/myeloid differentiation protein 2 complex were similar between biotin-sufficient and -deficient cells. No differences were detected in the activities of the NF-kappaB family or AP-1. The TNF-alpha induction by biotin deficiency was down-regulated by biotin supplementation in vitro and in vivo. These results indicate that biotin deficiency may up-regulate TNF-alpha production or that biotin excess down-regulates TNF-alpha production, suggesting that biotin status may influence inflammatory diseases.

Molecular events regulating messenger RNA stability in eukaryotes.

PubMed

Saini, K S; Summerhayes, I C; Thomas, P

1990-07-17

The regulation of mRNA turnover plays a major role in the overall control of gene expression. Transcriptional control of eukaryotic gene regulation by external and/or internal stimuli has received considerable attention and the purpose of this review is to highlight recent work elucidating the mechanisms underlying the steady-state levels of mRNAs in the cytoplasm. Protection of mRNA from the action of nucleases as it passes from the nucleus to the ribosomes for translation is achieved, at least in part, by its union with mRNA binding proteins and the presence of poly(A) tail. The half-life of a message represents a balance between the transcriptional activity and intracellular degradative processes. These properties can be modulated by the presence of specific nucleotide sequences in a mRNA along with cis- and trans-acting elements and accompanied by post-translational feed back mechanisms. Presently, various regulatory mechanisms involved in the mRNA decay process are ill-defined. The work described here illustrates the complexity of this emerging field of study and outlines its contribution to our understanding of gene regulation in eukaryotes.

Interleukin 6 deficiency modulates the hypothalamic expression of energy balance regulating peptides during pregnancy in mice.

PubMed

Pazos, Patricia; Lima, Luis; Casanueva, Felipe F; Diéguez, Carlos; García, María C

2013-01-01

Pregnancy is associated with hyperphagia, increased adiposity and multiple neuroendocrine adaptations. Maternal adipose tissue secretes rising amounts of interleukin 6 (IL6), which acts peripherally modulating metabolic function and centrally increasing energy expenditure and reducing body fat. To explore the role of IL6 in the central mechanisms governing dam's energy homeostasis, early, mid and late pregnant (gestational days 7, 13 and 18) wild-type (WT) and Il6 knockout mice (Il6-KO) were compared with virgin controls at diestrus. Food intake, body weight and composition as well as indirect calorimetry measurements were performed in vivo. Anabolic and orexigenic peptides: neuropeptide Y (Npy) and agouti-related peptide (Agrp); and catabolic and anorectic neuropeptides: proopiomelanocortin (Pomc), corticotrophin and thyrotropin-releasing hormone (Crh and Trh) mRNA levels were determined by in situ hybridization. Real time-PCR and western-blot were used for additional tissue gene expression and protein studies. Non-pregnant Il6-KO mice were leaner than WT mice due to a decrease in fat but not in lean body mass. Pregnant Il6-KO mice had higher fat accretion despite similar body weight gain than WT controls. A decreased fat utilization in absence of Il6 might explain this effect, as shown by increased respiratory exchange ratio (RER) in virgin Il6-KO mice. Il6 mRNA levels were markedly enhanced in adipose tissue but reduced in hypothalamus of mid and late pregnant WT mice. Trh expression was also stimulated at gestational day 13 and lack of Il6 blunted this effect. Conversely, in late pregnant mice lessened hypothalamic Il6 receptor alpha (Il6ra), Pomc and Crh mRNA were observed. Il6 deficiency during this stage up-regulated Npy and Agrp expression, while restoring Pomc mRNA levels to virgin values. Together these results demonstrate that IL6/IL6Ra system modulates Npy/Agrp, Pomc and Trh expression during mouse pregnancy, supporting a role of IL6 in the central regulation of body fat in this physiological state.

Long-term dose-dependent response of Mequindox on aldosterone, corticosterone and five steroidogenic enzyme mRNAs in the adrenal of male rats.

PubMed

Huang, Xian-Ju; Ihsan, Awais; Wang, Xu; Dai, Meng-Hong; Wang, Yu-Lian; Su, Shi-Jia; Xue, Xi-Juan; Yuan, Zong-Hui

2009-12-15

Mequindox (MEQ) is a synthetic quinoxaline 1,4-dioxides (QdNOs) derivative which can effectively improve growth and feed efficiency in animals. This study was to investigate the dose-dependent long-term toxicity in the adrenal of male rats exposed to 180 days of MEQ feed. Our data demonstrated that high doses of MEQ in the diet for 180 days led to adrenal damage and steroid hormone decrease, combined with sodium decrease and potassium increase in rat plasma. Significant changes of GSH and SOD in plasma were observed in the high doses (110, 275 mg/kg) groups. At the same doses, MEQ treatment down-regulated the mRNA levels of CYP11A1, CYP11B1 and CYP11B2 which located in mitochondria, but up-regulated mRNA levels of CYP21 and 3beta-HSD which located in endoplasmic reticulum. In conclusion, we reported the dose-dependent long-term toxicity of MEQ on adrenal gland in male rats, which raise awareness of its toxic effects to animals and consumers, and its mechanism may involve in oxidative stress and steroid hormone biosynthesis pathway.

Short-term dopaminergic regulation of GABA release in dopamine deafferented caudate-putamen is not directly associated with glutamic acid decarboxylase gene expression.

PubMed

O'Connor, W T; Lindefors, N; Brené, S; Herrera-Marschitz, M; Persson, H; Ungerstedt, U

1991-07-08

In vivo microdialysis and in situ hybridization were combined to study dopaminergic regulation of gamma-amino butyric acid (GABA) neurons in rat caudate-putamen (CPu). Potassium-stimulated GABA release in CPu was elevated following a dopamine deafferentation. Local perfusion with exogenous dopamine (50 microM) for 3 h via the microdialysis probe attenuated the potassium-stimulated increase in extracellular GABA in CPu. Expression of glutamic acid decarboxylase (GAD) mRNA was also increased in the dopamine deafferented CPu. However, local perfusion with dopamine had no significant attenuating effect on the increased GAD mRNA expression. These findings indicate that dopaminergic regulation of GABA neurons in the dopamine deafferented CPu includes both a short-term effect at the level of GABA release independent of changes in GAD mRNA expression and a long-term modulation at the level of GAD gene expression.

MicroRNA-9 promotes the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.

PubMed

Liu, D-Z; Chang, B; Li, X-D; Zhang, Q-H; Zou, Y-H

2017-09-01

The objective of the study was to investigate the role of microRNA-9 (miR-9) targeting forkhead box O1 (FOXO1) in the proliferation, migration, and invasion of breast cancer cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expressions of miR-9 and FOXO1 mRNA in breast cancer tissues, normal breast tissues, breast cancer cell lines, and normal breast epithelial cells. After the up-regulation of miR-9 expression, qRT-PCR and Western blotting were used to determine the expression of FOXO1. The luciferase reporter gene assay was used to validate the target gene. The CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay were used to investigate the changes in the proliferation, migration, and invasion of breast cancer cells, respectively. MicroRNA-9 expression was significantly up-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). FOXO1 mRNA and protein expressions were substantially down-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). There can be a negative correlation between miR-9 and FOXO1 mRNA in breast cancer. Luciferase reporter gene assay indicated that miR-9 can down-regulate FOXO1 expression at a post-transcriptional level through binding specifically to FOXO1 3'UTR. The results of CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay revealed that the inhibition of miR-9 can suppress MCF7 cell proliferation, migration, and invasion. Additionally, the expression of miR-9 increased significantly whilst that of FOXO1 decreased substantially as the disease progressed (P < 0.05). Our study provides evidence that miR-9 can promote the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.

[Effects of ingredients from Chinese herbs with nature of cold or hot on expression of TRPV1 and TRPM8].

