Expression of the 68-kilodalton neurofilament gene in aluminum intoxication

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

Muma, N.A.; Troncoso, J.C.; Hoffman, P.N.

1986-03-01

Intrathecal administration of aluminum salts induces accumulation of neurofilaments (NFs) in cell bodies and proximal axons of rabbit spinal motor neurons. Mechanisms leading to this pathological change are not well understood. Although impairments of NF transport have been demonstrated in this model, the hypothesis that NF accumulations are the result of an increase in NF synthesis needs to be explored. In rabbits, a large percentage of neurons develop accumulations of NFs following injections of aluminum lactate directly into the cisterna magna or into a reservoir placed in the lateral ventricle. To study levels of mRNA encoding cytoskeletal proteins, spinal cordmore » RNA was extracted, separated on a denaturing agarose gel, transferred to nitrocellulose paper, and hybridized to (/sup 32/P)-labeled cDNA clones encoding the mouse 68-kilodalton (kd) NF subunit and tubulin. Examining a constant amount of RNA, the radioactivity of labeled mRNA bands for the 68-kd NF subunit and for tubulin was decreased in spinal cords of aluminum-treated rabbits. These preliminary results will be followed up by in situ hybridization to determine levels of mRNA for tubulin and 68-kd NF subunit in affected and in normal spinal neurons. In conclusion, administration of aluminum decreased mRNA for the 608-kd NF protein in spinal neurons.« less

Alcohol Withdrawal-Induced Seizure Susceptibility is Associated with an Upregulation of CaV1.3 Channels in the Rat Inferior Colliculus

PubMed Central

Akinfiresoye, Luli R.; Allard, Joanne S.; Lovinger, David M.

2015-01-01

Background: We previously reported increased current density through L-type voltage-gated Ca2+ (CaV1) channels in inferior colliculus (IC) neurons during alcohol withdrawal. However, the molecular correlate of this increased CaV1 current is currently unknown. Methods: Rats received three daily doses of ethanol every 8 hours for 4 consecutive days; control rats received vehicle. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the CaV1.3 and CaV1.2 α1 subunits were measured. In separate experiments, rats were tested for their susceptibility to alcohol withdrawal–induced seizures (AWS) 3, 24, and 48 hours after alcohol withdrawal. Results: In the alcohol-treated group, AWS were observed 24 hours after withdrawal; no seizures were observed at 3 or 48 hours. No seizures were observed at any time in the control-treated rats. Compared to control-treated rats, the mRNA level of the CaV1.3 α1 subunit was increased 1.4-fold, 1.9-fold, and 1.3-fold at 3, 24, and 48 hours, respectively. In contrast, the mRNA level of the CaV1.2 α1 subunit increased 1.5-fold and 1.4-fold at 24 and 48 hours, respectively. At 24 hours, Western blot analyses revealed that the levels of the CaV1.3 and CaV1.2 α1 subunits increased by 52% and 32%, respectively, 24 hours after alcohol withdrawal. In contrast, the CaV1.2 and CaV1.3 α1 subunits were not altered at either 3 or 48 hours during alcohol withdrawal. Conclusions: Expression of the CaV1.3 α1 subunit increased in parallel with AWS development, suggesting that altered L-type CaV1.3 channel expression is an important feature of AWS pathogenesis. PMID:25556199

Adaptative decrease in expression of the mRNA for uncoupling protein and subunit II of cytochrome c oxidase in rat brown adipose tissue during pregnancy and lactation.

PubMed Central

Martin, I; Giralt, M; Viñas, O; Iglesias, R; Mampel, T; Villarroya, F

1989-01-01

Uncoupling-protein (UCP) mRNA expression is decreased to 15% of virgin control levels between days 10 and 15 of pregnancy, and remains at these low values during late pregnancy and lactation. Abrupt weaning of mid-lactating rats causes a slight but significant increase in UCP mRNA. Expression of mRNA for subunit II of cytochrome c oxidase (COII) decreased to half that of virgin control in late pregnancy and during lactation. Whereas COII mRNA expression is in step with the known modifications of brown-fat mitochondria content during the breeding cycle of the rat, UCP mRNA expression appears to be diminished much earlier than the mitochondrial proton-conductance-pathway activity. On the other hand, the reactivity of brown fat to increase expression of UCP and COII mRNAs in response to acute cold or noradrenaline treatment is not impaired during lactation. Images Fig. 1. Fig. 2. Fig. 3. PMID:2557014

The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

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

Watanabe, Kanako; Department of Anesthesiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501; Kanno, Takeshi

2008-03-07

The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression ofmore » the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.« less

[Effect of electroacupuncture on expression of ionotropic glutamate receptor subunits and their genes in lumbar segments of spinal cord in rats with neuropathic pain].

PubMed

Ma, Cheng; Yu, Li; Yan, Li-ping

2010-12-01

To observe the effect of electroacupuncture (EA) on the expression of ionotropic glutamate receptor (iGluR) subunits and their mRNAs in the lumbar segments of spinal cord in rats with neuropathic pain, so as to explore its underlying mechanism in relieving spinal hyperalgesia. Thirty SD rats were randomly divided into control, model, and EA groups, with 10 rats in each. The spared nerve injury (SNI) model was established by ligature of the sural nerve after cutting off the common peroneal nerve and anterior tibial nerve. EA (2 Hz, 1 mA) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) for 30 min, once daily for 7 days. Mechanical pain threshold was detected before and after modeling and before and after EA treatment. The expression levels of N-methyl-d-aspartic acid (NMDA) receptor subunits NR1 and NR 2 B,and AMPA receptor subunit GluR 1 of iGluR and their genes were assayed by Western blot and reverse transcription polymerase chain reaction (RT-PCR) separately. In comparison with control group, the mechanical pain thresholds were decreased significantly on day 2, 7 and day 14 following modeling in the model group (P < 0.05, P < 0.01). While compared with the model group, the pain threshold was increased considerably on day 14 in the EA group (P < 0.01). Compared with the control group, the expression levels of lumbar spinal cord NR 2 B and NR 2 B mRNA in the model group were increased significantly (P < 0.05), and those of lumbar spinal cord NR 1 and NR 1 mRNA, GluR 1 and GluR 1 mRNA in the model group increased slightly (P > 0.05). In comparison with the model group, the expression levels of lumbar spinal cord NR 2 B and NR 2 B mRNA in the EA group were downregulated remarkably (P < 0.05), and those of lumbar spinal cord NR 1 and NR 1 mRNA, GluR 1 and GluR 1 mRNA in the EA group down-regulated slightly (P > 0.05). EA can significantly suppress pain reaction in rats with neuropathic pain probably through down-regulating the expression of lumbar spinal cord NR 2 B protein and NR 2 B mRNA.

Exercise increases the plasma membrane content of the Na+ -K+ pump and its mRNA in rat skeletal muscles.

PubMed

Tsakiridis, T; Wong, P P; Liu, Z; Rodgers, C D; Vranic, M; Klip, A

1996-02-01

Muscle fibers adapt to ionic challenges of exercise by increasing the plasma membrane Na+-K+ pump activity. Chronic exercise training has been shown to increase the total amount of Na+-K+ pumps present in skeletal muscle. However, the mechanism of adaptation of the Na+-K+ pump to an acute bout of exercise has not been determined, and it is not known whether it involves alterations in the content of plasma membrane pump subunits. Here we examine the effect of 1 h of treadmill running (20 m/min, 10% grade) on the subcellular distribution and expression of Na+-K+ pump subunits in rat skeletal muscles. Red type I and IIa (red-I/IIa) and white type IIa and IIb (white-IIa/IIb) hindlimb muscles from resting and exercised female Sprague-Dawley rats were removed for subcellular fractionation. By homogenization and gradient centrifugation, crude membranes and purified plasma membranes were isolated and subjected to gel electrophoresis and immunoblotting by using pump subunit-specific antibodies. Furthermore, mRNA was isolated from specific red type I (red-I) and white type IIb (white-IIb) muscles and subjected to Northern blotting by using subunit-specific probes. In both red-I/IIa and white-IIa/IIb muscles, exercise significantly raised the plasma membrane content of the alpha1-subunit of the pump by 64 +/- 24 and 55 +/- 22%, respectively (P < 0.05), and elevated the alpha2-polypeptide by 43 +/- 22 and 94 +/- 39%, respectively (P < 0.05). No significant effect of exercise could be detected on the amount of these subunits in an internal membrane fraction or in total membranes. In addition, exercise significantly increased the alpha1-subunit mRNA in red-I muscle (by 50 +/- 7%; P < 0.05) and the beta2-subunit mRNA in white-IIb muscles (by 64 +/- 19%; P < 0.01), but the alpha2- and beta1-mRNA levels were unaffected in this time period. We conclude that increased presence of alpha1- and alpha2-polypeptides at the plasma membrane and subsequent elevation of the alpha1- and beta2-subunit mRNAs may be mechanisms by which acute exercise regulates the Na+-K+ pump of skeletal muscle.

N-Acetylcysteine-induced vasodilatation is modulated by KATP channels, Na+/K+-ATPase activity and intracellular calcium concentration: An in vitro study.

PubMed

Vezir, Özden; Çömelekoğlu, Ülkü; Sucu, Nehir; Yalın, Ali Erdinç; Yılmaz, Şakir Necat; Yalın, Serap; Söğüt, Fatma; Yaman, Selma; Kibar, Kezban; Akkapulu, Merih; Koç, Meryem İlkay; Seçer, Didem

2017-08-01

In this study, we aimed to investigate the role of ATP-sensitive potassium (K ATP ) channel, Na + /K + -ATPase activity, and intracellular calcium levels on the vasodilatory effect of N-acetylcysteine (NAC) in thoracic aorta by using electrophysiological and molecular techniques. Rat thoracic aorta ring preparations and cultured thoracic aorta cells were divided into four groups as control, 2mM NAC, 5mM NAC, and 10mM NAC. Thoracic aorta rings were isolated from rats for measurements of relaxation responses and Na + /K + -ATPase activity. In the cultured thoracic aorta cells, we measured the currents of K ATP channel, the concentration of intracellular calcium and mRNA expression level of K ATP channel subunits (KCNJ8, KCNJ11, ABCC8 and ABCC9). The relaxation rate significantly increased in all NAC groups compared to control. Similarly, Na + /K + - ATPase activity also significantly decreased in NAC groups. Outward K ATP channel current significantly increased in all NAC groups compared to the control group. Intracellular calcium concentration decreased significantly in all groups with compared control. mRNA expression level of ABCC8 subunit significantly increased in all NAC groups compared to the control group. Pearson correlation analysis showed that relaxation rate was significantly associated with K ATP current, intracellular calcium concentration, Na + /K + -ATPase activity and mRNA expression level of ABCC8 subunit. Our findings suggest that NAC relaxes vascular smooth muscle cells through a direct effect on K ATP channels, by increasing outward K+ flux, partly by increasing mRNA expression of K ATP subunit ABCC8, by decreasing in intracellular calcium and by decreasing in Na + /K + -ATPase activity. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Effects of recombinant gonadotropin hormones on the expression of vitellogenin, gonadotropin subunits and gonadotropin receptors in cinnamon clownfish, Amphiprion melanopus.

PubMed

Kim, Na Na; Habibi, Hamid R; Lee, Jehee; Choi, Cheol Young

2012-08-01

Gonadotropins (GTHs) are the key regulators of reproduction in vertebrates. The present study investigated autoregulatory effects of gonadotropins, using recombinant FSH (rFSH) and LH (rLH) in cinnamon clownfish (Amphiprion melanopus). Experiments were carried out to investigate the actions of cinnamon clownfish rFSH and rLH on expression of GTH subunits, GTH receptors, and vitellogenin (Vtg) mRNA in vivo and in vitro. Plasma estradiol-17β (E(2)) level was also measured in immature fish following treatments with rFSH and rLH. The results demonstrate increasing levels of GTH subunits, GTH-receptors, Vtg mRNA levels, as well as plasma E(2) levels following injection with rFSH and rLH. The findings support the hypothesis that LH and FSH stimulate reproduction, in part, by autoregulatory mechanisms leading to upregulation of GTH receptors and GTH hormone production in cinnamon clownfish. The results provide a framework for better understanding of the mechanisms of GTH-mediated control of reproduction in cinnamon clownfish and other vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

Elevated GRIA1 mRNA expression in Layer II/III and V pyramidal cells of the DLPFC in schizophrenia

PubMed Central

O’Connor, J.A.; Hemby, S.E.

2012-01-01

The functional integrity of the dorsolateral prefrontal cortex (DLPFC) is altered in schizophrenia leading to profound deficits in working memory and cognition. Growing evidence indicates that dysregulation of glutamate signaling may be a significant contributor to the pathophysiology mediating these effects; however, the contribution of NMDA and AMPA receptors in the mediation of this deficit remains unclear. The equivocality of data regarding ionotropic glutamate receptor alterations of subunit expression in the DLPFC of schizophrenics is likely reflective of subtle alterations in the cellular and molecular composition of specific neuronal populations within the region. Given previous evidence of Layer II/III and V pyramidal cell alterations in schizophrenia and the significant influence of subunit composition on NMDA and AMPA receptor function, laser capture microdissection combined with quantitative PCR was used to examine the expression of AMPA (GRIA1-4) and NMDA (GRIN1, 2A and 2B) subunit mRNA levels in Layer II/III and Layer V pyramidal cells in the DLPFC. Comparisons were made between individuals diagnosed with schizophrenia, bipolar disorder, major depressive disorder and controls (n=15/group). All subunits were expressed at detectable levels in both cell populations for all diseases as well as for the control group. Interestingly, GRIA1 mRNA was significantly increased in both cell types in the schizophrenia group compare to controls, while similar trends were observed in major depressive disorder (Layers II/III and V) and bipolar disorder (Layer V). These data suggest that increased GRIA1 subunit expression may contribute to schizophrenia pathology. PMID:17942280

Microgravity and Signaling Molecules in Rat Osteoblasts: Downstream of Receptor Tyrosine Kinase, G-Protein-Coupled Receptor, and Small GTP-Binding Proteins

NASA Technical Reports Server (NTRS)

Kumel, Yasuhiro; Shimokawa, Hitoyata; Morita, Sadao; Katano, Hisako; Akiyama, Hideo; Hirano, Masahiko; Ohya, Keiichi; Sams, Clarence F.; Whitson, Peggy A.

2005-01-01

Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized on board. The mRNA levels of the post-receptor signaling molecules were analyzed by quantitative RT-PCR. The G-protein alpha subunit G(alpha)q mRNA levels were elevated 3-fold by microgravity. G(alpha)q stimulates PLC(beta), and then PKC. PKC(delta) and PKC(theta) mRNA levels were increased 2- to 5-fold by microgravity The mRNA levels of SOS and Ras GRF were increased 4 to 5-fold by microgravity, while Ras GAP was not altered. Spaceflight-induced bone loss might be attributed to microgravity modulation of the signaling pathway in osteoblasts.

The postnatal 5-HT1A receptor regulates adult anxiety and depression differently via multiple molecules.

PubMed

Ishikawa, Chihiro; Shiga, Takashi

2017-08-01

Serotonin (5-HT) and the 5-HT 1A receptor during development are known to modulate anxiety and depression in later life. However, the brain mechanisms linking the postnatal 5-HT system and adult behavior remain unknown. Here, we examined the effects of pharmacological 5-HT 1A receptor activation during the postnatal period on anxiety and depression-like behavior in adult BALB/c male mice. To elucidate the underlying mechanisms, we measured mRNA expression of the 5-HT 1A receptor, brain-derived neurotrophic factor (BDNF), GABA A receptor subunits, and AMPA receptor subunits in the medial prefrontal cortex (mPFC), amygdala, and hippocampus. Treatment with the selective 5-HT reuptake inhibitor (fluoxetine) and 5-HT 1A receptor agonist (8-OH-DPAT) during the postnatal period decreased anxiety-like behavior in adulthood, whereas only 8-OH-DPAT treatment increased depression-like behavior. Concomitantly with the behavioral effects, postnatal treatment with fluoxetine and 8-OH-DPAT decreased the mRNA expression of the GABA A receptor α3 subunit in the mPFC and ventral hippocampus in adulthood, while 8-OH-DPAT, but not fluoxetine, decreased the mRNA expression of the 5-HT 1A receptor and BDNF in the mPFC and the GABA A receptor α2 subunit in the mPFC and ventral hippocampus. On the basis of the correlative changes between behavior and mRNA expression, these results suggest that the GABA A receptor α3 subunit in the mPFC and ventral hippocampus may regulate anxiety-like behavior. In contrast, depression-like behavior may be regulated by the 5-HT 1A receptor and BDNF in the mPFC and by the GABA A receptor α2 subunit in the mPFC and ventral hippocampus. In summary, activation of the 5-HT 1A receptor during the postnatal period may reduce anxiety levels, but increase depression levels during adulthood via different multiple molecules in the mPFC and ventral hippocampus. Copyright © 2017 Elsevier Inc. All rights reserved.

Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate-cysteine ligase subunits

PubMed Central

Sikalidis, Angelos K.; Mazor, Kevin M.; Lee, Jeong-In; Roman, Heather B.; Hirschberger, Lawrence L.; Stipanuk, Martha H.

2014-01-01

Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser51 phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent. PMID:24557597

Enhanced LPS-induced activation of IL-27 signalling in sarcoidosis.

PubMed

Ringkowski, Sabine; Loke, Joshua; Huang, Shuying; Ahmadzai, Hasib; Herth, Felix J F; Thomas, Paul S; Herbert, Cristan

2016-08-01

Granulomas in sarcoidosis have recently been described as containing Interleukin (IL)-27, one of the members of the IL-12 family of cytokines, which also includes IL-35. Levels of these cytokines and the IL-27 receptor subunits were hypothesised to differ between patients with sarcoidosis compared to healthy controls in peripheral blood. Using a cross-sectional study design, plasma and peripheral blood mononuclear cells (PBMC) were collected from patients and control subjects. Protein and mRNA (in PBMC) levels for IL-27 and IL-35 (IL27, EBI3, IL12A subunits) as well as IL-27 receptor (IL6ST and IL27RA subunits) were assessed spontaneously and following direct (LPS) and indirect (anti-CD3/28 activation beads) macrophage stimulation using RT- PCR, ELISA and flow cytometry. Following stimulation with LPS, PBMC of patients with sarcoidosis displayed significantly enhanced expression of IL27 and EBI3 mRNA (p = 0.020 and p = 0.037 respectively) compared to PBMCs from healthy controls. There was also significantly enhanced production of IL-27 by PBMC from patients with sarcoidosis compared to healthy controls in response to LPS stimulation (p = 0.027). IL6ST mRNA and IL6ST protein were significantly lower in patients with sarcoidosis (mRNA p = 0.0002; MFI p = 0.0015) whilst IL27RA protein levels were significantly higher in patients with sarcoidosis compared to healthy controls (MFI p < 0.0001). Plasma IL-35 protein levels did not differ between control and sarcoidosis subjects (p = 0.23). These results suggest there may be exaggerated activation of IL-27 signalling in response to LPS in sarcoidosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels

PubMed Central

Amândio, Ana Rita; Gaspar, Pedro; Whited, Jessica L.; Janody, Florence

2014-01-01

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth. PMID:24788460

Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

PubMed

Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

2005-01-01

Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P < .0001) with significant increases in COX activity in wake and sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P < .001). There was no difference between brain regions in the degree of increase in enzyme activity in wakefulness. Both COXI and COXIV mRNA were increased with wakefulness as compared to sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding protein; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; mRNA, messenger ribonucleic acid; NADH, nicotinamid adenine dinucleotide, reduced; NDII, NADH dehydrogenase subunit 2 mRNA; NRF, nuclear respiratory factor.

Loss of G2 subunit of vacuolar-type proton transporting ATPase leads to G1 subunit upregulation in the brain

PubMed Central

Kawamura, Nobuyuki; Sun-Wada, Ge-Hong; Wada, Yoh

2015-01-01

Vacuolar-type ATPase (V-ATPase) is a primary proton pump with versatile functions in various tissues. In nerve cells, V-ATPase is required for accumulation of neurotransmitters into secretory vesicles and subsequent release at the synapse. Neurons express a specific isoform (G2) of the G subunit of V-ATPase constituting the catalytic sector of the enzyme complex. Using gene targeting, we generated a mouse lacking functional G2 (G2 null), which showed no apparent disorders in architecture and behavior. In the G2-null mouse brain, a G1 subunit isoform, which is ubiquitously expressed in neuronal and non-neuronal tissues, accumulated more abundantly than in wild-type animals. This G1 upregulation was not accompanied by an increase in mRNA. These results indicate that loss of function of neuron-specific G2 isoform was compensated by an increase in levels of the G1 isoform without apparent upregulation of the G1 mRNA. PMID:26353914

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.

Rapid changes in ovarian mRNA induced by brief photostimulation in Siberian hamsters (Phodopus sungorus)

PubMed Central

Shahed, Asha; McMichael, Carling F.; Young, Kelly A.

2017-01-01

This study sought to characterize the rapid intraovarian mRNA response of key folliculogenic factors that may contribute to the restoration of folliculogenesis during 2-10 days of photostimulation in Siberian hamsters. Adult hamsters were exposed to short photoperiod (8L:16D) for 14 weeks (SD). A subset were then transferred to long photoperiod (16L:8D) for 2(PT day-2), 4(PT day-4), or 10 days (PT day-10). Quantitative real-time PCR was used to measure intraovarian mRNA expression of: gonadotropin releasing hormone (GnRH), follicle stimulating hormone β-subunit (FSHβ-subunit), luteinizing hormone β-subunit (LHβ-subunit), FSH and LH receptors, estrogen receptorsα and β (Esr1 and Esr2), matrix metalloproteinase (MMP)-2 and -9, anti-Müllerian hormone (AMH), inhibin-α subunit, fibroblast growth factor-2 (FGF-2) and proliferating cell nuclear antigen (PCNA). Compared to SD, plasma FSH concentrations increased on PT day-4 and the number of antral follicles and corpora lutea increased on PT day-10. FSHR and inhibin-α mRNA expression also increased on PT day-4, whereas LHR and proliferation marker PCNA both increased on PT day-10 as compared to SD. Esr1 mRNA increased on PT day-2 and remained significantly increased as compared to SD, whereas Esr1 mRNA increased only on PT day-2, similar to FGF-2 and MMP-2 results. No differences were observed in mRNA expression in ovarian GnRH, FSHβ- and LHβ-subunits, AMH, and MMP-9 mRNA with 2-10 days of photostimulation. Rapid increases in intraovarian FSHR and inhibin-α mRNA and antral follicle/corpora lutea numbers suggest that the ovary is primed to react quickly to the FSH released in response to brief periods of photostimulation. PMID:26174001

Intraovarian expression of GnRH-1 and gonadotropin mRNA and protein levels in Siberian hamsters during the estrus cycle and photoperiod induced regression/recrudescence

PubMed Central

Shahed, Asha; Young, Kelly A.

2010-01-01

The hypothalamic-pituitary-gonadal (HPG) axis is the key reproductive regulator in vertebrates. While gonadotropin releasing hormone (GnRH), follicle stimulating (FSH), and luteinizing (LH) hormones are primarily produced in the hypothalamus and pituitary, they can be synthesized in the gonads, suggesting an intraovarian GnRH-gonadotropin axis. Because these hormones are critical for follicle maturation and steroidogenesis, we hypothesized that this intraovarian axis may be important in photoperiod-induced ovarian regression/recrudescence in seasonal breeders. Thus, we investigated GnRH-1 and gonadotropin mRNA and protein expression in Siberian hamster ovaries during (1) the estrous cycle; where ovaries from cycling long day hamsters (LD;16L:8D) were collected at proestrus, estrus, diestrus I, and diestrus II and (2) during photoperiod induced regression/ recrudescence; where ovaries were collected from hamsters exposed to 14wks of LD, short days (SD;8L:16D), or 8wks post-transfer to LD after 14wks SD (PT). GnRH-1, LHβ, FSHβ, and common α subunit mRNA expression was observed in cycling ovaries. GnRH-1 expression peaked at diestrus I compared to other stages (p<0.05). FSHβ and LHβ mRNA levels peaked at proestrus and diestrus I (p<0.05), with no change in the α subunit across the cycle (p>0.05). SD exposure decreased ovarian mass and plasma estradiol concentrations (p<0.05) and increased GnRH-1, LHβ, FSHβ, and α subunit mRNA expression as compared to LD and, except for LH, compared to PT (p<0.05). GnRH and gonadotropin protein was also dynamically expressed across the estrous cycle and photoperiod exposure. The presence of cycling intraovarian GnRH-1 and gonadotropin mRNA suggests that these hormones may be locally involved in ovarian maintenance during SD regression and/or could potentially serve to prime ovaries for rapid recrudescence. PMID:20955709

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

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

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition.

PubMed

Doherty, Geoff P; Fogg, Mark J; Wilkinson, Anthony J; Lewis, Peter J

2010-12-01

Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β' subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β' and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β' and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β'. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

Age-related decline of the cytochrome c oxidase subunit expression in the auditory cortex of the mimetic aging rat model associated with the common deletion.

PubMed

Zhong, Yi; Hu, Yujuan; Peng, Wei; Sun, Yu; Yang, Yang; Zhao, Xueyan; Huang, Xiang; Zhang, Honglian; Kong, Weijia

2012-12-01

The age-related deterioration in the central auditory system is well known to impair the abilities of sound localization and speech perception. However, the mechanisms involved in the age-related central auditory deficiency remain unclear. Previous studies have demonstrated that mitochondrial DNA (mtDNA) deletions accumulated with age in the auditory system. Also, a cytochrome c oxidase (CcO) deficiency has been proposed to be a causal factor in the age-related decline in mitochondrial respiratory activity. This study was designed to explore the changes of CcO activity and to investigate the possible relationship between the mtDNA common deletion (CD) and CcO activity as well as the mRNA expression of CcO subunits in the auditory cortex of D-galactose (D-gal)-induced mimetic aging rats at different ages. Moreover, we explored whether peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) were involved in the changes of nuclear- and mitochondrial-encoded CcO subunits in the auditory cortex during aging. Our data demonstrated that d-gal-induced mimetic aging rats exhibited an accelerated accumulation of the CD and a gradual decline in the CcO activity in the auditory cortex during the aging process. The reduction in the CcO activity was correlated with the level of CD load in the auditory cortex. The mRNA expression of CcO subunit III was reduced significantly with age in the d-gal-induced mimetic aging rats. In contrast, the decline in the mRNA expression of subunits I and IV was relatively minor. Additionally, significant increases in the mRNA and protein levels of PGC-1α, NRF-1 and TFAM were observed in the auditory cortex of D-gal-induced mimetic aging rats with aging. These findings suggested that the accelerated accumulation of the CD in the auditory cortex may induce a substantial decline in CcO subunit III and lead to a significant decline in the CcO activity progressively with age despite compensatory increases of PGC-1α, NRF-1 and TFAM. Therefore, CcO may be a specific intramitochondrial site of age-related deterioration in the auditory cortex, and CcO subunit III might be a target in the development of presbycusis. Copyright © 2012 Elsevier B.V. All rights reserved.

Altered expression of genes involved in GABAergic transmission and neuromodulation of granule cell activity in the cerebellum of schizophrenia patients.

PubMed

Bullock, W Michael; Cardon, Karen; Bustillo, Juan; Roberts, Rosalinda C; Perrone-Bizzozero, Nora I

2008-12-01

Deficits in gamma-aminobutyric acid (GABA) signaling have been described in the prefrontal cortex, limbic system, and cerebellum in individuals with schizophrenia. The purpose of the present study was to further investigate cerebellar gene expression alterations as they relate to decreases in GABAergic transmission by examining the expression of GABAergic markers, N-methyl-d-aspartic-acid (NMDA) receptor subunits, and cerebellum neuromodulators in individuals with schizophrenia. Subjects were postmortem men with a diagnosis of schizophrenia (N=13) and a postmortem interval-matched non-psychiatric male comparison group (N=13). The authors utilized real-time-quantitative polymerase chain reaction (PCR) to measure mRNA levels of the following GABAergic markers: glutamic acid decarboxylase (GAD) 65 and 67; GABA plasma membrane transporter-1 (GAT-1); GABA type A (GABA(A)) receptor subunits alpha(6), beta(3), and delta; and parvalbumin. In addition, real-time-quantitative PCR was utilized to assess mRNA levels of the NMDA receptor (NR) subunits NR1, NR2-A, NR2-B, NR2-C, and NR2-D as well as the cerebellar neuromodulators glutamate receptor (GluR)-6, kainate-preferring glutamate receptor subunit-2 (KA2), metabotropic glutamate receptor (mGluR)-2 and mGluR3, and neuronal nitric oxide synthase. Measurements for mRNA levels were determined using lateral cerebellar hemisphere tissue from both schizophrenia and comparison subjects. Schizophrenia subjects showed significant decreases in mRNA levels of GAD(67), GAD(65), GAT-1, mGluR2, and neuronal nitric oxide synthase. Increases in GABA(A)-alpha(6 )and GABA(A)-delta as well as GluR6 and KA2 were also observed. Medication effects on the expression of the same genes were examined in rats treated with either haloperidol (Sprague-Dawley rats [N=16]) or clozapine (Long-Evans rats [N=20]). Both haloperidol and clozapine increased the levels of GAD(67) in the cerebellum and altered the expression of other cerebellar mRNAs. These findings suggest that GABA transmission is decreased in the cerebellar cortices in individuals with schizophrenia and additional gene expression changes may reflect an attempt to increase GABA neurotransmission at the cerebellar glomerulus.

Increased neuroinflammatory and arachidonic acid cascade markers, and reduced synaptic proteins, in the postmortem frontal cortex from schizophrenia patients

PubMed Central

Rao, Jagadeesh Sridhara; Kim, Hyung-Wook; Harry, Gaylia Jean; Rapoport, Stanley Isaac; Reese, Edmund Arthur

2013-01-01

Schizophrenia (SZ) is a progressive, neuropsychiatric disorder associated with cognitive impairment. A number of brain alterations have been linked to cognitive impairment, including neuroinflammation, excitotoxicity, increased arachidonic acid (AA) signaling and reduced synaptic protein. On this basis, we tested the hypothesis that SZ pathology is associated with these pathological brain changes. To do this, we examined postmortem frontal cortex from 10 SZ patients and 10 controls and measured protein and mRNA levels of cytokines, and astroglial, microglial, neuroinflammatory excitotoxic, AA cascade, apoptotic and synaptic markers. Mean protein and mRNA levels of interleukin-1β, tumor necrosis factor-α, glial acidic fibrillary protein (GFAP), a microglial marker CD11b, and nuclear factor kappa B subunits were significantly increased in SZ compared with control brain. Protein and mRNA levels of cytosolic and secretory phospholipase A2 and cyclooxygenase were significantly elevated in postmortem brains from SZ patients. N-methyl-D-aspartate receptor subunits 1 and 2B, inducible nitric oxide synthase and c-FOS were not significantly different. In addition, reduced protein and mRNA levels of brain-derived neurotrophic factor, synaptophysin and drebrin were found in SZ compared with control frontal cortex. Increased neuroinflammation and AA cascade enzyme markers with synaptic protein loss could promote disease progression and cognitive defects in SZ patients. Drugs that downregulate these changes might be considered for new therapies in SZ. PMID:23566496

Protective effect of hydroxytyrosol in arsenic-induced mitochondrial dysfunction in rat brain.

PubMed

Soni, Manisha; Prakash, Chandra; Sehwag, Sfurti; Kumar, Vijay

2017-07-01

The present study was planned to investigate the protective effect of hydroxytyrosol (HT) against arsenic (As)-induced mitochondrial dysfunction in rat brain. Rats exposed to sodium arsenite (25 ppm for 8 weeks) showed decreased mitochondrial complexes (I, II, IV) activities, mitochondrial superoxide dismutase (MnSOD), and catalase activities in brain mitochondria. As-treated rats showed reduced mRNA expression of complex I (ND-1, ND-2), IV (COX-1, COX-4) subunits, and uncoupling protein-2 (UCP-2). In addition to this, As exposure downregulated the protein expression of MnSOD. Administration of HT with As restored the enzymatic activities of mitochondrial complexes, MnSOD and catalase, increased the mRNA levels of complexes subunits and UCP-2 as well as proteins level of MnSOD. These results suggest that HT efficiently restores mitochondrial dysfunction in As neurotoxicity and might be used as potential mitoprotective agent in future. © 2017 Wiley Periodicals, Inc.

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

Effect of aldosterone on BK channel expression in mammalian cortical collecting duct

PubMed Central

Estilo, Genevieve; Liu, Wen; Pastor-Soler, Nuria; Mitchell, Phillip; Carattino, Marcelo D.; Kleyman, Thomas R.; Satlin, Lisa M.

2008-01-01

Apical large-conductance Ca2+-activated K+ (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K+ secretion. Dietary K+ loading for 10–14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K+ secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922–F932, 2005). To test whether this adaptation was mediated by a K+-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na+ (LS) or high-Na+ (HS) diet for 7–10 days to alter circulating levels of aldosterone but not serum K+ concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, α-splice variants, β-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na+ (JNa) and K+ (JK) transport by microperfusion; kidneys were processed for immunolocalization of BK α-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na+ and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK α-, β2-, and β4-subunit mRNA and localization of immunodetectable α-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from ∼1 to 5 nl·min−1·mm−1, the increase in JNa was greater in LS vs. HS rabbits, yet the flow-stimulated increase in JK was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K+ loading. PMID:18579708

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

PubMed

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

AMPA, NMDA and kainate glutamate receptor subunits are expressed in human peripheral blood mononuclear cells (PBMCs) where the expression of GluK4 is altered by pregnancy and GluN2D by depression in pregnant women.

PubMed

Bhandage, Amol K; Jin, Zhe; Hellgren, Charlotte; Korol, Sergiy V; Nowak, Krzysztof; Williamsson, Louise; Sundström-Poromaa, Inger; Birnir, Bryndis

2017-04-15

The amino acid glutamate opens cation permeable ion channels, the iGlu receptors. These ion channels are abundantly expressed in the mammalian brain where glutamate is the main excitatory neurotransmitter. The neurotransmitters and their receptors are being increasingly detected in the cells of immune system. Here we examined the expression of the 18 known subunits of the iGlu receptors families; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, N-methyl-d-aspartate (NMDA) and delta in human peripheral blood mononuclear cells (PBMCs). We compared the expression of the subunits between four groups: men, non-pregnant women, healthy pregnant women and depressed pregnant women. Out of 18 subunits of the iGlu receptors, mRNAs for 11 subunits were detected in PBMCs from men and non-pregnant women; AMPA: GluA3, GluA4, kainate: GluK2, GluK4, GluK5, NMDA: GluN1, GluN2C, GluN2D, GluN3A, GluN3B, and delta: GluD1. In the healthy and the depressed pregnant women, in addition, the delta GluD2 subunit was identified. The mRNAs for GluK4, GluK5, GluN2C and GluN2D were expressed at a higher level than other subunits. Gender, pregnancy or depression during pregnancy altered the expression of GluA3, GluK4, GluN2D, GluN3B and GluD1 iGlu subunit mRNAs. The greatest changes recorded were the lower GluA3 and GluK4 mRNA levels in pregnant women and the higher GluN2D mRNA level in healthy but not in depressed pregnant women as compared to non-pregnant individuals. Using subunit specific antibodies, the GluK4, GluK5, GluN1, GluN2C and GluN2D subunit proteins were identified in the PBMCs. The results show expression of specific iGlu receptor subunit in the PBMCs and support the idea of physiology-driven changes of iGlu receptors subtypes in the immune cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Na+, K+-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

PubMed

Hu, Yau-Chung; Chu, Keng-Fu; Yang, Wen-Kai; Lee, Tsung-Han

2017-10-01

The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na + , K + -ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1-β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1-β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1-β1 association in gill ionocytes of euryhaline teleosts.

Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma

NASA Technical Reports Server (NTRS)

Baracos, V. E.; DeVivo, C.; Hoyle, D. H.; Goldberg, A. L.

1995-01-01

Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.

Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice

PubMed Central

Fujisawa, Yukiko; Kato, Teruhisa; Ohki, Shizuka; Ishikawa, Atsushi; Kitano, Hidemi; Sasaki, Takuji; Asahi, Tadashi; Iwasaki, Yukimoto

1999-01-01

Transgenic rice containing an antisense cDNA for the α subunit of rice heterotrimeric G protein produced little or no mRNA for the subunit and exhibited abnormal morphology, including dwarf traits and the setting of small seeds. In normal rice, the mRNA for the α subunit was abundant in the internodes and florets, the tissues closely related to abnormality in the dwarf transformants. The position of the α-subunit gene was mapped on rice chromosome 5 by mapping with the restriction fragment length polymorphism. The position was closely linked to the locus of a rice dwarf mutant, Daikoku dwarf (d-1), which is known to exhibit abnormal phenotypes similar to those of the transformants that suppressed the endogenous mRNA for the α subunit by antisense technology. Analysis of the cDNAs for the α subunits of five alleles of Daikoku dwarf (d-1), ID-1, DK22, DKT-1, DKT-2, and CM1361–1, showed that these dwarf mutants had mutated in the coding region of the α-subunit gene. These results show that the G protein functions in the formation of normal internodes and seeds in rice. PMID:10377457

Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

PubMed

Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

2016-06-12

Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Short-term effects of hyposmotic shock on Na+/K+ -ATPase expression in gills of the euryhaline milkfish, Chanos chanos.

PubMed

Lin, Y M; Chen, C N; Yoshinaga, T; Tsai, S C; Shen, I D; Lee, T H

2006-03-01

Changes in expression of gill Na+/K+ -ATPase (NKA) on a short-term (96 h) time-course following hyposmotic shock (direct transfer to fresh water) of the euryhaline, marine milkfish were studied on gene, protein, and cell levels in this paper. Plasma osmolality and [Na+] responded with rapid declines in 3 h post-transfer yet, thereafter, remained constant. Plasma [Cl-] gradually fell to a significantly lower level at 6 h post-transfer. Gills responded to hyposmotic shock by a dual phase enhancement of NKA activity and protein abundance; (a) Before 24 h: NKA activity increased as early as 3 h and reached a maximum level from 6 to 12 h post-transfer coincided with the sustained lower levels of plasma osmolality, [Na+], and [Cl-] since 3 h post-transfer. This was followed by a gradual rise in alpha-subunit protein levels that peaked at 12 h post-transfer. Meanwhile, alpha-mRNA of NKA did no show significant change. (b) After 24 h: NKA activity as well as the amounts of alpha-subunit mRNA and protein increased significantly. Direct freshwater transfer induced a prompt and significant decrease of NKA immunoreactive (NKIR) cell abundance in filaments before 24 h, followed by a significant increase after 24 h due to their development in filaments and lamellae. Increased number of NKIR cells after 24 h of hyposmotic shock may occur in conjunction with rise of NKA activity as well as alpha-subunit mRNA and protein abundance. In conclusion, milkfish is able to avoid an excessive drop in plasma ions immediately upon hyposmotic shock and maintain plasma ions on a marginal lower level in fresh water. Notably, the initial increase in NKA activity (adjustive phase; 3-12 h) and delayed increase in NKA mRNA and protein abundance (regulatory phase; 48-96 h) indicate the importance of a higher level of the gill enzyme in milkfish upon hyposmotic shock.

Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

PubMed Central

Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

1992-01-01

The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

Endocrine regulation of gonadotropin and growth hormone gene transcription in fish.

PubMed

Melamed, P; Rosenfeld, H; Elizur, A; Yaron, Z

1998-06-01

The pituitary of a number of teleosts contains two gonadotropins (GtHs) which are produced in distinct populations of cells; the beta subunit of the GtH I being found in close proximity to the somatotrophs, while the II beta cells are more peripheral. In several species the GtH beta subunits are expressed at varying levels throughout the reproductive cycle, the I beta dominating in early maturing fish, after which the II beta becomes predominant. This suggests differential control of the beta subunit synthesis which may be regulated by both hypothalamic hormones and gonadal steroids. At ovulation and spawning, changes also occur in the somatotrophs, which become markedly more active, while plasma growth hormone (GH) levels increase. In a number of species, GnRH elevates either the I beta or the II beta mRNA levels, depending on the reproductive state of the fish. In tilapia, the GnRH effect on the II beta appears to be mediated through both cAMP-PKA and PKC pathways. GnRH also stimulates GH release in both goldfish and tilapia, but it increases the GH transcript levels only in goldfish; both GnRH and direct activation of PKC are ineffective in altering GH mRNA in tilapia pituitary cells. Dopamine (DA) does not alter II beta transcript levels in cultured tilapia pituitary cells, but increases GH mRNA levels in both rainbow trout and tilapia, in a PKA-dependent manner. This effect appears to be through interactions with Pit-1 and also by stabilizing the mRNA. Somatostatin (SRIF) does not alter GH transcript levels in either tilapia or rainbow trout, although it may alter GH synthesis by modulation of translation. Gonadal steroids appear to have differential effects on the transcription of the beta subunits. In tilapia, testosterone (T) elevates I beta mRNA levels in cells from immature or early maturing fish (in low doses), but depresses them in cells from late maturing fish and is ineffective in cells from regressed fish. Similar results were seen in early recrudescing male coho salmon injected with T or E2. T or E2 administered in vivo has dramatic stimulatory effects on the II beta transcript levels in immature fish of a number of species, while less powerful effects are seen in vitro. A response is also seen in cells from early maturing rainbow trout or tilapia, or regressed tilapia, but not in cells from late maturing or spawning fish. These results are substantiated by the finding that the promoter of the salmon II beta gene contains several estrogen responsive elements (EREs) which react and interact differently when exposed to varying levels of E2. In addition, activator protein-1 (AP-1) and steroidogenic factor-1 (SF-1) response elements are also found in the salmon II beta promoter; the AP-1 site is located close to a half ERE, while the SF-1 acts synergistically with the E2 receptor. The mRNA levels of both AP-1 and SP-1 are elevated, at least in mammals, by GnRH, suggesting possible sites for cross-talk between GnRH and steroid activated pathways. Reports of the effects of T or E2 on GH transcription differ. No effect is seen in vitro in pituitaries of tilapia, juvenile rainbow trout or common carp, but T does increase the transcript levels in pituitaries of both immature and mature goldfish. Reasons for these discrepancies are unclear, but other systemic hormones may be more instrumental than the gonadal steroids in regulating GH transcription. These include T3 which increases both GH mRNA levels and de novo synthesis (in tilapia and common carp) and insulin-like growth factor-I (IGF-I) which reduces GH transcript levels as well as inhibiting GH release.

Induced expression of hepatic N-methyl-D-aspartate receptor 2C subunit gene during liver enlargement induced by lead nitrate, a hepatocellular mitogen.

PubMed

Nemoto, Kiyomitsu; Ikeda, Ayaka; Hikida, Tokihiro; Kojima, Misaki; Degawa, Masakuni

2013-02-01

We previously demonstrated the super-induced expression of the Grin2c gene encoding the N-methyl-D-aspartate receptor 2C subunit during the development of liver enlargement with hepatocellular hypertrophy induced by phenobarbital, clofibrate, or piperonyl butoxide. In the present study, we assessed whether or not Grin2c gene expression was induced during the development of chemically induced liver enlargement with hyperplasia. Male Sprague-Dawley (SD) rats, stroke-prone spontaneously hypertensive rats (SHRSPs), and SHRSP's normotensive control, Wistar-Kyoto (WKY) rats, were administered lead nitrate (LN) (0.1 mmol/kg, single i.v.), a direct inducer of liver hyperplasia, and changes in the level of Grin2c mRNA in the liver were assessed by real-time RT-PCR. The level of hepatic Grin2c mRNA was significantly higher 6-48 hr after the injection in SD rats (about 30~40- and 70-fold over the control at 6~24 hr and 48 hr, respectively) and in WKY rats (about 20-fold over the control only at 12 hr), but was not significantly higher in SHRSPs. Such differences in LN-induced levels of Grin2c mRNA among SD rats, WKY rats, and SHRSPs were closely correlated with those in the previously reported increase in liver weight 48 hr after LN administration. The present findings suggest that the increase in the level of hepatic Grin2c mRNA relates to development of chemically induced liver enlargement with hyperplasia.

Triclosan-Evoked Neurotoxicity Involves NMDAR Subunits with the Specific Role of GluN2A in Caspase-3-Dependent Apoptosis.

PubMed

Szychowski, Konrad A; Wnuk, Agnieszka; Rzemieniec, Joanna; Kajta, Małgorzata; Leszczyńska, Teresa; Wójtowicz, Anna K

2018-04-19

Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitising products. A number of studies have shown the presence of TCS in different human tissues such as blood, adipose tissue, the liver, brain as well as in breast milk and urine. N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are widely expressed in the central nervous system and which play key roles in excitatory synaptic transmission. There is, however, no data on the involvement of NMDAR subunits in the apoptotic and neurotoxic effects of TCS. Our experiments are the first to show that TCS used at environmentally relevant concentrations evoked NMDA-dependent effects in neocortical neurons in primary cultures, as MK-801, an uncompetitive NMDA receptor antagonist, reduced the levels of TCS-induced ROS production as well as caspase-3 activity and LDH release. TCS caused a decrease in protein expression of all the studied NMDA receptor subunits (GluN1, GluN2A, GluN2B) that were measured at 3, 6 and 24 h post-treatment. However, at 48 h of the experiment, the level of the GluN1 subunit returned to the control level, and the levels of the other subunits showed a tendency to increase. In TCS-treated neocortical cells, protein profiles of NMDAR subunits measured up to 24 h were similar to mRNA expression of GluN1 and GluN2A, but not to GluN2B mRNA. In this study, cells transiently transfected with GluN1, GluN2A or GluN2B siRNA exhibited reduced levels of LDH release, which suggests the involvement of all of the studied NMDAR subunits in the neurotoxic action of TCS. According to our data, GluN1 and GluN2A were mainly responsible for neuronal cell death as evidenced by neutral red uptake, whereas GluN2A was involved in TCS-induced caspase-3-dependent apoptosis. We suggest that TCS-evoked apoptosis and neurotoxicity could be related to transient degradation of NMDAR subunits in mouse neurons. Furthermore, recycling of NMDAR subunits in response to TCS is possible. Because transfections with specific siRNA did not completely abolish the effects of TCS as compared to cells transfected with negative siRNA in this study, other NMDAR-independent mechanisms of TCS action are also possible.

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

Reduction in voltage-gated K+ channel activity in primary sensory neurons in painful diabetic neuropathy: role of brain-derived neurotrophic factor.

PubMed

Cao, Xue-Hong; Byun, Hee-Sun; Chen, Shao-Rui; Cai, You-Qing; Pan, Hui-Lin

2010-09-01

Abnormal hyperexcitability of primary sensory neurons plays an important role in neuropathic pain. Voltage-gated potassium (Kv) channels regulate neuronal excitability by affecting the resting membrane potential and influencing the repolarization and frequency of the action potential. In this study, we determined changes in Kv channels in dorsal root ganglion (DRG) neurons in a rat model of diabetic neuropathic pain. The densities of total Kv, A-type (IA) and sustained delayed (IK) currents were markedly reduced in medium- and large-, but not in small-, diameter DRG neurons in diabetic rats. Quantitative RT-PCR analysis revealed that the mRNA levels of IA subunits, including Kv1.4, Kv3.4, Kv4.2, and Kv4.3, in the DRG were reduced approximately 50% in diabetic rats compared with those in control rats. However, there were no significant differences in the mRNA levels of IK subunits (Kv1.1, Kv1.2, Kv2.1, and Kv2.2) in the DRG between the two groups. Incubation with brain-derived neurotrophic factor (BDNF) caused a large reduction in Kv currents, especially IA currents, in medium and large DRG neurons from control rats. Furthermore, the reductions in Kv currents and mRNA levels of IA subunits in diabetic rats were normalized by pre-treatment with anti-BDNF antibody or K252a, a TrkB tyrosine kinase inhibitor. In addition, the number of medium and large DRG neurons with BDNF immunoreactivity was greater in diabetic than control rats. Collectively, our findings suggest that diabetes primarily reduces Kv channel activity in medium and large DRG neurons. Increased BDNF activity in these neurons likely contributes to the reduction in Kv channel function through TrkB receptor stimulation in painful diabetic neuropathy.

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

PubMed Central

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms. PMID:26872259

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

PubMed

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

Motor Skills Training Enhances α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Subunit mRNA Expression in the Ipsilateral Sensorimotor Cortex and Striatum of Rats Following Intracerebral Hemorrhage.

PubMed

Tamakoshi, Keigo; Ishida, Kazuto; Kawanaka, Kentaro; Takamatsu, Yasuyuki; Tamaki, Hiroyuki

2017-10-01

We investigated the effects of acrobatic training (AT) on expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits in the sensorimotor cortex and striatum after intracerebral hemorrhage (ICH). Male Wistar rats were divided into 4 groups: ICH without AT (ICH), ICH with AT (ICH + AT), sham operation without AT (SHAM), and sham operation with AT (SHAM + AT). ICH was induced by collagenase injection into the left striatum. The ICH + AT group performed 5 acrobatic tasks daily on days 4-28 post ICH. Forelimb sensorimotor function was evaluated using the forelimb placing test. On days 14 and 29, mRNA expression levels of AMPAR subunits GluR1-4 were measured by real-time reverse transcription-polymerase chain reaction. Forelimb placing test scores were significantly higher in the ICH + AT group than in the ICH group. Expression levels of all AMPAR subunit mRNAs were significantly higher in the ipsilateral sensorimotor cortex of rats in the ICH + AT group than in that of rats in the ICH group on day 29. GluR3 and GluR4 expression levels were reduced in the ipsilateral striatum of rats in the ICH group compared with that of rats in the SHAM group on day 14. These changes may play a critical role in motor skills training-induced recovery after ICH. Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

DNA methylation regulates gabrb2 mRNA expression: developmental variations and disruptions in l-methionine-induced zebrafish with schizophrenia-like symptoms.

PubMed

Wang, L; Jiang, W; Lin, Q; Zhang, Y; Zhao, C

2016-11-01

Single nucleotide polymorphisms (SNPs) in the human type A gamma-aminobutyric acid (GABA) receptor β 2 subunit gene (GABRB2) have been associated with schizophrenia and quantitatively correlated with mRNA expression in the postmortem brain tissue of patients with schizophrenia. l-Methionine (MET) administration has been reported to cause a recrudescence of psychotic symptoms in patients with schizophrenia, and similar symptoms have been generated in MET-induced mice. In this study, a zebrafish animal model was used to evaluate the relationship between the gabrb2 mRNA expression and its promoter DNA methylation in developmental and MET-induced schizophrenia-like zebrafish. The results indicated developmental increases in global DNA methylation and decreases in gabrb2 promoter methylation in zebrafish. A significant increase in gabrb2 mRNA levels was observed after GABA was synthesized. Additionally, the MET-triggered schizophrenia-like symptoms in adult zebrafish, involving social withdrawal and cognitive dysfunction analyzed with social interaction and T-maze behavioral tests, were accompanied by significantly increased DNA methylation levels in the global genome and the gabrb2 promoter. Furthermore, the significant correlation between gabrb2 mRNA expression and gabrb2 promoter methylation observed in the developmental stages became non-significant in MET-triggered adult zebrafish. These findings demonstrate that gabrb2 mRNA expression is associated with DNA methylation varies by developmental stage and show that these epigenetic association mechanisms are disrupted in MET-triggered adult zebrafish with schizophrenia-like symptoms. In conclusion, these results provide plausible epigenetic evidence of the GABA A receptor β 2 subunit involvement in the schizophrenia-like behaviors and demonstrate the potential use of zebrafish models in neuropsychiatric research. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

PubMed

Zhang, Yixi; Wang, Xin; Yang, Baojun; Hu, Yuanyuan; Huang, Lixin; Bass, Chris; Liu, Zewen

2015-11-01

Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance. © 2015 International Society for Neurochemistry.

Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

NASA Astrophysics Data System (ADS)

Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

2003-06-01

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

PubMed

Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

2017-11-01

Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

The stargazin-related protein γ7 interacts with the mRNA binding protein hnRNP A2 and regulates the stability of specific mRNAs including CaV2.2

PubMed Central

Ferron, Laurent; Davies, Anthony; Page, Karen M.; Cox, David J.; Leroy, Jerôme; Waithe, Dominic; Butcher, Adrian J.; Sellaturay, Priya; Bolsover, Steven; Pratt, Wendy S.; Moss, Fraser J.; Dolphin, Annette C.

2009-01-01

The role(s) of the novel stargazin-like γ-subunit proteins remain controversial. We have shown previously that the neuron-specific γ7 suppresses the expression of certain calcium channels, particularly CaV2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of γ7 on CaV2.2 expression is via an increase in the degradation rate of CaV2.2 mRNA, and hence a reduction of CaV2.2 protein level. Furthermore, exogenous expression of γ7 in PC12 cells also decreased the endogenous CaV2.2 mRNA level. Conversely, knockdown of endogenous γ7 with short-hairpin RNAs produced a reciprocal enhancement of CaV2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed γ7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C-terminus of γ7 is essential for all its effects, and we show that γ7 binds directly via its C-terminus to a ribonucleoprotein (hnRNP A2), which also binds to a motif in CaV2.2 mRNA, and is associated with native CaV2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances CaV2.2 IBa and this enhancement is prevented by a concentration of γ7 that alone has no effect on IBa. The effect of γ7 is selective for certain mRNAs as it had no effect on α2δ-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride co-transporter, KCC1, which contains a similar hnRNP A2 binding motif to that in CaV2.2 mRNA. Our results indicate that γ7 plays a role in stabilizing CaV2.2 mRNA. PMID:18923037

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

PubMed

Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K

1989-06-01

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

LOOP IIId of the HCV IRES is essential for the structural rearrangement of the 40S-HCV IRES complex

PubMed Central

Angulo, Jenniffer; Ulryck, Nathalie; Deforges, Jules; Chamond, Nathalie; Lopez-Lastra, Marcelo; Masquida, Benoît; Sargueil, Bruno

2016-01-01

As obligatory intracellular parasites, viruses rely on cellular machines to complete their life cycle, and most importantly they recruit the host ribosomes to translate their mRNA. The Hepatitis C viral mRNA initiates translation by directly binding the 40S ribosomal subunit in such a way that the initiation codon is correctly positioned in the P site of the ribosome. Such a property is likely to be central for many viruses, therefore the description of host-pathogen interaction at the molecular level is instrumental to provide new therapeutic targets. In this study, we monitored the 40S ribosomal subunit and the viral RNA structural rearrangement induced upon the formation of the binary complex. We further took advantage of an IRES viral mutant mRNA deficient for translation to identify the interactions necessary to promote translation. Using a combination of structure probing in solution and molecular modeling we establish a whole atom model which appears to be very similar to the one obtained recently by cryoEM. Our model brings new information on the complex, and most importantly reveals some structural rearrangement within the ribosome. This study suggests that the formation of a ‘kissing complex’ between the viral RNA and the 18S ribosomal RNA locks the 40S ribosomal subunit in a conformation proficient for translation. PMID:26626152

Quantitative studies of mRNA recruitment to the eukaryotic ribosome.

PubMed

Fraser, Christopher S

2015-07-01

The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5' UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5' UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Effects of S-Adenosylmethionine and Its Combinations With Taurine and/or Betaine on Glutathione Homeostasis in Ethanol-induced Acute Hepatotoxicity

PubMed Central

Lee, Seo Yeon; Ko, Kwang Suk

2016-01-01

Background Exposure to ethanol abuse and severe oxidative stress are risk factors for hepatocarcinoma. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine on the level of glutathione (GSH), a powerful antioxidant in the liver, in acute hepatotoxicity induced by ethanol. Methods To examine the effects of SAMe and its combinations with taurine and/or betaine on ethanol-induced hepatotoxicity, AML12 cells and C57BL/6 mice were pretreated with SAMe, taurine, and/or betaine, followed by ethanol challenge. Cell viability was detected with an MTT assay. GSH concentration and mRNA levels of GSH synthetic enzymes were measured using GSH reductase and quantitative real-time reverse transcriptase-PCR. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with commercially available kits. Results Pretreatment of SAMe, with or without taurine and/or betaine, attenuated decreases in GSH levels and mRNA expression of the catalytic subunit of glutamate-cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis, in ethanol-treated cells and mice. mRNA levels of the modifier subunit of GCL and glutathione synthetase were increased in mice treated with SAMe combinations. SAMe, taurine, and/or betaine pretreatment restored serum ALT and AST levels to control levels in the ethanol-treated group. Conclusions Combinations of SAMe with taurine and/or betaine have a hepatoprotective effect against ethanol-induced liver injury by maintaining GSH homeostasis. PMID:27722142

Deficient brain RNA polymerase and altered nucleolar structure persists until day 8 after perinatal asphyxia of the rat.

PubMed

Kastner, Philomena; Mosgoeller, Wilhelm; Fang-Kircher, Susanne; Kitzmueller, Erwin; Kirchner, Liselotte; Hoeger, Harald; Seither, Peter; Lubec, Gert; Lubec, Barbara

2003-01-01

RNA polymerases (POL) are integral constituents of the protein synthesis machinery, with POL I and POL III coding for ribosomal RNA and POL II coding for protein. POL I is located in the nucleolus and transcribes class I genes, those that code for large ribosomal RNA. It has been reported that the POL system is seriously affected in perinatal asphyxia (PA) immediately after birth. Because POL I is necessary for protein synthesis and brain protein synthesis was shown to be deranged after hypoxic-ischemic conditions, we aimed to study whether POL derangement persists in a simple, well-documented animal model of graded global PA at the activity, mRNA, protein, and morphologic level until 8 d after the asphyctic insult. Nuclear POL I activity was determined according to a radiochemical method; mRNA steady state and protein levels of RPA4O-an essential subunit of POL I and III-were evaluated by blotting methods; and the POL I subunit polymerase activating factor-53 was evaluated using immunohistochemistry. Silver staining and transmission electron microscopy were used to examine the nucleolus. At the eighth day after PA, nuclear POL I decreased with the length of the asphyctic period, whereas mRNA and protein levels for RPA4O were unchanged. The subunit polymerase activating factor-53, however, was unambiguously reduced in several brain regions. Dramatic changes of nucleolar morphology were observed, the main finding being nucleolar disintegration at the electron microscopy level. We suggest that severe acidosis and/or deficient protein kinase C in the brain during the asphyctic period may be responsible for disintegration of the nucleolus as well as for decreased POL activity persisting until the eighth day after PA. The biologic effect may be that PA causes impaired RNA and protein synthesis, which has been already observed in hypoxic-ischemic states.

GABRA2 Alcohol Dependence Risk Allele is Associated with Reduced Expression of Chromosome 4p12 GABAA Subunit Genes in Human Neural Cultures.

PubMed

Lieberman, Richard; Kranzler, Henry R; Joshi, Pujan; Shin, Dong-Guk; Covault, Jonathan

2015-09-01

Genetic variation in a region of chromosome 4p12 that includes the GABAA subunit gene GABRA2 has been reproducibly associated with alcohol dependence (AD). However, the molecular mechanisms underlying the association are unknown. This study examined correlates of in vitro gene expression of the AD-associated GABRA2 rs279858*C-allele in human neural cells using an induced pluripotent stem cell (iPSC) model system. We examined mRNA expression of chromosome 4p12 GABAA subunit genes (GABRG1, GABRA2, GABRA4, and GABRB1) in 36 human neural cell lines differentiated from iPSCs using quantitative polymerase chain reaction and next-generation RNA sequencing. mRNA expression in adult human brain was examined using the BrainCloud and BRAINEAC data sets. We found significantly lower levels of GABRA2 mRNA in neural cell cultures derived from rs279858*C-allele carriers. Levels of GABRA2 RNA were correlated with those of the other 3 chromosome 4p12 GABAA genes, but not other neural genes. Cluster analysis based on the relative RNA levels of the 4 chromosome 4p12 GABAA genes identified 2 distinct clusters of cell lines, a low-expression cluster associated with rs279858*C-allele carriers and a high-expression cluster enriched for the rs279858*T/T genotype. In contrast, there was no association of genotype with chromosome 4p12 GABAA gene expression in postmortem adult cortex in either the BrainCloud or BRAINEAC data sets. AD-associated variation in GABRA2 is associated with differential expression of the entire cluster of GABAA subunit genes on chromosome 4p12 in human iPSC-derived neural cell cultures. The absence of a parallel effect in postmortem human adult brain samples suggests that AD-associated genotype effects on GABAA expression, although not present in mature cortex, could have effects on regulation of the chromosome 4p12 GABAA cluster during neural development. Copyright © 2015 by the Research Society on Alcoholism.

PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

PubMed

Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

2018-02-01

To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of epilepsy. Copyright © 2017. Published by Elsevier B.V.

Apigenin manipulates the ubiquitin-proteasome system to rescue estrogen receptor-β from degradation and induce apoptosis in prostate cancer cells.

PubMed

Singh, Vishal; Sharma, Vikas; Verma, Vikas; Pandey, Deepti; Yadav, Santosh K; Maikhuri, Jagdamba P; Gupta, Gopal

2015-12-01

To investigate apigenin (5,7,4-trihydroxyflavone), a dietary flavonoid with proteasome-inhibitory activity (desired for the management of multiple types of cancers), against FDA-approved anticancer proteasome inhibitor bortezomib in context to its effects on the tumor suppressor estrogen receptor-beta (ER-β) in prostate cancer cells. Prostate cancer (PC-3) cells were treated with either apigenin or bortezomib, and proliferation inhibition was correlated with proteasomal biochemistry, ER-degradation and cell apoptosis. Apigenin specifically inhibited only chymotrypsin-like activity of proteasome without affecting trypsin and caspase-like activities, which was in contrast to the non-specific inhibition of all the three activities by bortezomib. Apigenin selectively increased the protein levels of ER-β at 1.8 and 10.0 µM (without affecting mRNA levels) and preferentially accumulated ubiquitinated ER-β over ER-α in PC-3. Apigenin-treated cells exhibited increased ER-β interactions with ubiquitin-protein ligase E6AP, downregulated PSMA5 (α-5 subunit for assembly of 20S proteasome) without affecting PSMB1 (β-1 subunit), PSMB2 (β-2 subunit) and PSMB5 (β-5 subunit, whose overexpression by bortezomib causes drug resistance) of proteasome at mRNA levels. Caspase-3 activation in PC-3 by apigenin was dependent on caspase-8 activity but independent of mitochondrial membrane depolarization. The deubiquitinase USP14 activity, which antagonizes degradation of proteins via proteasome, was significantly increased by apigenin treatment. Apigenin selectively inhibits proteasomal degradation of tumor suppressor ER-β by specifically inhibiting chymotrypsin-like activity of proteasome, preventing its assembly via PSMA5 and inhibiting USP14 enzyme activity in prostate cancer cells, resulting in cancer cell apoptosis. Unlike bortezomib, apigenin's actions are subtle, precise, mechanistically distinct and capable of abstaining drug resistance.

Expression and activity of the 5'-adenosine monophosphate-activated protein kinase pathway in selected tissues during chicken embryonic development.

PubMed

Proszkowiec-Weglarz, M; Richards, M P

2009-01-01

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

Human Cytomegalovirus Strategies to Maintain and Promote mRNA Translation

PubMed Central

Vincent, Heather A.; Ziehr, Benjamin; Moorman, Nathaniel J.

2016-01-01

mRNA translation requires the ordered assembly of translation initiation factors and ribosomal subunits on a transcript. Host signaling pathways regulate each step in this process to match levels of protein synthesis to environmental cues. In response to infection, cells activate multiple defenses that limit viral protein synthesis, which viruses must counteract to successfully replicate. Human cytomegalovirus (HCMV) inhibits host defenses that limit viral protein expression and manipulates host signaling pathways to promote the expression of both host and viral proteins necessary for virus replication. Here we review key regulatory steps in mRNA translation, and the strategies used by HCMV to maintain protein synthesis in infected cells. PMID:27089357

Effect of progesterone on the expression of GABA(A) receptor subunits in the prefrontal cortex of rats: implications of sex differences and brain hemisphere.

PubMed

Andrade, Susie; Arbo, Bruno D; Batista, Bruna A M; Neves, Alice M; Branchini, Gisele; Brum, Ilma S; Barros, Helena M T; Gomez, Rosane; Ribeiro, Maria Flavia M

2012-12-01

Progesterone is a neuroactive hormone with non-genomic effects on GABA(A) receptors (GABA(A)R). Changes in the expression of GABA(A)R subunits are related to depressive-like behaviors in rats. Moreover, sex differences and depressive behaviors have been associated with prefrontal brain asymmetry in rodents and humans. Thus, our objective was to investigate the effect of progesterone on the GABA(A)R α1 and γ2 subunits mRNA expression in the right and left prefrontal cortex of diestrus female and male rats exposed to the forced swimming test (FST). Male and female rats (n = 8/group) were randomly selected to receive a daily dose of progesterone (0·4 mg·kg⁻¹) or vehicle, during two complete female estrous cycles (8-10 days). On the experiment day, male rats or diestrus female rats were euthanized 30 min after the FST. Our results showed that progesterone significantly increased the α1 subunit mRNA in both hemispheres of male and female rats. Moreover, there was an inverse correlation between depressive-like behaviors and GABA(A)R α1 subunit mRNA expression in the right hemisphere in female rats. Progesterone decreased the GABA(A)R γ2 mRNA expression only in the left hemisphere of male rats. Therefore, we conclude that the GABA(A) system displays an asymmetric distribution according to sex and that progesterone, at lower doses, presents an antidepressant effect after increasing the GABA(A) R α1 subunit expression in the right prefrontal cortex of female rats. Copyright © 2012 John Wiley & Sons, Ltd.

Altered emotionality, hippocampus-dependent performance and expression of NMDA receptor subunit mRNAs in chronically stressed mice.

PubMed

Costa-Nunes, João; Zubareva, Olga; Araújo-Correia, Margarida; Valença, Andreia; Schroeter, Careen A; Pawluski, Jodi L; Vignisse, Julie; Steinbusch, Hellen; Hermes, Denise; Phillipines, Marjan; Steinbusch, Harry M W; Strekalova, Tatyana

2014-01-01

N-Methyl-D-aspartate receptor (NMDAR)-mediated neurotransmission in the hippocampus is implicated in cognitive and emotional disturbances during stress-related disorders. Here, using quantitative RT-PCR, we investigated the hippocampal expression of NR2A, NR2B and NR1 subunit mRNAs in a mouse stress paradigm that mimics clinically relevant conditions of simultaneously affected emotionality and hippocampus-dependent functions. A 2-week stress procedure, which comprised ethologically valid stressors, exposure to a rat and social defeat, was applied to male C57BL/6J mice. For predation stress, mice were introduced into transparent containers that were placed in a rat home cage during the night; social defeat was applied during the daytime using aggressive CD1 mice. This treatment impaired hippocampus-dependent performance during contextual fear conditioning. A correlation between this behavior and food displacement performance was demonstrated, suggesting that burrowing behavior is affected by the stress procedure and is hippocampus-dependent. Stressed mice (n = 22) showed behavioral invigoration and anomalous anxiolytic-like profiles in the O-maze and brightly illuminated open field, unaltered short-term memory in the step-down avoidance task and enhanced aggressive traits, as compared to non-stressed mice (n = 10). Stressed mice showed increased basal serum corticosterone concentrations, hippocampal mRNA expression for the NR2A subunit of the NMDAR and in the NR2A/NR2B ratio; mRNA expression of NR2B and NR1 was unchanged. Thus, stress-induced aberrations in both hippocampal-dependent performance and emotional abnormalities are associated with alterations in hippocampal mRNA NR2A levels and the NR2A/NR2B ratio and not with mRNA expression of NR2B or NR1.

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

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.

GSK126 (EZH2 inhibitor) interferes with ultraviolet A radiation-induced photoaging of human skin fibroblast cells

PubMed Central

Qin, Haiyan; Zhang, Guang; Zhang, Lianbo

2018-01-01

Polycomb group genes (PcG) encode chromatin modification proteins that are involved in the epigenetic regulation of cell differentiation, proliferation and the aging processes. The key subunit of the PcG complex, enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), has a central role in a variety of mechanisms, such as the formation of chromatin structure, gene expression regulation and DNA damage. In the present study, ultraviolet A (UVA) was used to radiate human dermal fibroblasts in order to construct a photo-aged cell model. Subsequently, the cell viability assay, Hoechst staining, apoptosis detection using flow cytometry, senescence-associated β-galactosidase (SA-β-gal) staining and erythrocyte exclusion experiments were performed. GSK126, a histone methylation enzyme inhibitor of EZH2, was used as an experimental factor. Results suggested that GSK126 downregulated the mRNA expression levels of EZH2 and upregulated the mRNA expression levels of BMI-1. Notably, GSK126 affected the transcription of various photoaging-related genes and thus protected against photoaging induced by UVA radiation. PMID:29545866

Quantitative analysis of the expression and distribution of calcium channel alpha 1 subunit mRNA in the atria and ventricles of the rat heart.

PubMed

Larsen, Janice K; Mitchell, Jennifer W; Best, Philip M

2002-05-01

Two distinct calcium currents are present in mammalian cardiac myocytes. Utilizing quantitative RT-PCR methods, we have analysed the expression patterns and abundance of four calcium channel alpha 1 subunit mRNAs in different regions of the rat heart and compared them to the known density of calcium currents recorded from rat atria. Our results show that Ca(V)1.2 is the most abundant of the four alpha 1 subunit transcripts in the rat heart. The Ca(V)1.2 message is more abundant in ventricle than in atria and does not vary in expression as a function of developmental age. Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 mRNAs are 10-100 times less abundant than Ca(V)1.2. Interestingly, Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 are expressed in both atria and ventricle. The abundance of atrial Ca(V)3.1 mRNA does not change significantly during development and remains high in older animals. In contrast, levels of atrial Ca(V)3.2 mRNA are high in embryonic tissue and at 3- and 4-weeks postnatal but become undetectable at 5 weeks. Expression of atrial Ca(V)2.3 mRNA is highest at 4-weeks postnatal and then declines gradually. We have previously documented that the LVA calcium current density is highest within 4-5 weeks after birth and then declines gradually reaching less than 30% of its maximal value at 12-14 weeks. The complex relationship between atrial LVA current density and the abundance of Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 mRNA suggests that their contribution to the cardiac LVA current may vary as a function of postnatal age. Copyright 2002 Academic Press.

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

PubMed

Elson, G C; Graber, P; Losberger, C; Herren, S; Gretener, D; Menoud, L N; Wells, T N; Kosco-Vilbois, M H; Gauchat, J F

1998-08-01

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

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.

Differential expression of gill Na+,K+-ATPaseα - and β-subunits, Na+,K+,2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar

USGS Publications Warehouse

Nilsen, Tom O.; Ebbesson, Lars O.E.; Madsen, Steffen S.; McCormick, Stephen D.; Andersson, Eva; Bjornsson, Bjorn Thrandur; Prunet, Patrick; Stefansson, Sigurd O.

2007-01-01

This study examines changes in gill Na+,K+-ATPase (NKA) α- and β-subunit isoforms, Na+,K+,2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, and after seawater (SW) transfer in May/June. Gill NKA activity increased from February through April, May and June among both strains in freshwater (FW), with peak enzyme activity in the landlocked salmon being 50% below that of the anadromous fish in May and June. Gill NKA-α1b, -α3, -β1 and NKCC mRNA levels in anadromous salmon increased transiently, reaching peak levels in smolts in April/May, whereas no similar smolt-related upregulation of these transcripts occurred in juvenile landlocked salmon. Gill NKA-α1a mRNA decreased significantly in anadromous salmon from February through June, whereas α1a levels in landlocked salmon, after an initial decrease in April, remained significantly higher than those of the anadromous smolts in May and June. Following SW transfer, gill NKA-α1b and NKCC mRNA increased in both strains, whereas NKA-α1a decreased. Both strains exhibited a transient increase in gill NKA α-protein abundance, with peak levels in May. Gill α-protein abundance was lower in SW than corresponding FW values in June. Gill NKCC protein abundance increased transiently in anadromous fish, with peak levels in May, whereas a slight increase was observed in landlocked salmon in May, increasing to peak levels in June. Gill CFTR I mRNA levels increased significantly from February to April in both strains, followed by a slight, though not significant increase in May and June. CFTR I mRNA levels were significantly lower in landlocked than anadromous salmon in April/June. Gill CFTR II mRNA levels did not change significantly in either strain. Our findings demonstrates that differential expression of gill NKA-α1a, -α1b and -α3 isoforms may be important for potential functional differences in NKA, both during preparatory development and during salinity adjustments in salmon. Furthermore, landlocked salmon have lost some of the unique preparatory upregulation of gill NKA, NKCC and, to some extent, CFTR anion channel associated with the development of hypo-osmoregulatory ability in anadromous salmon.

Progesterone neuroprotection in the Wobbler mouse, a genetic model of spinal cord motor neuron disease.

PubMed

Gonzalez Deniselle, María Claudia; López-Costa, Juan José; Saavedra, Jorge Pecci; Pietranera, Luciana; Gonzalez, Susana L; Garay, Laura; Guennoun, Rachida; Schumacher, Michael; De Nicola, Alejandro F

2002-12-01

Motor neuron degeneration characterizes the spinal cord of patients with amyotrophic lateral sclerosis and the Wobbler mouse mutant. Considering that progesterone (PROG) provides neuroprotection in experimental ischemia and injury, its potential role in neurodegeneration was studied in the murine model. Two-month-old symptomatic Wobbler mice were left untreated or received sc a 20-mg PROG implant for 15 days. Both light and electron microscopy of Wobbler mice spinal cord showed severely affected motor neurons with profuse cytoplasmic vacuolation of the endoplasmic reticulum and/or Golgi apparatus and ruptured mitochondria with damaged cristae, a profile indicative of a type II cytoplasmic form of cell death. In contrast to untreated mice, neuropathology was less severe in Wobbler mice receiving PROG; including a reduction of vacuolation and of the number of vacuolated cells and better conservation of the mitochondrial ultrastructure. In biochemical studies, we determined the mRNA for the alpha3 subunit of Na,K-ATPase, a neuronal enzyme controlling ion fluxes, neurotransmission, membrane potential, and nutrient uptake. In untreated Wobbler mice, mRNA levels in motor neurons were reduced by half compared to controls, whereas PROG treatment of Wobbler mice restored the expression of alpha3 subunit Na,K-ATPase mRNA. Therefore, PROG was able to rescue motor neurons from degeneration, based on recovery of histopathological abnormalities and of mRNA levels of the sodium pump. However, because the gene mutation in Wobbler mice is still unknown, further studies are needed to unveil the action of PROG and the mechanism of neuronal death in this genetic model of neurodegeneration.

Cellular antioxidant effects of atorvastatin in vitro and in vivo.

PubMed

Wassmann, Sven; Laufs, Ulrich; Müller, Kirsten; Konkol, Christian; Ahlbory, Katja; Bäumer, Anselm T; Linz, Wolfgang; Böhm, Michael; Nickenig, Georg

2002-02-01

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert direct effects on vascular cells and beneficially influence endothelial dysfunction. Because reactive oxygen species (ROS) may lead to vascular damage and dysfunction, we investigated the effect of atorvastatin on ROS production and the underlying mechanisms in vitro and in vivo. Cultured rat aortic vascular smooth muscle cells were incubated with 10 micromol/L atorvastatin. Angiotensin II-induced and epidermal growth factor-induced ROS production were significantly reduced by atorvastatin (dichlorofluorescein fluorescence laser microscopy). Atorvastatin downregulated mRNA expression of the NAD(P)H oxidase subunit nox1, whereas p22phox mRNA expression was not significantly altered (reverse transcription-polymerase chain reaction, Northern analysis). Membrane translocation of rac1 GTPase, which is required for the activation of NAD(P)H oxidase, was inhibited by atorvastatin (Western blot). mRNA expression of superoxide dismutase isoforms and glutathione peroxidase was not modified by atorvastatin, whereas catalase expression was upregulated at mRNA and protein levels, resulting in an increased enzymatic activity. Effects of atorvastatin on ROS production and nox1, rac1, and catalase expression were inhibited by L-mevalonate but not by 25-hydroxycholesterol. In addition, spontaneously hypertensive rats were treated with atorvastatin for 30 days. ROS production in aortic segments was significantly reduced in statin-treated rats (lucigenin chemiluminescence). Treatment with atorvastatin reduced vascular mRNA expression of p22phox and nox1 and increased aortic catalase expression. mRNA expression of superoxide dismutases, glutathione peroxidase, and NAD(P)H oxidase subunits gp91phox, p40phox, p47phox, and p67phox remained unchanged. Translocation of rac1 from the cytosol to the cell membrane was also reduced in vivo. Thus, atorvastatin exerts cellular antioxidant effects in cultured rat vascular smooth muscle cells and in the vasculature of spontaneously hypertensive rats mediated by decreased expression of essential NAD(P)H oxidase subunits and by upregulation of catalase expression. These effects of atorvastatin may contribute to the vasoprotective effects of statins.

Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

NASA Technical Reports Server (NTRS)

Evans, G. L.; Morey-Holton, E.; Turner, R. T.