PubMed

Sui, Feng; Yang, Na; Zhang, Changbin; Du, Xinliang; Li, Lanfang; Weng, Xiaogang; Guo, Shuying; Huo, Hairu; Jiang, Tingliang

2010-06-01

To study the effects of the ingredients from Chinese herbs with the nature of cold or hot on the expression of TRPV1 and TRPM8. The effects of ingredients from herbs on primary culture DRG neurons are observed in vitro. The expression quantity of gene is detected by the method of real time PCR. the 2 (-deltadeltaCT) method is applied to analyze the data. Ingredients from herbs with the nature of cold up-regulate the expression level of TRPV1 and down-regulate that of TRPM8, especially under the temperature condition of 39 degrees C; while ingredients from herbs with the nature of hot up-regulate the expression level of TRPM8 and down-regulated that of TRPV1, which is more significant under the temperature condition of 19 degrees C. The regulatory changes of TRPV1 and TRPM8 mRNA expression induced by the chemical ingredients might be related to the cold and hot natures of the herbs from which the ingredients are extracted. And this could be one of the therapeutic mechanisms for the treatment of Chinese herbal medicines to cold- and heat-related diseases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lundasen, Thomas; Molecular Nutrition Unit, Department of Biosciences and Nutrition, NOVUM, Karolinska Institutet, Huddinge, SE-141 86 Stockholm; Hunt, Mary C.

The metabolic regulator fibroblast growth factor 21 (FGF21) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF21 is regulated by the peroxisome proliferator-activated receptor alpha (PPAR{alpha}). Fasting or treatment of mice with the PPAR{alpha} agonist Wy-14,643 induced FGF21 mRNA by 10-fold and 8-fold, respectively. In contrast, FGF21 mRNA was low in PPAR{alpha} deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF21. Obese ob/ob mice, known to have increased PPAR{alpha} levels, displayed 12-fold increased hepatic FGF21 mRNA levels. The potential importance of PPAR{alpha} formore » FGF21 expression also in human liver was shown by Wy-14,643 induction of FGF21 mRNA in human primary hepatocytes, and PPAR{alpha} response elements were identified in both the human and mouse FGF21 promoters. Further studies on the mechanisms of regulation of FGF21 by PPAR{alpha} in humans will be of great interest.« less

Identification of a Mg2+-sensitive ORF in the 5′-leader of TRPM7 magnesium channel mRNA

PubMed Central

Nikonorova, Inna A.; Kornakov, Nikolay V.; Dmitriev, Sergey E.; Vassilenko, Konstantin S.; Ryazanov, Alexey G.

2014-01-01

TRPM7 is an essential and ubiquitous channel-kinase regulating cellular influx of Mg2+. Although TRPM7 mRNA is highly abundant, very small amount of the protein is detected in cells, suggesting post-transcriptional regulation of trpm7 gene expression. We found that TRPM7 mRNA 5′-leader contains two evolutionarily conserved upstream open reading frames that act together to drastically inhibit translation of the TRPM7 reading frame at high magnesium levels and ensure its optimal translation at low magnesium levels, when the activity of the channel-kinase is most required. The study provides the first example of magnesium channel synthesis being controlled by Mg2+ in higher eukaryotes. PMID:25326319

Cloning of cardiac, kidney, and brain promoters of the feline ncx1 gene.

PubMed

Barnes, K V; Cheng, G; Dawson, M M; Menick, D R

1997-04-25

The Na+-Ca2+ exchanger (NCX1) plays a major role in calcium efflux and therefore in the control and regulation of intracellular calcium in the heart. The exchanger has been shown to be regulated at several levels including transcription. NCX1 mRNA levels are up-regulated in both cardiac hypertrophy and failure. In this work, the 5'-end of the ncx1 gene has been cloned to study the mechanisms that mediate hypertrophic stimulation and cardiac expression. The feline ncx1 gene has three exons that encode 5'-untranslated sequences that are under the control of three tissue-specific promoters. The cardiac promoter drives expression in cardiocytes, but not in mouse L cells. Although it contains at least one enhancer (-2000 to -1250 base pairs (bp)) and one or more negative elements (-1250 to -250 bp), a minimum promoter (-250 to +200 bp) is sufficient for cardiac expression and alpha-adrenergic stimulation.

Light-regulated protein and mRNA synthesis in root caps of maize

NASA Technical Reports Server (NTRS)

Feldman, L. J.; Piechulla, B.; Sun, P. S.

1988-01-01

Illumination of maize roots initiates changes in mRNA levels and in the activities of proteins within the root cap. Using Northern analysis we showed a 5-6 fold increase in the levels of three specific mRNAs and a 14-fold increase in plastid mRNA. This increase is rapid, occurring within 30 minutes of illumination. With prolonged periods of darkness following illumination, messages return to levels observed in dark, control caps. For two species of mRNA illumination results in a reduction in message levels. Light-stimulated increases in the levels of specific mRNAs are proportionally greater than are increases in the activities of corresponding proteins. We suggest that the light-stimulated increase in protein activity in root caps may be preceded by and occur as a consequence of enhanced levels of mRNA. Our work suggests that photomorphogenesis in roots could involve changes in the levels of a wide variety of mRNAs within the root cap.

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

PubMed

Manzardo, A M; Gunewardena, S; Butler, M G

2013-09-10

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling. Copyright © 2013 Elsevier B.V. All rights reserved.

miR-200a controls hepatic stellate cell activation and fibrosis via SIRT1/Notch1 signal pathway.

PubMed

Yang, Jing-Jing; Tao, Hui; Liu, Li-Ping; Hu, Wei; Deng, Zi-Yu; Li, Jun

2017-04-01

miR-200a has been established as a key regulator of HSC activation processes in liver fibrosis. Epigenetic silencing of miR-200a contributing to SIRT1 over-expression has been discussed in breast cancer; however, whether miR-200a controls SIRT1 gene expression in hepatic fibrosis is still unknown. We analyzed miR-200a regulation of SIRT1 expression in CCl 4 -induced liver fibrosis and TGF-β1-mediated activation of HSC. miR-200a, SIRT1, α-SMA, Col1A1, Notch1 and NICD expression were estimated by Western blotting, qRT-PCR and Immunohistochemistry. HSCs were transfected with miR-200a mimic, miR-200a inhibitor and SIRT1-RNAi. Luciferase reporter assays further confirmed the interaction between miR-200a and the SIRT1 mRNA 3'-UTR. Cell proliferation ability was assessed by MTT and cell cycle. We found that treatment activated HSC with miR-200a mimics, restored miR-200a expression and reduced SIRT1 levels. Conversely, treatment activated HSC with miR-200a inhibitors, decreased miR-200a expression and up-regulated SIRT1 levels. Restoration of miR-200a or the knockdown of SIRT1 prevented HSC activation and proliferation. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3'-UTR. Finally, HSC transfected with SIRT1-siRNA increased the levels of Notch1 protein and mRNA expression. Our study demonstrated that miR-200a regulates SIRT1/Notch1 expression during HSC activation and fibrosis.

Ecdysone receptor isoform-B mediates soluble trehalase expression to regulate growth and development in the mirid bug, Apolygus lucorum (Meyer-Dür).