1998-01-01

In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

PubMed

Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

PubMed

Harding, Louisa B; Schultz, Irvin R; da Silva, Denis A M; Ylitalo, Gina M; Ragsdale, Dave; Harris, Stephanie I; Bailey, Stephanie; Pepich, Barry V; Swanson, Penny

2016-09-01

It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17β-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to estrogenic activity of the WWTP effluents. These results suggest that lhb gene expression may be a sensitive index of acute exposure to estrogenic chemicals in juvenile coho salmon. Further work is needed to determine the kinetics and specificity of lhb induction to evaluate its utility as a potential indicator of estrogen exposure in immature fish. Published by Elsevier B.V.

Evidence against translational repression by the carboxyltransferase component of Escherichia coli acetyl coenzyme A carboxylase.

PubMed

Smith, Alexander C; Cronan, John E

2014-11-01

In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217-1227, 2010, doi:http://dx.doi.org/10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramer-mediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Evidence against Translational Repression by the Carboxyltransferase Component of Escherichia coli Acetyl Coenzyme A Carboxylase

PubMed Central

Smith, Alexander C.

2014-01-01

In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217–1227, 2010, doi:http://dx.doi.org/10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramer-mediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs. PMID:25157077

Changes in the role of the thyroid axis during metamorphosis of the Japanese eel, Anguilla japonica.

PubMed

Sudo, Ryusuke; Okamura, Akihiro; Kuroki, Mari; Tsukamoto, Katsumi

2014-08-01

To clarify the role of thyroid function during metamorphosis from leptocephalus to glass eel in the Japanese eel, we examined the histology of the thyroid gland and measured whole-body concentrations of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), and thyroid stimulating hormone β-subunit TSH (TSHβ) mRNA expression levels in five stages of artificially hatched eels (leptocephalus, early-metamorphosis, late-metamorphosis, glass eel, and elver). During metamorphosis, the inner colloid of thyroid follicles showed positive immunoreactivity for T4, and both T4 and T3 levels were significantly increased, whereas a small peak of TSHβ mRNA level was observed at the early-metamorphosis stage. Similarly, TSHβ mRNA levels were highest in the glass eel stage, and then decreased markedly in the elver stage. In contrast to TSHβ mRNA expression, thyroid hormones (both T4 and T3) increased further from the glass eel to elver stages. These results indicated that thyroid function in the Japanese eel was active both during and after metamorphosis. Therefore, the thyrotropic axis may play important roles not only in metamorphosis but also in subsequent inshore or upstream migrations. © 2014 Wiley Periodicals, Inc.

Changes in the mRNA levels of delayed rectifier potassium channels in human atrial fibrillation.

PubMed

Lai, L P; Su, M J; Lin, J L; Lin, F Y; Tsai, C H; Chen, Y S; Tseng, Y Z; Lien, W P; Huang, S K

1999-01-01

We measured mRNA levels of delayed rectifier potassium channels in human atrial tissue to investigate the mechanism of the shortening of the atrial effective refractory period and the loss of rate-adaptive shortening of the atrial effective refractory period in human atrial fibrillation. A total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The mRNA amounts of KVLQT1 (IKs), minK (beta-subunit of IKs), HERG (IKr), and KV1.5 (IKur) were measured by reverse transcription-polymerase chain reaction and normalized to the mRNA amount of GAPDH. We found that the mRNA levels of KV1.5, HERG and KVLQT1 were all significantly decreased in patients with persistent atrial fibrillation for more than 3 months. In contrast, the mRNA level of minK was significantly increased in patients with persistent atrial fibrillation for more than 3 months. We further showed that these changes were independent of the underlying cardiac disease, atrial filling pressure, gender and age. We also found that there was no spatial dispersion of mRNA levels among the four atrial sampling sites. Because the decrease in potassium currents results in a prolonged action potential, the shortening of the atrial effective refractory period in atrial fibrillation should be attributed to other factors. However, the decrease in IKs might contribute, at least in part, to the loss of rate-adaptive shortening of the atrial refractory period.

NADPH Oxidase Isoform 2 (NOX2) Is Involved in Drug Addiction Vulnerability in Progeny Developmentally Exposed to Ethanol.

PubMed

Contreras, Marcela L; de la Fuente-Ortega, Erwin; Vargas-Roberts, Sofía; Muñoz, Daniela C; Goic, Carolina A; Haeger, Paola A

2017-01-01

Ethanol exposure increases oxidative stress in developing organs, including the brain. Antioxidant treatment during maternal ethanol ingestion improves behavioral deficits in rodent models of fetal alcohol spectrum disorder (FASD). However, the impact of general antioxidant treatment in their adult offspring and the Specific Reactive Species (ROS)-dependent mechanism, are not fully understood. We hypothesized that pre and early postnatal ethanol exposure (PEE) modifies redox homeostasis, in particular NOX2 function during reward signaling in the mesocorticolimbic pathway, which reinforces the effects of alcohol. We developed a FASD rat model which was evaluated during adolescence (P21) and adulthood (P70). We first studied whether redox homeostasis is affected in PEE animals, by analyzing mRNA expression of SOD1, CAT, and Gpx1. We found that PEE reduced the mRNA levels of these three anti-oxidant enzymes in PFC and HIPP at P21 and in the VTA at P70. We also analyzed basal mRNA and protein expression of NOX2 subunits such as gp91phox, p22 phox, and p47 phox, in mesocorticolimbic brain areas of PEE rat brains. At P21, gp91 phox, and p47 phox levels in the VTA were decreased. At P70, gp91 phox mRNA levels was decreased in HIPP and both mRNA and protein levels were decreased in PFC. Since NOX2 is regulated by the N-methyl-D-aspartate Receptor (NMDAR), we analyzed NMDAR mRNA expression and found differential expression of NMDAR subunits (NR1 and NR2B) in the PFC that was age dependent, with levels decreased at P21 and increased at P70. The analysis also revealed decreased NR2B mRNA expression in HIPP and VTA at P70. Offspring from maternal ethanol users consumed 25% more ethanol in a free choice alcohol consumption test than control rats, and showed place preference for an alcohol-paired compartment. In vivo inhibition of NOX2 using apocynin in drinking water, or infusion of blocked peptide gp91 phox ds in the VTA normalized alcohol place preference, suggesting that NOX2 plays an important role in addictive like behavior. Taken together, PEE significantly affects the expression of antioxidant enzymes, NOX2, NMDAR in an age, and brain region dependent manner. Moreover, we demonstrate that NOX2 regulates alcohol seeking behavior.

Effects of gonadectomy and testosterone treatment on aquaporin expression in the kidney of normotensive and hypertensive rats.

PubMed

Loh, Su Yi; Giribabu, Nelli; Salleh, Naguib

2017-07-01

We tested the hypothesis that testosterone-induced increase in blood pressure was due to changes in aquaporin (AQP) expression in kidneys. In this study, expression level of kidney AQPs was investigated under testosterone influence. Adult normotensive Wistar Kyoto (WKY) and hypertensive SHR male and female rats underwent gonadectomy. For female rats, testosterone was given for six weeks duration, two weeks following ovariectomy via subcutaneous silastic implant. Mean arterial pressure (MAP) was measured in all the rats after eight weeks via carotid artery cannulation and the rats were then sacrificed and kidneys were harvested for analyses of AQP-1, 2, 3, 4, 6, and 7 mRNA and protein expressions by quantitative real-time PCR and Western blotting, respectively. Distribution of AQP subunits' protein in kidneys was observed by immunofluorescence. In male WKY rats, MAP, AQP-1, 2, 4, and 7 protein; and mRNA expression decreased however AQP-3 protein and mRNA expression increased following orchidectomy. The vice versa effects were observed in testosterone-treated ovariectomized female WKY rats. However, no changes in AQP-6 expression were observed. Meanwhile, in adult male SHR rats, MAP and expression level of all AQP subunits decreased following orchidectomy. The opposite effects were seen in ovariectomized female SHR rats following testosterone treatment. Immunofluorescence study showed AQP-1 and AQP-7 were distributed in the proximal convoluted tubules (PCT) while AQP-2, AQP-4, and AQP-6 were distributed in the collecting ducts (CDs). AQP-3 was distributed in the PCT and CD. In conclusion, changes in AQP subunit expression in kidneys could explain changes in blood pressure under testosterone influence. Impact statement This study provides fundamental understanding on the mechanisms underlying testosterone-induced increase in blood pressure which involve regulation of aquaporin channel subunits in the kidneys. A better understanding of this issue can help to explain the reason for higher blood pressure in males as compared to females and may explain the reason for higher blood pressure in females after menopause than females before menopause, the former most probably related to the changes in female androgen.

Glucosensing capacity in rainbow trout liver displays day-night variations possibly related to melatonin action.

PubMed

Conde-Sieira, Marta; Patiño, Marcos A López; Míguez, Jesús M; Soengas, José L

2012-09-01

To assess whether the glucosensing capacity in peripheral (liver and Brockmann bodies) and central (hypothalamus and hindbrain) locations of rainbow trout displays day-night variations in its response to changes in circulating glucose levels, we evaluated the response of parameters related to glucosensing [glucose, glycogen and glucose 6-phosphate levels, activities of glucokinase (GK), glycogen synthetase (GSase) and pyruvate kinase (PK), and mRNA abundance of GK, glucose transporter 2 (GLUT2), and K(ATP) channel subunits Kir6.x-like and sulfonylurea receptor (SUR)-like] in fish subjected to hyperglycemic treatment under night or day conditions. No day-night significant variations were noticed in the glucosensing capacity of the hypothalamus, hindbrain and Brockmann bodies. In contrast, a clear differential response was noticed in the liver, where glucose levels, GK activity (and mRNA levels) and GSase activity displayed increased values during the day in hyperglycemic fish compared with controls, and lower (GK mRNA levels) or non-existent (glucose, GK and GSase activities, and Kir6.x-like mRNA levels) values during the night. A similar decrease in parameters related to glucosensing in the liver was observed when fish under day conditions were treated with melatonin, suggesting a modulatory role of melatonin in day-night changes of the glucosensing response in the same tissue.

The eukaryote chaperonin CCT is a cold shock protein in Saccharomyces cerevisiae

PubMed Central

Somer, Lilach; Shmulman, Oshrit; Dror, Tali; Hashmueli, Sharon; Kashi, Yechezkel

2002-01-01

The eukaryotic Hsp60 cytoplasmic chaperonin CCT (chaperonin containing the T-complex polypeptide–1) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin. The present research focused mainly on the expression of the CCT subunits, CCTα and CCTβ, in yeast (Saccharomyces cerevisiae). Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock. In this study, messenger ribonucleic acid (mRNA) and protein levels of CCT subunits following exposure to low temperatures, were examined. The Northern blot analysis indicated a 3- to 4-fold increase in mRNA levels of CCTα and CCTβ genes after cold shock at 4°C. Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTα protein, which is expressed at 10°C, but not at 4°C. Transfer of 4°C cold-shocked cells to 10°C induced a 5-fold increase in the CCTα protein level. By means of fluorescent immunostaining and confocal microscopy, we found CCTα to be localized in the cortex and the cell cytoplasm of S. cerevisiae. Localization of CCTα was not affected at low temperatures. Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy. The induction pattern of the CCTα protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and the transition to growth at higher temperatures, as found for other Hsps during the recovery phase from heat shock. PMID:11892987

Building a bridge between neurobiology and mental illness.

PubMed

Costa, E

1992-10-01

GABA (gamma amino butyric acid) is the most abundant and important inhibitory transmitter in mammalian CNS. It counterbalances the glutamate mediated neuronal excitation. Abnormalities of the interaction of these two transmitters might change the mechanisms of neuronal group selection that according to Edelman [Neural Darwinism. Basic Books, New York] play a role in mediating several brain functions including cognition processes. Indeed imbalances in GABAergic functions were shown to elicit psychoses. They can be obtained by administration of drugs that affect synthesis, metabolism and uptake of GABA and thereby cause a persistent stimulation of GABAA receptors or perhaps by genetic abnormalities in DNA transcription, pre-mRNA splicing, mRNA translation and posttranslation modifications of GABAA receptor subunits. The complexities in the regulation of GABAA receptor subunit structure, synthesis, assembly and the brain location of specific mRNA encoding for these subunits are investigated with in situ mRNA hybridization specific for subunits of GABAA receptors. The role of the variability resulting from the complexities in the regulation of GABAA receptor allosteric modulation by drugs and putative endogenous allosteric modulators of GABA action at GABAA receptors is discussed. This discussion gives relevance to the possibility that genetic abnormalities in the expression of proteins participating in GABAergic function are to be considered as a possible target of the genetic defects operative in psychoses. In line with this thinking, it is suggested that partial allosteric modulators (partial agonists) of GABAA receptors and the phosphothioate or methylphosphonate analogs antisense to specific mRNA oligonucleotides that mediate the expression of genetic information concerning GABAA and glutamate receptor subunits may become valuable tools in psychiatric research. Perhaps in the future these studies might generate new ideas useful in the therapy of genetically determined psychiatric illness.

Distinct functions of neuromedin u and neuromedin s in orange-spotted grouper.

PubMed

Li, Shuisheng; Xiao, Ling; Liu, Qiongyu; Zheng, Binbin; Chen, Huapu; Liu, Xiaochun; Zhang, Yong; Lin, Haoran

2015-10-01

Neuromedin U (NMU) and neuromedin S (NMS) play inhibitory roles in the regulation of food intake and energy homeostasis in mammals. However, their functions are not clearly established in teleost fish. In the present study, nmu and nms homologs were identified in several fish species. Subsequently, their cDNA sequences were cloned from the orange-spotted grouper (Epinephelus coioides). Sequence analysis showed that the orange-spotted grouper Nmu proprotein contains a 21-amino acid mature Nmu peptide (Nmu-21). The Nms proprotein lost the typical mature Nms peptide, but it retains a putative 34-amino acid peptide (Nmsrp). In situ hybridization revealed that nmu- and nms-expressing cells are mainly localized in the hypothalamic regions associated with appetite regulation. Food deprivation decreased the hypothalamic nmu mRNA levels but induced an increase of nms mRNA levels. Periprandial expression analysis showed that hypothalamic expression of nmu increased significantly at 3 h post-feeding, while nms expression was elevated at the normal feeding time. I.p. injection of synthetic Nmu-21 peptide suppressed the hypothalamic neuropeptide y (npy) expression, while Nmsrp administration significantly increased the expression of npy and orexin in orange-spotted grouper. Furthermore, the mRNA levels of LH beta subunit (lhβ) and gh in the pituitary were significantly down-regulated after Nmu-21 peptide administration, while Nmsrp was able to significantly stimulate the expression of FSH beta subunit (fshβ), prolactin (prl), and somatolaction (sl). Our results indicate that nmu and nms possess distinct neuroendocrine functions and pituitary functions in the orange spotted grouper. © 2015 Society for Endocrinology.

Regulation of PSMB5 Protein and β Subunits of Mammalian Proteasome by Constitutively Activated Signal Transducer and Activator of Transcription 3 (STAT3)

PubMed Central

Vangala, Janakiram Reddy; Dudem, Srikanth; Jain, Nishant; Kalivendi, Shasi V.

2014-01-01

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome. PMID:24627483

A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

PubMed Central

Méthot, N; Song, M S; Sonenberg, N

1996-01-01

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3. PMID:8816444

Cis-Regulatory Variants Affect CHRNA5 mRNA Expression in Populations of African and European Ancestry

PubMed Central

Wang, Jen-Chyong; Spiegel, Noah; Bertelsen, Sarah; Le, Nhung; McKenna, Nicholas; Budde, John P.; Harari, Oscar; Kapoor, Manav; Brooks, Andrew; Hancock, Dana; Tischfield, Jay; Foroud, Tatiana; Bierut, Laura J.; Steinbach, Joe Henry; Edenberg, Howard J.; Traynor, Bryan J.; Goate, Alison M.

2013-01-01

Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels. PMID:24303001

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

NASA Technical Reports Server (NTRS)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

PubMed

Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

2016-07-01

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

PubMed Central

Pánek, Josef; Kolář, Michal; Vohradský, Jiří; Shivaya Valášek, Leoš

2013-01-01

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs. PMID:23804757

Cup regulates oskar mRNA stability during oogenesis.

PubMed

Broyer, Risa M; Monfort, Elena; Wilhelm, James E

2017-01-01

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

Effect of thyroid status on the expression of metabolic enzymes during chronic stimulation.

PubMed

Hood, D A; Simoneau, J A; Kelly, A M; Pette, D

1992-10-01

The effect of thyroid status on the expression of cytochrome c oxidase (CYTOX) and the activities of citrate synthase (CS) and phosphofructokinase (PFK) were examined in chronically stimulated (10 Hz; 35 days) and contralateral, nonstimulated rat tibialis anterior muscle of hypothyroid, hyperthyroid, and euthyroid animals. Stimulation increased CYTOX activity by 2.7-, 3.2-, and 4.9-fold in hyperthyroid, euthyroid, and hypothyroid animals, respectively, to similar absolute values. CS displayed similar increases. Stimulation reduced PFK activity in hypothyroid and euthyroid animals to 45% and 60% of control values. This effect was abolished with hyperthyroidism. Thus stimulation and thyroid hormone act antagonistically on PFK activity. Stimulation increased CYTOX subunit III (mitochondrially encoded) mRNA by 2.5- and 2.9-fold in hyperthyroid and euthyroid animals. Similar increases were observed in the nuclear-encoded mRNAs of CYTOX subunit VIc in euthyroid muscle. In hyperthyroid and euthyroid conditions, the mRNA changes paralleled the increases in enzyme activity. In hypothyroid muscle, the increase in mRNA was less for subunit VIc than III, suggesting that hypothyroidism upsets the coordinate expression of nuclear and mitochondrial genes. Further, the increases in CYTOX activity exceeded that of both subunit mRNAs in hypothyroid muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Cervia, Davide, E-mail: d.cervia@unitus.it; Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano; Catalani, Elisabetta

Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected.more » Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ► Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ► Er-1 increases the T-cell production of specific cytokines. ► Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ► The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.« less

Local expression of interferon-alpha and interferon receptors in cervical intraepithelial neoplasia.

PubMed

Tirone, Nelson R; Peghini, Bethanea C; Barcelos, Ana Cristina M; Murta, Eddie F C; Michelin, Marcia A

2009-12-01

The present study evaluated mRNA expression of interferon-alpha (IFN-alpha), IFN-alpha receptor subunits (IFNAR-1 and IFNAR-2) and an IFN-stimulated gene encoding the enzyme 2',5'-oligoadenylate synthetase (2'5'OAS) in biopsies on patients with varying grades of cervical intraepithelial neoplasia (CIN I, II and III). Uterine cervix biopsies were collected from women with CIN I, II and III (n = 28) and controls without CIN lesions or human papilloma virus (HPV) infection (n = 17). The presence of high and low-risk HPV DNA was determined using hybrid capture. The mRNA levels of IFNAR-1, IFNAR-2, IFN-alpha and 2'5'OAS were determined by RT-PCR with specific primers. The control group exhibited a greater frequency of IFNAR-1 expression (10/17; 58.3%) than the CIN samples (4/28; 14.2%) (P = 0.0018), while, the expression of IFNAR-2 was also greater in the control samples (11/17; 64.7%) than in the patients with lesions (2/28; 7.1%) (P = 0.0018). Importantly, simultaneous expression of both receptors was observed only in the control group (8/17; 47.0%) (P = 0.0001). Among the CIN samples, there was one case of low expression of mRNA of IFNAR-1 and IFNAR-2. IFN-alpha was present in 14.2% (4/28) of the CIN samples but was not expressed in the control group. mRNA 2'5'OAS were expressed in 28.5% (8/28) of the CIN samples and 11.7% (2/17) of the control samples (not statistically significant). Fifty percent (14/28) of the CIN samples were positive for HPV DNA. Cervical biopsy samples from control women or those without neoplasia or HPV infection displayed higher IFN-alpha receptor expression than those with CIN, while simultaneous expression of both IFN-alpha receptor subunits was found only in the control group. There was no significant difference in mRNA expression of IFN-alpha and 2'5'OAS between the control and CIN groups. Then we concluded that the samples obtained from patients with CIN present low levels of the IFN-alpha receptor mRNA.

Reductions in the Cardiac Transient Outward K+ Current Ito Caused by Chronic β-Adrenergic Receptor Stimulation Are Partly Rescued by Inhibition of Nuclear Factor κB.

PubMed

Panama, Brian K; Korogyi, Adam S; Aschar-Sobbi, Roozbeh; Oh, Yena; Gray, Charles B B; Gang, Hongying; Brown, Joan Heller; Kirshenbaum, Lorrie A; Backx, Peter H

2016-02-19

The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory β-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of β-adrenergic receptors (β-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic β-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic β-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by β-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as β-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic β-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential senescence capacities in meibomian gland carcinoma and basal cell carcinoma.

PubMed

Zhang, Leilei; Huang, Xiaolin; Zhu, Xiaowei; Ge, Shengfang; Gilson, Eric; Jia, Renbing; Ye, Jing; Fan, Xianqun

2016-03-15

Meibomian gland carcinoma (MGC) and basal cell carcinoma (BCC) are common eyelid carcinomas that exhibit highly dissimilar degrees of proliferation and prognoses. We address here the question of the differential mechanisms between these two eyelid cancers that explain their different outcome. A total of 102 confirmed MGC and 175 diagnosed BCC cases were analyzed. Twenty confirmed MGC and twenty diagnosed BCC cases were collected to determine the telomere length, the presence of senescent cells, and the expression levels of the telomere capping shelterin complex, P53, and the E3 ubiquitin ligase Siah1. Decreased protein levels of the shelterin subunits, shortened telomere length, over-expressed Ki-67, and Bcl2 as well as mutations in P53 were detected both in MGC and BCC. It suggests that the decreased protein levels of the shelterin complex and the shortened telomere length contribute to the tumorigenesis of MGC and BCC. However, several parameters distinguish MGC from BCC samples: (i) the mRNA level of the shelterin subunits decreased in MGC but it increased in BCC; (ii) P53 was more highly mutated in MGC; (iii) Siah1 mRNA was over-expressed in BCC; (iv) BCC samples contain a higher level of senescent cells; (v) Ki-67 and Bcl2 expression were lower in BCC. These results support a model where a preserved P53 checkpoint in BCC leads to cellular senescence and reduced tumor proliferation as compared to MGC. © 2015 UICC.

Dietary vitamin E deficiency does not affect global and specific DNA methylation patterns in rat liver.

PubMed

Fischer, Alexandra; Gaedicke, Sonja; Frank, Jan; Döring, Frank; Rimbach, Gerald

2010-10-01

The aim of the present study was to determine the effects of a 6-month dietary vitamin E (VE) deficiency on DNA methylation and gene expression in rat liver. Two enzymes, 5-α-steroid reductase type 1 (SRD5A1) and the regulatory subunit of γ-glutamylcysteinyl synthetase (GCLM), which are differentially expressed on the mRNA level, were analysed for promoter methylation in putative cytosine-phospho-guanine (CpG) island regions located at the 5' end using base-specific cleavage and matrix-assisted laser desorption ionisation time-of-flight MS. A twofold increase in the mRNA level of SRD5A1 gene and a twofold decrease in the mRNA level of GCLM gene in VE-deficient animals were not associated with different CpG methylation of the analysed promoter region. Furthermore, global DNA methylation was not significantly different in these two groups. Thus, the present results indicate that the VE-induced regulation of SRD5A1 and GCLM in rat liver is not directly mediated by changes in promoter DNA methylation.

Multiple isoforms for the catalytic subunit of PKA in the basal fungal lineage Mucor circinelloides.

PubMed

Fernández Núñez, Lucas; Ocampo, Josefina; Gottlieb, Alexandra M; Rossi, Silvia; Moreno, Silvia

2016-12-01

Protein kinase A (PKA) activity is involved in dimorphism of the basal fungal lineage Mucor. From the recently sequenced genome of Mucor circinelloides we could predict ten catalytic subunits of PKA. From sequence alignment and structural prediction we conclude that the catalytic core of the isoforms is conserved, and the difference between them resides in their amino termini. This high number of isoforms is maintained in the subdivision Mucoromycotina. Each paralogue, when compared to the ones form other fungi is more homologous to one of its orthologs than to its paralogs. All of these fungal isoforms cannot be included in the class I or II in which fungal protein kinases have been classified. mRNA levels for each isoform were measured during aerobic and anaerobic growth. The expression of each isoform is differential and associated to a particular growth stage. We reanalyzed the sequence of PKAC (GI 20218944), the only cloned sequence available until now for a catalytic subunit of M. circinelloides. PKAC cannot be classified as a PKA because of its difference in the conserved C-tail; it shares with PKB a conserved C2 domain in the N-terminus. No catalytic activity could be measured for this protein nor predicted bioinformatically. It can thus be classified as a pseudokinase. Its importance can not be underestimated since it is expressed at the mRNA level in different stages of growth, and its deletion is lethal. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Chemical and structural characterization of a model Post-Termination Complex (PoTC) for the ribosome recycling reaction: Evidence for the release of the mRNA by RRF and EF-G

PubMed Central

Iwakura, Nobuhiro; Yokoyama, Takeshi; Quaglia, Fabio; Mitsuoka, Kaoru; Mio, Kazuhiro; Shigematsu, Hideki; Shirouzu, Mikako; Kaji, Akira; Kaji, Hideko

2017-01-01

A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the biochemical measurement of bound tRNA and mRNA. Using this substrate, we establish that the release of tRNA, release of mRNA and splitting of ribosomal subunits occur during the recycling reaction. Order of these events is tRNA release first followed by mRNA release and splitting almost simultaneously. Moreover, we demonstrate that IF3 is not involved in any of the recycling reactions but simply prevents the re-association of split ribosomal subunits. Our finding demonstrates that the important function of RRF includes the release of mRNA, which is often missed by the use of a short ORF with the Shine-Dalgarno sequence near the termination site. PMID:28542628

Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs

PubMed Central

Pachernegg, Svenja; Joshi, Illah; Muth-Köhne, Elke; Pahl, Steffen; Münster, Yvonne; Terhag, Jan; Karus, Michael; Werner, Markus; Ma-Högemeier, Zhan-Lu; Körber, Christoph; Grunwald, Thomas; Faissner, Andreas; Wiese, Stefan; Hollmann, Michael

2013-01-01

Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs. PMID:24348335

Effects of atorvastatin and losartan on monocrotaline-induced pulmonary artery remodeling in rats.

PubMed

Xie, Liangdi; Lin, Peisen; Xie, Hong; Xu, Changsheng

2010-01-01

Structural remodeling of pulmonary artery plays an important role in maintaining sustained pulmonary arterial hypertension (PAH). The anti-remodeling effects of statins have been reported in systemic hypertension. In this study, we studied the effects of atovastatin (Ato) or losartan (Los) in monocrotaline (MCL)-induced pulmonary artery remodeling using a rat model. Forty Sprague-Dawley (SD) rats were randomly assigned into four groups (n = 10): normal control (Ctr), PAH, PAH treated with Los, and PAH treated with Ato. We found that in the Los- or Ato-treated group, the mean pulmonary arterial pressure, right heart hypertrophy index, ratio of wall/lumen thickness (WT%), as well as the wall/lumen area (WA%) were significantly reduced compared to the PAH group. Also in pulmonary arteries dissected from rats in the Ato- or Los-treated group, in both mRNA and protein levels, the expression of α1C subunit of voltage-gated calcium channel (Ca(v)α1c) was downregulated, while sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA-2a) and inositol 1,4,5 triphosphate receptor 1 (IP3R-1) upregulated. However, the mRNA level of RyR-3 subunit of calcium regulating channel was increased, whereas its protein level was reduced in the treated groups. Our results suggest that atorvastatin or losartan may regress the remodeling of the pulmonary artery in pulmonary hypertensive rats, with differential expression of calcium regulating channels.

An Abnormal Nitric Oxide Metabolism Contributes to Brain Oxidative Stress in the Mouse Model for the Fragile X Syndrome, a Possible Role in Intellectual Disability

PubMed Central

Lima-Cabello, Elena; Garcia-Guirado, Francisco; Calvo-Medina, Rocio; el Bekay, Rajaa; Perez-Costillas, Lucia; Quintero-Navarro, Carolina; Sanchez-Salido, Lourdes

2016-01-01

Background. Fragile X syndrome is the most common genetic cause of mental disability. Although many research has been performed, the mechanism underlying the pathogenesis is unclear and needs further investigation. Oxidative stress played major roles in the syndrome. The aim was to investigate the nitric oxide metabolism, protein nitration level, the expression of NOS isoforms, and furthermore the activation of the nuclear factor NF-κB-p65 subunit in different brain areas on the fragile X mouse model. Methods. This study involved adult male Fmr1-knockout and wild-type mice as controls. We detected nitric oxide metabolism and the activation of the nuclear factor NF-κBp65 subunit, comparing the mRNA expression and protein content of the three NOS isoforms in different brain areas. Results. Fmr1-KO mice showed an abnormal nitric oxide metabolism and increased levels of protein tyrosine nitrosylation. Besides that, nuclear factor NF-κB-p65 and inducible nitric oxide synthase appeared significantly increased in the Fmr1-knockout mice. mRNA and protein levels of the neuronal nitric oxide synthase appeared significantly decreased in the knockout mice. However, the epithelial nitric oxide synthase isoform displayed no significant changes. Conclusions. These data suggest the potential involvement of an abnormal nitric oxide metabolism in the pathogenesis of the fragile X syndrome. PMID:26788253

Scutellarin protects against Aβ-induced learning and memory deficits in rats: involvement of nicotinic acetylcholine receptors and cholinesterase

PubMed Central

Guo, Li-li; Guan, Zhi-zhong; Wang, Yong-lin

2011-01-01

Aim: To examine the protective effects of scutellarin (Scu) on rats with learning and memory deficit induced by β-amyloid peptide (Aβ). Methods: Fifty male Wistar rats were randomly divided into 5 groups: control, sham operation, Aβ, Aβ+Scu, and Aβ+piracetam groups. Aβ25–35 was injected into the lateral ventricle (10 μg each side). Scu (10 mg/2 mL) or piracetam (10 mg/2 mL was intragastrically administered per day for 20 consecutive days following Aβ treatment. Learning and memory was assessed with Morris water maze test. The protein and mRNA levels of nicotinic acetylcholine receptor (nAChR) α4, α7, and β2 subunits in the brain were examined using Western blotting and real-time PCR, respectively. The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the brain and plasma were measured using Ellman's colorimetric method. Results: In Aβ group, the escape latency period and first platform cross was significantly increased, and the total number of platform crossings was significantly decreased, as compared with the control and the sham operation groups. Both Scu and piracetam treatment significantly reduced the escape latency period and time to cross platform, and increased the number of platform crosses, but there were no significant differences between Aβ+Scu and Aβ+piracetam groups. In Aβ group, the protein levels of nAChR α4 and α7 subunits in the cerebral cortex were significantly decreased by 42%–47% and 58%–61%, respectively, as compared to the control and the sham operation groups. Scu treatment caused upregulation of α4 and α7 subunit proteins by around 24% and 30%, respectively, as compared to Aβ group, but there were no significant differences between Aβ+Scu and Aβ+piracetam groups. The protein level of nAChR β2 subunit had no significant difference among different groups. The mRNA levels of nAChR α4, α7, and β2 subunits were not significantly changed. In Aβ group, the activities of AChE and BuChE in the brain were significantly increased, but were significantly decreased in the plasma, as compared to the control and the sham operation groups. Scu or piracetam treatment restored the activities in brain and plasma nearly to the levels in the control group. Conclusion: The results suggest that Scu may rescue some of the deleterious effects of Aβ, possibly by stimulating nAChR protein translation and regulating cholinesterase activity. PMID:21986571

Effect of One Month Duration Ketogenic and non-Ketogenic High Fat Diets on Mouse Brain Bioenergetic Infrastructure

PubMed Central

Selfridge, J. Eva; Wilkins, Heather M.; Lezi, E; Carl, Steven M.; Koppel, Scott; Funk, Eric; Fields, Timothy; Lu, Jianghua; Tang, Ee Phie; Slawson, Chad; Wang, WenFang; Zhu, Hao; Swerdlow, Russell H.

2014-01-01

Diet composition may affect energy metabolism in a tissue-specific manner. Using C57Bl/6J mice, we tested the effect of ketosis-inducing and non-inducing high fat diets on genes relevant to brain bioenergetic infrastructures, and on proteins that constitute and regulate that infrastructure. At the end of a one-month study period the two high fat diets appeared to differentially affect peripheral insulin signaling, but brain insulin signaling was not obviously altered. Some bioenergetic infrastructure parameters were similarly impacted by both high fat diets, while other parameters were only impacted by the ketogenic diet. For both diets, mRNA levels for CREB, PGC1α, and NRF2 increased while NRF1, TFAM, and COX4I1 mRNA levels decreased. PGC1β mRNA increased and TNFα mRNA decreased only with the ketogenic diet. Brain mtDNA levels fell in both the ketogenic and non-ketogenic high fat diet groups, although TOMM20 and COX4I1 protein levels were maintained, and mRNA and protein levels of the mtDNA-encoded COX2 subunit were also preserved. Overall, the pattern of changes observed in mice fed ketogenic and non-ketogenic high fat diets over a one month time period suggests these interventions enhance some aspects of the brain’s aerobic infrastructure, and may enhance mtDNA transcription efficiency. Further studies to determine which diet effects are due to changes in brain ketone body levels, fatty acid levels, glucose levels, altered brain insulin signaling, or other factors such as adipose tissue-associated hormones are indicated. PMID:25104046

Expression of gonadotropin subunits in roach (Rutilus rutilus, Cyprinidae) infected with plerocercoids of the tapeworm Ligula intestinalis (Cestoda).

PubMed

Trubiroha, Achim; Wuertz, Sven; Frank, Sabrina N; Sures, Bernd; Kloas, Werner

2009-11-01

Plerocercoids of the tapeworm Ligula intestinalis (Cestoda: Bothriocephalidea) have been reported to inhibit gametogenesis of their intermediate fish hosts. However, mechanistic studies are rare and the proximate cues leading to impaired reproduction still remain unknown. In the present study we investigated the effects of infection by L. intestinalis on reproductive parameters of roach (Rutilus rutilus, Cyprinidae), a common fish host of this parasite. Field studies on roach demonstrated that in both genders infection prevented gonad development. As revealed by quantitative PCR, infection was accompanied by essentially lower pituitary expression of follicle-stimulating hormone beta-subunit (FSHbeta) and luteinizing hormone beta-subunit (LHbeta) mRNA compared with uninfected roach, providing clear evidence for gonadotropin-insufficiency as the cause of arrested gametogenesis. Under controlled laboratory conditions infected roach showed lower mRNA levels of FSHbeta but not of LHbeta, despite histology revealing similar gonad stages as in uninfected conspecifics. These findings indicate the involvement of FSH rather than LH in mediating effects of infection early during gonad development in roach. Moreover, the impact of L. intestinalis on reproductive parameters of roach appeared to be independent of the parasite burden. Together, these data provide valuable information on the role of FSH and LH as mediators of parasite-induced sterilization in a vertebrate and implicate the selective inhibition of host reproduction by L. intestinalis as a natural source of endocrine disruption in fish.

Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

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

Chuang, J.L.; Fisher, C.R.; Chuang, D.T.

1994-08-01

The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertionmore » generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.« less

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation.

PubMed

Chennupati, Vijaykumar; Veiga, Diogo Ft; Maslowski, Kendle M; Andina, Nicola; Tardivel, Aubry; Yu, Eric Chi-Wang; Stilinovic, Martina; Simillion, Cedric; Duchosal, Michel A; Quadroni, Manfredo; Roberts, Irene; Sankaran, Vijay G; MacDonald, H Robson; Fasel, Nicolas; Angelillo-Scherrer, Anne; Schneider, Pascal; Hoang, Trang; Allam, Ramanjaneyulu

2018-04-02

Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.

The expression characteristics of mt-ND2 gene in chicken.

PubMed

Zhang, Wenwen; Hou, Lingling; Wang, Ting; Lu, Weiwei; Tao, Yafei; Chen, Wen; Du, Xiaohui; Huang, Yanqun

2016-09-01

Subunit 2 of NADH dehydrogenase (ND2) is encoded by the mt-ND2 gene and plays a critical role in controlling the production of the mitochondrial reactive oxygen species. Our study focused on exploring the mt-ND2 tissue expression patterns and the effects of energy restriction and dietary fat (linseed oil, corn oil, sesame oil or lard) level (2.5% and 5%) on its expression in chicken. The results showed that mt-ND2 gene was expressed in the 15 tissues of hybrid chickens with the highest level in heart and lowest level in pancreas tissue; 30% energy restriction did not significantly affect mt-ND2 mRNA level in chicken liver tissue. Both the mt-ND2 mRNA levels in chicken pectoralis (p < 0.05) and hepatic tissues (p < 0.05) at 42 d-old were affected by the type of dietary fats in 5% level, while not in abdominal fat tissues. The expression of mt-ND2 in hepatic tissues was down-regulated with chicken age (p < 0.01). The interactive effect of dietary fat types with chicken age (p < 0.05) was significant on mt-ND2 mRNA level. The study demonstrated that mt-ND2 gene was extensively expressed in tissues, and the expression was affected by dietary fat types and chicken age.

The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signaling peptides in ovariectomized growing rats

NASA Technical Reports Server (NTRS)

Cavolina, J. M.; Evans, G. L.; Harris, S. A.; Zhang, M.; Westerlind, K. C.; Turner, R. T.

1997-01-01

A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-alpha2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-beta, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.

Studies of Ribonucleotide Reductase in Crucian Carp—An Oxygen Dependent Enzyme in an Anoxia Tolerant Vertebrate

PubMed Central

Sandvik, Guro K.; Tomter, Ane B.; Bergan, Jonas; Zoppellaro, Giorgio; Barra, Anne-Laure; Røhr, Åsmund K.; Kolberg, Matthias; Ellefsen, Stian

2012-01-01

The enzyme ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR), this radical originates from a tyrosyl radical formed in reaction with oxygen (O2) and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius) is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia), a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii), obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA), brain derived neurotrophic factor (BDNF) and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR) measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp. PMID:22916159

Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the β-subunit of the amiloride-sensitive epithelial sodium channel

PubMed Central

Pradervand, Sylvain; Barker, Pierre M.; Wang, Qing; Ernst, Stephen A.; Beermann, Friedrich; Grubb, Barbara R.; Burnier, Michel; Schmidt, Andrea; Bindels, Rene J. M.; Gatzy, John T.; Rossier, Bernard C.; Hummler, Edith

1999-01-01

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (α-, β-, and γ-subunits). To study the role of the β-subunit in vivo, we analyzed mice in which the βENaC gene locus was disrupted. These mice showed low levels of βENaC mRNA expression in kidney (≈1%), lung (≈1%), and colon (≈4%). In homozygous mutant βENaC mice, no βENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na+ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult βENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as βENaC m/m mice showed no weight loss, normal plasma Na+ and K+ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, βENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that βENaC is required for Na+ conservation during salt deprivation. PMID:9990093

An inversion of 25 base pairs causes feline GM2 gangliosidosis variant.