PubMed

Tan, Y-A; Xiao, L-B; Zhao, J; Xiao, Y-F; Sun, Y; Bai, L-X

2015-12-01

Ecdysone receptor (EcR) is the hormonal receptor of ecdysteroids and strictly regulates growth and development in insects. However, the action mechanism of EcR is not very clear. In this study, the cDNA of EcR isoform-B was cloned from Apolygus lucorum (AlEcR-B) and its expression profile was investigated. We reduced AlEcR-B mRNA expression using systemic RNA interference in vivo, and obtained knockdown specimens. Examination of these specimens indicated that AlEcR-B is required for nymphal survival, and that reduced expression is associated with longer development time and lower nymphal weight. To investigate the underlying molecular mechanism of the observed suppression effects, we selected trehalase for a detailed study. Transcript encoding soluble trehalase (AlTre-1) was up-regulated by 20-hydroxyecdysone and in agreement with the mRNA expression of AlEcR-B. The expression profile of AlTre-1, soluble trehalase activity and translated protein level in the midgut of surviving nymphs were down-regulated, compared with controls, after the knockdown expression of AlEcR-B. By contrast, membrane-bound trehalase activity, the related gene expression and translated protein level remained at their initial levels. However, trehalose content significantly increased and the glucose content significantly decreased under the same conditions. We propose that AlEcR-B controls normal carbohydrate metabolism by mediating the expression of AlTre-1 to regulate the growth and development in A. lucorum, which provide an extended information into the functions of AlEcR-B. © 2015 The Royal Entomological Society.

Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels: NEW TARGETS OF ALDOSTERONE IN CARDIOMYOCYTES.

PubMed

Sabourin, Jessica; Bartoli, Fiona; Antigny, Fabrice; Gomez, Ana Maria; Benitah, Jean-Pierre

2016-06-17

Store-operated Ca(2+) entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca(2+) influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca(2+)]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels

PubMed Central

Sabourin, Jessica; Bartoli, Fiona; Antigny, Fabrice; Gomez, Ana Maria; Benitah, Jean-Pierre

2016-01-01

Store-operated Ca2+ entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca2+ influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca2+]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway. PMID:27129253

Creatine uptake in mouse hearts with genetically altered creatine levels

PubMed Central

Hove, Michiel ten; Makinen, Kimmo; Sebag-Montefiore, Liam; Hunyor, Imre; Fischer, Alexandra; Wallis, Julie; Isbrandt, Dirk; Lygate, Craig; Neubauer, Stefan

2008-01-01

Creatine plays an important role in energy metabolism in the heart. Cardiomyocytes accumulate creatine via a specific creatine transporter (CrT), the capacity of which is reduced in the failing heart, resulting in lower myocardial creatine concentration. Therefore, to gain insight into how the CrT is regulated, we studied two mouse models of severely altered myocardial creatine levels. Cardiac creatine uptake levels were measured in isolated hearts from creatine-free guanidinoacetate-N-methyl transferase knock out (GAMT−/−) mice and from mice overexpressing the myocardial CrT (CrT-OE) using 14C-radiolabeled creatine. CrT mRNA levels were measured using real time RT-PCR and creatine levels with HPLC. Hearts from GAMT−/− mice showed a 7-fold increase in Vmax of creatine uptake and a 1.4-fold increase in CrT mRNA levels. The increase in Cr uptake and in CrT mRNA levels, however, was almost completely prevented when mice were fed a creatine supplemented diet, indicating that creatine uptake is subject to negative feedback regulation. Cardiac creatine uptake levels in CrT-OE mice were increased on average 2.7-fold, showing a considerable variation, in line with a similar variation in creatine content. Total CrT mRNA levels correlated well with myocardial creatine content (r = 0.67; p < 0.0001) but endogenous CrT mRNA levels did not correlate at all with myocardial creatine content (r = 0.01; p = 0.96). This study shows that creatine uptake can be massively upregulated in the heart, by almost an order of magnitude and that this upregulation is subject to feedback inhibition. In addition, our results strongly suggest that CrT activity is predominantly regulated by mechanisms other than alterations in gene expression. PMID:18602925

Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.

PubMed

Voynow, J A; Young, L R; Wang, Y; Horger, T; Rose, M C; Fischer, B M

1999-05-01

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

Gene expression levels of heat shock proteins in the soleus and plantaris muscles of rats after hindlimb suspension or spaceflight.

PubMed

Ishihara, Akihiko; Fujino, Hidemi; Nagatomo, Fumiko; Takeda, Isao; Ohira, Yoshinobu

2008-12-01

Gene expression levels of heat shock proteins (HSPs) in the slow-twitch soleus and fast-twitch plantaris muscles of rats were determined after hindlimb suspension or spaceflight. Male rats were hindlimb-suspended for 14 d or exposed to microgravity for 9 d. The mRNA expression levels of HSP27, HSP70, and HSP84 in the hindlimb-suspended and microgravity-exposed groups were compared with those in the controls. The mRNA expression levels of the 3 HSPs in the soleus muscle under normal conditions were higher compared with those in the plantaris muscle. The mRNA expression levels of the 3 HSPs in the soleus muscle were inhibited by hindlimb suspension and spaceflight. The mRNA expression levels of the 3 HSPs in the plantaris muscle did not change after hindlimb suspension. It is suggested that the mRNA expression levels of the 3 HSPs are regulated by the mechanical and neural activity levels, and therefore the decreased mRNA expression levels of HSPs in the slow-twitch muscle following hindlimb suspension and spaceflight are related to a reduction in the mechanical and neural activity levels.

Actions of hypoxia on catecholamine synthetic enzyme mRNA expression before and after development of adrenal innervation in the sheep fetus

PubMed Central

Adams, M B; McMillen, I C

2000-01-01

We have investigated adrenal mRNA expression of the catecholamine synthetic enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) following acute hypoxia in fetal sheep before (< 105 days gestation, n = 20) and after (> 125 days gestation, n = 20) the development of adrenal innervation and following pretreatment with the nicotinic receptor anatgonist hexamethonium (n = 12). Total RNA was extracted from fetal adrenal glands collected at specific time points at 3-20 h after the onset of either hypoxia (∼50% reduction in fetal arterial oxygen saturation (SO2) for 30 min), or normoxia. Before 105 days, there was a decrease in adrenal TH mRNA expression at 20 h after hypoxia and adrenal TH mRNA expression was directly related to the changes in arterial PO2 measured during normoxia and hypoxia. After 125 days, adrenal TH mRNA levels were suppressed for up to 12 h following hypoxia. In both age groups, adrenal PNMT mRNA expression increased at 3-5 h after hypoxia and was inversely related to the changes in fetal arterial PO2 during normoxia or hypoxia. After 125 days, the administration of hexamethonium (25 mg kg−1, I. V.) reduced TH mRNA but not PNMT mRNA expression after normoxia. After hexamethonium pretreatment, there was no significant change in either adrenal TH or PNMT mRNA expression following hypoxia. We conclude that acute hypoxia differentially regulates adrenal TH and PNMT mRNA expression in the fetal sheep both before and after the development of adrenal innervation. After the development of adrenal innervation, however, the effect of acute hypoxia upon adrenal TH and PNMT mRNA expression is dependent upon neurogenic input acting via nicotinic receptors. PMID:11118487

Analysis of thyroid hormone receptor {beta}A mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Opitz, Robert; Lutz, Ilka; Nguyen, Ngoc-Ha

2006-04-01

Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors {alpha} (TR{alpha}) and {beta}A (TR{beta}A) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TR{alpha} gene expression whereas a marked up-regulation of TR{beta}A mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TR{beta}A mRNA in head and tail tissue withinmore » 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TR{beta}A mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TR{beta}A gene expression analysis for detection of agonistic and antagonistic effects on T3 action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TR{beta}A expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TR{beta}A mRNA up-regulation. Results of this study suggest that TR{beta}A mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.« less

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

PubMed

Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

2018-01-01

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P < 0.05 and more than 5.96% genes presented very strong correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

Endothelial glucocorticoid receptor promoter methylation according to dexamethasone sensitivity

PubMed Central

Mata-Greenwood, Eugenia; Jackson, P Naomi; Pearce, William J; Zhang, Lubo

2016-01-01

We have previously shown that in vitro sensitivity to dexamethasone (DEX) stimulation in human endothelial cells is positively regulated by the glucocorticoid receptor (NR3C1, GR). The present study determined the role of differential GR transcriptional regulation in glucocorticoid sensitivity. We studied 25 human umbilical vein endothelial cells (HUVECs) that had been previously characterized as DEX-sensitive (n = 15), or resistant (n = 10). Real-time PCR analysis of GR 5′UTR mRNA isoforms showed that all HUVECs expressed isoforms 1B, 1C, 1D, 1F, and 1H, and isoforms 1B and 1C were predominantly expressed. DEX-resistant cells expressed higher basal levels of the 5′UTR mRNA isoforms 1C and 1D, but lower levels of the 5′UTR mRNA isoform 1F than DEX-sensitive cells. DEX treatment significantly decreased GRα and GR-1C mRNA isoform expression in DEX-resistant cells only. Reporter luciferase assays indicated that differential GR mRNA isoform expression was not due to differential promoter usage between DEX-sensitive and DEX-resistant cells. Analysis of promoter methylation, however, showed that DEX-sensitive cells have higher methylation levels of promoter 1D and lower methylation levels of promoter 1F than DEX-resistant cells. Treatment with 5-aza-2-deoxycytidine abolished the differential 5′UTR mRNA isoform expression between DEX-sensitive and DEX-resistant cells. Finally, both GRα overexpression and 5-aza-2-deoxycytidine treatment eliminated the differences between sensitivity groups to DEX-mediated downregulation of endothelial nitric oxide synthase (NOS3), and upregulation of plasminogen activator inhibitor 1 (SERPINE1). In sum, human endothelial GR 5′UTR mRNA expression is regulated by promoter methylation with DEX-sensitive and DEX-resistant cells having different GR promoter methylation patterns. PMID:26242202

A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Yuen; Wang, Yuan; Feng, Jinyan

2015-04-03

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within itsmore » 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.« less

Excessive innate immune response and mutant D222G/N in severe A (H1N1) pandemic influenza.

PubMed

Berdal, Jan-Erik; Mollnes, Tom E; Wæhre, Torgun; Olstad, Ole K; Halvorsen, Bente; Ueland, Thor; Laake, Jon H; Furuseth, May T; Maagaard, Anne; Kjekshus, Harald; Aukrust, Pål; Jonassen, Christine M

2011-10-01

Explore the role of viral factors and immune response in patients with severe pandemic pdmH1N1 illness without significant co-morbidity. Seven patients with pdmH1N1 influenza, bilateral chest X-rays infiltrates, requiring mechanical ventilator support were consecutively recruited. Seven age- and gender-matched healthy individuals served as controls. Four patients were viremic, two with the mutant D222G/N pdmH1N1.Microarray analyses of peripheral blood leukocytes suggested a marked granulocytes activation, but no up-regulation of inflammatory cytokine mRNA. Patients with severe pdmH1NI had a marked systemic complement activation, and in contrast to the lack of cytokine mRNA up-regulation in blood leukocytes, plasma levels of a broad range of inflammatory mediators, including IP-10, and mediators involved in pulmonary remodelling were markedly elevated. Patients with mutant virus had particularly high IP-10 levels, and the most pronounced complement activation. In severe pdmH1N1, viremia was common and the D222G/N mutant was found in half of the viremic patients. Host immune response was characterized by strong activation of the innate immune system, including complement and granulocytes activation, increased serum levels of inflammation and pulmonary remodelling markers, possibly contributing to the observed tissue damage. However, few patients were included and further studies are needed to characterize the immune response in severe pdmH1N1 infection. Copyright © 2011 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway

PubMed Central

Lin, Fei-xiang; Du, Shi-xin; Liu, De-zhong; Hu, Qin-xiao; Yu, Guo-yong; Wu, Chu-cheng; Zheng, Gui-zhou; Xie, Da; Li, Xue-dong; Chang, Bo

2016-01-01

Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway. PMID:27904711

Uridine protects cortical neurons from glucose deprivation-induced death: possible role of uridine phosphorylase.

PubMed

Choi, Ji Woong; Shin, Chan Young; Choi, Min Sik; Yoon, Seo Young; Ryu, Jong Hoon; Lee, Jae-Chul; Kim, Won-Ki; El Kouni, Mahmoud H; Ko, Kwang Ho

2008-06-01

We previously reported that uridine blocked glucose deprivation-induced death of immunostimulated astrocytes by preserving ATP levels. Uridine phosphorylase (UPase), an enzyme catalyzing the reversible phosphorylation of uridine, was involved in this effect. Here, we tried to expand our previous findings by investigating the uridine effect on the brain and neurons using in vivo and in vitro ischemic injury models. Orally administrated uridine (50-200 mg/kg) reduced middle cerebral artery occlusion (1.5 h)/reperfusion (22 h)-induced infarct in mouse brain. Additionally, in the rat brain subjected to the same ischemic condition, UPase mRNA and protein levels were up-regulated. Next, we employed glucose deprivation-induced hypoglycemia in mixed cortical cultures of neurons and astrocytes as an in vitro model. Cells were deprived of glucose and, two hours later, supplemented with 20 mM glucose. Under this condition, a significant ATP loss followed by death was observed in neurons but not in astrocytes, which were blocked by treatment with uridine in a concentration-dependent manner. Inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine blocked the uridine effect. Similar to our in vivo data, UPase expression was up-regulated by glucose deprivation in mRNA as well as protein levels. Additionally, 5-(phenylthio)acyclouridine, a specific inhibitor of UPase, prevented the uridine effect. Finally, the uridine effect was shown only in the presence of astrocytes. Taken together, the present study provides the first evidence that uridine protects neurons against ischemic insult-induced neuronal death, possibly through the action of UPase.

Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway.

PubMed

Lin, Fei-Xiang; Du, Shi-Xin; Liu, De-Zhong; Hu, Qin-Xiao; Yu, Guo-Yong; Wu, Chu-Cheng; Zheng, Gui-Zhou; Xie, Da; Li, Xue-Dong; Chang, Bo

2016-01-01

Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway.

Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268.

PubMed

Kimoto, Sohei; Bazmi, H Holly; Lewis, David A

2014-09-01

Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

Cinnamon extract regulates plasma levels of adipose-derived factors and expression of multiple genes related to carbohydrate metabolism and lipogenesis in adipose tissue of fructose-fed rats.

PubMed

Qin, B; Polansky, M M; Anderson, R A

2010-03-01

We reported earlier that dietary cinnamon extract (CE) improves systemic insulin sensitivity and dyslipidemia by enhancing insulin signaling. In the present study, we have examined the effects of CE on several biomarkers including plasma levels of adipose-derived adipokines, and the potential molecular mechanisms of CE in epididymal adipose tissue (EAT). In Wistar rats fed a high-fructose diet (HFD) to induce insulin resistance, supplementation with a CE (Cinnulin PF, 50 mg/kg daily) for 8 weeks reduced blood glucose, plasma insulin, triglycerides, total cholesterol, chylomicron-apoB48, VLDL-apoB100, and soluble CD36. CE also inhibited plasma retinol binding protein 4 (RBP4) and fatty acid binding protein 4 (FABP4) levels. CE-induced increases in plasma adiponectin were not significant. CE did not affect food intake, bodyweight, and EAT weight. In EAT, there were increases in the insulin receptor ( IR) and IR substrate 2 ( IRS2) mRNA, but CE-induced increases in mRNA expression of IRS1, phosphoinositide-3-kinase, AKT1, glucose transporters 1 and 4 , and glycogen synthase 1 expression and decreased trends in mRNA expression of glycogen synthase kinase 3beta were not statistically significant. CE also enhanced the mRNA levels of ADIPOQ, and inhibited sterol regulatory element binding protein-1c mRNA levels. mRNA and protein levels of fatty acid synthase and FABP4 were inhibited by CE and RBP4, and CD36 protein levels were also decreased by CE. These results suggest that CE effectively ameliorates circulating levels of adipokines partially mediated via regulation of the expression of multiple genes involved in insulin sensitivity and lipogenesis in the EAT.

Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells.

PubMed

Tang, Li-li; Wang, Rui; Tang, Xi-can

2005-06-01

To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). After being treated with 10 micromol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. AChE activity, mRNA and protein expression were measured by the Ellman method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Treatment of PC12 cells with 10 micromol/L HupA for 48 h markedly increased the number of neurite-bearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 micromol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 micromol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

PubMed

Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

2014-03-28

Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression. Copyright © 2014 Elsevier Inc. All rights reserved.

Human endogenous retrovirus expression is inversely related with the up-regulation of interferon-inducible genes in the skin of patients with lichen planus.

PubMed

Nogueira, Marcelle Almeida de Sousa; Gavioli, Camila Fátima Biancardi; Pereira, Nátalli Zanete; de Carvalho, Gabriel Costa; Domingues, Rosana; Aoki, Valéria; Sato, Maria Notomi

2015-04-01

Lichen planus (LP) is a common inflammatory skin disease of unknown etiology. Reports of a common transactivation of quiescent human endogenous retroviruses (HERVs) support the connection of viruses to the disease. HERVs are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancer and autoimmune diseases. We explored the transcriptional activity of HERV sequences as well as the antiviral restriction factor and interferon-inducible genes in the skin from LP patients and healthy control (HC) donors. The study included 13 skin biopsies from patients with LP and 12 controls. Real-time PCR assay identified significant decrease in the HERV-K gag and env mRNA expression levels in LP subjects, when compared to control group. The expressions of HERV-K18 and HERV-W env were also inhibited in the skin of LP patients. We observed a strong correlation between HERV-K gag with other HERV sequences, regardless the down-modulation of transcripts levels in LP group. In contrast, a significant up-regulation of the cytidine deaminase APOBEC 3G (apolipoprotein B mRNA-editing), and the GTPase MxA (Myxovirus resistance A) mRNA expression level was identified in the LP skin specimens. Other transcript expressions, such as the master regulator of type I interferon-dependent immune responses, STING (stimulator of interferon genes) and IRF-7 (interferon regulatory factor 7), IFN-β and the inflammassome NALP3, had increased levels in LP, when compared to HC group. Our study suggests that interferon-inducible factors, in addition to their role in innate immunity against exogenous pathogens, contribute to the immune control of HERVs. Evaluation of the balance between HERV and interferon-inducible factor expression could possibly contribute to surveillance of inflammatory/malignant status of skin diseases.

Expression of protease-activated receptor (PAR)-2, but not other PARs, is regulated by inflammatory cytokines in rat astrocytes.

PubMed

Sokolova, Elena; Aleshin, Stepan; Reiser, Georg

2012-02-01

Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS) and are believed to play an important role in normal brain functioning as well as in development of various inflammatory and neurodegenerative disorders. Pathological conditions cause altered expression of PARs in brain cells and therefore altered responsiveness to PAR activation. The exact mechanisms of regulation of PAR expression are not well studied. Here, we evaluated in rat astrocytes the influence of LPS, pro-inflammatory cytokines TNFα and IL-1β and continuous PAR activation by PAR agonists on the expression levels of PARs. These stimuli are important in inflammatory and neurological disorders, where their levels are increased. We report that LPS as well as cytokines TNFα and IL-1β affected only the PAR-2 level, but their effects were opposite. LPS and TNFα increased the functional expression of PAR-2, whereas IL-1β down-regulated the functional response of PAR-2. Agonists of PAR-1 specifically increased mRNA level of PAR-2, but not protein level. Transcript levels of other PARs were not changed after PAR-1 activation. Stimulation of the cells with PAR-2 or PAR-4 agonists did not alter PAR levels. We found that up-regulation of PAR-2 is dependent on PKC activity, mostly via its Ca²⁺-sensitive isoforms. Two transcription factors, NFκB and AP-1, are involved in up-regulation of PAR-2. These findings provide new information about the regulation of expression of PAR subtypes in brain cells. This is of importance for targeting PARs, especially PAR-2, for the treatment of CNS disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

Estradiol and progesterone regulate the expression of insulin-like growth factor-I receptor and insulin-like growth factor binding protein-2 in the hypothalamus of adult female rats.

PubMed

Cardona-Gómez, G P; Chowen, J A; Garcia-Segura, L M

2000-06-05

Gonadal hormones interact with insulin-like growthfactor-I (IGF-I) to regulate synaptic plasticity during the estrous cycle in the rat mediobasal hypothalamus. It has been proposed that tanycytes, specialized glial cells lining the ventral region of the third ventricle, may regulate the availability of IGF-I to hypothalamic neurons. IGF-I levels in tanycytes fluctuate during the estrous cycle. Furthermore, estrogen administration to ovariectomized rats increases IGF-I levels in tanycytes, while progesterone, injected simultaneously with estrogen, blocks the estrogen-induced increase of IGF-I levels in tanycytes. To test whether hormonal regulation of IGF-I receptor (IGF-IR) and IGF binding protein-2 (IGFBP-2) may be involved in the accumulation of IGF-I in tanycytes, we assessed the effect of ovarian hormones on the levels of these molecules in the mediobasal hypothalamus of adult female rats. Ovariectomized animals were treated with either oil, estrogen, progesterone, or estrogen and progesterone simultaneously and then killed 6 or 24 h later. Some neurons, some astrocytes, and many tanycytes in the mediobasal hypothalamus were found by confocal microscopy to be immunoreactive for IGF-IR. IGFBP-2 immunoreactivity was restricted almost exclusively to tanycytes and ependymal cells and was colocalized with IGF-IR immunoreactivity in tanycytes. By electron microscope immunocytochemistry using colloidal gold labeling, IGF-IR and IGFBP-2 immunoreactivities were observed in the microvilli of tanycytes in the lumen of the third ventricle. IGF-IR and IGFBP-2 immunoreactive levels on the apical surface of tanycytes were significantly decreased by the administration of progesterone, either alone or in the presence of estradiol. IGF-IR levels in the mediobasal hypothalamus, measured by Western blotting, were not significantly affected by the separate administration of estradiol or progesterone to ovariectomized rats. However, the simultaneous administration of both hormones resulted in a marked decrease in IGF-IR protein levels. Estradiol administration to ovariectomized rats increased IGFBP-2 immunoreactive levels in the hypothalamus. While progesterone did not significantly affect IGFBP-2 expression, the simultaneous injection of estradiol and progesterone resulted in a marked decrease in IGFBP-2 protein levels. The effect of estradiol on IGFBP-2 was observed both in protein and mRNA levels, suggesting a transcriptional regulation. However, the simultaneous administration of progesterone and estradiol had different effects on IGF-IR protein and IGF-IR mRNA levels, as well as on IGFBP-2 protein and IGFBP-2 mRNA levels, suggesting a postranscriptional action. These findings indicate that estradiol and progesterone regulate the expression of IGF-IR and IGFBP-2 in the mediobasal hypothalamus of adult female rats. Regulation of the hypothalamic IGF-I system by ovarian hormones may be physiologically relevant for neuroendocrine regulation and for synaptic plasticity during the estrous cycle. These results do not support the hypothesis that estrogen-induced accumulation of IGF-I by tanycytes is mediated by the hormonal regulation of IGF-IR. However, estrogen-induced up-regulation of IGFBP-2 and progesterone-induced down-regulation of IGF-IR and IGFBP-2 levels in the apical plasma membrane of tanycytes may be involved in the fluctuation of IGF-I levels in the mediobasal hypothalamus during the estrous cycle. Copyright 2000 John Wiley & Sons, Inc.