PubMed

Martin, Douglas R; Krum, Barbara K; Varadarajan, G S; Hathcock, Terri L; Smith, Bruce F; Baker, Henry J

2004-05-01

In G(M2) gangliosidosis variant 0, a defect in the beta-subunit of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52) causes abnormal accumulation of G(M2) ganglioside and severe neurodegeneration. Distinct feline models of G(M2) gangliosidosis variant 0 have been described in both domestic shorthair and Korat cats. In this study, we determined that the causative mutation of G(M2) gangliosidosis in the domestic shorthair cat is a 25-base-pair inversion at the extreme 3' end of the beta-subunit (HEXB) coding sequence, which introduces three amino acid substitutions at the carboxyl terminus of the protein and a translational stop that is eight amino acids premature. Cats homozygous for the 25-base-pair inversion express levels of beta-subunit mRNA approximately 190% of normal and protein levels only 10-20% of normal. Because the 25-base-pair inversion is similar to mutations in the terminal exon of human HEXB, the domestic shorthair cat should serve as an appropriate model to study the molecular pathogenesis of human G(M2) gangliosidosis variant 0 (Sandhoff disease).

Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics.

PubMed

Jin, Zhe; Bhandage, Amol K; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

2014-01-01

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence.

Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics

PubMed Central

Jin, Zhe; Bhandage, Amol K.; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R.; Birnir, Bryndis

2014-01-01

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence. PMID:25278838

Ribosomal proteins L5 and L11 co-operatively inactivate c-Myc via RNA-induced silencing complex.

PubMed

Liao, J-M; Zhou, X; Gatignol, A; Lu, H

2014-10-09

Oncogene MYC is highly expressed in many human cancers and functions as a global regulator of ribosome biogenesis. Previously, we reported that ribosomal protein (RP) L11 binds to c-Myc and inhibits its transcriptional activity in response to ribosomal stress. Here, we show that RPL5, co-operatively with RPL11, guides the RNA-induced silencing complex (RISC) to c-Myc mRNA and mediates the degradation of the mRNA, consequently leading to inhibition of c-Myc activity. Knocking down of RPL5 induced c-Myc expression at both mRNA and protein levels, whereas overexpression of RPL5 suppressed c-Myc expression and activity. Immunoprecipitation revealed that RPL5 binds to 3'UTR of c-Myc mRNA and two subunits of RISC, TRBP (HIV-1 TAR RNA-binding protein) and Ago2, mediating the targeting of c-Myc mRNA by miRNAs. Interestingly, RPL5 and RPL11 co-resided on c-Myc mRNA and suppressed c-Myc expression co-operatively. These findings uncover a mechanism by which these two RPs can co-operatively suppress c-Myc expression, allowing a tightly controlled ribosome biogenesis in cells.

Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

PubMed

Tilignac, Thomas; Temparis, Sandrine; Combaret, Lydie; Taillandier, Daniel; Pouch, Marie-Noëlle; Cervek, Matjaz; Cardenas, Diana M; Le Bricon, Thierry; Debiton, Eric; Samuels, Susan E; Madelmont, Jean-Claude; Attaix, Didier

2002-05-15

Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.

Regulatory effect of acetyl-l-carnitine on expression of lenticular antioxidant and apoptotic genes in selenite-induced cataract.

PubMed

Elanchezhian, R; Sakthivel, M; Geraldine, P; Thomas, P A

2010-03-30

Differential expression of apoptotic genes has been demonstrated in selenite-induced cataract. Acetyl-l-carnitine (ALCAR) has been shown to prevent selenite cataractogenesis by maintaining lenticular antioxidant enzyme and redox system components at near normal levels and also by inhibiting lenticular calpain activity. The aim of the present experiment was to investigate the possibility that ALCAR also prevents selenite-induced cataractogenesis by regulating the expression of antioxidant (catalase) and apoptotic [caspase-3, early growth response protein-1 (EGR-1) and cytochrome c oxidase subunit I (COX-I)] genes. The experiment was conducted on 9-day-old Wistar rat pups, which were divided into normal, cataract-untreated and cataract-treated groups. Putative changes in gene expression in whole lenses removed from the rats were determined by measuring mRNA transcript levels of the four genes by RT-PCR analysis, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The expression of lenticular caspase-3 and EGR-1 genes appeared to be upregulated, as inferred by detecting increased mRNA transcript levels, while that of COX-I and catalase genes appeared to be downregulated (lowered mRNA transcript levels) in the lenses of cataract-untreated rats. However, in rats treated with ALCAR, the lenticular mRNA transcript levels were maintained at near normal (control) levels. These results suggest that ALCAR may prevent selenite-induced cataractogenesis by preventing abnormal expression of lenticular genes governing apoptosis.

Selective activation of α7 nicotinic acetylcholine receptor by PHA-543613 improves Aβ25-35-mediated cognitive deficits in mice.

PubMed

Sadigh-Eteghad, S; Talebi, M; Mahmoudi, J; Babri, S; Shanehbandi, D

2015-07-09

Agonists of α7 nicotinic acetylcholine receptors (nAChRs) are currently being considered as therapeutic approaches for managing cognitive deficits in Alzheimer's disease (AD). Present study was designed to evaluate the effect of α7 nAChR selective activation by PHA-543613 (PHA) on beta-amyloid (Aβ)25-35-mediated cognitive deficits in mice. For this purpose, PHA (1mg/kg, i.p.), a selective α7 nAChR agonist, and galantamine (Gal) (3mg/kg, s.c.), an acetylcholine-esterase inhibitor (AChEI) effects on α7 nAChR were tested in Aβ25-35-received (intracerebroventricular, 10 nmol) mice model of AD. Methyllycaconitine (MLA) (1mg/kg, i.p.), a α7 nAChR antagonist, was used for receptor blockage effects evaluation. Working and reference memory in animals was assessed by the Morris water maze (MWM) task. The mRNA and protein levels of α7 subunit were analyzed by real-time PCR and Western blotting, respectively. PHA and Gal, ameliorate Aβ-impaired working and reference memory. However, Gal had less effect than PHA in this regard. Pretreatment with MLA reverses both Gal and PHA effects in MWM. PHA and Gal treatment prevent Aβ-induced α7 subunit protein reduction, but Gal has lesser effect than PHA. This effect blocked by pretreatment with MLA. In neither the pretreatment nor treatment group, the mRNA levels of nAChR α7 subunit were significantly changed. Therefore, α7 nAChR activation, reduces Aβ-induced cognitive deficits and increases the α7 protein level and subsequent neuron survival. However, blockage of receptor, increases Aβ toxicity and cognitive impairment and reduces the α7 nAChR protein level and flowing neuroprotection. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Effects of troxerutin on cognitive deficits and glutamate cysteine ligase subunits in the hippocampus of streptozotocin-induced type 1 diabetes mellitus rats.

PubMed

Zhang, Songyun; Li, Hongyan; Zhang, Lihui; Li, Jie; Wang, Ruiying; Wang, Mian

2017-02-15

Increasing evidence demonstrates an association between diabetes and hippocampal neuron damage. This study aimed to determine the effects of troxerutin on cognitive deficits and glutamate cysteine ligase subunits (GCLM and GCLC) in the hippocampus of streptozotocin-induced type 1 diabetes mellitus (T1DM) rats. At 12weeks after streptozotocin injection, T1DM rats were randomly divided into 4 groups (n=15 each group) to receive no treatment (T1DM), saline (T1DM+saline), alpha-lipoic acid (T1DM+alpha-lipoic acid), and troxerutin (T1DM+troxerutin), respectively, for 6weeks. Meanwhile, 10 control animals (NC group) were assessed in parallel. Learning performance was evaluated by the Morris water maze. After treatment, hippocampi were collected for pathological examination by hematoxylin and eosin (H&E) staining. Next, hippocampal superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and glutathione (GSH) levels were assessed. Finally, glutamate cysteine ligase catalytic (GCLC) and glutamate cysteine ligase modifier (GCLM) subunit mRNA and protein levels were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. Compared with T1DM and T1DM+saline groups, escape latency was overtly reduced in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. Significantly increased GCLM and GCLC mRNA levels, GCLC protein amounts, SOD activity, and GSH levels, and reduced MDA amounts were observed in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. In T1DM and T1DM+saline groups, H&E staining showed less pyramidal cells in the hippocampus, with disorganized layers, karyopyknosis, decreased endochylema, and cavitation, effects relieved in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. Troxerutin alleviates oxidative stress and promotes learning in streptozotocin-induced T1DM rats, a process involving GCLC expression. Copyright © 2016 Elsevier B.V. All rights reserved.

Epigenetic regulation of dorsal raphe GABA(B1a) associated with isolation-induced abnormal responses to social stimulation in mice.

PubMed

Araki, Ryota; Hiraki, Yosuke; Nishida, Shoji; Kuramoto, Nobuyuki; Matsumoto, Kinzo; Yabe, Takeshi

2016-02-01

In isolation-reared mice, social encounter stimulation induces locomotor hyperactivity and activation of the dorsal raphe nucleus (DRN), suggesting that dysregulation of dorsal raphe function may be involved in abnormal behaviors. In this study, we examined the involvement of dorsal raphe GABAergic dysregulation in the abnormal behaviors of isolation-reared mice. We also studied an epigenetic mechanism underlying abnormalities of the dorsal raphe GABAergic system. Both mRNA and protein levels of GABA(B1a), a GABA(B) receptor subunit, were increased in the DRN of isolation-reared mice, compared with these levels in group-reared mice. In contrast, mRNA levels for other GABAergic system-related genes (GABA(A) receptor α1, β2 and γ2 subunits, GABA(B) receptor 1b and 2 subunits, and glutamate decarboxylase 67 and 65) were unchanged. Intra-DRN microinjection of 0.06 nmol baclofen (a GABA(B) receptor agonist) exacerbated encounter-induced hyperactivity and aggressive behavior, while microinjection of 0.3 nmol phaclofen (a GABA(B) receptor antagonist) attenuated encounter-induced hyperactivity and aggressive behavior in isolation-reared mice. Furthermore, microinjection of 0.06 nmol baclofen elicited encounter-induced hyperactivity in group-reared mice. Neither baclofen nor phaclofen affected immobility time in the forced swim test and hyperactivity in a novel environment of isolation reared mice. Bisulfite sequence analyses revealed that the DNA methylation level of the CpG island around the transcription start site (TSS) of GABA(B1a) was decreased in the DRN of isolation-reared mice. Chromatin immunoprecipitation analysis showed that histone H3 was hyperacetylated around the TSS of GABA(B1a) in the DRN of isolation-reared mice. These findings indicate that an increase in dorsal raphe GABA(B1a) expression via epigenetic regulation is associated with abnormal responses to social stimulation such as encounter-induced hyperactivity and aggressive behavior in isolation-reared mice. Copyright © 2015 Elsevier Ltd. All rights reserved.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

PubMed

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

Interferon-alpha receptor 1 mRNA expression in peripheral blood mononuclear cells is associated with response to interferon-alpha therapy of patients with chronic hepatitis C.

PubMed

Massirer, K B; Hirata, M H; Silva, A E B; Ferraz, M L G; Nguyen, N Y; Hirata, R D C

2004-05-01

Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha 2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/beta-actin-mRNA ratio (mean +/- SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 +/- 0.15; N = 5; P < 0.05) compared to non-responders (0.35 +/- 0.17; N = 12) and controls (0.30 +/- 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.

Posttranscriptional Control of Photosynthetic mRNA Decay under Stress Conditions Requires 3′ and 5′ Untranslated Regions and Correlates with Differential Polysome Association in Rice1[W][OA

PubMed Central

Park, Su-Hyun; Chung, Pil Joong; Juntawong, Piyada; Bailey-Serres, Julia; Kim, Youn Shic; Jung, Harin; Bang, Seung Woon; Kim, Yeon-Ki; Do Choi, Yang; Kim, Ju-Kon

2012-01-01

Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3′ and 5′ untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3′ and 5′ untranslated regions and correlates with differential polysome association. PMID:22566494

Identification of a single p19 gene and three p40 paralogues in grass carp (Ctenopharyngodon idellus): Their potential for the formation of interleukin 23 and inducible expression in vitro and in vivo.

PubMed

Wang, Xinyan; Qin, Lei; Du, Linyong; Chen, Di; Zhang, Anying; Yang, Kun; Zhou, Hong

2017-12-01

Interleukin (IL-) 23, a member of IL-12 family, is a composite cytokine with the subunits of p19 and p40. Although IL-12 and IL-23 share the p40 subunit, they play vastly different roles in immune regulation. In teleost, much emphasis has been placed on the identification of IL-12, but evidence for the existence of IL-23 is still lacking. In the present study, a p19 gene and three p40 paralogues were isolated and identified from grass carp, suggesting multiple assembly of IL-23 molecules in fish species. To address this issue, the existence of different p19/p40 heterodimers were examined by Co-Immunoprecipitation (Co-IP) assay, showing that only co-expression of p19 and each p40 subunit could produce the soluble proteins corresponding to three IL-23 isoforms. Additionally, bacterial infection could up-regulate the mRNA expression of p19, p40a and p40b but not p40c in head kidney, indicating distinct expression patterns of three p40 paralogues. Moreover, in vitro experiments demonstrated that both B-cell stimulator, LPS and T-cell mitogen, PHA markedly increased the mRNA levels of p19 and three p40 paralogues in grass carp periphery blood lymphocytes (PBLs). The simultaneous up-regulation of mRNA expression of p19 and p40 paralogues in response to immune stimuli supports the idea that p19 may form heterodimeric molecules with three p40 subunits in grass carp under immune activation. These findings for the first time highlight the potential of p19 and p40 for dimerization in fish, particularly the existence of three IL-23 isoforms as soluble heterodimeric cytokines in grass carp, thereby providing the basis for further investigating the function of IL-23 in fish immunity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Increased Glutamate Receptor and Transporter Expression in the Cerebral Cortex and Striatum of Gcdh -/- Mice: Possible Implications for the Neuropathology of Glutaric Acidemia Type I

PubMed Central

Lagranha, Valeska Lizzi; Matte, Ursula; de Carvalho, Talita Giacomet; Seminotti, Bianca; Pereira, Carolina Coffi; Koeller, David M.; Woontner, Michael; Goodman, Stephen I.; de Souza, Diogo Onofre Gomes; Wajner, Moacir

2014-01-01

We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh -/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh -/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh -/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh -/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh -/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh -/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh -/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh -/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I. PMID:24594605

Increased glutamate receptor and transporter expression in the cerebral cortex and striatum of gcdh-/- mice: possible implications for the neuropathology of glutaric acidemia type I.

PubMed

Lagranha, Valeska Lizzi; Matte, Ursula; de Carvalho, Talita Giacomet; Seminotti, Bianca; Pereira, Carolina Coffi; Koeller, David M; Woontner, Michael; Goodman, Stephen I; de Souza, Diogo Onofre Gomes; Wajner, Moacir

2014-01-01

We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh-/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh-/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh-/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh-/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh-/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh-/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh-/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh-/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I.

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line.

PubMed

Moeslinger, Thomas; Friedl, Roswitha; Spieckermann, Paul Gerhard

2006-06-20

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.

Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome

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

Hamidian, Arash; Stedingk, Kristoffer von; Munksgaard Thorén, Matilda

2015-06-05

Hypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leadsmore » to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma. - Highlights: • Transcriptional control of HIF-2α is restricted to neural cell-derived tumors. • Enhanced transcription of HIF2A is not due to increased mRNA stability. • Chemical stabilization of the HIF-α subunits leads to increased HIF2A transcription. • ERRα regulates HIF2A mRNA expression in neuroblastoma. • High expression of ESRRA correlates to poor outcome in neuroblastoma.« less

Changes in growth, carbon and nitrogen enzyme activity and mRNA accumulation in the halophilic microalga Dunaliella viridis in response to NaCl stress

NASA Astrophysics Data System (ADS)

Wang, Dongmei; Wang, Weiwei; Xu, Nianjun; Sun, Xue

2016-12-01

Many species of microalga Dunaliella exhibit a remarkable tolerance to salinity and are therefore ideal for probing the effects of salinity. In this work, we assessed the effects of NaCl stress on the growth, activity and mRNA level of carbon and nitrogen metabolism enzymes of D. viridis. The alga could grow over a salinity range of 0.44 mol L-1 to 3.00 mol L-1 NaCl, but the most rapid growth was observed at 1.00 mol L-1 NaCl, followed by 2.00 mol L-1 NaCl. Paralleling these growth patterns, the highest initial and total Rubisco activities were detected in the presence of 1.00 mol L-1 NaCl, decreasing to 37.33% and 26.39% of those values, respectively, in the presence of 3.00 mol L-1 NaCl, respectively. However, the highest extracellular carbonic anhydrase (CA) activity was measured in the presence of 2.00 mol L-1 NaCl, followed by 1.00 mol L-1 NaCl. Different from the two carbon enzymes, nitrate reductase (NR) activity showed a slight change under different NaCl concentrations. At the transcriptional level, the mRNAs of Rubisco large subunit ( rbcL), and small subunit ( rbcS), attained their highest abundances in the presence of 1.00 and 2.00 mol L-1 NaCl, respectively. The CA mRNA accumulation was induced from 0.44 mol L-1 to 3.00 mol L-1 NaCl, but the NR mRNA showed the decreasing tendency with the increasing salinity. In conclusion, the growth and carbon fixation enzyme of Rubisco displayed similar tendency in response to NaCl stress, CA was proved be salt-inducible within a certain salinity range and NR showed the least effect by NaCl in D. viridis.

Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein

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

Higgins, Larry G.; Cavin, Christophe; Itoh, Ken

2008-02-01

Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase 1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor 2) than those lacking the transcription factor. Basal levels of mRNAs for NQO1, GSTA1,more » UGT1A6 and GCLC were lower in tissues from nrf2{sup -/-} mice than from nrf2{sup +/+} mice, but modest induction occurred in the mutant animals. Treatment of mouse embryonic fibroblasts (MEFs) from nrf2{sup +/+} mice with either coffee or the coffee-specific diterpenes cafestol and kahweol (C + K) increased NQO1 mRNA up to 9-fold. MEFs from nrf2{sup -/-} mice expressed less NQO1 mRNA than did wild-type MEFs, but NQO1 was induced modestly by coffee or C + K in the mutant fibroblasts. Transfection of MEFs with nqo1-luciferase reporter constructs showed that induction by C + K was mediated primarily by Nrf2 and required the presence of an antioxidant response element in the 5'-upstream region of the gene. Luciferase reporter activity did not increase following treatment of MEFs with 100 {mu}mol/l furan, suggesting that this ring structure within C + K is insufficient for gene induction. Priming of nrf2{sup +/+} MEFs, but not nrf2{sup -/-} MEFs, with C + K conferred 2-fold resistance towards acrolein.« less

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

PubMed

Kashimata, M; Gresik, E W

1997-02-01

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

ATP sensitive K+ channel subunits (Kir6.1, Kir6.2) are the candidate mediators regulating ameliorating effects of pulsed magnetic field on aortic contractility in diabetic rats.

PubMed

Ocal, Isil; Yilmaz, Mehmet B; Kocaturk-Sel, Sabriye; Tufan, Turan; Erkoc, Mehmet A; Comertpay, Gamze; Oksuz, Hale; Barc, Esma D

2018-05-01

Diabetes mellitus is a metabolic disease that causes increased morbidity and mortality in developed and developing countries. With recent advancements in technology, alternative treatment methods have begun to be investigated in the world. This study aims to evaluate the effect of pulsed magnetic field (PMF) on vascular complications and contractile activities of aortic rings along with Kir6.1 and Kir6.2 subunit expressions of ATP-sensitive potassium channels (K ATP ) in aortas of controlled-diabetic and non-controlled diabetic rats. Controlled-diabetic and non-controlled diabetic adult male Wistar rats were exposed to PMF for a period of 6 weeks according to the PMF application protocol (1 h/day; intensity: 1.5 mT; consecutive frequency: 1, 10, 20, and 40 Hz). After PMF exposure, body weight and blood glucose levels were measured. Then, thoracic aorta tissue was extracted for relaxation-contraction and Kir6.1 and Kir6.2 expression experiments. Blood plasma glucose levels, body weight, and aortic ring contraction percentage decreased in controlled-diabetic rats but increased in non-controlled diabetic rats. PMF therapy repressed Kir6.1 mRNA expression in non-controlled diabetic rats but not in controlled diabetic rats. Conversely, Kir6.2 mRNA expressions were repressed both in controlled diabetic and non-controlled diabetic rats by PMF. Our findings suggest that the positive therapeutic effects of PMF may act through (K ATP ) subunits and may frequently occur in insulin-free conditions. Bioelectromagnetics. 39:299-311, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

The alpha subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression.

PubMed

Dagenais, A; Kothary, R; Berthiaume, Y

1997-09-01

Sodium reabsorption by the amiloride-sensitive sodium channel of epithelial cells plays a crucial role in the management of ionic composition and fluid volume in the body. In the respiratory system, sodium transport is involved in the clearance of pulmonary edema and of liquid secreted during fetal life at birth. We have cloned a partial cDNA of the alpha subunit of the mouse amiloride-sensitive sodium channel (alpha mENaC). In the region of comparison, the mouse alpha subunit shows 92% identity at the DNA level and 95% identity at the amino acid level with the rat sequence. The kidneys, lungs, and distal colon are major sites of expression of a 3.5-kb alpha mENaC mRNA. During mouse development, alpha mENaC transcripts appear late during gestation (d 17.5) and are expressed continuously thereafter. In the distal colon, a short 1.2-kb mRNA deleted of the 5' part of the transcript is detected during gestation and is replaced gradually by the mature 3.5-kb transcript after birth. Alpha mENaC and alpha1 Na+-K+-ATPase mRNAs have an expression profile that is modulated similarly during development for a given tissue. The expression of alpha mENaC transcripts increases transiently in the lungs at birth (2.5-fold), as for alpha1 Na+-K+-ATPase mRNAs (1.5-fold), suggesting that the expression of several components of the sodium transport system is modulated in the lungs at that time. In the kidney, there is no significant increase of alpha mENaC and alpha1 Na+-K+-ATPase mRNAs in newborns.

Dual effects of ouabain on the regulation of proliferation and apoptosis in human umbilical vein endothelial cells: involvement of Na(+)-K(+)-ATPase α-subunits and NF-κB.

PubMed

Ren, Yan-Ping; Zhang, Ming-Juan; Zhang, Ting; Huang, Ruo-Wen

2014-01-01

To elucidate the effect of ouabain on the regulation of proliferation and apoptosis of HUVECs and involvement of different Na(+)-K(+)-ATPase α-subunits and NF-κB. HUVECs were isolated by collagenase perfusion, and MTT assays and cell cycle analysis were performed to study proliferation. NF-κB expression and function were examined by immunohistochemical staining and western blotting. Na(+)-K(+)-ATPase activity was determined by measuring released ouabain inhibitable inorganic phosphate (Pi). The expression of different α-subunits was investigated by real RT-PCR, western blotting and cell immunofluorescence. 0.3 nM ouabain treatment for 0.5 h triggered the proliferation of HUVECs, peaking at 1-2 h. At 1.8 nM for 0.5 h, ouabain induced an increase of cell proliferation for a short time, and then triggered a decrease after 1 h. Cell cycle analysis show that 37% of HUVECs were in G2/M phase of the cell cycle following incubation with 1.8 nM ouabain, compared with 18% with 0.3 nM ouabain. NF-κB activity was assessed by western blot analysis of IκB expression, which was significantly reduced with 0.3 nM ouabain treatment; there was no different between 1.8 nM ouabain treatment and untreated cells. Na(+)-K(+)-ATPase activity in HUVECs was markedly reduced after treatment with 0.3 nM and 1.8 nM ouabain. Real RT-PCR and western blotting indicated that Na(+)-K(+)-ATPase α1-subunit mRNA expression levels increased after 0.3 nM ouabain treatment and decreased after 1.8 nM ouabain treatment. However, α2- and α3-subunit mRNA decreased after 0.3 nM ouabain treatment and increased after 1.8 nM ouabain treatment. Ouabain at different concentrations caused dual effects on proliferation and apoptosis in HUVECs.

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

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

Feng, Guoxing; Shi, Hui; Li, Jiong

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 atmore » the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells through targeting P4HA1 mRNA.« less

Ribosome rearrangements at the onset of translational bypassing

PubMed Central

Agirrezabala, Xabier; Samatova, Ekaterina; Klimova, Mariia; Zamora, Miguel; Gil-Carton, David; Rodnina, Marina V.; Valle, Mikel

2017-01-01

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap. PMID:28630923

Gravity and Skeletal Growth

NASA Technical Reports Server (NTRS)

Morey-Holton, Emily; Turner, Russell T.

1999-01-01

Two simultaneous experiments were performed using 5-week-old male Sprague Dawley rats; in one study, the rats were flown in low earth orbit; in the other study, the hindlimbs of the growing rats were elevated to prevent weight bearing. Following 9 d of unloading, weight bearing was restored for 4, 28, and 76 hrs. Afterwards, additional hindlimb unloading experiments were performed to evaluate the skeletal response to 0, 2, 4, 6, 8, 10, 12, 16, and 24 hrs of restored weight bearing following 7 d of unloading. Cancellous and cortical bone histomorphometry were evaluated in the left tibia at the proximal metaphysis and in the left femur at mid-diaphysis, respectively. Steady-state mRNA levels for bone matrix proteins and skeletal signaling peptides were determined in total cellular RNA extracted from trabeculae from the right proximal tibiametaphysis and periosteum from the right femur. Spaceflight and hindlimb unloading each resulted in cancellous osteopenia, as well as a tendency towards decreased periosteal bone formation. Both models for skeletal unloading resulted in site specific reductions in mRNA levels for transforming growth factor-beta (sub 1) (TGF-beta) osteocalcin (OC), and prepro-alpha (I) subunit of type 1 collagen (collagen) and little or no changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAP) and insulin-like growth factor I (IGF-I). Restoration of normal weight bearing resulted in transient increases in mRNA levels for the bone matrix proteins and TGF-beta in the proximal metaphysis and periosteum and no changes in either GAP or IGF-I mRNA levels. The timecourse for the response differed between the two skeletal compartments; the tibial metaphysis responded much more quickly to reloading. These results suggest that the skeletal adaptation to acute physiological changes in mechanical usage are mediated, in part, by changes in mRNA levels for bone matrix proteins and TGF-beta.

Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats.

PubMed

Chen, Qiyi; Li, Ning; Zhu, Weiming; Li, Weiqin; Tang, Shaoqiu; Yu, Wenkui; Gao, Tao; Zhang, Juanjuan; Li, Jieshou

2011-06-03

Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome system. However, whether insulin can alleviate the degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system under septic condition is unclear. This paper confirmed that mRNA and protein levels of the ubiquitin-proteasome system were upregulated and molecular markers of skeletal muscle proteolysis (tyrosine and 3-methylhistidine) simultaneously increased in the skeletal muscle of septic rats. Septic rats were infused with insulin at a constant rate of 2.4 mU.kg-1.min-1 for 8 hours. Concentrations of mRNA and proteins of the ubiquitin-proteasome system and molecular markers of skeletal muscle proteolysis were mildly affected. When the insulin infusion dose increased to 4.8 mU.kg-1.min-1, mRNA for ubiquitin, E2-14 KDa, and the C2 subunit were all sharply downregulated. At the same time, the levels of ubiquitinated proteins, E2-14KDa, and the C2 subunit protein were significantly reduced. Tyrosine and 3-methylhistidine decreased significantly. We concluded that the ubiquitin-proteasome system is important skeletal muscle hypercatabolism in septic rats. Infusion of insulin can reverse the detrimental metabolism of skeletal muscle by inhibiting the ubiquitin-proteasome system, and the effect is proportional to the insulin infusion dose.

Rice protein improves oxidative stress by regulating glutathione metabolism and attenuating oxidative damage to lipids and proteins in rats.

PubMed

Yang, Lin; Chen, Jia-Hou; Xu, Tong; Zhou, Ai-Shen; Yang, Hong-Kun

2012-10-05

To evaluate the effects of rice protein (RP) on glutathione metabolism and oxidative damage. Seven-week-old male Wistar rats were fed diets containing casein and RP without cholesterol for 3weeks. Plasma and liver lipid levels, hepatic accumulation of total glutathione (T-GSH), oxidized glutathione (GSSG), reduced glutathione (GSH), malondialdehyde (MDA) and protein carbonyl (PCO) were measured. In the liver, the total antioxidative capacity (T-AOC), mRNA levels of glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modulatory subunit (GCLM), and the activities of hepatic catalase (CAT), total superoxide dismutase (T-SOD), γ-glutamylcysteine synthetase (γ-GCS), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSHPx) were also measured. T-AOC, GCLC and GCLM mRNA levels, antioxidative enzyme activities (T-SOD and CAT) and glutathione metabolism related enzyme activities (γ-GCS, GST, GR and GSHPx) were effectively stimulated by RP feeding compared to casein, and RP significantly reduced the hepatic accumulation of MDA and PCO in rats. These results indicate that lipid-lowering activity was induced by RP feeding. The present study demonstrates that RP improves oxidative stress primarily through enzymatic and non-enzymatic antioxidative defense mechanisms, reflected by enhancing the antioxidative status and attenuating the oxidative damage to lipids and proteins. These results suggest that RP can prevent hyperlipidemia in part through modifying glutathione metabolism, and sulfur amino acids may be the main modulator of this antioxidative mechanism. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes

PubMed Central

McKinney, Jeffrey; Guerrier-Takada, Cecilia; Wesolowski, Donna; Altman, Sidney

2001-01-01

Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not. PMID:11381134

Localization of mRNA for CHRNA7 in human fetal brain.

PubMed

Agulhon, C; Abitbol, M; Bertrand, D; Malafosse, A

1999-08-02

The aim of this study was to determine the regional distribution in situ of the mRNA for the alpha 7 subunit of the neuronal nicotinic acetylcholine receptor in human fetal brain. We found high levels of alpha 7 gene expression in nuclei that receive sensory information, such as those of the neocortex and hippocampus, the thalamic nuclei, the reticular thalamic nucleus, the pontine nuclei and the superior olive complex. These data support a possible regulatory function for alpha 7-containing receptors in sensory processing, which may be involved in the pathological physiology of schizophrenia and autism. Early alpha 7 gene expression is also consistent with a morphogenetic role for alpha 7 receptors in central nervous system development.

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma.

PubMed

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named "hTERT messenger ribonucleic acid (mRNA)" the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0-160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control-untreated group. The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent ( P < 0.05) and not in time-dependent manner ( P > 0.05), in vitro . Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM.

CCR4-NOT deadenylates mRNA associated with RNA-induced silencing complexes in human cells.

PubMed

Piao, Xianghua; Zhang, Xue; Wu, Ligang; Belasco, Joel G

2010-03-01

MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of "off-target" mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.

[Expression of proteasome subunits PSMB5 and PSMB9 mRNA in hippocampal neurons in experimental diabetes mellitus: link with apoptosis and necrosis].

PubMed

Lebid', Iu V; Dosenko, V Ie; Skybo, H H

2010-01-01

There is a huge body of evidence showing that long-termed diabetes mellitus is followed with hippocampal dysfunction. The goal of this work was to investigate the expression of proteasome subunits PSMB5 and PSMB9 mRNA in CA1, CA2 and CA3 areas of hippocampus in parallel with processes of cell death (apoptosis and necrosis) in development dynamics of streptozotocine-induced diabetes. We have studied hippocampal neurons using chromatine dye Hoechst-33342 and immunohistochemical detection of apoptotic cell death marker caspase-3. At day 3 and 7 after injection of streptozotocine we have performed visualization of caspase-3-immunopositive neurons showing signs of neurodegeneration in hippocampal sections using confocal microscope Olympus FV1000. The rate of proteasome subunits PSMB5 and PSMB9 mRNA expression was determined with RT-PCR. The results indicated elevation of PSMB9 mRNA content (from 4807 +/- 0.392 arbU up to 20,023 +/- 4949 arbU on day 3 and up to 20,253 +/- 5141 arbU on day 7). A maximal number of cells with signs of chromatin condensation was observed at day 3 and day 7 in CA2 and CA3 area (11.51% and 12.49% respectively). That indicates an intensification of proapoptotic processes. Summarizing the results presented above we can conclude that during the first week of diabetes mellitus development, hippocampal cells undergo the process of impairment and degeneration.

Cardioprotective Effect of Danshensu against Ischemic/Reperfusion Injury via c-Subunit of ATP Synthase Inhibition

PubMed Central

Zhao, JingYi; Fan, Zixuan; Bao, Jiadi; Sun, Dawei; Sun, Chun

2017-01-01

Mitochondrial permeability transition pore (MPTP) opening is the main culprit of ischemic/reperfusion (IR) injury. It is reported that c-subunit of ATP synthase is the core component of MPTP. Danshensu (DSS), a monomer isolated from the traditional Chinese herb Danshen, has showed cardioprotective effect against IR injury through unknown mechanism. In this study, rat hearts were suspended in Langendorff instrument and perfused with Krebs-Henseleit (KH) buffer containing DSS for 60 minutes, followed by 30 minutes of global ischemia. Parameters including heart rate, left ventricular developed pressure, and the rate of left ventricle diastolic pressure change were recorded to assess their cardiac function. All these indexes were improved in DSS group. The rate of cardiomyocytes apoptosis and MPTP opening were both inhibited in DSS group. In addition, DSS administration leads to downregulation of c-subunit of ATP synthase in both mRNA and protein levels. Consistently, when c-subunit of ATP synthase was overexpressed in H9C2 cells through pcDNA3/5G1 plasmid transfection, MPTP opening was enhanced when the cardioprotective effect of DSS also tapers. In conclusion, DSS could alleviate cardiac IR injury via inhibiting c-subunit of ATP synthase expression. PMID:29250127

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Characterisation of imidacloprid resistance in the bird cherry-oat aphid, Rhopalosiphum padi, a serious pest on wheat crops.

PubMed

Wang, Kang; Zhang, Meng; Huang, Yanna; Yang, Zhuolin; Su, Sha; Chen, Maohua

2018-06-01

Rhopalosiphum padi is a destructive insect pest of wheat worldwide. Studies have shown that R. padi has developed resistance to different insecticides, including imidacloprid. We studied the mechanisms conferring resistance to imidacloprid at the biochemical and molecular levels. An R. padi imidacloprid-resistant (IM-R) strain and a susceptible (SS) strain were established. Fitness analysis using life-tables showed that the IM-R strain had obvious disadvantages in several parameters, indicating reduced fitness. Profiles of cross-resistance of IM-R and SS to seven insecticides were detected. Both synergistic and enzyme activity data suggested that P450 plays a role in resistance. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) genes CYP6CY3-1 and CYP6CY3-2 were significantly increased in the IM-R strain. No target-site mutation within the nicotinic acetylcholine receptor (nAChR) subunits was detected in the IM-R strain. Interestingly, the expression levels of the nAChR α1, α2, α3, α7-2, and β1 subunit genes were significantly decreased, suggesting that down-regulation of these subunits may be involved in resistance. Multiple mechanisms confer imidacloprid resistance in R. padi. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Maternal plasma levels of cell-free β-HCG mRNA as a prenatal diagnostic indicator of placenta accrete.

PubMed

Zhou, J; Li, J; Yan, P; Ye, Y H; Peng, W; Wang, S; Wang, X Tong

2014-09-01

Several biomarkers, including maternal serum creatinine kinase and α-fetoprotein, have been described as potential tools for the diagnosis of placental abnormalities. This study aimed to determine whether maternal plasma mRNA levels of the β subunit of human chorionic gonadotropin (β-HCG) could predict placenta accreta prenatally. Sixty-eight singleton pregnant women with prior cesarean deliveries (CDs) were classified into three groups: normal placentation (35 women, control group); placenta previa alone (21 women, placenta previa group); and both placenta previa and placenta accreta (12 women, placenta previa/accreta group). Maternal plasma concentrations of cell-free β-HCG mRNA were measured by real-time reverse-transcription polymerase chain reaction and were expressed as multiples of the median (MoM). Cell-free β-HCG mRNA concentrations (MoM, range) were significantly higher in women with placenta accreta (3.65, 2.78-7.19) than in women with placenta previa (0.94, 0.00-2.97) or normal placentation (1.00, 0.00-2.69) (Steel-Dwass test, P < 0.01 and P < 0.01, respectively). In the placenta previa/accreta group, the concentration of cell-free β-HCG mRNA was significantly higher among women who underwent CDs with hysterectomy (4.41, 3.49-7.19) than among women whose CDs did not result in hysterectomy (3.20, 2.78-3.70) (Mann-Whitney U test, P = 0.012). An increased level of cell-free β-HCG mRNA in the maternal plasma of women with placenta accreta may arise from direct uteroplacental transfer of cell-free placental mRNA molecules. The concentration of cell-free β-HCG mRNA in maternal plasma may be applicable to the prenatal diagnosis of placenta accreta, especially to identify women with placenta accreta likely to require hysterectomy. Copyright © 2014 Elsevier Ltd. All rights reserved.

NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

PubMed

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-07-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

The beneficial effects of betaine on dysfunctional adipose tissue and N6-methyladenosine mRNA methylation requires the AMP-activated protein kinase α1 subunit.

PubMed

Zhou, Xihong; Chen, Jingqing; Chen, Jin; Wu, Weiche; Wang, Xinxia; Wang, Yizhen

2015-12-01

The current study was conducted to determine whether betaine could improve fatty acid oxidation, mitochondrial function and N6-methyladenosine (m(6)A) mRNA methylation in adipose tissue in high-fat-induced mice and how AMP-activated protein kinase α1 subunit (AMPKα1) was involved. AMPKα1 knockout mice and wild-type mice were fed either a low-fat diet, high-fat diet or high-fat diet supplemented with betaine in the drinking water for 8weeks. Our results showed that mitochondrial genes (PGC1α) and β-oxidation-related genes (CPT1a) at protein level were increased in wild-type mice supplemented with betaine when compared with those in mice with high-fat diet. Betaine also decreased FTO expression and improved m(6)A methylation in adipose tissue of wild-type mice with high-fat diet. However, betaine failed to exert the abovementioned effects in AMPKα1 knockout mice. In adipocytes isolated from mice with high-fat diet, betaine treatment increased lipolysis and lipid oxidation. Moreover, betaine decreased FTO expression and increased m(6)A methylation. However, while AMPKα1 was knockdown, no remarkable changes in adipocytes were observed under betaine treatment. Our results indicated that betaine supplementation rectified mRNA hypomethylation and high FTO expression induced by high-fat diet, which may contribute to its beneficial effects on impaired adipose tissue function. Our results suggested that the AMPKα1 subunit is required for the beneficial effects of betaine on dysfunctional adipose tissue and m(6)A methylation. These results may provide the foundation for a mechanism that links m(6)A methylation status in RNA, AMPKα1 phosphorylation and dysfunctional adipose tissue induced by high-fat diet. Copyright © 2015 Elsevier Inc. All rights reserved.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

PubMed

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-10-15

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. © 2016. Published by The Company of Biologists Ltd.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

PubMed Central

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-01-01

ABSTRACT The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. PMID:27543059

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

NASA Technical Reports Server (NTRS)

Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

1999-01-01

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit

PubMed Central

Lobato-Álvarez, Jorge A.; Roldán, María L.; López-Murillo, Teresa del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

2016-01-01

Na+, K+-ATPase, or the Na+ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na+ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na+, K+-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na+ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na+, K+-ATPase in RPE cells depends on the expression of the β2 subunit. PMID:27774068

The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit.

PubMed

Lobato-Álvarez, Jorge A; Roldán, María L; López-Murillo, Teresa Del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

2016-01-01

Na + , K + -ATPase, or the Na + pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β 1 subunit of Na + , K + -ATPase plays an important role in this mechanism because homotypic β 1 -β 1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na + pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β 2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na + , K + -ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na + pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β 2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β 2 subunit. qPCR results showed a time-dependent increase in the level of β 2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β 2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α 2 subunit in that domain. Our results demonstrate that the apical polarization of Na + , K + -ATPase in RPE cells depends on the expression of the β 2 subunit.

Effects of chemotherapy on gene expression of lingual taste receptors in patients with head and neck cancer.

PubMed

Tsutsumi, Rie; Goda, Masakazu; Fujimoto, Chisa; Kanno, Kyoko; Nobe, Misaki; Kitamura, Yoshiaki; Abe, Koji; Kawai, Misako; Matsumoto, Hideki; Sakai, Tohru; Takeda, Noriaki

2016-03-01

We aimed to test the hypothesis that chemotherapy changes the gene expression of taste receptors in the tongue to induce dysgeusia in patients with head and neck cancer. Prospective observation study. We enrolled 21 patients who received chemoradiotherapy and five patients who underwent radiotherapy for head and neck cancer. The messenger RNA (mRNA) levels of the taste receptor subunits T1R1, T1R2, T1R3, and T2R5 were measured in lingual mucosa scrapings obtained with a small spatula. The perception thresholds of umami, sweet, and bitter tastes were assessed by the whole mouth gustatory test. In four patients with severe stomatitis induced by chemoradiotherapy, the mRNA levels of T1R1, T1R2, T1R3, and T2R5 in the lingual mucosa were significantly decreased. However, in 17 patients with mild/moderate stomatitis, the mRNA levels of T1R3 were significantly and transiently decreased, whereas those of T1R1 and T1R2 remained unchanged and those of T2R5 mRNA were significantly and transiently increased after chemotherapy. There was a significant negative correlation between the perception thresholds of umami or sweet tastes and lingual mRNA levels of T1R3 in patients with mild/moderate stomatitis after chemotherapy. Although the perception threshold of bitter taste remained unchanged, lingual mRNA levels of T2R5 were significantly increased in patients who complained of phantogeusia after chemotherapy. Chemotherapy specifically changed the gene expression of T1R3 and T2R5 in head and neck cancer patients with mild/moderate stomatitis, resulting in both dysgeusia of umami and sweet tastes as well as phantogeusia. 4. Laryngoscope, 126:E103-E109, 2016. © 2015 The American Laryngological, Rhinological and Otological Society, Inc.

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.

Long-distance transport of mRNA via parenchyma cells and phloem across the host-parasite junction in Cuscuta.

PubMed

David-Schwartz, Rakefet; Runo, Steven; Townsley, Brad; Machuka, Jesse; Sinha, Neelima

2008-01-01

It has been shown that the parasitic plant dodder (Cuscuta pentagona) establishes a continuous vascular system through which water and nutrients are drawn. Along with solutes, viruses and proteins, mRNA transcripts are transported from the host to the parasite. The path of the transcripts and their stability in the parasite have yet to be revealed. To discover the route of mRNA transportation, the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to locally amplify host transcript within parasitic tissue. The stability of host mRNA molecules was also checked by monitoring specific transcripts along the growing dodder thread. Four mRNAs, alpha and beta subunits of PYROPHOSPHATE (PPi)-DEPENDENT PHOSPHOFRUCTOKINASE (LePFP), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and GIBBERELLIC ACID INSENSITIVE (LeGAI), were found to move from host (tomato (Solanum lycopersicum)) to dodder. LePFP mRNA was localized to the dodder parenchyma cells and to the phloem. LePFP transcripts were found in the growing dodder stem up to 30 cm from the tomato-dodder connection. These results suggest that mRNA molecules are transferred from host to parasite via symplastic connections between parenchyma cells, move towards the phloem, and are stable for a long distance in the parasite. This may allow developmental coordination between the parasite and its host.

Lower KV7.5 Potassium Channel Subunit Expression in an Animal Model of Paroxysmal Dystonia.

PubMed

Sander, Svenja E; Diwan, Mustansir; Raymond, Roger; Nobrega, José N; Richter, Angelika

2016-01-01

Dystonia is a hyperkinetic disabling movement disorder. In the dt(sz) hamster, a model of paroxysmal dystonia, pronounced antidystonic effects of the KV7.2-5 potassium channel opener retigabine and aggravation of dystonia by a selective KV7.2-5 blocker indicated a pathophysiological role of an abnormal expression of KV7 channels. We therefore investigated the expression of KV7 subunits in brains of dystonic hamsters. While KV7.2 and KV7.3 subunits were unaltered, lower KV7.5 mRNA levels became evident in motor areas and in limbic structures of dystonic hamsters. The KV7.2/3 subunit-preferring channel opener N-(6-chloropyridin-3-yl)-3,4- difluorobenzamide (ICA 27243; 10-30 mg/kg i.p.) failed to reduce the severity of dystonia in mutant hamsters, suggesting that the previously observed antidystonic action of retigabine is mediated by the activation of KV7.5 channels. The experiments indicate a functional relevance for KV7.5 channels in paroxysmal dystonia. We suggest that compounds highly selective for subtypes of KV7 channels, i.e. for KV7.5, may provide new therapeutic approaches.

Effects of spaceflight on murine skeletal muscle gene expression

PubMed Central

Allen, David L.; Bandstra, Eric R.; Harrison, Brooke C.; Thorng, Seiha; Stodieck, Louis S.; Kostenuik, Paul J.; Morony, Sean; Lacey, David L.; Hammond, Timothy G.; Leinwand, Leslie L.; Argraves, W. Scott; Bateman, Ted A.; Barth, Jeremy L.

2009-01-01

Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85α, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-γ coactivator-1α and the transcription factor PPAR-α were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type. PMID:19074574

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

NASA Technical Reports Server (NTRS)

Krumenacker, J. S.; Hyder, S. M.; Murad, F.

2001-01-01

Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

Molecular and neurochemical biomarkers in Arctic beluga whales (Delphinapterus leucas) were correlated to brain mercury and selenium concentrations.

PubMed

Ostertag, Sonja K; Shaw, Alyssa C; Basu, Niladri; Chan, Hing Man

2014-10-07

Mercury (Hg) concentrations have increased in western Arctic beluga whales (Delphinapterus leucas) since the industrial revolution. Methylmercruy (MeHg) is a known neurotoxicant, yet little is known about the risk of exposure for beluga whales. Selenium (Se) has been linked to demethylation of MeHg in cetaceans, but its role in attenuating Hg toxicity in beluga whales is poorly understood. The objective of this study is to explore relationships between Hg and Se concentrations and neurochemical biomarkers in different brain regions of beluga whales in order to assess potential neurotoxicological risk of Hg exposure in this population. Brain tissue was sampled from hunter-harvested beluga whales from the western Canadian Arctic in 2008 and 2010. Neurochemical and molecular biomarkers were measured with radioligand binding assays and quantitative PCR, respectively. Total Hg (HgT) concentration ranged from 2.6-113 mg kg(-1) dw in temporal cortex. Gamma-amminobutyric acid type A receptor (GABAA-R) binding in the cerebellum was negatively associated with HgT, MeHg and total Se (SeT) concentrations (p ≤ 0.05). The expression of mRNA for GABAA-R subunit α2 was negatively associated with HgT and MeHg (p ≤ 0.05). Furthermore, GABAA-R binding was positively correlated to mRNA expression for GABAA-R α2 subunit, and negatively correlated to the expression of mRNA for GABAA-R α4 subunit (p ≤ 0.05). The expression of N-methyl-d-aspartate receptor (NMDA-R) subunit 2b mRNA expression was negatively associated with iHglabile concentration in the cerebellum (p ≤ 0.05). Variation of molecular and/or biochemical components of the GABAergic and glutamatergic signaling pathways were associated with MeHg exposure in beluga whales. Our results show that MeHg exposure is associated with neurochemical variation in the cerebellum of beluga whales and Se may partially protect from MeHg-associated neurotoxicity.

The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans.

PubMed

Szekeres, M; Droux, M; Buchanan, B B

1991-03-01

The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form.

Transient Hippocampal Down-Regulation of Kv1.1 Subunit mRNA during Associative Learning in Rats

ERIC Educational Resources Information Center

Kourrich, Said; Manrique, Christine; Salin, Pascal; Mourre, Christiane

2005-01-01

Voltage-gated potassium channels (Kv) are critically involved in learning and memory processes. It is not known, however, whether the expression of the Kv1.1 subunit, constituting Kv1 channels, can be specifically regulated in brain areas important for learning and memory processing. Radioactive in situ hybridization was used to evaluate the…

Rice G-protein subunits qPE9-1 and RGB1 play distinct roles in abscisic acid responses and drought adaptation.

PubMed

Zhang, Dong-Ping; Zhou, Yong; Yin, Jian-Feng; Yan, Xue-Jiao; Lin, Sheng; Xu, Wei-Feng; Baluška, František; Wang, Yi-Ping; Xia, Yi-Ji; Liang, Guo-hua; Liang, Jian-Sheng

2015-10-01

Heterotrimeric GTP-binding protein (G-protein)-mediated abscisic acid (ABA) and drought-stress responses have been documented in numerous plant species. However, our understanding of the function of rice G-protein subunits in ABA signalling and drought tolerance is limited. In this study, the function of G-protein subunits in ABA response and drought resistance in rice plants was explored. It was found that the transcription level of qPE9-1 (rice Gγ subunit) gradually decreased with increasing ABA concentration and the lack of qPE9-1 showed an enhanced drought tolerance in rice plants. In contrast, mRNA levels of RGB1 (rice Gβ subunit) were significantly upregulated by ABA treatment and the lack of RGB1 led to reduced drought tolerance. Furthermore, the results suggested that qPE9-1 negatively regulates the ABA response by suppressing the expression of key transcription factors involved in ABA and stress responses, while RGB1 positively regulates ABA biosynthesis by upregulating NCED gene expression under both normal and drought stress conditions. Taken together, it is proposed that RGB1 is a positive regulator of the ABA response and drought adaption in rice plants, whereas qPE9-1 is modulated by RGB1 and functions as a negative regulator in the ABA-dependent drought-stress responses. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA.

PubMed

Marchetto, G S; Henry, H L

1997-02-01

The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10(-7) M 1,25(OH)2D3 for 24h resulted in a 20-30% decrease in PKI mRNA. 1,25(OH)2D3 treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)2D3 directly and tissue-specifically downregulates PKI mRNA in the chick kidney.

β1-Adrenergic blocker bisoprolol reverses down-regulated ion channels in sinoatrial node of heart failure rats.

PubMed

Du, Yuan; Zhang, Junbo; Xi, Yutao; Wu, Geru; Han, Ke; Huang, Xin; Ma, Aiqun; Wang, Tingzhong

2016-06-01

Bisoprolol, an antagonist of β1-adrenergic receptors, is effective in reducing the morbidity and mortality in patients with heart failure (HF). It has been found that HF is accompanied with dysfunction of the sinoatrial node (SAN). However, whether bisoprolol reverses the decreased SAN function in HF and how the relevant ion channels in SAN change were relatively less studied. SAN function and messenger RNA (mRNA) expression of sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits were assessed in sham-operated rats, abdominal arterio-venous shunt (volume overload)-induced HF rats, and bisoprolol- treated HF rats. SAN cells of rats were isolated by laser capture microdissection. Quantitative real-time PCR analysis was used to quantify mRNA expression of sodium channels and HCN channel subunits in SAN. Intrinsic heart rate declined and sinus node recovery time prolonged in HF rats, indicating the suppressed SAN function, which could be improved by bisoprolol treatment. Nav1.1, Nav1.6, and HCN4 mRNA expressions were reduced in SAN in HF rats compared with that in control rats. Treatment with bisoprolol could reverse both the SAN function and the Nav1.1, Nav1.6, and HCN4 mRNA expression partially. These data indicated that bisoprolol is effective in HF treatment partially due to improved SAN function by reversing the down-regulation of sodium channels (Nav1.1 and Nav1.6) and HCN channel (HCN4) subunits in SAN in failing hearts.

Prenatal exposure of testosterone prevents SDN-POA neurons of postnatal male rats from apoptosis through NMDA receptor.

PubMed

Hsu, H K; Yang, R C; Shih, H C; Hsieh, Y L; Chen, U Y; Hsu, C

2001-11-01

The role of N-methyl-D-aspartate (NMDA) receptor in mediating the effect of testosterone exposure prenatally on neuronal apoptosis in the sexual dimorphic nucleus of the preoptic area (SDN-POA) of rats was studied. The endogenous testosterone was diminished by prenatal stress (PNS) or simulated by testosterone exposure (TE) to understand the effect of testosterone on NR(1) (a functional subunit protein of NMDA receptor) expression and neuronal apoptosis. To further study whether the testosterone, after being converted into estradiol, modulates NR(1) expression, 4-androstein-4-ol-3,17-dione (ATD; an aromatase inhibitor) was used to block the conversion of estradiol from testosterone. The expressions of the NR(1) mRNA and NR(1) subunit protein were quantified by RT-PCR and western blotting analysis, respectively. In addition, a noncompetitive antagonist of NMDA receptor, MK-801, was used to find out whether blockage of NMDA receptor affects the naturally occurring apoptosis in SDN-POA. The results showed the following. 1) Expression of perinatal NR(1) subunit protein in the central part of the medial preoptic area of male rats was significantly higher than that of females, especially on postnatal days 1 and 3. 2) The testosterone level of male fetuses on embryonic day 18 was significantly higher than that of females, while the testosterone level of TE females or PNS males was similar to that of intact males or intact females, respectively. 3) The apoptotic incidence of intact male rats was significantly less than that of females, and the apoptosis was stimulated by PNS in male or inhibited by TE in female. 4) The expression of NR(1) subunit protein could be inhibited by PNS or ATD-treatment in male, while stimulated by TE in female. 5) NR(1) mRNA showed no significant difference among intact male, PNS male, ATD-treated male, TE female and intact female rats. 6) The low apoptotic incidence of male rats was significantly increased when NMDA receptor was blocked by MK-801. These results suggest that testosterone, after being converted to estradiol, may prevent the SDN-POA neurons of male rats from apoptosis through enhancing the expression of NR(1) at the posttranscriptional level.

The tapeworm Ligula intestinalis (Cestoda: Pseudophyllidea) inhibits LH expression and puberty in its teleost host, Rutilus rutilus.

PubMed

Carter, V; Pierce, R; Dufour, S; Arme, C; Hoole, D

2005-12-01

The tapeworm Ligula intestinalis occurs in the body cavity of its cyprinid second intermediate host, in this study the roach Rutilus rutilus, and inhibits host gonadal development. The mechanism by which infected fish are prevented from reproducing is unknown. Comparison of parameters, such as body length and weight, and condition factor and age, between infected and uninfected individuals, indicated only minor effects of parasitism on growth and condition. In contrast, seasonal gonadal development, as observed in uninfected fish, did not occur in infected fish, and gonads remained small and blocked at the primary oocyte stage in female roach. As immature ovaries and testes are still present, the parasite is presumed to act upon the brain-pituitary-gonadal axis of the fish to inhibit further development of reproductive organs. We investigated the Ligula/fish interaction at the level of the pituitary gland by determination of gonadotrophin (LH) content using a heterologous RIA for carp (Cyprinus carpio) LHbeta subunit. The results indicated that the pituitary glands of infected roach contained approximately 50% less LH than non-infected fish. After the cloning and sequencing of roach LHbeta subunit, we measured roach LHbeta mRNA levels by real-time RT-PCR. A corresponding 50% reduction in LHbeta mRNA pituitary levels was determined. These results reflect a significant and measurable effect of parasitism on the pituitary gland, and lend support to the hypothesis that excretory/secretory products released from the parasite interact with the brain-pituitary-gonadal axis of the fish host and thus inhibit gonadal development.

Monosialotetrahexosylganglioside Inhibits the Expression of p-CREB and NR2B in the Auditory Cortex in Rats with Salicylate-Induced Tinnitus.

PubMed

Song, Rui-Biao; Lou, Wei-Hua

2015-01-01

This study investigated the effects of monosialotetrahexosylganglioside (GM1) on the expression of N-methyl-D-aspartate receptor subunit 2B (NR2B) and phosphorylated (p)-cyclic AMP response element-binding protein (CREB) in the auditory cortex of rats with tinnitus. Tinnitus-like behavior in rats was tested with the gap prepulse inhibition of acoustic startle paradigm. We then investigated the NR2B mRNA and protein and p-CREB protein levels in the auditory cortex of tinnitus rats compared with normal rats. Rats treated for 4 days with salicylate exhibited tinnitus. NR2B mRNA and protein and p-CREB protein levels were upregulated in these animals, with expression returning to normal levels 14 days after cessation of treatment; baseline levels of NR2B and p-CREB were also restored by GM1 administration. These data suggest that chronic salicylate administration induces tinnitus via upregulation of p-CREB and NR2B expression, and that GM1 can potentially be used to treat tinnitus.

Activin B is produced early in antral follicular development and suppresses thecal androgen production

PubMed Central

Young, J M; Henderson, S; Souza, C; Ludlow, H; Groome, N; McNeilly, A S

2012-01-01

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1–3 to >6 mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3–4 mm, and TCs more inhibin α mRNA at >4 mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1–3 mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s). PMID:22450673

Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

PubMed Central

Umaer, Khan; Ciganda, Martin

2014-01-01

Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention. PMID:24706018

Human pDCs display sex-specific differences in type I interferon subtypes and interferon α/β receptor expression.

PubMed

Ziegler, Susanne M; Beisel, Claudia; Sutter, Kathrin; Griesbeck, Morgane; Hildebrandt, Heike; Hagen, Sven H; Dittmer, Ulf; Altfeld, Marcus

2017-02-01

The outcomes of many diseases differ between women and men, with women experiencing a higher incidence and more severe pathogenesis of autoimmune and some infectious diseases. It has been suggested that this is partially due to activation of plasmacytoid dendritic cells (pDCs), the main producers of interferon (IFN)-α, in response to toll-like receptor (TLR)7 stimulation. We investigated the induction of type I IFN (IFN-I) subtypes upon TLR7 stimulation on isolated pDCs. Our data revealed a sex-specific differential expression of IFN-Is, with pDCs from females showing a significantly higher mRNA expression of all 13 IFN-α subtypes. In addition, pDCs from females had higher levels of IFN-β mRNA after stimulation, indicating that sex differences in IFN-I production by pDCs were mediated by a signaling event upstream of the first loop of IFN-I mRNA transcription. Furthermore, the surface expression levels of the common IFN-α/β receptor subunit 2 were significantly higher on pDCs from females in comparison to males. These data indicate that higher IFN-α production is already established at the mRNA level and propose a contribution of higher IFN-α/β receptor 2 expression on pDCs to the immunological differences in IFN-I production observed between females and males. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans.

PubMed Central

Szekeres, M; Droux, M; Buchanan, B B

1991-01-01

The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form. Images PMID:1705544

Expression Levels of Myostatin and Matrix Metalloproteinase 14 mRNAs in Uterine Leiomyoma are Correlated With Dysmenorrhea.

PubMed

Tsigkou, Anastasia; Reis, Fernando M; Ciarmela, Pasquapina; Lee, Meng H; Jiang, Bingjie; Tosti, Claudia; Shen, Fang-Rong; Shi, Zhendan; Chen, You-Guo; Petraglia, Felice

2015-12-01

Uterine leiomyoma is the most common benign neoplasm of female reproductive system, found in about 50% of women in reproductive age. The mechanisms of leiomyoma growth include cell proliferation, which is modulated by growth factors, and deposition of extracellular matrix (ECM). Activin A and myostatin are growth factors that play a role in proliferation of leiomyoma cells. Matrix metalloproteinases (MMPs) are known for their ability to remodel the ECM in different biological systems. The aim of this study was to evaluate the expression levels of activin βA-subunit, myostatin, and MMP14 messenger RNAs (mRNAs) in uterine leiomyomas and the possible correlation of these factors with clinical features of the disease. Matrix metalloproteinase 14 was highly expressed in uterine leiomyoma and correlated with myostatin and activin A mRNA expression. Moreover, MMP14 and myostatin mRNA expression correlated significantly and directly with the intensity of dysmenorrhea. Overall, the present findings showed that MMP14 mRNA is highly expressed in uterine leiomyoma, where it correlates with the molecular expression of growth factors and is further increased in cases of intense dysmenorrhea. © The Author(s) 2015.

[Schizophrenia and cortical GABA neurotransmission].

PubMed

Hashimoto, Takanori; Matsubara, Takuro; Lewis, David A

2010-01-01

Individuals with schizophrenia show disturbances in a number of brain functions that regulate cognitive, affective, motor, and sensory processing. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related molecules. First, mRNA levels for the 67-kilodalton isoform of glutamic acid decarboxylase (GAD67), an enzyme principally responsible for GABA synthesis, and the GABA membrane transporter GAT1, which regulates the reuptake of synaptically released GABA, are decreased in a subset of GABA neurons. Second, affected GABA neurons include those that express the calcium-binding protein parvalbumin (PV), because PV mRNA levels are decreased in the prefrontal cortex of subjects with schizophrenia and GAD67 mRNA is undetectable in almost half of PV-containing neurons. These changes are accompanied by decreased GAT1 expression in the presynaptic terminals of PV-containing neurons and by increased postsynaptic GABA-A receptor alpha2 subunit expression at the axon initial segments of pyramidal neurons. These findings indicate decreased GABA synthesis/release by PV-containing GABA neurons and compensatory changes at synapses formed by these neurons. Third, another subset of GABA neurons that express the neuropeptide somatostatin (SST) also appear to be affected because their specific markers, SST and neuropeptide Y mRNAs, are decreased in a manner highly correlated with the decreases in GAD67 mRNA. Finally, mRNA levels for GABA-A receptor subunits for synaptic (alpha1 and gamma2) and extra-synaptic (delta) receptors are decreased, indicating alterations in both synaptic and extra-synaptic GABA neurotransmission. Together, this pattern of changes indicates that the altered GABA neurotransmission is specific to PV-containing and SST-containing GABA neuron subsets and involves both synaptic and extra-synaptic GABA-A receptors. Our recent analyses demonstrated that this pattern exists across diverse cortical areas including the prefrontal, anterior cingulate, primary motor, and primary visual cortices. GABA neurotransmission by PV-containing and SST-containing neurons is important for the generation of cortical oscillatory activities in the gamma (30-100 Hz) and theta (4-7 Hz) bands, respectively. These oscillatory activities have been proposed to play critical roles in regulating the efficiency of information transfer between neurons and neuronal networks in the cortex. Altered cortical GABA neurotransmission appears to contribute to disturbances in diverse functions through affecting the generation of cortical oscillations in schizophrenia.

Changes in PEP carboxylase, rubisco and rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress.

PubMed

Leitao, Louis; Maoret, Jean-José; Biolley, Jean-Philippe

2007-01-01

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

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.

Progesterone modulation of alpha5 nAChR subunits influences anxiety-related behavior during estrus cycle.

PubMed

Gangitano, D; Salas, R; Teng, Y; Perez, E; De Biasi, M

2009-06-01

Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the alpha5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and alpha5 subunit null mice (alpha5(-/-)) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, alpha5(-/-) behaved like wild-type controls. In the EPM, female alpha5(-/-) mice displayed an anxiolytic-like phenotype, while male alpha5(-/-) mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolytic-like phenotype, female alpha5(-/-) mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of alpha5, we treated cultured NTera 2 cells with progesterone and found that alpha5 protein levels were upregulated. In addition, brain levels of alpha5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic-like in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in alpha5(-/-) females. Thus, alpha5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of alpha5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.

The NMDA Receptor Subunit NR2b: Effects on LH Release and GnRH Gene Expression in Young and Middle-aged Female Rats, with Modulation by Estradiol

PubMed Central

Maffucci, Jacqueline A.; Walker, Deena M.; Ikegami, Aiko; Woller, Michael J.; Gore, Andrea C.

2008-01-01

The loss of reproductive capacity during aging involves changes in the neural regulation of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons controlling reproduction. This neuronal circuitry includes glutamate receptors on GnRH neurons. Previously, we reported an increase in the expression of the NR2b subunit protein of the NMDA receptor on GnRH neurons in middle-aged compared to young female rats. Here, we examined the functional implications of the NR2b subunit on the onset of reproductive aging, using an NR2b-specific antagonist ifenprodil. Young (3–5 mos.) and middle-aged (10–13 mos.) female rats were ovariectomized (OVX), 17β-estradiol (E2) or vehicle (cholesterol) treated, and implanted with a jugular catheter. Serial blood sampling was undertaken every 10 minutes for 4 hours, with ifenprodil (10mg/kg) or vehicle injected (i.p.) after one hour of baseline sampling. The pulsatile release of pituitary LH and levels of GnRH mRNA in hypothalamus were quantified as indices of the reproductive axis. Our results showed effects of ifenprodil on both endpoints. In OVX rats given cholesterol, neither age nor ifenprodil had any effects on LH release. In E2-treated rats, aging was associated with significant decreases in pulsatile LH release. Additionally, ifenprodil stimulated parameters of pulsatile LH release in both young and middle-aged animals. Ifenprodil had few effects on GnRH mRNA; the only significant effect of ifenprodil was found in the middle-aged, cholesterol group. Together, these findings support a role for the NR2b subunit of the NMDAR in GnRH/LH regulation. Because most of these effects were exhibited on pituitary LH release in the absence of a concomitant change in GnRH gene expression, it is likely that NMDA receptors containing the NR2b subunit plays a role in GnRH-induced LH release, independent of de novo GnRH gene expression. PMID:18025808

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA*

PubMed Central

Wigington, Callie P.; Morris, Kevin J.; Newman, Laura E.; Corbett, Anita H.

2016-01-01

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function. PMID:27563065

Icaritin requires Phosphatidylinositol 3 kinase (PI3K)/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

PubMed Central

ZHANG, Zong-Kang; LI, Jie; LIU, Jin; GUO, Baosheng; LEUNG, Albert; ZHANG, Ge; ZHANG, Bao-Ting

2016-01-01

Counteracting muscle atrophy induced by mechanical unloading/inactivity is of great clinical need and challenge. A therapeutic agent that could counteract muscle atrophy following mechanical unloading in safety is desired. This study showed that natural product Icaritin (ICT) could increase the phosphorylation level of Phosphatidylinositol 3 kinase (PI3K) at p110 catalytic subunit and promote PI3K/Akt signaling markers in C2C12 cells. This study further showed that the high dose ICT treatment could significantly attenuate the decreases in the phosphorylation level of PI3K at p110 catalytic subunit and its downstream markers related to protein synthesis, and inhibit the increases in protein degradation markers at mRNA and protein levels in rat soleus muscle following 28-day hindlimb unloading. In addition, the decreases in soleus muscle mass, muscle fiber cross-sectional area, twitch force, specific force, contraction time and half relaxation time could be significantly attenuated by the high dose ICT treatment. The low dose ICT treatment could moderately attenuate the above changes induced by unloading. Wortmannin, a specific inhibitor of PI3K at p110 catalytic subunit, could abolish the above effects of ICT in vitro and in vivo, indicating that PI3K/Akt signaling could be required by ICT to counteract skeletal muscle atrophy following mechanical unloading. PMID:26831566

Phylogenetic analysis of fungal heterotrimeric G protein-encoding genes and their expression during dimorphism in Mucor circinelloides.

PubMed

Valle-Maldonado, Marco Iván; Jácome-Galarza, Irvin Eduardo; Díaz-Pérez, Alma Laura; Martínez-Cadena, Guadalupe; Campos-García, Jesús; Ramírez-Díaz, Martha Isela; Reyes-De la Cruz, Homero; Riveros-Rosas, Héctor; Díaz-Pérez, César; Meza-Carmen, Víctor

2015-12-01

In fungi, heterotrimeric G proteins are key regulators of biological processes such as mating, virulence, morphology, among others. Mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric G protein subunits in the fungal kingdom: twelve Gα (McGpa1-12), three Gβ (McGpb1-3), and three Gγ (McGpg1-3). Phylogenetic analysis of fungal Gα showed that they are divided into four distinct groups as reported previously. Fungal Gβ and Gγ are also divided into four phylogenetic groups, and to our understanding this is the first report of a phylogenetic classification for fungal Gβ and Gγ subunits. Almost all genes that encode putative heterotrimeric G subunits in M. circinelloides are differentially expressed during dimorphic growth, except for McGpg1 (Gγ) that showed very low mRNA levels at all developmental stages. Moreover, several of the subunits are expressed in a similar pattern and at the same level, suggesting that they constitute discrete complexes. For example, McGpb3 (Gβ), and McGpg2 (Gγ), are co-expressed during mycelium growth, and McGpa1, McGpb2, and McGpg2, are co-expressed during yeast development. These findings provide the conceptual framework to study the biological role of these genes during M. circinelloides morphogenesis. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62▿

PubMed Central

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-01-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus. PMID:21543505

Proteasomes remain intact, but show early focal alteration in their composition in a mouse model of amyotrophic lateral sclerosis.

PubMed

Kabashi, Edor; Agar, Jeffrey N; Hong, Yu; Taylor, David M; Minotti, Sandra; Figlewicz, Denise A; Durham, Heather D

2008-06-01

In amyotrophic lateral sclerosis caused by mutations in Cu/Zn-superoxide dismutase (SOD1), altered solubility and aggregation of the mutant protein implicates failure of pathways for detecting and catabolizing misfolded proteins. Our previous studies demonstrated early reduction of proteasome-mediated proteolytic activity in lumbar spinal cord of SOD1(G93A) transgenic mice, tissue particularly vulnerable to disease. The purpose of this study was to identify any underlying abnormalities in proteasomal structure. In lumbar spinal cord of pre-symptomatic mice [postnatal day 45 (P45) and P75], normal levels of structural 20S alpha subunits were incorporated into 20S/26S proteasomes; however, proteasomal complexes separated by native gel electrophoresis showed decreased immunoreactivity with antibodies to beta3, a structural subunit of the 20S proteasome core, and beta5, the subunit with chymotrypsin-like activity. This occurred prior to increase in beta5i immunoproteasomal subunit. mRNA levels were maintained and no association of mutant SOD1 with proteasomes was identified, implicating post-transcriptional mechanisms. mRNAs also were maintained in laser captured motor neurons at a later stage of disease (P100) in which multiple 20S proteins are reduced relative to the surrounding neuropil. Increase in detergent-insoluble, ubiquitinated proteins at P75 provided further evidence of stress on mechanisms of protein quality control in multiple cell types prior to significant motor neuron death.

A core subunit of the RNA-processing/degrading exosome specifically influences cuticular wax biosynthesis in Arabidopsis.

PubMed

Hooker, Tanya S; Lam, Patricia; Zheng, Huanquan; Kunst, Ljerka

2007-03-01

The cuticle is an extracellular matrix composed of cutin polyester and waxes that covers aerial organs of land plants and protects them from environmental stresses. The Arabidopsis thaliana cer7 mutant exhibits reduced cuticular wax accumulation and contains considerably lower transcript levels of ECERIFERUM3/WAX2/YORE-YORE (CER3/WAX2/YRE), a key wax biosynthetic gene. We show here that CER7 protein is a putative 3'-5' exoribonuclease homologous to yeast Ribonuclease PH45 (RRP45p), a core subunit of the RNA processing and degrading exosome that controls the expression of CER3/WAX2/YRE. We propose that CER7 acts by degrading a specific mRNA species encoding a negative regulator of CER3/WAX2/YRE transcription. A second RRP45p homolog found in Arabidopsis, designated At RRP45a, is partially functionally redundant with CER7, and complete loss of RRP45 function in Arabidopsis is lethal. To our knowledge, CER7 is currently the only example of a core exosomal subunit specifically influencing a cellular process.

The Chinese herbal medicine FTZ attenuates insulin resistance via IRS1 and PI3K in vitro and in rats with metabolic syndrome

PubMed Central

2014-01-01

Background Insulin resistance plays an important role in the development of metabolic syndrome (MS). Fu Fang Zhen Zhu Tiao Zhi formula (FTZ), a Chinese medicinal decoction, has been used to relieve hyperlipidemia, atherosclerosis and other symptoms associated with metabolic disorders in the clinic. Methods To evaluate the effect of FTZ on insulin resistance, HepG2 cells were induced with high insulin as a model of insulin resistance and treated with FTZ at one of three dosages. Next, the levels of glucose content, insulin receptor substrate1 (IRS1) protein expression and phosphatidylinositol 3-kinase (PI3K) subunit p85 mRNA expression were measured. Alternatively, MS was induced in rats via gavage feeding of a high-fat diet for four consecutive weeks followed by administration of FTZ for eight consecutive weeks. Body weight and the plasma levels of lipids, insulin and glucose were evaluated. Finally, the expression of PI3K p85 mRNA in adipose tissue of rats was measured. Results Our results revealed that FTZ attenuated glucose content and up-regulated the expression of PI3K p85 mRNA and IRS1 protein in insulin-resistant HepG2 cells in vitro. Moreover, FTZ reduced body weight and the plasma concentrations of triacylglycerol, cholesterol, fasting glucose and insulin in insulin resistant MS rats. FTZ also elevated the expression of PI3K p85 mRNA in the adipose tissues of MS rats. Conclusion FTZ attenuated MS symptoms by decreasing the plasma levels of glucose and lipids. The underlying mechanism was attenuation of the reduced expression of PI3K p85 mRNA and IRS1 protein in both insulin-resistant HepG2 cells and MS rats. PMID:24555840

γ-Amino-butyric acid (GABA) receptor subunit and transporter expression in the gonad and liver of the fathead minnow (Pimephales promelas).

PubMed

Biggs, Katie; Seidel, Jason S; Wilson, Alex; Martyniuk, Christopher J

2013-09-01

γ-Amino-butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate central nervous system. GABA receptors and synthesizing enzymes have also been localized to peripheral tissues including the liver, oviduct, uterus and ovary of mammals but the distribution and role of GABA in peripheral tissues of fish has not been fully investigated. The objectives of this study were to (1) determine if mRNA encoding GABA synthesizing enzymes (glutamic acid decarboxylase 65 and 67; gad65 and gad67), GABA transporters, and GABAA receptor subunits are localized to liver and gonad of fathead minnow (Pimephales promelas) (FHM) (2) investigate the effects of GABA on ovarian 17β-estradiol (E2) production, and (3) measure transcript responses in the ovary after in vitro incubation to GABA. Real-time PCR assays were developed for gad65, gad67, vesicular GABA transporter (vgat) and GABA transporter 1 (gat1), and select GABAA receptor subunits (gabra1, gabra5, gabrb1, gabrb2, gabrg1, gabrg2). All transcripts were localized to the brain as expected; however transcripts were also detected in the liver, ovary, and testis of FHMs. In the female liver, gad65 mRNA was significantly higher in expression compared to the male liver. Transcripts for gad67 were the highest in the brain>gonad>liver and in the gonads, gad67 was significantly higher in expression than gad65 mRNA. In the liver and gonad, the relative abundance of the subunits followed a general trend of gabrb1>gabrb2=gabrg1=gabrg2>gabra1=gabra5. To explore the effects of GABA in the ovary, tissue explants from reproductive female FHMs were treated with GABA (10(-10), 10(-8) and 10(-6)M) for 12h. GABA had no significant effect on 17β-estradiol production or on mRNA abundance for genes involved in ovarian steroidogenesis (e.g., 11βhsd, cyp17, cyp19a). There was a significant decrease in estrogen receptor 2a (esr2a) mRNA with 10(-10)M GABA. This study begins to investigate the GABA system in non-neural tissues of teleost fish and addresses the broader topic regarding the peripheral roles of neurotransmitters. Copyright © 2013 Elsevier Inc. All rights reserved.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

PubMed

Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F; Larson, Victoria S; Valasek, Leos; Hinnebusch, Alan G; Lorsch, Jon R

2013-02-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

The Contrasting Role of the Mediator Subunit MED30 in the Progression of Bladder Cancer.

PubMed

Syring, Isabella; Weiten, Richard; Müller, Tim; Schmidt, Doris; Steiner, Susanne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg

2017-12-01

The Mediator complex is a key regulator of gene transcription, and several studies have demonstrated altered expression of particular subunits in diverse human diseases, especially cancer. To date, nothing is known about the role of MED30 in bladder cancer. We, therefore, performed an RNA expression and survival analysis of the subunit MED30 in 537 samples of bladder cancer by using the database cBioPortal. To validate these data on the protein level, we practiced immunohistochemical staining against MED30 on a tissue microarray containing 210 samples of all tumour stages and performed survival analyses. For functional analysis, the siRNA-mediated knockdown of MED30 was performed in the cell lines T24 and TCCSUP followed by proliferation, migration, and invasion assays. On the mRNA and protein levels, higher expression of MED30 is associated with better patient survival. In accordance with this, advanced T- and N-stages showed lower expression of MED30. In contrast, knockdown of MED30 led to reduction of the tumour parameters proliferation, migration, and invasion in the BCa cell lines. MED30 appears to be integrated in the progression of the urothelial tumour in the bladder. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

PubMed

Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

2013-01-01

The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

Plasticity of the reproductive axis caused by social status change in an african cichlid fish: I. Pituitary gonadotropins.