Insulin immuno-neutralization decreases food intake in chickens without altering hypothalamic mRNA levels for genes involved in regulation of food intake and metabolism

USDA-ARS?s Scientific Manuscript database

Chickens are characterized by rather unique glucose homeostasis, with relatively high blood glucose levels, reduced glucose sensitivity of pancreatic cells, and large resistance to exogenous insulin. In mammals, insulin regulates blood glucose level but also plays a key role in appetite regulation ...

Expression of Fushi tarazu factor 1 homolog and Pit-1 genes in the pituitaries of pre-spawning chum and sockeye salmon.

PubMed

Higa, M; Ando, H; Urano, A

2001-06-01

Fushi tarazu factor-1 (FTZ-F1) and Pit-1 are major pituitary transcription factors, controlling expression of genes coding for gonadotropin (GTH) subunits and growth hormone/prolactin/somatolactin family hormone, respectively. As a first step to investigate physiological factors regulating gene expression of these transcription factors, we determined their mRNA levels in the pituitaries of chum salmon (Oncorhynchus keta) at different stages of sexual maturation. FTZ-F1 gene expression was increased in males at the stage before spermiation, where the levels of GTH alpha and IIbeta subunit mRNAs were elevated. Pit-1 mRNA showed maximum levels at the final stage of sexual maturation in both sexes, when expression of somatolactin gene peaked. To clarify whether gonadotropin-releasing hormone (GnRH) is involved in these increases in FTZ-F1 and Pit-1 gene expression, we examined effects of GnRH analog (GnRHa) administration on their gene expression in maturing sockeye salmon (Oncorhynchus nerka). GnRHa stimulated Pit-1 gene expression in females only, but failed to stimulate FTZ-F1 gene expression in both sexes. The up-regulated expression of FTZ-F1 and Pit-1 genes at the pre-spawning stages suggest that the two transcription factors have roles in sexual maturation of salmonids. Physiological factors regulating gene expression of FTZ-F1 and Pit-1 are discussed in this review.

Regulation of low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase expression by Zingiber officinale in the liver of high-fat diet-fed rats.

PubMed

Nammi, Srinivas; Kim, Moon S; Gavande, Navnath S; Li, George Q; Roufogalis, Basil D

2010-05-01

Zingiber officinale has been used to control lipid disorders and reported to possess remarkable cholesterol-lowering activity in experimental hyperlipidaemia. In the present study, the effect of a characterized and standardized extract of Zingiber officinale on the hepatic lipid levels as well as on the hepatic mRNA and protein expression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was investigated in a high-fat diet-fed rat model. Rats were treated with an ethanol extract of Zingiber officinale (400 mg/kg) extract along with a high-fat diet for 6 weeks. The extract of Zingiber officinale significantly decreased hepatic triglyceride and tended to decrease hepatic cholesterol levels when administered over 6 weeks to the rats fed a high-fat diet. We found that in parallel, the extract up-regulated both LDL receptor mRNA and protein level and down-regulated HMG-CoA reductase protein expression in the liver of these rats. The metabolic control of body lipid homeostasis is in part due to enhanced cholesterol biosynthesis and reduced expression of LDL receptor sites following long-term consumption of high-fat diets. The present results show restoration of transcriptional and post-transcriptional changes in low-density lipoprotein and HMG CoA reductase by Zingiber officinale administration with a high-fat diet and provide a rational explanation for the effect of ginger in the treatment of hyperlipidaemia.

Experimental colitis triggers the release of substance P and calcitonin gene-related peptide in the urinary bladder via TRPV1 signaling pathways

PubMed Central

Pan, Xiao-Qing; Gonzalez, Jessica A.; Chang, Shaohua; Chacko, Samuel; Wein, Alan J.; Malykhina, Anna P.

2010-01-01

Clinical data provides evidence of high level of co-morbidity among genitourinary and gastrointestinal disorders characterized by chronic pelvic pain. The objective of this study was to test the hypothesis that colonic inflammation can impact the function of the urinary bladder via activation of TRPV1 signaling pathways followed by alterations in gene and protein expression of Substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory neurons and in the bladder. Inflammation was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS, 12.5 mg/kg) and desensitization of TRPV1 receptors was evoked by intracolonic resiniferatoxin (RTX, 10−7 M). mRNA and protein concentrations of CGRP and SP were measured at 3, 5 and 30 days. RTX instillation in the colon caused 3-fold up-regulation of SP mRNA in the urinary bladder at day 5 (n=7, p≤0.05) followed by 35-fold increase at day 30 (n=5, p≤0.05). Likewise, TNBS colitis triggered 15.8-fold up-regulation of SP mRNA one month after TNBS (n=5, p≤0.05). Desensitization of colonic TRPV1 receptors prior to TNBS abolished SP increase in the urinary bladder. RTX led to 4.3-fold increase of CGRP mRNA at day 5 (n=7, p≤0.05 to control) in the bladder followed by 28-fold increase at day 30 post-RTX (n=4, p≤0.05). Colitis did not alter CGRP concentration during acute phase, however, at day 30 mRNA level was increased by 17.8±6.9 fold (n=5, p≤0.05) in parallel with 4-fold increase in CGRP protein (n=5, p≤0.01) in the detrusor. Protein concentration of CGRP in the spinal cord was diminished by 45–65% (p≤0.05) during colitis. RTX pretreatment did not affect CGRP concentration in the urinary bladder, however, caused a reduction in CGRP release from lumbosacral DRG neurons during acute phase (3 and 5 days post-TNBS). Our results clearly demonstrate that colonic inflammation triggers the release of pro-inflammatory neuropeptides SP and CGRP in the urinary bladder via activation of TRPV1 signaling mechanisms enunciating the neurogenic nature of pelvic organ cross-sensitization. PMID:20501335

Effect of dietary copper addition on lipid metabolism in rabbits

PubMed Central

Lei, Liu; Xiaoyi, Sui; Fuchang, Li

2017-01-01

ABSTRACT The present study was conducted to investigate the effect of copper supplementation on lipid metabolism in rabbits. Our study showed dietary copper addition (5-45 mg/kg) increased body mass gain, but decreased fat and liver weights compared with those in the control group (P < 0.05). Copper (45 mg/kg) addition significantly increased the skeletal muscle weight, but inhibited cytoplasmic lipid accumulation in liver, skeletal muscle and adipose tissue (P < 0.05). Compared with the control group, dietary copper addition (45 mg/kg) significantly increased plasma triglyceride levels but decreased very low density lipoprotein levels (P < 0.05). Copper treatment significantly increased gene expression of carnitine palmitoyltransferase (CPT) 1, CPT2 and peroxisome proliferator-activated receptor (PPAR) a in liver (P < 0.05). In skeletal muscle, CPT1, CPT2, fatty acid transport protein, fatty acid-binding protein, and PPARa mRNA as well as phosphorylated AMP-activated protein kinase (AMPK) levels were significantly up-regulated by copper treatment (P < 0.05). Rabbits receiving copper supplementation had higher CPT1, CPT2, PPARa and hormone-sensitive lipase mRNA levels in adipose tissue (P < 0.05). In conclusion, copper promoted skeletal muscle growth and reduced fat accretion. PPARa signaling in liver, skeletal muscle and adipose tissues and AMPK signaling in skeletal muscle tissue were involved in the regulation of lipid metabolism by copper. PMID:28747869

Increased CSF aquaporin-4, and interleukin-6 levels in dogs with idiopathic communicating internal hydrocephalus and a decrease after ventriculo-peritoneal shunting.