PubMed

Maruska, Karen P; Levavi-Sivan, Berta; Biran, Jakob; Fernald, Russell D

2011-01-01

Social position in a dominance hierarchy is often tightly coupled with fertility. Consequently, an animal that can recognize and rapidly take advantage of an opportunity to rise in rank will have a reproductive advantage. Reproduction in all vertebrates is controlled by the brain-pituitary-gonad axis, and in males of the African cichlid fish Astatotilapia burtoni, GnRH1 neurons at the apex of this axis are under social control. However, little is known about how quickly social information is transformed into functional reproductive change, or about how socially controlled changes in GnRH1 neurons influence downstream actions of the brain-pituitary-gonad axis. We created an opportunity for reproductively suppressed males to ascend in status and then measured how quickly the perception of this opportunity caused changes in mRNA and protein levels of the pituitary gonadotropins. mRNA levels of the β-subunits of LH and FSH rose rapidly in the pituitary 30 min after suppressed males perceived an opportunity to ascend. In contrast, mRNA levels of GnRH receptor-1 remained unchanged during social transition but were higher in stable dominant compared with subordinate males. In the circulation, levels of both LH and FSH were also quickly elevated. There was a positive correlation between mRNA in the pituitary and circulating protein levels for LH and FSH, and both gonadotropins were positively correlated with plasma 11-ketotestosterone. Our results show that the pituitary is stimulated extremely rapidly after perception of social opportunity, probably to allow suppressed males to quickly achieve reproductive success in a dynamic social environment.

Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

PubMed

Durrieu-Gaillard, Stéphanie; Dumay-Odelot, Hélène; Boldina, Galina; Tourasse, Nicolas J; Allard, Delphine; André, Fabrice; Macari, Françoise; Choquet, Armelle; Lagarde, Pauline; Drutel, Guillaume; Leste-Lasserre, Thierry; Petitet, Marion; Lesluyes, Tom; Lartigue-Faustin, Lydia; Dupuy, Jean-William; Chibon, Frédéric; Roeder, Robert G; Joubert, Dominique; Vagner, Stéphan; Teichmann, Martin

2018-01-01

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

Hyperexcitability of bladder afferent neurons associated with reduction of Kv1.4 α-subunit in rats with spinal cord injury.

PubMed

Takahashi, Ryosuke; Yoshizawa, Tsuyoshi; Yunoki, Takakazu; Tyagi, Pradeep; Naito, Seiji; de Groat, William C; Yoshimura, Naoki

2013-12-01

To clarify the functional and molecular mechanisms inducing hyperexcitability of C-fiber bladder afferent pathways after spinal cord injury we examined changes in the electrophysiological properties of bladder afferent neurons, focusing especially on voltage-gated K channels. Freshly dissociated L6-S1 dorsal root ganglion neurons were prepared from female spinal intact and spinal transected (T9-T10 transection) Sprague Dawley® rats. Whole cell patch clamp recordings were performed on individual bladder afferent neurons. Kv1.2 and Kv1.4 α-subunit expression levels were also evaluated by immunohistochemical and real-time polymerase chain reaction methods. Capsaicin sensitive bladder afferent neurons from spinal transected rats showed increased cell excitability, as evidenced by lower spike activation thresholds and a tonic firing pattern. The peak density of transient A-type K+ currents in capsaicin sensitive bladder afferent neurons from spinal transected rats was significantly less than that from spinal intact rats. Also, the KA current inactivation curve was displaced to more hyperpolarized levels after spinal transection. The protein and mRNA expression of Kv1.4 α-subunits, which can form transient A-type K+ channels, was decreased in bladder afferent neurons after spinal transection. Results indicate that the excitability of capsaicin sensitive C-fiber bladder afferent neurons is increased in association with reductions in transient A-type K+ current density and Kv1.4 α-subunit expression in injured rats. Thus, the Kv1.4 α-subunit could be a molecular target for treating overactive bladder due to neurogenic detrusor overactivity. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension.

PubMed

Afolayan, Adeleye J; Eis, Annie; Alexander, Maxwell; Michalkiewicz, Teresa; Teng, Ru-Jeng; Lakshminrusimha, Satyan; Konduri, Girija G

2016-01-01

Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen. Copyright © 2016 the American Physiological Society.

The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

PubMed

Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A

2015-05-19

The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. © 2015 Authors.

Fate of mRNA following disaggregation of brain polysomes after administration of (+)-lysergic acid diethylamide in vivo.

PubMed

Mahony, J B; Brown, I R

1979-11-22

Intravenous injection of (+)-lysergic acid diethylamide into young rabbits induced a transient brain-specific disaggregation of polysomes to monosomes. Investigation of the fate of mRNA revealed that brain poly(A+)mRNA was conserved. In particular, mRNA coding for brain-specific S100 protein was not degraded, nor was it released into free ribonucleoprotein particles. Following the (+)-lysergic acid diethylamide-induced disaggregation of polysomes, mRNA shifted from polysomes and accumulated on monosomes. Formation of a blocked monosome complex, which contained intact mRNA and 40-S plus 60-S ribosomal subunits but lacked nascent peptide chains, suggested that (+)-lysergic acid diethylamide inhibited brain protein synthesis at a specific stage of late initiation or early elongation.

Combined Effects of Simultaneous Exposure to Caffeine and Cocaine in the Mouse Striatum.

PubMed

Muñiz, Javier A; Gomez, Gimena; González, Betina; Rivero-Echeto, María Celeste; Cadet, Jean Lud; García-Rill, Edgar; Urbano, Francisco J; Bisagno, Veronica

2016-05-01

Caffeine is the world's most popular psychoactive drug and is also an active adulterant found in many drugs of abuse, including seized cocaine samples. Despite several studies which examine the effects of caffeine or cocaine administered as single agents, little data are available for these agents when given in combination. The purpose of the present study was to determine if combined intake of both psychostimulants can lead to maladaptive changes in striatal function. Mice were injected with a binge regimen (intermittent treatment for 13 days) of caffeine (3 × 5 mg/kg), cocaine (3 × 10 mg/kg), or combined administration. We found that chronic caffeine potentiated locomotion induced by cocaine and that both caffeine-treated groups showed sensitization. Striatal tissue was obtained 24 h and 7 days after last injection (withdrawal) for immunohistochemistry and mRNA expression. Our results show that combined intake of both psychostimulants can increase GFAP immunoreactivity in the striatum at both times post treatment. Gene expression analysis, targeted at dopamine, adenosine, and glutamate receptor subunit genes, revealed significant transcript down-regulation in the dorsal striatum of AMPA, NMDA, D1 and D2 receptor subunit mRNA expression in the group that received combined treatment, but not after individual administration. At withdrawal, we found increased D1 receptor mRNA expression along with increased A1, AMPA, NMDA, and metabotropic subunit expression. A2A mRNA showed decreased expression after both times in all experimental groups. Our study provides evidence that there are striatal alterations mediated by combined caffeine and cocaine administration, and highlights negative outcomes of chronic intake of both psychostimulants.

Short- and long-term salinity challenge, Osmoregulatory ability, and (Na+, K+)-ATPase KINETICS AND α-SUBUNIT mRNA expression in the gills of the thinstripe hermit CRAB Clibanarius symmetricus.

PubMed

Faleiros, Rogério O; Garçon, Daniela P; Lucena, Malson N; McNamara, John C; Leone, Francisco A

2018-06-19

The evolutionary history of the Crustacea reveals ample adaptive radiation and the subsequent occupation of many osmotic niches resulting from physiological plasticity in their osmoregulatory mechanisms. We evaluate osmoregulatory ability in the intertidal, thinstripe hermit crab Clibanarius symmetricus after short-term exposure (6 h) or long-term acclimation (10 days) to a wide salinity range, also analyzing kinetic behavior and α-subunit mRNA expression of the gill (Na + , K + )-ATPase. The crab strongly hyper-regulates its hemolymph at 5 and 15‰S (Salinity, g L -1 ) but weakly hyper-regulates up to ≈27‰S. After 6 h exposure to 35‰S and 45‰S, C. symmetricus slightly hypo-regulates its hemolymph, becoming isosmotic after 10 days acclimation to these salinities. (Na + , K + )-ATPase specific activity decreases with increasing salinity for both exposure periods, reflecting physiological adjustment to isosmoticity. At low salinities, the gill enzyme exhibits a single, low affinity ATP binding site. However, at elevated salinities, a second, high affinity, ATP binding site appears, independently of exposure time. (Na + , K + )-ATPase α-subunit mRNA expression increases only after 10 days acclimation to 5‰S. Our findings suggest that hemolymph hyper-regulation is effected by alterations in enzyme activity during short-term exposure, but is sustained by increased mRNA expression during long-term acclimation. The decrease in gill (Na + , K + )-ATPase activity seen as a consequence of increasing salinity appears to underlie biochemical adjustments to hemolymph isosmoticity as hypo-regulatory ability diminishes. Copyright © 2018. Published by Elsevier Inc.

Congenitally learned helpless rats show abnormalities in intracellular signaling.

PubMed

Kohen, Ruth; Neumaier, John F; Hamblin, Mark W; Edwards, Emmeline

2003-03-15

Affective disorders and the drugs used to treat them lead to changes in intracellular signaling. We used a genetic animal model to investigate to what extent changes in intracellular signal transduction confer a vulnerability to mood or anxiety disorders. Levels of gene expression in a selectively bred strain of rats with a high vulnerability to develop congenitally learned helplessness (cLH), a strain highly resistant to the same behavior (cNLH) and outbred Sprague-Dawley (SD) control animals were compared using quantitative reverse transcription polymerase chain reaction. Congenitally learned helpless animals had a 24%-30% reduced expression of the cyclic adenosine monophosphate response element binding protein messenger ribonucleic acid (mRNA) in the hippocampus and a 40%-41% increased level of the antiapoptotic protein bcl-2 mRNA in the prefrontal cortex compared to cNLH and SD rats. Other significant changes included changes in the expression levels of the alpha catalytic subunit of protein kinase A, glycogen synthase kinase 3beta, and protein kinase C epsilon. Congenitally learned helpless animals show evidence of altered signal transduction and regulation of apoptosis compared to cNLH and SD control animals.

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

PubMed

Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

2016-01-01

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria. © 2016 American Society of Plant Biologists. All Rights Reserved.

Physiological and molecular endocrine changes in maturing wild sockeye salmon, Oncorhynchus nerka, during ocean and river migration.

PubMed

Flores, A M; Shrimpton, J M; Patterson, D A; Hills, J A; Cooke, S J; Yada, T; Moriyama, S; Hinch, S G; Farrell, A P

2012-01-01

Maturing adult sockeye salmon Oncorhynchus nerka were intercepted while migrating in the ocean and upstream in freshwater over a combined distance of more than 1,300 km to determine physiological and endocrine changes associated with ionoregulation. Sockeye migrating through seawater and freshwater showed consistent declines in gill Na+/K+ -ATPase (NKA) activity, plasma osmolality and plasma chloride concentration. In contrast, plasma sodium concentration became elevated in seawater as fish approached the river mouth and was then restored after sockeye entered the river. Accompanying the movement from seawater to freshwater was a significant increase in mRNA for the NKA α1a subunit in the gill, with little change in the α1b subunit. Potential endocrine signals stimulating the physiological changes during migration were assessed by measuring plasma cortisol and prolactin (Prl) concentrations and quantifying mRNA extracted from the gill for glucocorticoid receptors 1 and 2 (GR1 and GR2), mineralocorticoid receptor (MR), growth hormone 1 receptor (GH1R), and prolactin receptor (PrlR). Plasma cortisol and prolactin concentrations were high in seawater suggesting a preparatory endocrine signal before freshwater entry. Generally, the mRNA expression for GR1, GR2 and MR declined during migration, most notably after fish entered freshwater. In contrast, PrlR mRNA increased throughout migration, particularly as sockeye approached the spawning grounds. A highly significant association existed between gill PrlR mRNA and gill NKA α1a mRNA. GH1R mRNA also increased significantly, but only after sockeye had migrated beyond tidal influence in the river and then again just before the fish reached the spawning grounds. These findings suggest that cortisol and prolactin stimulate ionoregulation in the gill as sockeye salmon adapt to freshwater.

Association of EGFR mutation or ALK rearrangement with expression of DNA repair and synthesis genes in never-smoker women with pulmonary adenocarcinoma.

PubMed

Ren, Shengxiang; Chen, Xiaoxia; Kuang, Peng; Zheng, Limou; Su, Chunxia; Li, Jiayu; Li, Bing; Wang, Yongshen; Liu, Lu; Hu, Qiong; Zhang, Jie; Tang, Liang; Li, Xuefei; Zhou, Caicun; Schmid-Bindert, Gerald

2012-11-15

Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement may predict the outcome of targeted drug therapy and also are associated with the efficacy of chemotherapy in patients with nonsmall cell lung cancer (NSCLC). The authors of this report investigated the relation of EGFR mutation or ALK rearrangement status and the expression of DNA repair or synthesis genes, including excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1), thymidylate synthetase (TS), and breast cancer-early onset (BRCA1), as a potential explanation for these observations. In total, 104 resected lung adenocarcinomas from women who were nonsmokers were analyzed concurrently for EGFR mutations, ALK rearrangements, and mRNA expression of the ERCC1, RRM1, TS, and BRCA1 genes. EGFR mutations were detected with a proprietary detection kit, ALK rearrangements were detected by polymerase chain reaction analysis, and genetic mRNA expression was detected by real-time polymerase chain reaction analysis. Of 104 patients, 73 (70.2%) had EGFR mutations, and 10 (9.6%) had ALK rearrangements. ERCC1 mRNA levels in patients who had EGFR mutations were 3.44 ± 1.94 × 10(-3) , which were significantly lower than the levels in patients who were positive for ALK rearrangements and in patients who were negative for both biomarkers (4.60 ± 1.95 × 10(-3) and 4.95 ± 2.33 × 10(-3) , respectively; P = .010). However, TS mRNA levels were significantly lower in patients who had EGFR mutations (1.15 ± 1.38 × 10(-3) vs 2.69 ± 3.97 × 10(-3) ; P = .006) or ALK rearrangements (1.21 ± 0.78 × 10(-3) vs 2.69 ± 3.97 × 10(-3) ; P = .020) than in patients who were negative for both biomarkers. NSCLC specimens that harbored activating EGFR mutations were more likely to express low ERCC1 and TS mRNA levels, whereas patients with NSCLC who had ALK rearrangement were more likely to express low TS mRNA levels. Copyright © 2012 American Cancer Society.

Magnolol and honokiol regulate the calcium-activated potassium channels signaling pathway in Enterotoxigenic Escherichia coli-induced diarrhea mice.

PubMed

Deng, Yanli; Han, Xuefeng; Tang, Shaoxun; Xiao, Wenjun; Tan, Zhiliang; Zhou, Chuanshe; Wang, Min; Kang, Jinghe

2015-05-15

To explore the regulatory mechanisms of magnolol and honokiol on calcium-activated potassium channels signaling pathway in Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea mice, the concentrations of serum chloride ion (Cl(-)), sodium ion (Na(+)), potassium ion (K(+)) and calcium ion (Ca(2+)) were measured. Additionally, the mRNA expressions of calmodulin 1 (CaM), calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) and beta subunit (CaMKIIβ), ryanodine receptor 1, inositol 1,4,5-trisphosphate receptors (IP3 receptors), protein kinases C (PKC), potassium intermediate/small conductance calcium-activated channels (SK) and potassium large conductance calcium-activated channels(BK)were determined. A diarrhea mouse model was established using ETEC suspensions (3.29×10(9)CFU/ml) at a dosage of 0.02ml/g live body weight (BW). Magnolol or honokiol was intragastrically administered at dosages of 100 (M100 or H100), 300 (M300 or H300) and 500 (M500 or H500) mg/kg BW according to a 3×3 factorial arrangement. Magnolol and honokiol increased the Cl(-) and K(+) concentrations, further, upregulated the CaM, BKα1 and BKβ3 mRNA levels but downregulated the IP3 receptors 1, PKC, SK1, SK2, SK3, SK4 and BKβ4 mRNA expressions. Magnolol and honokiol did not alter the CaMKIIα, CaMKIIβ, ryanodine receptor 1, IP3 receptor 2, IP3 receptor 3, BKβ1 and BKβ2 mRNA expressions. These results clarify that magnolol and honokiol, acting through Ca(2+) channel blockade, inhibit the activation of IP3 receptor 1 to regulate the IP3-Ca(2+) store release, activate CaM to inhibit SK channels, and effectively suppress PKC kinases to promote BKα1 and BKβ3 channels opening and BKβ4 channel closing, which modulates the intestinal ion secretion. Copyright © 2015 Elsevier B.V. All rights reserved.

Captopril reduces cardiac inflammatory markers in spontaneously hypertensive rats by inactivation of NF-kB

PubMed Central

2010-01-01

Background Captopril is an angiotensin-converting enzyme (ACE) inhibitor widely used in the treatment of arterial hypertension and cardiovascular diseases. Our objective was to study whether captopril is able to attenuate the cardiac inflammatory process associated with arterial hypertension. Methods Left ventricle mRNA expression and plasma levels of pro-inflammatory (interleukin-1β (IL-1β) and IL-6) and anti-inflammatory (IL-10) cytokines, were measured in spontaneously hypertensive rats (SHR) and their control normotensive, Wistar-Kyoto (WKY) rats, with or without a 12-week treatment with captopril (80 mg/Kg/day; n = six animals per group). To understand the mechanisms involved in the effect of captopril, mRNA expression of ACE, angiotensin II type I receptor (AT1R) and p22phox (a subunit of NADPH oxidase), as well as NF-κB activation and expression, were measured in the left ventricle of these animals. Results In SHR, the observed increases in blood pressures, heart rate, left ventricle relative weight, plasma levels and cardiac mRNA expression of IL-1β and IL-6, as well as the reductions in the plasma levels and in the cardiac mRNA expression of IL-10, were reversed after the treatment with captopril. Moreover, the mRNA expressions of ACE, AT1R and p22phox, which were enhanced in the left ventricle of SHR, were reduced to normal values after captopril treatment. Finally, SHR presented an elevated cardiac mRNA expression and activation of the transcription nuclear factor, NF-κB, accompanied by a reduced expression of its inhibitor, IκB; captopril administration corrected the observed changes in all these parameters. Conclusion These findings show that captopril decreases the inflammation process in the left ventricle of hypertensive rats and suggest that NF-κB-driven inflammatory reactivity might be responsible for this effect through an inactivation of NF-κB-dependent pro-inflammatory factors. PMID:20462420

Constitutive Activation of the G-Protein Subunit G[alpha]s within Forebrain Neurons Causes PKA-Dependent Alterations in Fear Conditioning and Cortical "Arc" mRNA Expression

ERIC Educational Resources Information Center

Kelly, Michele P.; Cheung, York-Fong; Favilla, Christopher; Siegel, Steven J.; Kanes, Stephen J.; Houslay, Miles D.; Abel, Ted

2008-01-01

Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G[alpha]s…

Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis.

PubMed Central

Li, S J; Cronan, J E

1993-01-01

Acetyl coenzyme A (CoA) carboxylase catalyzes the synthesis of malonyl-CoA, the first intermediate of fatty acid synthesis. The Escherichia coli enzyme is encoded by four subunits located at three different positions on the E. coli chromosome. The accBC genes lie in a small operon at min 72, whereas accA and accD are located at min 4.3 and 50, respectively. We examined the expression of the genes that encode the E. coli acetyl-CoA carboxylase subunits (accA, accBC, and accD) under a variety of growth conditions by quantitative Northern (RNA) blot analysis. We found a direct correlation between the levels of transcription of the acc genes and the rate of cellular growth. Consistent results were also obtained upon nutritional upshift and downshift experiments and upon dilution of stationary-phase cultures into fresh media. We also determined the 5' end of the accA and accD mRNAs by primer extension and did transcriptional fusion analysis of the previously reported accBC promoter. Several interesting features were found in the promoter regions of these genes, including a bent DNA sequence and an open reading frame within the unusually long leader mRNA of the accBC operon, potential stem-loop structures in the accA and accD mRNA leader regions, and a stretch of GC-rich sequences followed by AT-rich sequences common to all three promoters. In addition, both accA and accD are located in complex gene clusters. For example, the accA promoter was localized within the upstream polC gene (which encodes the DNA polymerase III catalytic subunit), suggesting that additional regulatory mechanisms exist. Images PMID:7678242

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression.

PubMed

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Kandimalla, Ramesh J L; Bal, Amanjit; Gill, Kiran Dip

2013-12-01

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10mg/kgb.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits-NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. © 2013.

Protein Kinase A Regulatory Subunits in Human Adipose Tissue

PubMed Central

Mantovani, Giovanna; Bondioni, Sara; Alberti, Luisella; Gilardini, Luisa; Invitti, Cecilia; Corbetta, Sabrina; Zappa, Marco A.; Ferrero, Stefano; Lania, Andrea G.; Bosari, Silvano; Beck-Peccoz, Paolo; Spada, Anna

2009-01-01

OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects. RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively. RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects. CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity. PMID:19095761

Molecular characterization of hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) from Taiwan voles (Microtus kikuchii).

PubMed

Jiang, Yi-Fan; Chou, Chung-Hsi; Lin, En-Chung; Chiu, Chih-Hsien

2011-02-01

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that senses and adapts cells to hypoxic environmental conditions. HIF-1 is composed of an oxygen-regulated α subunit (HIF-1α) and a constitutively expressed β subunit (HIF-1β). Taiwan voles (Microtus kikuchii) are an endemic species in Taiwan, found only in mountainous areas greater than 2000m above sea level. In this study, the full-length HIF-1α cDNA was cloned and sequenced from liver tissues of Taiwan voles. We found that HIF-1α of Taiwan voles had high sequence similarity to HIF-1α of other species. Sequence alignment of HIF-1α functional domains indicated basic helix-loop-helix (bHLH), PER-ARNT-SIM (PAS) and C-terminal transactivation (TAD-C) domains were conserved among species, but sequence variations were found between the oxygen-dependent degradation domains (ODDD). To measure Taiwan vole HIF-1α responses to hypoxia, animals were challenged with cobalt chloride, and HIF-1α mRNA and protein expression in brain, lung, heart, liver, kidney, and muscle was assessed by quantitative RT-PCR and Western blot analysis. Upon induction of hypoxic stress with cobalt chloride, an increase in HIF-1α mRNA levels was detected in lung, heart, kidney, and muscle tissue. In contrast, protein expression levels showed greater variation between individual animals. These results suggest that the regulation of HIF-1α may be important to the Taiwan vole under cobalt chloride treatments. But more details regarding the evolutionary effect of environmental pressure on HIF-1α primary sequence, HIF-1α function and regulation in Taiwan voles remain to be identified. Copyright © 2010 Elsevier Inc. All rights reserved.

mRNA bound to the 30S subunit is a HigB toxin substrate

PubMed Central

Schureck, Marc A.; Maehigashi, Tatsuya; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

2016-01-01

Activation of bacterial toxins during stress results in cleavage of mRNAs in the context of the ribosome. These toxins are thought to function as global translational inhibitors yet recent studies suggest each may have distinct mRNA specificities that result in selective translation for bacterial survival. Here we demonstrate that mRNA in the context of a bacterial 30S subunit is sufficient for ribosome-dependent toxin HigB endonucleolytic activity, suggesting that HigB interferes with the initiation step of translation. We determined the X-ray crystal structure of HigB bound to the 30S, revealing that two solvent-exposed clusters of HigB basic residues directly interact with 30S 16S rRNA helices 18, 30, and 31. We further show that these HigB residues are essential for ribosome recognition and function. Comparison with other ribosome-dependent toxins RelE and YoeB reveals that each interacts with similar features of the 30S aminoacyl (A) site yet does so through presentation of diverse structural motifs. PMID:27307497

Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

PubMed Central

Llácer, Jose L.; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R.; Hinnebusch, Alan G.; Ramakrishnan, V.

2015-01-01

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition. PMID:26212456

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

Interleukin-2 and subunit alpha of its soluble receptor in autoimmune Addison's disease--an association study and expression analysis.

PubMed

Fichna, Marta; Żurawek, Magdalena; Bratland, Eirik; Husebye, Eystein S; Kasperlik-Załuska, Anna; Czarnocka, Barbara; Januszkiewicz-Lewandowska, Danuta; Nowak, Jerzy

2015-03-01

Autoimmune Addison's disease (AAD) results from T cell-mediated destruction of the adrenal cortex, commonly accompanied by autoantibodies to 21-hydroxylase (21OH). In order to gain insight into the obscure aetiology of this disease, we investigated the roles of the IL2 and IL2RA genes, encoding interleukin-2 and subunit alpha of its receptor (IL2Ra), respectively. The association of AAD with IL2 and IL2RA polymorphisms (rs6822844, rs2069762, rs3136534, rs11594656, rs3118470 and rs2104286) was tested in 223 patients and 672 healthy controls. Functional studies consisted of gene expression analysis in cultured PBMCs exposed to 21OH and evaluation of serum interleukin by ELISA assays. The frequency of the minor C allele of rs3136534 was significantly decreased in AAD subjects compared to controls (OR 0.71; 95%CI 0.561-0.887; p = 0.003). Only AAD cells responded to 21OH with an elevated IL2 and IL2RA mRNA synthesis (p = 0.004 and p = 0.009 versus controls, respectively), paralleled by increased supernatant levels of both cytokines (p = 0.031 and p = 0.001 versus controls). IL2 mRNA level in 21OH-stimulated AAD PBMCs correlated negatively with age (p = 0.036) and positively with serum antibodies to 21OH (p = 0.006). Carriers of the rs2104286 AA genotype demonstrated higher IL2RA mRNA (p = 0.022) and soluble IL2Ra secretion (p = 0.029) upon 21OH stimulation. Serum interleukin-2 in AAD subjects was significantly higher compared to controls (4.61 ± 4.3 versus 1.71 ± 3.2 pg/mL, p < 0.001), whereas sIL2Ra levels remained similar in both groups (p = 0.885). In conclusion, the study reveals an association between AAD and IL2 locus. It confirms specific 21OH-directed reactivity of the peripheral AAD lymphocytes, which display increased synthesis of interleukin-2 and sIL2Ra.

The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway.

PubMed

Jeong, Ji-Hye; Nam, Yeon-Ju; Kim, Seok-Yong; Kim, Eung-Gook; Jeong, Jooyoung; Kim, Hyong Kyu

2007-09-01

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.

Pomegranate protects liver against cecal ligation and puncture-induced oxidative stress and inflammation in rats through TLR4/NF-κB pathway inhibition.

PubMed

Makled, Mirhan N; El-Awady, Mohammed S; Abdelaziz, Rania R; Atwan, Nadia; Guns, Emma T; Gameil, Nariman M; Shehab El-Din, Ahmed B; Ammar, Elsayed M

2016-04-01

Acute liver injury secondary to sepsis is a major challenge in intensive care unit. This study was designed to investigate potential protective effects of pomegranate against sepsis-induced acute liver injury in rats and possible underlying mechanisms. Pomegranate was orally given (800mg/kg/day) for two weeks before sepsis induction by cecal ligation and puncture (CLP). Pomegranate improved survival and attenuated liver inflammatory response, likely related to downregulation of mRNA expression of toll like recptor-4, reduced nuclear translocation and DNA binding activity of proinflammatory transcription factor NF-κB subunit p65, decreased mRNA and protein expression of tumor necrosis factor-alpha and reduction in myeloperoxidase activity and mRNA expression. Pomegranate also decreased CLP-induced oxidative stress as reflected by decreased malondialdehyde content, and increased reduced glutathione level and superoxide dismutase activity. These results confirm the antiinflammatory and antioxidant effects of pomegranate in CLP-induced acute liver injury mediated through inhibiting TLR4/NF-κB pathway, lipid peroxidation and neutrophil infiltration. Copyright © 2016 Elsevier B.V. All rights reserved.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

PubMed Central

Walker, Sarah E.; Zhou, Fujun; Mitchell, Sarah F.; Larson, Victoria S.; Valasek, Leos; Hinnebusch, Alan G.; Lorsch, Jon R.

2013-01-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B’s domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome’s mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment. PMID:23236192

How does a scanning ribosomal particle move along the 5'-untranslated region of eukaryotic mRNA? Brownian Ratchet model.

PubMed

Spirin, Alexander S

2009-11-17

A model of the ATP-dependent unidirectional movement of the 43S ribosomal initiation complex (=40S ribosomal subunit + eIF1 + eIF1A + eIF2.GTP.Met-tRNA(i) + eIF3) during scanning of the 5'-untranslated region of eukaryotic mRNA is proposed. The model is based on the principles of molecular Brownian ratchet machines and explains several enigmatic data concerning the scanning complex. In this model, the one-dimensional diffusion of the ribosomal initiation complex along the mRNA chain is rectified into the net-unidirectional 5'-to-3' movement by the Feynman ratchet-and-pawl mechanism. The proposed mechanism is organized by the heterotrimeric protein eIF4F (=eIF4A + eIF4E + eIF4G), attached to the scanning ribosomal particle via eIF3, and the RNA-binding protein eIF4B that is postulated to play the role of the pawl. The energy for the useful work of the ratchet-and-pawl mechanism is supplied from ATP hydrolysis induced by the eIF4A subunit: ATP binding and its hydrolysis alternately change the affinities of eIF4A for eIF4B and for mRNA, resulting in the restriction of backward diffusional sliding of the 43S ribosomal complex along the mRNA chain, while stochastic movements ahead are allowed.

Opiate physical dependence and N-methyl-D-aspartate receptors.

PubMed

Noda, Yukihiro; Nabeshima, Toshitaka

2004-10-01

The present review focused the involvement of N-methyl-D-aspartate (NMDA) receptors in morphine physical dependence. The increased levels of extracellular glutamate, NMDA receptor zeta subunit (NR1) mRNA, NMDA receptor epsilon 1 subunit (NR2A) protein, phosphorylated Ca(2+)/calmodulin kinase II (p-CaMKII) protein, c-fos mRNA, c-Fos protein, are observed in the specific brain areas of mice and/or rats showing signs of naloxone-precipitated withdrawal. In preclinical and clinical studies, a variety of NMDA receptor antagonists and pretreatment with an antisense oligonucleotide of the NR1 have been reported to inhibit the development, expression and/or maintenance of opiate physical dependence. In contrast to data obtained in adult animals, NMDA receptor antagonists are neither effective in blocking the development of opiate dependence nor the expression of opiate withdrawal in neonatal rats. In the NMDA receptor-deficient mice, the NR2A knockout mice show the marked loss of typical withdrawal abstinence behaviors precipitated by naloxone. The rescue of NR2A protein by electroporation into the nucleus accumbens of NR2A knockout mice reverses the loss of abstinence behaviors. The activation of CaMKII and increased expression of c-Fos protein in the brain of animals with naloxone-precipitated withdrawal syndrome are prevented by NMDA receptor antagonists, whereas the increased levels of extracellular glutamate are not prevented by them. These findings indicate that glutamatergic neurotransmission at the NMDA receptor site contributes to the development, expression and maintenance of opiate dependence, and suggest that NMDA receptor antagonists may be a useful adjunct in the treatment of opiate dependence.

Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

PubMed

Okada, Hirokazu; Schittenhelm, Ralf B; Straessle, Anna; Hafen, Ernst

2015-01-01

The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

Altered Sex Hormone Concentrations and Gonadal mRNA Expression Levels of Activin Signaling Factors in Hatchling Alligators From a Contaminated Florida Lake

PubMed Central

MOORE, BRANDON C.; KOHNO, SATOMI; COOK, ROBERT W.; ALVERS, ASHLEY L.; HAMLIN, HEATHER J.; WOODRUFF, TERESA K.; GUILLETTE, LOUIS J.

2014-01-01

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin βA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly. PMID:20166196

Sexually dimorphic expression of gonadotropin subunits in the pituitary of protogynous honeycomb grouper (Epinephelus merra): evidence that follicle-stimulating hormone (FSH) induces gonadal sex change.

PubMed

Kobayashi, Yasuhisa; Alam, Mohammad Ashraful; Horiguchi, Ryo; Shimizu, Akio; Nakamura, Masaru

2010-06-01

Recent studies have suggested that the hypothalamic-pituitary-gonadal axis is involved in gonadal sex change in sex-changing teleosts. However, its underlying mechanism remains largely unknown. In this study, we focused on the distinct roles of two gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), in the protogynous hermaphrodite teleost, honeycomb grouper (Epinephelus merra). First, we investigated the expression pattern of mRNAs for GTH subunits (cga, fshb, and lhb) in the pituitaries from fish at the different sexual phases. Real-time RT-PCR analyses showed that fhsb mRNA levels in the female pituitary were low. However, fshb transcripts increased dramatically in association with testis development. In contrast, levels of cga and lhb mRNAs did not significantly vary during sex change. In addition, immunohistochemical observations of Fshb- and Lhb-producing cells in the pituitary, through the use of specific antibodies for detections of teleost GTH subunits, were consistent with sexually dimorphic expression of Fshb. In order to identify the role of GTH in gonad of honeycomb grouper, we treated females with bovine FSH (50 or 500 ng/fish) or LH (500 ng/fish) in vivo. After 3 wk, FSH treatments induced female-to-male sex change and up-regulated endogenous androgen levels and fshb transcripts, whereas LH treatment had no effect on sex change. These results suggest that FSH may trigger the female-to-male sex change in honeycomb grouper.

Agomelatine, a MT1/MT2 melatonergic receptor agonist with serotonin 5-HT2C receptor antagonistic properties, suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

PubMed

Hyeon, Jin-Yi; Choi, Eun-Young; Choe, So-Hui; Park, Hae Ryoun; Choi, Jeom-Il; Choi, In Soon; Kim, Sung-Jo

2017-10-01

This study was performed in an attempt to examine the influence of agomelatine in mitigating the generation of proinflammatory mediators in RAW264.7 murine macrophages exposed to lipopolysaccharide (LPS) obtained from Prevotella intermedia, a gram-negative anaerobic bacterium that is related with various types of periodontal diseases, and the molecular mechanisms behind its effects. LPS from P. intermedia strain ATCC 25611 was prepared employing the conventional phenol-water procedure. Conditioned culture media were analyzed for the levels of nitric oxide (NO), interleukin-1β (IL-1β) and IL-6. Real-time PCR analysis was carried out to determine the mRNA levels of inducible NO synthase (iNOS), IL-1β, IL-6 and SOCS1. Protein expression levels were evaluated by immunoblot test. NF-κB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-κB subunits were analyzed utilizing the ELISA-based kits. Agomelatine was found to down-regulate significantly the generation of iNOS-derived NO, IL-1β and IL-6 as well as the expression of their mRNAs in cells activated with P. intermedia LPS. Agomelatine decreased NF-κB-dependent SEAP release caused by P. intermedia LPS. Agomelatine did not inhibit NF-κB transcription induced by LPS at the level of IκB-α degradation. Instead, LPS-induced nuclear translocation and DNA binding of NF-κB p50 subunit was blocked by agomelatine. P. intermedia LPS-elicited activation of STAT1 and STAT3 was reduced notably by co-treatment with agomelatine. Agomelatine showed a tendency to enhance mRNA level of SOCS1 in LPS-activated cells as well. Agomelatine merits further evaluation to reveal its usefulness on the host modulation of periodontal disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

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

Jiang, Meisheng; Tran, V.T.; Fong, H.K.W.

1991-05-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha}more » protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.« less

Structure of a human cap-dependent 48S translation pre-initiation complex

PubMed Central

Eliseev, Boris; Yeramala, Lahari; Leitner, Alexander; Karuppasamy, Manikandan; Raimondeau, Etienne; Huard, Karine; Alkalaeva, Elena; Aebersold, Ruedi

2018-01-01

Abstract Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition. PMID:29401259

Molecular and functional characterization of seven Na+/K+-ATPase β subunit paralogs in Senegalese sole (Solea senegalensis Kaup, 1858).

PubMed

Armesto, Paula; Infante, Carlos; Cousin, Xavier; Ponce, Marian; Manchado, Manuel

2015-04-01

In the present work, seven genes encoding Na(+),K(+)-ATPase (NKA) β-subunits in the teleost Solea senegalensis are described for the first time. Sequence analysis of the predicted polypeptides revealed a high degree of conservation with those of other vertebrate species and maintenance of important motifs involved in structure and function. Phylogenetic analysis clustered the seven genes into four main clades: β1 (atp1b1a and atp1b1b), β2 (atp1b2a and atp1b2b), β3 (atp1b3a and atp1b3b) and β4 (atp1b4). In juveniles, all paralogous transcripts were detected in the nine tissues examined albeit with different expression patterns. The most ubiquitous expressed gene was atp1b1a whereas atp1b1b was mainly detected in osmoregulatory organs (gill, kidney and intestine), and atp1b2a, atp1b2b, atp1b3a, atp1b3b and atp1b4 in brain. An expression analysis in three brain regions and pituitary revealed that β1-type transcripts were more abundant in pituitary than the other β paralogs with slight differences between brain regions. Quantification of mRNA abundance in gills after a salinity challenge showed an activation of atp1b1a and atp1b1b at high salinity water (60 ppt) and atp1b3a and atp1b3b in response to low salinity (5 ppt). Transcriptional analysis during larval development showed specific expression patterns for each paralog. Moreover, no differences in the expression profiles between larvae cultivated at 10 and 35 ppt were observed except for atp1b4 with higher mRNA levels at 10 than 35 ppt at 18 days post hatch. Whole-mount in situ hybridization analysis revealed that atp1b1b was mainly localized in gut, pronephric tubule, gill, otic vesicle, and chordacentrum of newly hatched larvae. All these data suggest distinct roles of NKA β subunits in tissues, during development and osmoregulation with β1 subunits involved in the adaptation to hyperosmotic conditions and β3 subunits to hypoosmotic environments. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of irradiation and semistarvation on rat thyrotropin beta subunit messenger ribonucleic acid, pituitary thyrotropin content, and thyroid hormone levels

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

Litten, R.Z.; Carr, F.E.; Fein, H.G.

1990-01-01

The effect of radiation-induced anorexia on serum thyrotropin (TSH), pituitary TSH-{beta} mRNA, pituitary TSH content, serum thyroxine (T{sub 4}), and serum 3,5,3{prime}-triiodothyronine (T{sub 3}) was investigated using feed-matched controls. Rats received 10 Gy gamma whole-body irradiation and were examined 1-3 days postirradiation. Feed-matched and untreated controls were also studied. The average food intake of the irradiated and feed-matched groups was approximately 18% of the untreated controls. Over the three day period both the irradiated and feed-matched groups lost a significant amount of body weight. The serum T{sub 4} levels of both the irradiated and feed-matched groups were not significantly differentmore » from each other, but were significantly depressed when compared to the untreated control group. The serum TSH and T{sub 3} were, however, significantly greater in the irradiated than the feed-matched groups at day 3 posttreatment. To determine if the difference in the serum TSH level between the two groups was due to a pretranslational alteration in TSH production, we measured the TSH-{beta} mRNA using an RNA blot hybridization assay. We found that the TSH-{beta} mRNA level was the same in the irradiated and feed-matched groups, suggesting that the mechanism responsible for the radiation-induced increase in the serum TSH level is posttranscriptional. Pituitary TSH content in the irradiated rats was significantly less than in pair-fed controls, suggesting that irradiation may permit enhanced secretion of stored hormone.« less

A Transcript-Specific eIF3 Complex Mediates Global Translational Control of Energy Metabolism.

PubMed

Shah, Meera; Su, Dan; Scheliga, Judith S; Pluskal, Tomáš; Boronat, Susanna; Motamedchaboki, Khatereh; Campos, Alexandre Rosa; Qi, Feng; Hidalgo, Elena; Yanagida, Mitsuhiro; Wolf, Dieter A

2016-08-16

The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears to be conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Genetics of bacteria that utilize one carbon compounds: Final report, March 1, 1982-February 29, 1988

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

Hanson, R.S.

Broad host range plasmid vectors useful for cloning genes from bacteria that grow on methane and methanol were constructed. We have cloned and mapped nineteen genes required for the growth of Methylobacterium organophilum strain XX on methanol. Nineteen genes were found in seven linkage groups on the M. organophilum genome and were separated by 40 kb or more. Eleven genes were required for the synthesis of methanol dehydrogenase (MDH) and were located in three unlinked gene clusters. The MDH structural gene was localized on a 2.5 kb DNA fragment. The gene was sequenced and contains a 175 bp untranslated leadermore » sequence, a signal sequence and the structural gene. MDH messenger RNA (mRNA) has a half life of approximately 20 min. and is present at approximately 2% of the cellular mRNA. The structural gene for the ..gamma.. subunit of methane monoxygenases has been cloned from Methylosporovibrio. Methane monooxygenase subunits have been purified by Prof. J. Lipscomb's laboratory and are being sequenced to construct DNA probes to identify cloned subunit genes. New facultative methylotrophic bacteria were isolated and characterized. Several amino acid auxotrophs have been isolated. 11 refs.« less

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

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

Soeda, Junpei; Morgan, Maelle; McKee, Chad

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells inmore » the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. {alpha}1 and {alpha}3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers' blood is pro-fibrogenic, through actions on hHSCs expressed nAChRs. Therefore, CS, via its nicotine content, may worsen liver fibrosis. Moreover, nicotinic receptor antagonists may have utility as novel anti-fibrotic agents.« less

Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation.

PubMed

Wasserman, Michael R; Alejo, Jose L; Altman, Roger B; Blanchard, Scott C

2016-04-01

Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

Fragile X Mental Retardation Protein Regulates the Levels of Scaffold Proteins and Glutamate Receptors in Postsynaptic Densities*

PubMed Central

Schütt, Janin; Falley, Katrin; Richter, Dietmar; Kreienkamp, Hans-Jürgen; Kindler, Stefan

2009-01-01

Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1–3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3′-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients. PMID:19640847

Excess Testosterone Exposure Alters Hypothalamic-Pituitary-Testicular Axis Dynamics and Gene Expression in Sheep Fetuses

PubMed Central

Amodei, Rebecka; Gribbin, Kyle P.; Corder, Keely; Stormshak, Fred; Estill, Charles T.

2016-01-01

Prenatal exposure to excess androgen may result in impaired adult fertility in a variety of mammalian species. However, little is known about what feedback mechanisms regulate gonadotropin secretion during early gestation and how they respond to excess T exposure. The objective of this study was to determine the effect of exogenous exposure to T on key genes that regulate gonadotropin and GnRH secretion in fetal male lambs as compared with female cohorts. We found that biweekly maternal testosterone propionate (100 mg) treatment administered from day 30 to day 58 of gestation acutely decreased (P < .05) serum LH concentrations and reduced the expression of gonadotropin subunit mRNA in both sexes and the levels of GnRH receptor mRNA in males. These results are consistent with enhanced negative feedback at the level of the pituitary and were accompanied by reduced mRNA levels for testicular steroidogenic enzymes, suggesting that Leydig cell function was also suppressed. The expression of kisspeptin 1 mRNA, a key regulator of GnRH neurons, was significantly greater (P < .01) in control females than in males and reduced (P < .001) in females by T exposure, indicating that hypothalamic regulation of gonadotropin secretion was also affected by androgen exposure. Although endocrine homeostasis was reestablished 2 weeks after maternal testosterone propionate treatment ceased, additional differences in the gene expression of GnRH, estrogen receptor-β, and kisspeptin receptor (G protein coupled receptor 54) emerged between the treatment cohorts. These changes suggest the normal trajectory of hypothalamic-pituitary axis development was disrupted, which may, in turn, contribute to negative effects on fertility later in life. PMID:27673555

Chronic lithium treatment up-regulates cell surface Na(V)1.7 sodium channels via inhibition of glycogen synthase kinase-3 in adrenal chromaffin cells: enhancement of Na(+) influx, Ca(2+) influx and catecholamine secretion after lithium withdrawal.

PubMed

Yanagita, Toshihiko; Maruta, Toyoaki; Nemoto, Takayuki; Uezono, Yasuhito; Matsuo, Kiyotaka; Satoh, Shinya; Yoshikawa, Norie; Kanai, Tasuku; Kobayashi, Hideyuki; Wada, Akihiko

2009-09-01

In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, we have previously reported that lithium chloride (LiCl) inhibits function of Na(+) channels independent of glycogen synthase kinase-3 (GSK-3) (Yanagita et al., 2007). Here, we further examined the effects of chronic lithium treatment on Na(+) channels. LiCl treatment (1-30 mM, > or = 12 h) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by approximately 32% without altering the affinity of [(3)H]STX binding. This increase was prevented by cycloheximide and actinomycin D. SB216763 and SB415286 (GSK-3 inhibitors) also increased cell surface [(3)H]STX binding by approximately 31%. Simultaneous treatment with LiCl and SB216763 or SB415286 did not produce an increased effect on [(3)H]STX binding compared with either treatment alone. LiCl increased Na(+) channel alpha-subunit mRNA level by 32% at 24 h. LiCl accelerated alpha-subunit gene transcription by 35% without altering alpha-subunit mRNA stability. In LiCl-treated cells, LiCl inhibited veratridine-induced (22)Na(+) influx as in untreated cells. However, washout of LiCl after chronic treatment enhanced veratridine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion by approximately 30%. Washout of LiCl after 24 h treatment shifted concentration-response curve of veratridine upon (22)Na(+) influx upward, without altering its EC(50) value. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced (22)Na(+) influx by two-fold in untreated and LiCl-treated cells. Whole-cell patch-clamp analysis indicated that I-V curve and steady-state inactivation/activation curves were comparable between untreated and LiCl-treated cells. Thus, GSK-3 inhibition by LiCl up-regulated cell surface Na(V)1.7 via acceleration of alpha-subunit gene transcription, enhancing veratridine-induced Na(+) influx, Ca(2+) influx and catecholamine secretion.

Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

PubMed Central

Hinnebusch, Alan G.

2011-01-01

Summary: The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5′ end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNAiMet) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an “open” conformation of the 40S subunit with Met-tRNAiMet bound in a low-affinity state conducive to scanning and in triggering rearrangement into a “closed” conformation incompatible with scanning, which features Met-tRNAiMet more tightly bound to the “P” site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded “landing pad” for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site. PMID:21885680

Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

PubMed Central

Occhipinti, Laura; Chang, Yiming; Altvater, Martin; Menet, Anna M.; Kemmler, Stefan; Panse, Vikram G.

2013-01-01

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel. PMID:23907389

The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1

PubMed Central

Drapier, Dominique; Suzuki, Hideki; Levy, Haim; Rimbault, Blandine; Kindle, Karen L.; Stern, David B.; Wollman, Francis-André

1998-01-01

Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the α-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-α can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter. PMID:9625716

Expression and regulation of glucocorticoid-induced leucine zipper in the developing anterior pituitary gland.

PubMed

Ellestad, Laura E; Malkiewicz, Stefanie A; Guthrie, H David; Welch, Glenn R; Porter, Tom E

2009-02-01

The expression profile of glucocorticoid-induced leucine zipper (GILZ) in the anterior pituitary during the second half of embryonic development in the chick is consistent with in vivo regulation by circulating corticosteroids. However, nothing else has been reported about the presence of GILZ in the neuroendocrine system. We sought to characterize expression and regulation of GILZ in the chicken embryonic pituitary gland and determine the effect of GILZ overexpression on anterior pituitary hormone levels. Pituitary GILZ mRNA levels increased during embryogenesis to a maximum on the day of hatch, and decreased through the first week after hatch. GILZ expression was rapidly upregulated by corticosterone in embryonic pituitary cells. To determine whether GILZ regulates hormone gene expression in the developing anterior pituitary, we overexpressed GILZ in embryonic pituitary cells and measured mRNA for the major pituitary hormones. Exogenous GILZ increased prolactin mRNA above basal levels, but not as high as that in corticosterone-treated cells, indicating that GILZ may play a small role in lactotroph differentiation. The largest effect we observed was a twofold increase in FSH beta subunit in cells transfected with GILZ but not treated with corticosterone, suggesting that GILZ may positively regulate gonadotroph development in a manner not involving glucocorticoids. In conclusion, this is the first report to characterize avian GILZ and examine its regulation in the developing neuroendocrine system. We have shown that GILZ is upregulated by glucocorticoids in the embryonic pituitary gland and may regulate expression of several pituitary hormones.

The Amelioration of N-Acetyl-p-Benzoquinone Imine Toxicity by Ginsenoside Rg3: The Role of Nrf2-Mediated Detoxification and Mrp1/Mrp3 Transports

PubMed Central

Gum, Sang Il; Cho, Min Kyung

2013-01-01

Previously, we found that Korean red ginseng suppressed acetaminophen (APAP)-induced hepatotoxicity via alteration of its metabolic profile involving GSTA2 induction and that ginsenoside Rg3 was a major component of this gene induction. In the present study, therefore, we assessed the protective effect of Rg3 against N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolic intermediate of APAP. Excess NAPQI resulted in GSH depletion with increases in the ALT and AST activities in H4IIE cells. Rg3 pretreatment reversed GSH depletion by NAPQI. Rg3 resulted in increased mRNA levels of the catalytic and modulatory subunit of glutamate cysteine ligase (GCL), the rate-limiting steps in GSH synthesis and subsequently increased GSH content. Rg3 increased levels of nuclear Nrf2, an essential transcriptional factor of these genes. The knockdown or knockout of the Nrf2 gene abrogated the inductions of mRNA and protein by Rg3. Abolishment of the reversal of GSH depletion by Rg3 against NAPQI was observed in Nrf2-deficient cells. Rg3 induced multidrug resistance-associated protein (Mrp) 1 and Mrp3 mRNA levels, but not in Nrf2-deficient cells. Taken together, these results demonstrate that Rg3 is efficacious in protecting hepatocytes against NAPQI insult, due to GSH repletion and coordinated gene regulations of GSH synthesis and Mrp family genes by Nrf2. PMID:23766864

Dizocilpine (MK-801) induces distinct changes of N-methyl-D-aspartic acid receptor subunits in parvalbumin-containing interneurons in young adult rat prefrontal cortex.

PubMed

Xi, Dong; Zhang, Wentong; Wang, Huai-Xing; Stradtman, George G; Gao, Wen-Jun

2009-11-01

N-methyl-D-aspartic acid receptor (NMDAR) hypofunction has long been implicated in schizophrenia and NMDARs on gamma-aminobutyric acid (GABA)ergic interneurons are proposed to play an essential role in the pathogenesis. However, controversial results have been reported regarding the regulation of NMDAR expression, and direct evidence of how NMDAR antagonists act on specific subpopulations of prefrontal interneurons is missing. We investigated the effects of the NMDAR antagonist dizocilpine (MK-801) on the expression of NMDAR subtypes in the identified interneurons in young adult rat prefrontal cortex (PFC) by using laser microdissection and real-time polymerase chain reaction, combined with Western blotting and immunofluorescent staining. We found that MK-801 induced distinct changes of NMDAR subunits in the parvalbumin-immunoreactive (PV-ir) interneurons vs. pyramidal neurons in the PFC circuitry. The messenger RNA (mRNA) expression of all NMDAR subtypes, including NR1 and NR2A to 2D, exhibited inverted-U dose-dependent changes in response to MK-801 treatment in the PFC. In contrast, subunit mRNAs of NMDARs in PV-ir interneurons were significantly down-regulated at low doses, unaltered at medium doses, and significantly decreased again at high doses, suggesting a biphasic dose response to MK-801. The differential effects of MK-801 in mRNA expression of NMDAR subunits were consistent with the protein expression of NR2A and NR2B subunits revealed with Western blotting and double immunofluorescent staining. These results suggest that PV-containing interneurons in the PFC exhibit a distinct responsiveness to NMDAR antagonism and that NMDA antagonist can differentially and dose-dependently regulate the functions of pyramidal neurons and GABAergic interneurons in the prefrontal cortical circuitry.

Growth Factor-Induced Mobilization of Cardiac Progenitor Cells Reduces the Risk of Arrhythmias, in a Rat Model of Chronic Myocardial Infarction

PubMed Central

Graiani, Gallia; Rossi, Stefano; Agnetti, Aldo; Stillitano, Francesca; Lagrasta, Costanza; Baruffi, Silvana; Berni, Roberta; Frati, Caterina; Vassalle, Mario; Squarcia, Umberto; Cerbai, Elisabetta; Macchi, Emilio; Stilli, Donatella; Quaini, Federico; Musso, Ezio

2011-01-01

Heart repair by stem cell treatment may involve life-threatening arrhythmias. Cardiac progenitor cells (CPCs) appear best suited for reconstituting lost myocardium without posing arrhythmic risks, being commissioned towards cardiac phenotype. In this study we tested the hypothesis that mobilization of CPCs through locally delivered Hepatocyte Growth Factor and Insulin-Like Growth Factor-1 to heal chronic myocardial infarction (MI), lowers the proneness to arrhythmias. We used 133 adult male Wistar rats either with one-month old MI and treated with growth factors (GFs, n = 60) or vehicle (V, n = 55), or sham operated (n = 18). In selected groups of animals, prior to and two weeks after GF/V delivery, we evaluated stress-induced ventricular arrhythmias by telemetry-ECG, cardiac mechanics by echocardiography, and ventricular excitability, conduction velocity and refractoriness by epicardial multiple-lead recording. Invasive hemodynamic measurements were performed before sacrifice and eventually the hearts were subjected to anatomical, morphometric, immunohistochemical, and molecular biology analyses. When compared with untreated MI, GFs decreased stress-induced arrhythmias and concurrently prolonged the effective refractory period (ERP) without affecting neither the duration of ventricular repolarization, as suggested by measurements of QTc interval and mRNA levels for K-channel α-subunits Kv4.2 and Kv4.3, nor the dispersion of refractoriness. Further, markers of cardiomyocyte reactive hypertrophy, including mRNA levels for K-channel α-subunit Kv1.4 and β-subunit KChIP2, interstitial fibrosis and negative structural remodeling were significantly reduced in peri-infarcted/remote ventricular myocardium. Finally, analyses of BrdU incorporation and distribution of connexin43 and N-cadherin indicated that cytokines generated new vessels and electromechanically-connected myocytes and abolished the correlation of infarct size with deterioration of mechanical function. In conclusion, local injection of GFs ameliorates electromechanical competence in chronic MI. Reduced arrhythmogenesis is attributable to prolongation of ERP resulting from improved intercellular coupling via increased expression of connexin43, and attenuation of unfavorable remodeling. PMID:21445273

Down-regulation of activity and expression of three transport-related proteins in the gills of the euryhaline green crab, Carcinus maenas, in response to high salinity acclimation.

PubMed

Jillette, Nathaniel; Cammack, Lauren; Lowenstein, Margaret; Henry, Raymond P

2011-02-01

The euryhaline green crab, Carcinus maenas, undergoes an annual cycle of salinity exposure, having to adapt to low salinity during its annual spring migration into estuaries, and then having to re-adapt to high salinity when it moves off-shore at the end of summer. Most studies have focused on low salinity acclimation, the activation of osmoregulatory mechanisms, and the induction of transport protein and transport-related enzyme activity and gene expression. In this study we followed the changes in hemolymph osmolality, carbonic anhydrase activity, and mRNA expression of three proteins through a complete cycle of low (15 ppt) and high (32 ppt) salinity acclimation. One week of low salinity acclimation resulted in hemolymph osmoregulation and a four-fold induction of branchial carbonic anhydrase activity. Relative mRNA expression increased for two CA isoforms (CAc 100-fold, and CAg 7-fold) and the α-subunit of the Na/K-ATPase (8-fold). Upon re-exposure to high salinity, hemolymph osmolality increased to 32 ppt acclimated levels by 6 h, and mRNA levels returned to high salinity, baseline levels within 1 week. However, CA activity remained unchanged in response to high salinity exposure for the first week and then gradually declined to baseline levels over 4 weeks. The relative timing of these changes suggests that while whole-organism physiological adaptations and regulation at the gene level can be very rapid, changes at the level of protein expression and turnover are much slower. It is possible that the high metabolic cost of protein synthesis and/or processing could be the underlying reason for long biological life spans of physiologically important proteins. Published by Elsevier Inc.

MC EMiNEM maps the interaction landscape of the Mediator.

PubMed

Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C; Martin, Dietmar E; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

2012-01-01

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

PubMed Central

Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

2012-01-01

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

PubMed

Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

2014-06-01

The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster. Copyright © 2014 Elsevier Ltd. All rights reserved.

Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate

PubMed Central

2012-01-01

Background During neocortical development, multiple voltage- and ligand-gated ion channels are differentially expressed in neurons thereby shaping their intrinsic electrical properties. One of these voltage-gated ion channels, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel and its current Ih, is an important regulator of neuronal excitability. Thus far, studies on an early Ih appearance in rodent neocortex are missing or conflicting. Therefore, we focused our study on perinatal neocortical Ih and its properties. Results In the perinatal rat neocortex we observed a rapid increase in the number of neurons exhibiting Ih. Perinatal Ih had unique properties: first, a pronounced cAMP sensitivity resulting in a marked shift of the voltage sufficient for half-maximum activation of the current towards depolarized voltages and second, an up to 10 times slower deactivation at physiological membrane potentials when compared to the one at postnatal day 30. The combination of these features was sufficient to suppress membrane resonance in our in silico and in vitro experiments. Although all four HCN subunits were present on the mRNA level we only detected HCN4, HCN3 and HCN1 on the protein level at P0. HCN1 protein at P0, however, appeared incompletely processed. At P30 glycosilated HCN1 and HCN2 dominated. By in silico simulations and heterologous co-expression experiments of a ‘slow’ and a ‘fast’ Ih conducting HCN channel subunit in HEK293 cells, we mimicked most characteristics of the native current, pointing to a functional combination of subunit homo- or heteromeres. Conclusion Taken together, these data indicate a HCN subunit shift initiated in the first 24 hours after birth and implicate a prominent perinatal role of the phylogenetically older HCN3 and/or HCN4 subunits in the developing neocortex. PMID:22694806

Potential down-regulation of salivary gland AQP5 by LPS via cross-coupling of NF-kappaB and p-c-Jun/c-Fos.

PubMed

Yao, Chenjuan; Purwanti, Nunuk; Karabasil, Mileva Ratko; Azlina, Ahmad; Javkhlan, Purevjav; Hasegawa, Takahiro; Akamatsu, Tetsuya; Hosoi, Toru; Ozawa, Koichiro; Hosoi, Kazuo

2010-08-01

The mRNA and protein levels of aquaporin (AQP)5 in the parotid gland were found to be potentially decreased by lipopolysaccharide (LPS) in vivo in C3H/HeN mice, but only weakly in C3H/HeJ, a TLR4 mutant mouse strain. In the LPS-injected mice, pilocarpine-stimulated saliva production was reduced by more than 50%. In a tissue culture system, the LPS-induced decrease in the AQP5 mRNA level was blocked completely by pyrrolidine dithiocarbamate, MG132, tyrphostin AG126, SP600125, and partially by SB203580, which are inhibitors for IkappaB kinase, 26S proteasome, ERK1/2, JNK, and p38 MAPK, respectively. In contrast, the expression of AQP1 mRNA was down-regulated by LPS and such down-regulation was blocked only by SP600125. The transcription factors NF-kappaB (p65 subunit), p-c-Jun, and c-Fos were increased by LPS given in vivo, whereas the protein-binding activities of the parotid gland extract toward the sequences for NF-kappaB but not AP-1-responsive elements present at the promoter region of the AQP5 gene were increased by LPS injection. Co-immunoprecipitation by using antibody columns suggested the physical association of the three transcription factors. These results suggest that LPS-induced potential down-regulation of expression of AQP5 mRNA in the parotid gland is mediated via a complex(es) of these two classes of transcription factors, NF-kappaB and p-c-Jun/c-Fos.

Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

NASA Astrophysics Data System (ADS)

Shao, Zhanru; Liu, Fuli; Li, Qiuying; Yao, Jianting; Duan, Delin

2014-03-01

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica ( SJ-rbc). It contained an open reading frame for a large subunit gene ( SJ — rbcL) of 1 467 bp, a small subunit gene ( SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl- β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

Momordica charantia Inhibits Inflammatory Responses in Murine Macrophages via Suppression of TAK1.

PubMed

Yang, Woo Seok; Yang, Eunju; Kim, Min-Jeong; Jeong, Deok; Yoon, Deok Hyo; Sung, Gi-Ho; Lee, Seungihm; Yoo, Byong Chul; Yeo, Seung-Gu; Cho, Jae Youl

2018-01-01

Momordica charantia known as bitter melon is a representative medicinal plant reported to exhibit numerous pharmacological activities such as antibacterial, antidiabetic, anti-inflammatory, anti-oxidant, antitumor, and hypoglycemic actions. Although this plant has high ethnopharmacological value for treating inflammatory diseases, the molecular mechanisms by which it inhibits the inflammatory response are not fully understood. In this study, we aim to identify the anti-inflammatory mechanism of this plant. To this end, we studied the effects of its methanol extract (Mc-ME) on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Specifically, we evaluated nitric oxide (NO) production, mRNA expression of inflammatory genes, luciferase reporter gene activity, and putative molecular targets. Mc-ME blocked NO production in a dose-dependent manner in RAW264.7 cells; importantly, no cytotoxicity was observed. Moreover, the mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were decreased by Mc-ME treatment in a dose-dependent manner. Luciferase assays and nuclear lysate immunoblotting analyses strongly indicated that Mc-ME decreases the levels of p65 [a nuclear factor (NF)-[Formula: see text]B subunit] and c-Fos [an activator protein (AP)-1 subunit]. Whole lysate immunoblotting assays, luciferase assays, and overexpression experiments suggested that transforming growth factor [Formula: see text]-activated kinase 1 (TAK1) is targeted by Mc-ME, thereby suppressing NF-[Formula: see text]B and AP-1 activity via downregulation of extracellular signal-regulated kinases (ERKs) and AKT. These results strongly suggest that Mc-ME exerts its anti-inflammatory activity by reducing the action of TAK1, which also affects the activation of NF-[Formula: see text]B and AP-1.

The Duration of Nicotine Withdrawal-Associated Deficits in Contextual Fear Conditioning Parallels Changes in Hippocampal High Affinity Nicotinic Acetylcholine Receptor Upregulation

PubMed Central

Gould, Thomas J.; Portugal, George S.; André, Jessica M.; Tadman, Matthew P.; Marks, Michael J.; Kenney, Justin W.; Yildirim, Emre; Adoff, Michael

2012-01-01

A predominant symptom of nicotine withdrawal is cognitive deficits, yet understanding of the neural basis for these deficits is limited. Withdrawal from chronic nicotine disrupts contextual learning in mice and this deficit is mediated by direct effects of nicotine in the hippocampus. Chronic nicotine treatment upregulates nicotinic acetylcholine receptors (nAChR); however, it is unknown whether upregulation is related to the observed withdawal-induced cognitive deficits. If a relationship between altered learning and nAChR levels exists, changes in nAChR levels after cessation of nicotine treatment should match the duration of learning deficits. To test this hypothesis, mice were chronically administered 6.3 mg/kg/day (freebase) nicotine for 12 days and trained in contextual fear conditioning on day 11 or between 1 to 16 days after withdrawal of treatment. Changes in [125I]-epibatidine binding at cytisine-sensitive and cytisine-resistant nAChRs and chronic nicotine-related changes in α4, α7, and β2 nAChR subunit mRNA expression were assessed. Chronic nicotine had no behavioral effect but withdrawal produced deficits in contextual fear conditioning that lasted 4 days. Nicotine withdrawal did not disrupt cued fear conditioning. Chronic nicotine upregulated hippocampal cytisine-sensitive nAChR binding; upregulation continued after cessation of nicotine administration and the duration of upregulation during withdrawal paralleled the duration of behavioral changes. Changes in binding in cortex and cerebellum did not match behavioral changes. No changes in α4, α7, and β2 subunit mRNA expression were seen with chronic nicotine. Thus, nicotine withdrawal-related deficits in contextual learning are time-limited changes that are associated with temporal changes in upregulation of high-affinity nAChR binding. PMID:22285742

The ClC-3 chloride channel and osmoregulation in the European sea bass, Dicentrarchus labrax.

PubMed

Bossus, Maryline; Charmantier, Guy; Blondeau-Bidet, Eva; Valletta, Bianca; Boulo, Viviane; Lorin-Nebel, Catherine

2013-07-01

Dicentrarchus labrax migrates between sea (SW), brackish and fresh water (FW) where chloride concentrations and requirements for chloride handling change: in FW, fish absorb chloride and restrict renal losses; in SW, they excrete chloride. In this study, the expression and localization of ClC-3 and Na(+)/K(+)-ATPase (NKA) were studied in fish adapted to SW, or exposed to FW from 10 min to 30 days. In gills, NKA-α1 subunit expression transiently increased from 10 min and reached a stabilized intermediate expression level after 24 h in FW. ClC-3 co-localized with NKA in the basolateral membrane of mitochondria-rich cells (MRCs) at all conditions. The intensity of MRC ClC-3 immunostaining was significantly higher (by 50 %) 1 h after the transfer to FW, whereas the branchial ClC-3 protein expression was 30 % higher 7 days after the transfer as compared to SW. This is consistent with the increased number of immunopositive MRCs (immunostained for NKA and ClC-3). However, the ClC-3 mRNA expression was significantly lower in FW gills. In the kidney, after FW transfer, a transient decrease in NKA-α1 subunit expression was followed by significantly higher stable levels from 24 h. The low ClC-3 protein expression detected at both salinities was not observed by immunocytochemistry in the SW kidney; ClC-3 was localized in the basal membrane of the collecting ducts and tubules 7 and 30 days after transfer to FW. Renal ClC-3 mRNA expression, however, seemed higher in SW than in FW. The potential role of this chloride channel ClC-3 in osmoregulatory and osmosensing mechanisms is discussed.

Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon.

PubMed

Bourchookarn, Apichai; Havanapan, Phattara-Orn; Thongboonkerd, Visith; Krittanai, Chartchai

2008-03-01

A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI-TOF MS and nanoLC-ESI-MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.

Different Hypothalamic Nicotinic α7 Receptor Expression and Response to Low Nicotine Dose in Alcohol-Preferring and Alcohol-Avoiding Rats.

PubMed

Nuutinen, Saara; Panula, Pertti; Salminen, Outi

2016-02-01

The aim of this study was to examine possible differences in nicotinic acetylcholine receptors and responses in rats with genetic preference or avoidance for alcohol. This was done by using 2 rat lines with high alcohol preference (Alko Alcohol [AA]) or alcohol avoidance (Alko Non-Alcohol [ANA]). Locomotor activity was measured following nicotine and histamine H3 receptor (H3R) antagonist treatment. In situ hybridization and receptor ligand binding experiments were used in drug-naïve animals to examine the expression of different α nicotinic receptor subunits. The AA rats were found to be more sensitive to the stimulatory effect of a low dose of nicotine than ANA rats, which were not significantly activated. Combination of histamine H3R antagonist, JNJ-39220675, and nicotine resulted to similar locomotor activation as nicotine alone. To further understand the mechanism underlying the difference in nicotine response in AA and ANA rats, we studied the expression of α5, α6, and α7 nicotinic receptor subunits in specific brain areas of AA and ANA rats. We found no differences in the expression of α5 nicotinic receptor subunits in the medial habenula and hippocampus or in α6 subunit in the ventral tegmental area and substantia nigra. However, the level of α7 nicotinic receptor subunit mRNA was significantly lower in the tuberomamillary nucleus of posterior hypothalamus of alcohol-preferring AA rats than in alcohol-avoiding ANA rats. Also the hypothalamic [125I-α-bungarotoxin binding was lower in AA rats indicating lower levels of α7 nicotinic receptors. The lower expression and receptor binding of α7 nicotinic receptors in the tuberomamillary nucleus of AA rats suggest a difference in the regulation of brain histamine neurons between the rat lines since the α7 nicotinic receptors are located in histaminergic neurons. Stronger nicotine-induced locomotor response, mediated partially via α7 receptors, and previously described high alcohol consumption in AA rats could be explained by the found difference in tuberomamillary α7 receptor levels. Copyright © 2016 by the Research Society on Alcoholism.

Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP).

PubMed

Lin, Guiting; Fandel, Thomas M; Shindel, Alan W; Wang, Guifang; Banie, Lia; Ning, Hongxiu; Lue, Tom F; Lin, Ching-Shwun

2011-07-01

To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra. Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of β-actin. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity. MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P = 0.09]. MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P = 0.001]. Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to β-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra. In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone. © 2010 THE AUTHORS. BJU INTERNATIONAL © 2010 BJU INTERNATIONAL.

The absence of genes for cytochrome c oxidase and reductase subunits in maxicircle kinetoplast DNA of the respiration-deficient plant trypanosomatid Phytomonas serpens.

PubMed

Nawathean, P; Maslov, D A

2000-08-01

By completing the sequencing of the maxicircle conserved region in the kinetoplast DNA of Phytomonas serpens, we showed that the genes for subunits I and II (COI and COII) of cytochrome c oxidase in this organism were missing. We had previously shown that the genes for cytochrome c oxidase subunit III and apocytochrome b were also missing. These deletions did not affect the structure or expression of the remaining genes. Partial editing of the mRNA for NADH dehydrogenase subunit 8, previously found in strain IG from insects, was complete in two other strains isolated from plants. The appearance of a novel maxicircle gene for MURF2 block I gRNA, which substitutes for the gene missing due to the COII gene deletion, may illustrate a general mechanism for the origin of gRNAs.

Altered Expression of Human Smooth Muscle Myosin Phosphatase Targeting (MYPT) Isovariants with Pregnancy and Labor.

PubMed

Lartey, Jon; Taggart, Julie; Robson, Stephen; Taggart, Michael

2016-01-01

Myosin light-chain phosphatase is a trimeric protein that hydrolyses phosphorylated myosin II light chains (MYLII) to cause relaxation in smooth muscle cells including those of the uterus. A major component of the phosphatase is the myosin targeting subunit (MYPT), which directs a catalytic subunit to dephosphorylate MYLII. There are 5 main MYPT family members (MYPT1 (PPP1R12A), MYPT2 (PPP1R12B), MYPT3 (PPP1R16A), myosin binding subunit 85 MBS85 (PPP1R12C) and TIMAP (TGF-beta-inhibited membrane-associated protein (PPP1R16B)). Nitric oxide (NO)-mediated smooth muscle relaxation has in part been attributed to activation of the phosphatase by PKG binding to a leucine zipper (LZ) dimerization domain located at the carboxyl-terminus of PPP1R12A. In animal studies, alternative splicing of PPP1R12A can lead to the inclusion of a 31-nucleotide exonic segment that generates a LZ negative (LZ-) isovariant rendering the phosphatase less sensitive to NO vasodilators and alterations in PPP1R12ALZ- and LZ+ expression have been linked to phenotypic changes in smooth muscle function. Moreover, PPP1R12B and PPP1R12C, but not PPP1R16A or PPP1R16B, have the potential for LZ+/LZ- alternative splicing. Yet, by comparison to animal studies, the information on human MYPT genomic sequences/mRNA expressions is scant. As uterine smooth muscle undergoes substantial remodeling during pregnancy we were interested in establishing the patterns of expression of human MYPT isovariants during this process and also following labor onset as this could have important implications for determining successful pregnancy outcome. We used cross-species genome alignment, to infer putative human sequences not available in the public domain, and isovariant-specific quantitative PCR, to analyse the expression of mRNA encoding putative LZ+ and LZ- forms of PPP1R12A, PPP1R12B and PPP1R12C as well as canonical PPP1R16A and PPP1R16B genes in human uterine smooth muscle from non-pregnant, pregnant and in-labor donors. We found a reduction in the expression of PPP1R12A, PPP1R12BLZ+, PPP1R16A and PPP1R16B mRNA in late pregnancy (not-in-labor) relative to non-pregnancy. PPP1R12ALZ+ and PPP1R12ALZ- mRNA levels were similar in the non-pregnant and pregnant not in labor groups. There was a further reduction in the uterine expression of PPP1R12ALZ+, PPP1R12CLZ+ and PPP1R12ALZ- mRNA with labor relative to the pregnant not-in-labor group. PPP1R12A, PPP1R12BLZ+, PPP1R16A and PPP1R16B mRNA levels were invariant between the not in labor and in-labor groups. MYPT proteins are crucial determinants of smooth muscle function. Therefore, these alterations in human uterine smooth muscle MYPT isovariant expression during pregnancy and labor may be part of the important molecular physiological transition between uterine quiescence and activation.

Extraribosomal L13a Is a Specific Innate Immune Factor for Antiviral Defense

PubMed Central

Poddar, Darshana; Basu, Abhijit; Kour, Ravinder; Verbovetskaya, Valentina

2014-01-01

ABSTRACT We report a novel extraribosomal innate immune function of mammalian ribosomal protein L13a, whereby it acts as an antiviral agent. We found that L13a is released from the 60S ribosomal subunit in response to infection by respiratory syncytial virus (RSV), an RNA virus of the Pneumovirus genus and a serious lung pathogen. Unexpectedly, the growth of RSV was highly enhanced in L13a-knocked-down cells of various lineages as well as in L13a knockout macrophages from mice. In all L13a-deficient cells tested, translation of RSV matrix (M) protein was specifically stimulated, as judged by a greater abundance of M protein and greater association of the M mRNA with polyribosomes, while general translation was unaffected. In silico RNA folding analysis and translational reporter assays revealed a putative hairpin in the 3′untranslated region (UTR) of M mRNA with significant structural similarity to the cellular GAIT (gamma-activated inhibitor of translation) RNA hairpin, previously shown to be responsible for assembling a large, L13a-containing ribonucleoprotein complex that promoted translational silencing in gamma interferon (IFN-γ)-activated myeloid cells. However, RNA-protein interaction studies revealed that this complex, which we named VAIT (respiratory syncytial virus-activated inhibitor of translation) is functionally different from the GAIT complex. VAIT is the first report of an extraribosomal L13a-mediated, IFN-γ-independent innate antiviral complex triggered in response to virus infection. We provide a model in which the VAIT complex strongly hinders RSV replication by inhibiting the translation of the rate-limiting viral M protein, which is a new paradigm in antiviral defense. IMPORTANCE The innate immune mechanisms of host cells are diverse in nature and act as a broad-spectrum cellular defense against viruses. Here, we report a novel innate immune mechanism functioning against respiratory syncytial virus (RSV), in which the cellular ribosomal protein L13a is released from the large ribosomal subunit soon after infection and inhibits the translation of a specific viral mRNA, namely, that of the matrix protein M. Regarding its mechanism, we show that the recognition of a specific secondary structure in the 3′ untranslated region of the M mRNA leads to translational arrest of the mRNA. We also show that the level of M protein in the infected cell is rate limiting for viral morphogenesis, providing a rationale for L13a to target the M mRNA for suppression of RSV growth. Translational silencing of a viral mRNA by a deployed ribosomal protein is a new paradigm in innate immunity. PMID:24899178

Extraribosomal l13a is a specific innate immune factor for antiviral defense.

PubMed

Mazumder, Barsanjit; Poddar, Darshana; Basu, Abhijit; Kour, Ravinder; Verbovetskaya, Valentina; Barik, Sailen

2014-08-01

We report a novel extraribosomal innate immune function of mammalian ribosomal protein L13a, whereby it acts as an antiviral agent. We found that L13a is released from the 60S ribosomal subunit in response to infection by respiratory syncytial virus (RSV), an RNA virus of the Pneumovirus genus and a serious lung pathogen. Unexpectedly, the growth of RSV was highly enhanced in L13a-knocked-down cells of various lineages as well as in L13a knockout macrophages from mice. In all L13a-deficient cells tested, translation of RSV matrix (M) protein was specifically stimulated, as judged by a greater abundance of M protein and greater association of the M mRNA with polyribosomes, while general translation was unaffected. In silico RNA folding analysis and translational reporter assays revealed a putative hairpin in the 3'untranslated region (UTR) of M mRNA with significant structural similarity to the cellular GAIT (gamma-activated inhibitor of translation) RNA hairpin, previously shown to be responsible for assembling a large, L13a-containing ribonucleoprotein complex that promoted translational silencing in gamma interferon (IFN-γ)-activated myeloid cells. However, RNA-protein interaction studies revealed that this complex, which we named VAIT (respiratory syncytial virus-activated inhibitor of translation) is functionally different from the GAIT complex. VAIT is the first report of an extraribosomal L13a-mediated, IFN-γ-independent innate antiviral complex triggered in response to virus infection. We provide a model in which the VAIT complex strongly hinders RSV replication by inhibiting the translation of the rate-limiting viral M protein, which is a new paradigm in antiviral defense. The innate immune mechanisms of host cells are diverse in nature and act as a broad-spectrum cellular defense against viruses. Here, we report a novel innate immune mechanism functioning against respiratory syncytial virus (RSV), in which the cellular ribosomal protein L13a is released from the large ribosomal subunit soon after infection and inhibits the translation of a specific viral mRNA, namely, that of the matrix protein M. Regarding its mechanism, we show that the recognition of a specific secondary structure in the 3' untranslated region of the M mRNA leads to translational arrest of the mRNA. We also show that the level of M protein in the infected cell is rate limiting for viral morphogenesis, providing a rationale for L13a to target the M mRNA for suppression of RSV growth. Translational silencing of a viral mRNA by a deployed ribosomal protein is a new paradigm in innate immunity. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

PubMed

Pasion, S G; Brown, G W; Brown, L M; Ray, D S

1994-12-01

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis.