PubMed

Schmidt, Martin J; Rummel, Christoph; Hauer, Jessica; Kolecka, Malgorzata; Ondreka, Nele; McClure, Vanessa; Roth, Joachim

2016-06-29

Studies in animal models, in which internal hydrocephalus has been induced by obstructing the cerebrospinal fluid pathways, have documented an up-regulation of the concentrations of aquaporin-4 (AQP4) in the brain. In this study, the concentrations of aquaporin-1 (AQP1), AQP1, AQP4 and interleukin-6 (IL-6) were determined in the CSF of dogs with idiopathic communicating hydrocephalus before and after the reduction of intraventricular volume following ventriculo-peritoneal shunt (VP-shunt) treatment. The concentrations of AQP4 and IL-6 were increased in the cerebrospinal fluid of dogs with hydrocephalus compared to controls. Both parameters significantly decreased after surgical treatment, accompanied by decrease of ventricular size and the clinical recovery of the dogs. AQP1 was not detectable in CSF. Brain AQP4 up-regulation might be a compensatory response in dogs with hydrocephalus. Future determination of AQP4 at the mRNA and protein level in brain tissue is warranted to substantiate this hypothesis.

Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, P.-Y.; Hsu, S.-P.; Liang, Y.-C.

2008-05-15

Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstratedmore » that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest.« less

Ursolic Acid Attenuates Diabetic Mesangial Cell Injury through the Up-Regulation of Autophagy via miRNA-21/PTEN/Akt/mTOR Suppression

PubMed Central

Lu, Xinxing; Fan, Qiuling; Xu, Li; Li, Lin; Yue, Yuan; Xu, Yanyan; Su, Yan; Zhang, Dongcheng; Wang, Lining

2015-01-01

Objective To investigate the effect of ursolic acid on autophagy mediated through the miRNA-21-targeted phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in rat mesangial cells cultured under high glucose (HG) conditions. Methods Rat glomerular mesangial cells were cultured under normal glucose, HG, HG with the PI3K inhibitor LY294002 or HG with ursolic acid conditions. Cell proliferation and hypertrophy were assayed using an MTT assay and the ratio of total protein to cell number, respectively. The miRNA-21 expression was detected using RT-qPCR. The expression of phosphatase and tensin homolog (PTEN)/AKT/mTOR signaling signatures, autophagy-associated protein and collagen I was detected by western blotting and RT-qPCR. Autophagosomes were observed using electron microscopy. Results Compared with mesangial cells cultured under normal glucose conditions, the cells exposed to HG showed up-regulated miRNA-21 expression, down-regulated PTEN protein and mRNA expression, up-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and down-regulated LC3II expression. Ursolic acid and LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation, down-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and up-regulated LC3II expression. However, LY294002 did not affect the expression of miRNA-21 and PTEN. Ursolic acid down-regulated miRNA-21 expression and up-regulated PTEN protein and mRNA expression. Conclusions Ursolic acid inhibits the glucose-induced up-regulation of mesangial cell miRNA-21 expression, up-regulates PTEN expression, inhibits the activation of PI3K/Akt/mTOR signaling pathway, and enhances autophagy to reduce the accumulation of the extracellular matrix and ameliorate cell hypertrophy and proliferation. PMID:25689721

Intravenous infusion of hexamethonium and atropine but not propranolol diminishes apolipoprotein A-IV gene expression in rat ileum.

PubMed

Sonoyama, K; Tajima, K; Fujiwara, R; Kasai, T

2000-03-01

To clarify the role of neural factors in the regulation of apolipoprotein (apo) A-IV expression in the small intestine, we investigated the effect of neural blockers on mRNA levels of apo A-IV in rat small intestine. Either ganglionic blocker (hexamethonium), cholinergic blocker (atropine) or beta-adrenergic blocker (propranolol) was infused intravenously to unrestrained conscious rats for 8 h, and then total RNA was isolated from the small intestine and analyzed using Northern hybridization. Apo A-IV mRNA levels in the ileum were significantly lower in hexamethonium- or atropine-infused rats than in saline- (control) or propranolol-infused rats. Immunoblot analysis showed no difference in plasma apo A-IV concentrations between hexamethonium- and saline-infused groups. The lower mRNA levels of apo A-IV in the ileum of hexamethonium-infused rats were observed even in bile-drained rats, indicating that the lower expression was not due to any changes in bile availability. The ileal apo A-IV mRNA levels were significantly higher in rats infused with lipid emulsion into the ileum than in rats infused with glucose-saline, and the concomitant infusion of intravenous hexamethonium did not affect the higher levels of apo A-IV mRNA. These results suggest that the basal expression of the ileal A-IV gene is at least partially regulated in a site-specific manner by cholinergic neurons.

Conserved small mRNA with an unique, extended Shine-Dalgarno sequence

PubMed Central

Hahn, Julia; Migur, Anzhela; von Boeselager, Raphael Freiherr; Kubatova, Nina; Kubareva, Elena; Schwalbe, Harald

2017-01-01

ABSTRACT Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level. PMID:27834614

The response of the natriuretic peptide system to water deprivation in the desert rodent, Notomys alexis.

PubMed

Heimeier, Rachel A; Donald, John A

2006-02-01

Natriuretic peptides (NPs) are regulatory molecules that cause cGMP-mediated diuresis and natriuresis in mammals. Accordingly, it is interesting to consider their role in desert-adapted animals in which water is often limited. This study investigated the response of the natriuretic peptide (NP) system to varying periods of water deprivation (WD) in the Australian desert rodent species, Notomys alexis. It was hypothesised that the expression of the NP system will be down-regulated in water-deprived N. alexis compared to water-replete animals. The plasma levels of ANP were significantly reduced after 3 days of WD, but were unaffected by 7, 14 and 28 days of WD. Water deprivation for 3, 7, 14 days had a variable effect on the mRNA expression of ANP, CNP, NPR-A, NPR-B, and NPR-C, and a uniform down-regulation was not observed. However, after 28 days of WD, mRNA expression was similar to water-replete animals, except for NPR-A. Surprisingly, 7 and 14 days of WD caused an up-regulation in the ability of ANP to stimulate cGMP; this also occurred at 14 days for CNP. Taken together, the mRNA expression and peptide mediated guanylyl cyclase activity data after WD were in the opposite direction to what was predicted. Interestingly, after 28 days of WD, most parameters were similar to those of water-replete animals, which indicates that a down-regulation of the NP system is not part of the physiological response to an absence of free water in N. alexis.

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

PubMed Central

Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

2014-01-01

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

Modulatory effects of steroid hormones, oxytocin, arachidonic acid, forskolin and cyclic AMP on the expression of aquaporin 1 and aquaporin 5 in the porcine uterus during placentation.

PubMed

Skowronska, A; Mlotkowska, P; Okrasa, S; Nielsen, S; Skowronski, M T

2016-04-01

Aquaporins (AQPs) are proteins forming trans-membrane channels responsible for water transport. AQP1 and AQP5 are present in structures of the female reproductive system. In the uterus, these AQPs are involved in water movement between the intraluminal, interstitial and capillary compartments and their uterine expression is essential throughout the pregnancy, including its early stages. Thus, the study aimed to assess the influence of P4 (progesterone), E2 (estradiol), OT (oxytocin), AA (arachidonic acid), cAMP and FSK (forskolin) on the AQP1 and AQP5 mRNA and protein expression in the uterine tissue of gilts on Days 30 - 32 of gestation (the placentation period), following short (3 h) and long (24 h) incubations. Steroid hormones influenced the expression of AQP1 and AQP5; E2 up-regulated, but P4 down-regulated mRNAs of these AQPs, whereas the protein level of studied AQPs was increased by both steroids. OT treatment decreased AQP1 (after 24 h), but increased AQP5 (after 3 h) mRNA expression. Treatment with AA significantly reduced the AQP1 expression at the mRNA level, but stimulated at the protein level. The expression of AQP5 mRNA and protein was stimulated by AA. FSK markedly decreased AQP1 mRNA, but increased of AQP5 after 3-h incubation. In turn, cAMP stimulated and inhibited transcription of AQP5 after 3- and 24-h incubations, respectively. Immunohistochemical analysis confirmed the uterine localization of AQP1 in the apical and basal membranes of endothelial cells and AQP5 in the apical membranes of epithelial cells under control condition. Treatments with P4, E2, AA, cAMP or FSK have caused additional appearance of AQP5 labeling in the basolateral membranes of epithelial cells. These results suggest a participation of steroid hormones (P4 and E2), AA derivatives and cAMP in controlling the expression of AQP1 and AQP5 as well as the distribution of AQP5 in the uterine tissue of pregnant gilts during placentation (Days 30 - 32 of gestation).

The comparison of the rejuvenation effects on the skin of Wistar rats between 10600 nm CO2 fractional laser and retinoic acid.

PubMed

Qu, Y; Ma, W-Y; Sun, Q

2017-04-01

The fractional laser and topical retinoic acid treatment have been applied for skin rejuvenation; however, the possible molecular mechanism of promoting remodeling of dermis is not clearly. Here we aimed to compare the effects of 10600 nm CO2 fractional laser and topical retinoic acid formulation on the skin collagen proliferation of Wistar rats, and to further explore the possible molecular mechanism of promoting remodeling of dermis. The hair on the back of Wistar rats was removed, and the back was divided equally into four regions with the cross-streaking method: A (the control group), B (the retinoic acid group), C (retinoic acid and fractional laser combination treatment group), and D (the fractional laser group). Specimens were collected at 3rd day and in 1-8 weeks after CO2 fractional laser irradiation; then they were used for detection of the changes of dermis thickness and content of hydroxyproline in the four regions of the rats' back. Real-time PCR method was used to detect the dynamic changes of the expression level of type III procollagen mRNA and the expression levels of miR-29a, Akt and transforming growth factor-β (TGF-β) mRNA at 3rd week in the skin tissue of Wistar rats. The thickness of dermis, content of hydroxyproline and expression level of type III procollagen mRNA in the treatment groups (B, C, and D) were found all significantly increased compared with those in the control group (A) (p<0.05); at 3rd week, up-regulation of Akt and TGF-β mRNA expression and down-regulation of miR-29a mRNA expression were observed in the treatment groups (B, C, and D). The difference in the combination treatment group (C) was the most significant (p<0.05). These results demonstrate that retinoic acid formulation and CO2 fractional laser both can promote collagen proliferation and reconstruction, with the skin rejuvenation efficacy in group C > group D > group B. miR-29a/Akt/TGF-β signal pathways may play a certain role in the promotion of collagen synthesis and proliferation.

Maternal distress associates with placental genes regulating fetal glucocorticoid exposure and IGF2: Role of obesity and sex.

PubMed

Mina, Theresia H; Räikkönen, Katri; Riley, Simon C; Norman, Jane E; Reynolds, Rebecca M

2015-09-01

Maternal emotional distress symptoms, including life satisfaction, anxiety and depressed mood, are worse in Severely Obese (SO) than lean pregnancy and may alter placental genes regulating fetal glucocorticoid exposure and placental growth. We hypothesised that the associations between increased maternal distress symptoms and changes in placental gene expression including IGF2 and genes regulating fetal glucocorticoid exposure are more pronounced in SO pregnancy. We also considered whether there were sex-specific effects. Placental mRNA levels of 11β-HSDs, NR3C1-α, NR3C2, ABC transporters, mTOR and the IGF2 family were measured in term placental samples from 43 lean (BMI≤25kg/m(2)) and 50 SO (BMI≥40kg/m(2)) women, in whom distress symptoms were prospectively evaluated during pregnancy. The mRNA levels of genes with a similar role in regulating fetal glucocorticoid exposure were strongly inter-correlated. Increased maternal distress symptoms associated with increased NR3C2 and IGF2 isoform 1(IGF2-1) in both lean and SO group (p≤0.05). Increased distress was associated with higher ABCB1 and ABCG2 mRNA levels in SO but lower ABCB1 and higher 11β-HSD1 mRNA levels in lean (p≤0.05) suggesting a protective adaptive response in SO placentas. Increased maternal distress associated with reduced mRNA levels of ABCB1, ABCG2, 11β-HSD2, NR3C1-α and IGF2-1 in placentas of female but not male offspring. The observed sex differences in placental responses suggest greater vulnerability of female fetuses to maternal distress with potentially greater fetal glucocorticoid exposure and excess IGF2. Further studies are needed to replicate these findings and to test whether this translates to potentially greater negative outcomes of maternal distress in female offspring in early childhood. Copyright © 2015 Elsevier Ltd. All rights reserved.

Induction of Anthocyanin Accumulation by Cytokinins in Arabidopsis thaliana.

PubMed Central

Deikman, J.; Hammer, P. E.

1995-01-01

Arabidopsis thaliana plants treated with exogenous cytokinins accumulate anthocyanin pigments. We have characterized this response because it is potentially useful as a genetic marker for cytokinin responsiveness. Levels of mRNAs for four genes of the anthocyanin biosynthesis pathway, phenylalanine ammonia lyase 1 (PAL1), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) were shown to increase coordinately in response to benzyladenine (BA). However, nuclear run-on transcription experiments suggested that although CHS and DFR are controlled by BA at the transcriptional level, PAL1 and CHI are controlled by BA posttranscriptionally. CHS mRNA levels increased within 2 h of BA spray application, and peaked by 3 h. Levels of PAL1 mRNA did not increase within 6 h of BA spray. We also showed that PAL1, CHS, CHI, and DFR mRNA levels fluctuate during a 24-h period and appear to be controlled by a circadian clock. The relation between cytokinin regulation and light regulation of CHS gene transcription is discussed. PMID:12228453

Food restriction attenuates oxidative stress in brown adipose tissue of striped hamsters acclimated to a warm temperature.

PubMed

Zhang, Ji-Ying; Zhao, Xiao-Ya; Wang, Gui-Ying; Wang, Chun-Ming; Zhao, Zhi-Jun

2016-05-01

It has been suggested that the up-regulation of uncoupling proteins (UCPs) decreases reactive oxygen species (ROS) production, in which case there should be a negative relationship between UCPs expression and ROS levels. In this study, the effects of temperature and food restriction on ROS levels and metabolic rate, UCP1 mRNA expression and antioxidant levels were examined in the brown adipose tissue (BAT) of the striped hamsters (Cricetulus barabensis). The metabolic rate and food intake of hamsters which had been restricted to 80% of ad libitum food intake, and acclimated to a warm temperature (30°C), decreased significantly compared to a control group. Hydrogen peroxide (H2O2) levels were 42.9% lower in food restricted hamsters than in the control. Malonadialdehyde (MDA) levels of hamsters acclimated to 30°C that were fed ad libitum were significantly higher than those of the control group, but 60.1% lower than hamsters that had been acclimated to the same temperature but subject to food restriction. There were significantly positive correlations between H2O2 and, MDA levels, catalase activity, and total antioxidant capacity. Cytochrome c oxidase activity and UCP1 mRNA expression significantly decreased in food restricted hamsters compared to the control. These results suggest that warmer temperatures increase oxidative stress in BAT by causing the down-regulation of UCP1 expression and decreased antioxidant activity, but food restriction may attenuate the effects. Copyright © 2016 Elsevier Ltd. All rights reserved.