PubMed

Wilkins, Heather M; Harris, Janna L; Carl, Steven M; E, Lezi; Lu, Jianghua; Eva Selfridge, J; Roy, Nairita; Hutfles, Lewis; Koppel, Scott; Morris, Jill; Burns, Jeffrey M; Michaelis, Mary L; Michaelis, Elias K; Brooks, William M; Swerdlow, Russell H

2014-12-15

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Deregulation of DNA-dependent protein kinase catalytic subunit contributes to human hepatocarcinogenesis development and has a putative prognostic value.

PubMed

Evert, M; Frau, M; Tomasi, M L; Latte, G; Simile, M M; Seddaiu, M A; Zimmermann, A; Ladu, S; Staniscia, T; Brozzetti, S; Solinas, G; Dombrowski, F; Feo, F; Pascale, R M; Calvisi, D F

2013-11-12

The DNA-repair gene DNA-dependent kinase catalytic subunit (DNA-PKcs) favours or inhibits carcinogenesis, depending on the cancer type. Its role in human hepatocellular carcinoma (HCC) is unknown. DNA-dependent protein kinase catalytic subunit, H2A histone family member X (H2AFX) and heat shock transcription factor-1 (HSF1) levels were assessed by immunohistochemistry and/or immunoblotting and qRT-PCR in a collection of human HCC. Rates of proliferation, apoptosis, microvessel density and genomic instability were also determined. Heat shock factor-1 cDNA or DNA-PKcs-specific siRNA were used to explore the role of both genes in HCC. Activator protein 1 (AP-1) binding to DNA-PKcs promoter was evaluated by chromatin immunoprecipitation. Kaplan-Meier curves and multivariate Cox model were used to study the impact on clinical outcome. Total and phosphorylated DNA-PKcs and H2AFX were upregulated in HCC. Activated DNA-PKcs positively correlated with HCC proliferation, genomic instability and microvessel density, and negatively with apoptosis and patient's survival. Proliferation decline and massive apoptosis followed DNA-PKcs silencing in HCC cell lines. Total and phosphorylated HSF1 protein, mRNA and activity were upregulated in HCC. Mechanistically, we demonstrated that HSF1 induces DNA-PKcs upregulation through the activation of the MAPK/JNK/AP-1 axis. DNA-dependent protein kinase catalytic subunit transduces HSF1 effects in HCC cells, and might represent a novel target and prognostic factor in human HCC.

Vacuolar H+-ATPase Is Expressed in Response to Gibberellin during Tomato Seed Germination1

PubMed Central

Cooley, Michael B.; Yang, Hong; Dahal, Peetambar; Mella, R. Alejandra; Downie, A. Bruce; Haigh, Anthony M.; Bradford, Kent J.

1999-01-01

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA4+7. Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V0 membrane sector of vacuolar H+-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V1 sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds. PMID:10594121

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation

NASA Technical Reports Server (NTRS)

Jagoe, R. Thomas; Lecker, Stewart H.; Gomes, Marcelo; Goldberg, Alfred L.

2002-01-01

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli.

PubMed Central

Bae, Y M; Holmgren, E; Crawford, I P

1989-01-01

We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzyme of the tryptophan pathway. Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons. This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment. We designate the fused gene trpE(G). The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control. The location of the promoter was determined by S1 nuclease protection, using Rhizobium mRNA. Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained. One of these was a C----T change at position -9 in the -10 region. The +1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site. Images PMID:2656657

[Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon].

PubMed

Molotkov, M V; Graĭfer, D M; Popugaeva, E A; Bulygin, K N; Meshchaninova, M I; Ven'iaminova, A G; Karpova, G G

2007-01-01

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

NASA Astrophysics Data System (ADS)

Fagerquist, Clifton K.; Zaragoza, William J.

2015-05-01

We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z ~7819 and m/z ~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D ↔ 16 N and 24D ↔ 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD because both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.

Expression of a functional asialoglycoprotein receptor in human renal proximal tubular epithelial cells.

PubMed

Seow, Ying-ying T; Tan, Michelle G K; Woo, Keng Thye

2002-07-01

The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake. Copyright 2002 S. Karger AG, Basel

Enduring abolishment of remote but not recent expression of conditioned fear by the blockade of calcium-permeable AMPA receptors before extinction training.

PubMed

Zelena, Dóra; Mikics, Éva; Balázsfi, Diána; Varga, János; Klausz, Barbara; Urbán, Eszter; Sipos, Eszter; Biró, László; Miskolczi, Christina; Kovács, Krisztina; Ferenczi, Szilamér; Haller, József

2016-06-01

Calcium-permeable (GluA2 subunit-free) AMPA receptors (CP-AMPAR) play prominent roles in fear extinction; however, no blockers of these receptors were studied in tests relevant to extinction learning so far. The CP-AMPAR antagonist IEM-1460 was administered once before extinction trainings, which were started either 1 or 28 days after fear conditioning (FC). We used a mild extinction protocol that durably decreased but did not abolish conditioned fear. The messenger RNA (mRNA) expression of GluA1 and GluA2 subunits were investigated at both time points in the ventromedial prefrontal cortex (vmPFC) and amygdala. IEM-1460 transiently facilitated extinction 1 day after conditioning, but learned fear spontaneously recovered 4 weeks later. When the extinction protocol was applied 28 days after training, IEM-1460 enhanced extinction memory, moreover abolished conditioned fear for at least a month. The expression of GluA1 and GluA2 mRNAs was increased at both time points in the vmPFC. In the basolateral and central amygdala, the GluA1/GluA2 mRNA ratio increased, suggesting a shift towards the preponderance of GluA1 over GluA2 expression. AMPAR blockade lastingly enhanced the extinction of remote but not recent fear memories. Time-dependent changes in AMPA receptor subunit mRNA expression may explain the differential effects of CP-AMPAR blockade on recent and remote conditioned fear, further supporting the notion that the mechanisms maintaining learned fear change over time. Our findings suggest clinical implications for CP-AMPAR blockers, particularly for acquired anxieties (e.g., post-traumatic stress disorder) which have a slow onset and are durable.

Altered peroxisome-proliferator activated receptors expression in human endometrial cancer.

PubMed

Knapp, Paweł; Chabowski, Adrian; Błachnio-Zabielska, Agnieszka; Jarząbek, Katarzyna; Wołczyński, Sławomir

2012-01-01

Peroxisome proliferator-activated receptors (PPARs) belong to a family of nuclear hormone receptors acting as transcriptional factors, recently involved also in carcinogenesis. Present study was undertaken to evaluate the presence and subcellular localization of different PPAR isoforms (α, β, γ) in healthy endometrial tissue (n = 10) and endometrial carcinoma (FIGO I, endometrioides type, G1, n = 35). We sought to analyze PPARs mRNA content as well as protein immunohistochemical expression that was further quantified by Western Blot technique. For both PPARα and PPARβ, protein expression was significantly higher in endometrial cancers compared to normal endometrial mucosa. In opposite, PPARγ protein expression was lower in endometrial cancer cells. In each case, immunohistochemical reaction was confined to the perinuclear and/or nuclear region. At the transcriptional level, the content of mRNA of all PPAR subunits did not follow the protein pattern of changes. These results provide evidence for altered PPAR's protein expression and disregulation of posttranslational processes in endometrial cancers.

Differential mitochondrial DNA and gene expression in inherited retinal dysplasia in miniature Schnauzer dogs.

PubMed

Appleyard, Greg D; Forsyth, George W; Kiehlbauch, Laura M; Sigfrid, Kristen N; Hanik, Heather L J; Quon, Anita; Loewen, Matthew E; Grahn, Bruce H

2006-05-01

To investigate the molecular basis of inherited retinal dysplasia in miniature Schnauzers. Retina and retinal pigment epithelial tissues were collected from canine subjects at the age of 3 weeks. Total RNA isolated from these tissues was reverse transcribed to make representative cDNA pools that were compared for differences in gene expression by using a subtractive hybridization technique referred to as representational difference analysis (RDA). Expression differences identified by RDA were confirmed and quantified by real-time reverse-transcription PCR. Mitochondrial morphology from leukocytes and skeletal muscle of normal and affected miniature Schnauzers was examined by transmission electron microscopy. RDA screening of retinal pigment epithelial cDNA identified differences in mRNA transcript coding for two mitochondrial (mt) proteins--cytochrome oxidase subunit 1 and NADH dehydrogenase subunit 6--in affected dogs. Contrary to expectations, these identified sequences did not contain mutations. Based on the implication of mt-DNA-encoded proteins by the RDA experiments we used real-time PCR to compare the relative amounts of mt-DNA template in white blood cells from normal and affected dogs. White blood cells of affected dogs contained less than 30% of the normal amount of two specific mtDNA sequences, compared with the content of the nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) reference gene. Retina and RPE tissue from affected dogs had reduced mRNA transcript levels for the two mitochondrial genes detected in the RDA experiment. Transcript levels for another mtDNA-encoded gene as well as the nuclear-encoded mitochondrial Tfam transcription factor were reduced in these tissues in affected dogs. Mitochondria from affected dogs were reduced in number and size and were unusually electron dense. Reduced levels of nuclear and mitochondrial transcripts in the retina and RPE of miniature Schnauzers affected with retinal dysplasia suggest that the pathogenesis of the disorder may arise from a lowered energy supply to the retina and RPE.

Dietary Exposure to 2,2′,4,4′-Tetrabromodiphenyl Ether (PBDE-47) Alters Thyroid Status and Thyroid Hormone–Regulated Gene Transcription in the Pituitary and Brain

PubMed Central

Lema, Sean C.; Dickey, Jon T.; Schultz, Irvin R.; Swanson, Penny

2008-01-01

Background Polybrominated diphenyl ether (PBDE) flame retardants have been implicated as disruptors of the hypothalamic-pituitary-thyroid axis. Animals exposed to PBDEs may show reduced plasma thyroid hormone (TH), but it is not known whether PBDEs impact TH-regulated pathways in target tissues. Objective We examined the effects of dietary exposure to 2,2′,4,4′-tetrabromodiphenyl ether (PBDE-47)—commonly the highest concentrated PBDE in human tissues—on plasma TH levels and on gene transcripts for glycoprotein hormone α-subunit (GPHα) and thyrotropin β-subunit (TSHβ) in the pituitary gland, the autoinduced TH receptors α and β in the brain and liver, and the TH-responsive transcription factor basic transcription element-binding protein (BTEB) in the brain. Methods Breeding pairs of adult fathead minnows (Pimephales promelas) were given dietary PBDE-47 at two doses (2.4 μg/pair/day or 12.3 μg/pair/day) for 21 days. Results Minnows exposed to PBDE-47 had depressed plasma thyroxine (T4), but not 3,5,3′-triiodothyronine (T3). This decline in T4 was accompanied by elevated mRNA levels for TStHβ (low dose only) in the pituitary. PBDE-47 intake elevated transcript for TH receptor αin the brain of females and decreased mRNA for TH receptor β in the brain of both sexes, without altering these transcripts in the liver. In males, PBDE-47 exposure also reduced brain transcripts for BTEB. Conclusions Our results indicate that dietary exposure to PBDE-47 alters TH signaling at multiple levels of the hypothalamic-pituitary-thyroid axis and provide evidence that TH-responsive pathways in the brain may be particularly sensitive to disruption by PBDE flame retardants. PMID:19079722

Voltage-gated calcium channel and antisense oligonucleotides thereto

NASA Technical Reports Server (NTRS)

Friedman, Peter A. (Inventor); Duncan, Randall L. (Inventor); Hruska, Keith A. (Inventor); Barry, Elizabeth L. R. (Inventor)

1998-01-01

An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

Stress and transcriptional regulation of tick ferritin HC.

PubMed

Mulenga, A; Simser, J A; Macaluso, K R; Azad, A F

2004-08-01

We previously identified a partial Dermacentor variabilis cDNA encoding ferritin HC (HC) subunit homolog (DVFER) that was differentially upregulated in Rickettsia montanensis infected ticks (Mulenga et al., 2003a). We have used rapid amplification of cDNA ends to clone full-length DVFER cDNA and its apparent ortholog from the wood tick, D. andersoni (DAFER), both of which show high sequence similarity to vertebrate than insect ferritin. Both DVFER and DAFER contain the stem-loop structure of a putative iron responsive element in the 5' untranslated region (nucleotide positions, 16-42) and the feroxidase centre loop typical for vertebrate ferritin HC subunits. Quantitative Western and Northern blotting analyses of protein and RNA from unfed and partially fed whole tick as well as dissected tick tissues demonstrated that DVFER is constitutively and ubiquitously expressed. Based on densitometric analysis of detected protein and mRNA bands, DVFER is predominantly expressed in the midgut, and to a lesser extent in the salivary glands, ovary and fatbody. Sham treatment (mechanical injury) and Escherichia coli challenge of D. variabilis ticks stimulated statistically significant (approximately 1.5- and approximately 3.0-fold, respectively) increases in DVFER mRNA abundance over time point matched naive control ticks. These data suggest that DVFER mRNA is nonspecifically up regulated in response to mechanical injury or bacterial infection induced stress.

Extensive frameshift at all AGG and CCC codons in the mitochondrial cytochrome c oxidase subunit 1 gene of Perkinsus marinus (Alveolata; Dinoflagellata).

PubMed

Masuda, Isao; Matsuzaki, Motomichi; Kita, Kiyoshi

2010-10-01

Diverse mitochondrial (mt) genetic systems have evolved independently of the more uniform nuclear system and often employ modified genetic codes. The organization and genetic system of dinoflagellate mt genomes are particularly unusual and remain an evolutionary enigma. We determined the sequence of full-length cytochrome c oxidase subunit 1 (cox1) mRNA of the earliest diverging dinoflagellate Perkinsus and show that this gene resides in the mt genome. Apparently, this mRNA is not translated in a single reading frame with standard codon usage. Our examination of the nucleotide sequence and three-frame translation of the mRNA suggest that the reading frame must be shifted 10 times, at every AGG and CCC codon, to yield a consensus COX1 protein. We suggest two possible mechanisms for these translational frameshifts: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons or specialized tRNAs recognizing non-triplet codons, AGGY and CCCCU. Regardless of the mechanism, active and efficient machinery would be required to tolerate the frameshifts predicted in Perkinsus mitochondria. To our knowledge, this is the first evidence of translational frameshifts in protist mitochondria and, by far, is the most extensive case in mitochondria.

Using analogy role-play activity in an undergraduate biology classroom to show central dogma revision.

PubMed

Takemura, Masaharu; Kurabayashi, Mario

2014-01-01

For the study of biology in an undergraduate classroom, a classroom exercise was developed: an analogy role-play to learn mechanisms of gene transcription and protein translation (central dogma). To develop the central dogma role-play exercise, we made DNA and mRNA using paper sheets, tRNA using a wire dress hanger, and amino acids using Lego® blocks (Lego System A/S, Denmark). Students were studying in the course of mathematics, physics, or chemistry, so biology was not among their usual studies. In this exercise, students perform the central dogma role-play and respectively act out nuclear matrix proteins, a transcription factor, an RNA polymerase II, an mRNA transport protein, nuclear pore proteins, a large ribosomal subunit, a small ribosomal subunit, and several amino-acyl tRNA synthetases. Questionnaire results obtained after the activity show that this central dogma role-play analogy holds student interest in the practical molecular biological processes of transcription and translation. © 2014 The International Union of Biochemistry and Molecular Biology.

Cortical NMDA receptor expression in human chronic alcoholism: influence of the TaqIA allele of ANKK1.

PubMed

Ridge, Justin P; Dodd, Peter R

2009-10-01

Real-time RT-PCR normalized to GAPDH was used to assay N-methyl-D-aspartate (NMDA) receptor NR1, NR2A and NR2B subunit mRNA in human autopsy cortex tissue from chronic alcoholics with and without comorbid cirrhosis of the liver and matched controls. Subunit expression was influenced by the subject's genotype. The TaqIA polymorphism selectively modulated NMDA receptor mean transcript expression in cirrhotic-alcoholic superior frontal cortex, in diametrically opposite ways in male and female subjects. Genetic make-up may differentially influence vulnerability to brain damage by altering the excitation: inhibition balance, particularly in alcoholics with comorbid cirrhosis of the liver. The TaqIA polymorphism occurs within the poorly characterised ankyrin-repeat containing kinase 1 (ANKK1) gene. Using PCR, ANKK1 mRNA transcript was detected in inferior temporal, occipital, superior frontal and primary motor cortex of control human brain. ANKK1 expression may mediate the influence of the TaqIA polymorphism on phenotype.

Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution

NASA Astrophysics Data System (ADS)

Quade, Nick; Boehringer, Daniel; Leibundgut, Marc; van den Heuvel, Joop; Ban, Nenad

2015-07-01

Hepatitis C virus (HCV), a widespread human pathogen, is dependent on a highly structured 5'-untranslated region of its mRNA, referred to as internal ribosome entry site (IRES), for the translation of all of its proteins. The HCV IRES initiates translation by directly binding to the small ribosomal subunit (40S), circumventing the need for many eukaryotic translation initiation factors required for mRNA scanning. Here we present the cryo-EM structure of the human 40S ribosomal subunit in complex with the HCV IRES at 3.9 Å resolution, determined by focused refinement of an 80S ribosome-HCV IRES complex. The structure reveals the molecular details of the interactions between the IRES and the 40S, showing that expansion segment 7 (ES7) of the 18S rRNA acts as a central anchor point for the HCV IRES. The structural data rationalizes previous biochemical and genetic evidence regarding the initiation mechanism of the HCV and other related IRESs.

MC EMiNEM Maps the Interaction Landscape of the Mediator

PubMed Central

Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C.; Martin, Dietmar E.; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

2012-01-01

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors. PMID:22737066

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma

PubMed Central

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named “hTERT messenger ribonucleic acid (mRNA)” the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. Materials and Methods: U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0–160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control–untreated group. Results: The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent (P < 0.05) and not in time-dependent manner (P > 0.05), in vitro. Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. Conclusion: With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM. PMID:28706881

Conjugated linoleic acid synthesis-related protein proteasome subunit α 5 (PSMA5) is increased by vaccenic acid treatment in goat mammary tissue.

PubMed

Jin, Y C; Li, Z H; Hong, Z S; Xu, C X; Han, J A; Choi, S H; Yin, J L; Zhang, Q K; Lee, K B; Kang, S K; Song, M K; Kim, Y J; Kang, H S; Choi, Y J; Lee, H G

2012-08-01

This study was conducted to identify proteins associated with the endogenous synthesis of conjugated linoleic acid (CLA) from trans-vaccenic acid (TVA; trans-11 C18:1, a precursor for CLA endogenous synthesis) in mammary tissues. Six lactating goats were divided into 2 groups. One group was given an intravenous bolus injection of TVA (150mg) twice daily over 4 d; the other group received saline injections. Treatment with TVA increased the concentration of cis-9,trans-11 CLA and TVA in goat milk. Additionally, TVA treatment increased the expression of stearoyl-CoA desaturase (SCD) in mammary tissue. Using 2-dimensional gel electrophoresis and electrospray ionization quadrupole time-of-flight mass spectrometry, 3 proteins affected by infusions of TVA were identified. Proteasome (prosome, macropain) subunit α type 5 (PSMA5) was upregulated, whereas peroxiredoxin-1 and translationally controlled tumor protein 1 were downregulated in TVA-treated animals compared with the vehicle-injected controls. Only the effect of TVA on PSMA5 could be confirmed by Western blot analysis. To further explore the regulation of PSMA5 in mammary epithelial cells when TVA is converted into CLA, we used a differentiated bovine mammary epithelial cell line treated with TVA for 6h. Changes in cis-9,trans-11 CLA concentrations and mRNA expression patterns of both SCD and PSMA5 were monitored. The concentration of cis-9,trans-11 CLA increased after TVA treatment. The mRNA expression level of PSMA5 was significantly elevated to 6h, but SCD mRNA expression only increased in 2h after TVA treatment. These results indicate that PSMA5 is highly expressed in goat mammary tissue and bovine mammary epithelial cells when TVA is converted into CLA. Our data suggest that PSMA5 protein is associated with CLA biosynthesis in mammary tissue. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Regulation of c-Myc mRNA by L11 in Response to UV and Gamma irradiation

DTIC Science & Technology

2011-10-01

release of L11 from the nucleolus to the nucleoplasm, where it binds to c-Myc protein, and to the cytoplasm, where it binds to c-myc mRNA. We also found...rRNA and ribosomal proteins (RPs), rRNA processing, and the as- sembly of the mature ribosome subunits in the nucleolus fol- lowed by their transport...from the nucleolus or from intact ribosomes to suppress MDM2 (68). However, whether L11 suppresses c-Myc in response to ribosomal stress is not known

Structure of ratcheted ribosomes with tRNAs in hybrid states

PubMed Central

Julián, Patricia; Konevega, Andrey L.; Scheres, Sjors H. W.; Lázaro, Melisa; Gil, David; Wintermeyer, Wolfgang; Rodnina, Marina V.; Valle, Mikel

2008-01-01

During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G. PMID:18971332

Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.

PubMed Central

LaPolla, R J; Mayne, K M; Davidson, N

1984-01-01

A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the delta subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector lambda 10 from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the delta clone was selected by hybridization with cDNA encoding the gamma subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR delta and gamma subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo gamma subunit cDNA, but not with delta. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences. Images PMID:6096870

Orchestrated Regulation of Nogo Receptors, Lotus, AMPA Receptors and BDNF in an ECT Model Suggests Opening and Closure of a Window of Synaptic Plasticity

PubMed Central

Nordgren, Max; Karlsson, Tobias; Svensson, Maria; Koczy, Josefin; Josephson, Anna; Olson, Lars; Tingström, Anders; Brené, Stefan

2013-01-01

Electroconvulsive therapy (ECT) is an efficient and relatively fast acting treatment for depression. However, one severe side effect of the treatment is retrograde amnesia, which in certain cases can be long-term. The mechanisms behind the antidepressant effect and the amnesia are not well understood. We hypothesized that ECT causes transient downregulation of key molecules needed to stabilize synaptic structure and to prevent Ca2+ influx, and a simultaneous increase in neurotrophic factors, thus providing a short time window of increased structural synaptic plasticity. Here we followed regulation of NgR1, NgR3, LOTUS, BDNF, and AMPA subunits GluR1 and GluR2 flip and flop mRNA levels in hippocampus at 2, 4, 12, 24, and 72 hours after a single episode of induced electroconvulsive seizures (ECS) in rats. NgR1 and LOTUS mRNA levels were transiently downregulated in the dentate gyrus 2, 4, 12 and 4, 12, 24 h after ECS treatment, respectively. GluR2 flip, flop and GluR1 flop were downregulated at 4 h. GluR2 flip remained downregulated at 12 h. In contrast, BDNF, NgR3 and GluR1 flip mRNA levels were upregulated. Thus, ECS treatment induces a transient regulation of factors important for neuronal plasticity. Our data provide correlations between ECS treatment and molecular events compatible with the hypothesis that both effects and side effects of ECT may be caused by structural synaptic rearrangements. PMID:24244357

Orchestrated regulation of Nogo receptors, LOTUS, AMPA receptors and BDNF in an ECT model suggests opening and closure of a window of synaptic plasticity.

PubMed

Nordgren, Max; Karlsson, Tobias; Svensson, Maria; Koczy, Josefin; Josephson, Anna; Olson, Lars; Tingström, Anders; Brené, Stefan

2013-01-01

Electroconvulsive therapy (ECT) is an efficient and relatively fast acting treatment for depression. However, one severe side effect of the treatment is retrograde amnesia, which in certain cases can be long-term. The mechanisms behind the antidepressant effect and the amnesia are not well understood. We hypothesized that ECT causes transient downregulation of key molecules needed to stabilize synaptic structure and to prevent Ca2+ influx, and a simultaneous increase in neurotrophic factors, thus providing a short time window of increased structural synaptic plasticity. Here we followed regulation of NgR1, NgR3, LOTUS, BDNF, and AMPA subunits GluR1 and GluR2 flip and flop mRNA levels in hippocampus at 2, 4, 12, 24, and 72 hours after a single episode of induced electroconvulsive seizures (ECS) in rats. NgR1 and LOTUS mRNA levels were transiently downregulated in the dentate gyrus 2, 4, 12 and 4, 12, 24 h after ECS treatment, respectively. GluR2 flip, flop and GluR1 flop were downregulated at 4 h. GluR2 flip remained downregulated at 12 h. In contrast, BDNF, NgR3 and GluR1 flip mRNA levels were upregulated. Thus, ECS treatment induces a transient regulation of factors important for neuronal plasticity. Our data provide correlations between ECS treatment and molecular events compatible with the hypothesis that both effects and side effects of ECT may be caused by structural synaptic rearrangements.

Molecular determinants of Cytochrome C oxidase IV mRNA axonal trafficking

PubMed Central

Kar, Amar N.; Vargas, Jose Norberto S.; Chen, Cai-Yun; Kowalak, Jeffrey A; Gioio, Anthony E.; Kaplan, Barry B.

2017-01-01

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3′ untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA. PMID:28161363

RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity

PubMed Central

Tsunoda, Honami; Suzuki, Yuji; Makino, Amane; Ishida, Hiroyuki

2012-01-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (RBCS) is encoded by a nuclear RBCS multigene family in many plant species. The contribution of the RBCS multigenes to accumulation of Rubisco holoenzyme and photosynthetic characteristics remains unclear. T-DNA insertion mutants of RBCS1A (rbcs1a-1) and RBCS3B (rbcs3b-1) were isolated among the four Arabidopsis RBCS genes, and a double mutant (rbcs1a3b-1) was generated. RBCS1A mRNA was not detected in rbcs1a-1 and rbcs1a3b-1, while the RBCS3B mRNA level was suppressed to ∼20% of the wild-type level in rbcs3b-1 and rbcs1a3b-1 leaves. As a result, total RBCS mRNA levels declined to 52, 79, and 23% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. Rubisco contents showed declines similar to total RBCS mRNA levels, and the ratio of Rubisco-nitrogen to total nitrogen was 62, 78, and 40% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. The effects of RBCS1A and RBCS3B mutations in rbcs1a3b-1 were clearly additive. The rates of CO2 assimilation at ambient CO2 of 40 Pa were reduced with decreased Rubisco contents in the respective mutant leaves. Although the RBCS composition in the Rubisco holoenzyme changed, the CO2 assimilation rates per unit of Rubisco content were the same irrespective of the genotype. These results clearly indicate that RBCS1A and RBCS3B contribute to accumulation of Rubisco in Arabidopsis leaves and that these genes work additively to yield sufficient Rubisco for photosynthetic capacity. It is also suggested that the RBCS composition in the Rubisco holoenzyme does not affect photosynthesis under the present ambient [CO2] conditions. PMID:22223809

RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity.

PubMed

Izumi, Masanori; Tsunoda, Honami; Suzuki, Yuji; Makino, Amane; Ishida, Hiroyuki

2012-03-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (RBCS) is encoded by a nuclear RBCS multigene family in many plant species. The contribution of the RBCS multigenes to accumulation of Rubisco holoenzyme and photosynthetic characteristics remains unclear. T-DNA insertion mutants of RBCS1A (rbcs1a-1) and RBCS3B (rbcs3b-1) were isolated among the four Arabidopsis RBCS genes, and a double mutant (rbcs1a3b-1) was generated. RBCS1A mRNA was not detected in rbcs1a-1 and rbcs1a3b-1, while the RBCS3B mRNA level was suppressed to ∼20% of the wild-type level in rbcs3b-1 and rbcs1a3b-1 leaves. As a result, total RBCS mRNA levels declined to 52, 79, and 23% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. Rubisco contents showed declines similar to total RBCS mRNA levels, and the ratio of Rubisco-nitrogen to total nitrogen was 62, 78, and 40% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. The effects of RBCS1A and RBCS3B mutations in rbcs1a3b-1 were clearly additive. The rates of CO(2) assimilation at ambient CO(2) of 40 Pa were reduced with decreased Rubisco contents in the respective mutant leaves. Although the RBCS composition in the Rubisco holoenzyme changed, the CO(2) assimilation rates per unit of Rubisco content were the same irrespective of the genotype. These results clearly indicate that RBCS1A and RBCS3B contribute to accumulation of Rubisco in Arabidopsis leaves and that these genes work additively to yield sufficient Rubisco for photosynthetic capacity. It is also suggested that the RBCS composition in the Rubisco holoenzyme does not affect photosynthesis under the present ambient [CO(2)] conditions.

Regulation of hepatic Na+/K+-ATPase in obese female and male rats: involvement of ERK1/2, AMPK, and Rho/ROCK.

PubMed

Stanimirovic, Julijana; Obradovic, Milan; Panic, Anastasija; Petrovic, Voin; Alavantic, Dragan; Melih, Irena; Isenovic, Esma R

2018-03-01

In this study, we assessed whether the disturbed regulation of sodium/potassium-adenosine-triphosphatase (Na + /K + -ATPase) occurs as a consequence of obesity-induced IR in sex-specific manner. We also assessed whether alterations of IRS/PI3K/Akt, ERK1/2, AMPKα, and RhoA/ROCK signaling cascades have an important role in this pathology. Female and male Wistar rats (150-200 g, 8 weeks old) were fed a standard laboratory diet or a high-fat (HF) diet (42% fat) for 10 weeks. The activity of hepatic Na + /K + -ATPase and Rho, and the association of IRS-1/p85 were assessed in liver. Furthermore, the protein level of α 1 Na + /K + -ATPase in plasma membrane fractions, and protein levels of IRS-1, PI3K-p85, -p110, RhoA, ROCK1, ROCK2, ERK1/2, AMPKα, ERα, and ERβ in liver lysates were assessed. The expression of hepatic α 1 Na + /K + -ATPase mRNA was also analyzed by qRT-PCR. The results show that HF-fed female rats exhibited an increase in hepatic ERK1/2 (p < 0.05) and AMPKα (p < 0.05) phosphorylation levels, unchanged level of Na + /K + -ATPase α 1 mRNA, decreased level of Na + /K + -ATPase activity (p < 0.05), and decreased α 1 Na + /K + -ATPase protein expression (p < 0.01). In liver of HF-fed male rats, results show decreased levels of Na + /K + -ATPase activity (p < 0.01), both protein and mRNA of α 1 subunit (p < 0.05), but significant increase in Rho activity (p < 0.05). Our results indicate significant sex differences in α 1 Na + /K + -ATPase mRNA expression and activation of ERK1/2, AMPKα, and Rho in the liver. Exploring the sex-specific factors and pathways that promote obesity-related diseases may lead to a better understanding of pathogenesis and discovering new therapeutic targets.

Consequences of Late-Stage Non-Small-Cell Lung Cancer Cachexia on Muscle Metabolic Processes.

PubMed

Murton, Andrew J; Maddocks, Matthew; Stephens, Francis B; Marimuthu, Kanagaraj; England, Ruth; Wilcock, Andrew

2017-01-01

The loss of muscle is common in patients with advanced non-small-cell lung cancer (NSCLC) and contributes to the high morbidity and mortality of this group. The exact mechanisms behind the muscle loss are unclear. To investigate this, 4 patients with stage IV NSCLC who met the clinical definitions for sarcopenia and cachexia were recruited, along with 4 age-matched healthy volunteers. After an overnight fast, biopsy specimens were obtained from the vastus lateralis, and the key components associated with inflammation and the control of muscle protein, carbohydrate, and fat metabolism were assessed. Compared with the healthy volunteers, significant increases in mRNA levels for interleukin-6 and NF-κB signaling and lower intramyocellular lipid content in slow-twitch fibers were observed in NSCLC patients. Although a significant decrease in phosphorylation of the mechanistic target of rapamycin (mTOR) signaling protein 4E-BP1 (Ser 65 ) was observed, along with a trend toward reduced p70 S6K (Thr 389 ) phosphorylation (P = .06), no difference was found between groups for the mRNA levels of MAFbx (muscle atrophy F box) and MuRF1 (muscle ring finger protein 1), chymotrypsin-like activity of the proteasome, or protein levels of multiple proteasome subunits. Moreover, despite decreases in intramyocellular lipid content, no robust changes in mRNA levels for key proteins involved in insulin signaling, glycolysis, oxidative metabolism, or fat metabolism were observed. These findings suggest that examining the contribution of suppressed mTOR signaling in the loss of muscle mass in late-stage NSCLC patients is warranted and reinforces our need to understand the potential contribution of impaired fat metabolism and muscle protein synthesis in the etiology of cancer cachexia. Copyright © 2016 Elsevier Inc. All rights reserved.

The ly-6 protein, lynx1, is an endogenous inhibitor of nicotinic signaling in airway epithelium.

PubMed

Fu, Xiao Wen; Rekow, Stephen S; Spindel, Eliot R

2012-10-15

Our laboratory has previously reported that bronchial epithelial cells (BEC) express a regulatory cascade of classic neurotransmitters and receptors that communicate in an almost neuronal-like manner to achieve physiological regulation. In this paper we show that the similarity between neurotransmitter signaling in neurons and BEC extends to the level of transmitter receptor allosteric modulators. Lynx1 is a member of the ly-6/three-finger superfamily of proteins, many of which modulate receptor signaling activity. Lynx1 specifically has been shown to modulate nicotinic acetylcholine receptor (nAChR) function in neurons by altering receptor sensitivity and desensitization. We now report that lynx1 forms a complex with α7 nAChR in BEC and serves to negatively regulate α7 downstream signaling events. Treatment of primary cultures of BEC with nicotine increased levels of nAChR subunits and that increase was potentiated by lynx1 knockdown. Lynx1 knockdown also potentiated the nicotine-induced increase in GABA(A) receptors (GABA(A)R) and MUC5AC mRNA expression, and that effect was blocked by α7 antagonists and α7 knockdown. In parallel with the increases in nAChR, GABA(A)R, and mucin mRNA levels, lynx1 knockdown also increased levels of p-Src. Consistent with this, inhibition of Src signaling blocked the ability of the lynx1 knockdown to increase basal and nicotine-stimulated GABA(A)R and mucin mRNA expression. Thus lynx1 appears to act as a negative modulator of α7 nAChR-induced events by inhibiting Src activation. This suggests that lynx1 agonists or mimetics are a potentially important therapeutic target to develop new therapies for smoking-related diseases characterized by increased mucin expression.

Integrator complex plays an essential role in adipose differentiation

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

Otani, Yuichiro; Nakatsu, Yusuke; Sakoda, Hideyuki

2013-05-03

Highlights: •IntS6 and IntS11 are subunits of the Integrator complex. •Expression levels of IntS6 and IntS11 were very low in 3T3-L1 fibroblast. •IntS6 and IntS11 were upregulated during adipose differentiation. •Suppression of IntS6 or IntS11 expression inhibited adipose differentiation. -- Abstract: The dynamic process of adipose differentiation involves stepwise expressions of transcription factors and proteins specific to the mature fat cell phenotype. In this study, it was revealed that expression levels of IntS6 and IntS11, subunits of the Integrator complex, were increased in 3T3-L1 cells in the period when the cells reached confluence and differentiated into adipocytes, while being reducedmore » to basal levels after the completion of differentiation. Suppression of IntS6 or IntS11 expression using siRNAs in 3T3-L1 preadipocytes markedly inhibited differentiation into mature adipocytes, based on morphological findings as well as mRNA analysis of adipocyte-specific genes such as Glut4, perilipin and Fabp4. Although Pparγ2 protein expression was suppressed in IntS6 or IntS11-siRNA treated cells, adenoviral forced expression of Pparγ2 failed to restore the capacity for differentiation into mature adipocytes. Taken together, these findings demonstrate that increased expression of Integrator complex subunits is an indispensable event in adipose differentiation. Although further study is necessary to elucidate the underlying mechanism, the processing of U1, U2 small nuclear RNAs may be involved in cell differentiation steps.« less

Expression of membrane-bound and cytosolic guanylyl cyclases in the rat inner ear.

PubMed

Seebacher, T; Beitz, E; Kumagami, H; Wild, K; Ruppersberg, J P; Schultz, J E

1999-01-01

Membrane-bound guanylyl cyclases (GCs) are peptide hormone receptors whereas the cytosolic isoforms are receptors for nitric oxide. In the inner ear, the membrane-bound GCs may be involved in the regulation of fluid homeostasis and the cytosolic forms possibly play a role in signal processing and regulation of local blood flow. In this comprehensive study, we examined, qualitatively and quantitatively, the transcription pattern of all known GC isoforms in the inner ear from rat by RT-PCR. The tissues used were endolymphatic sac, stria vascularis, organ of Corti, organ of Corti outer hair cells, cochlear nerve, Reissner's membrane, vestibular dark cells, and vestibular sensory cells. We show that multiple particulate (GC-A, GC-B, GC-D, GC-E, GC-F and GC-G) and several subunits of the heterodimeric cytosolic GCs (alpha1, alpha2, beta1 and beta2) are expressed, albeit at highly different levels. GC-C was not found. GC-A and the soluble subunits alpha1 and beta1 were transcribed ubiquitously. GC-B was present in all tissues except stria vascularis, which contained GC-A and traces of GC-E and GC-G. GC-B was by far the predominant membrane-bound isoform in the organ of Corti (86%), Reissner's membrane (75%) and the vestibulum (80%). Surprisingly, GC-E, a retinal isoform, was detected in significant amounts in the cochlear nerve (8%) and in the organ of Corti (4%). Although the cytosolic GC is a heterodimer composed of an alpha and a beta subunit, the mRNA transcription of these subunits was not stoichiometric. Particularly in the vestibulum, the transcription of the beta1 subunits was at least four-fold higher than of the alpha1 subunit. The data are compatible with earlier suggestions that membrane receptor GCs may be involved in the control of inner ear electrolyte and fluid composition whereas NO-stimulated GC isoforms mainly participate in the regulation of blood flow and supporting cell physiology.

Ribosomal Protein Rps26 Influences 80S Ribosome Assembly in Saccharomyces cerevisiae.

PubMed

Belyy, Alexander; Levanova, Nadezhda; Tabakova, Irina; Rospert, Sabine; Belyi, Yury

2016-01-01

The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5' untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62-K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62-K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62-K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62-K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62-K70 segment of Rps26 and the 5' untranslated region of mRNA. The data suggested a specific role of the Y62-K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62-K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5' untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62-K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast.

Amicoumacin A inhibits translation by stabilizing mRNA interaction with the ribosome

PubMed Central

Polikanov, Yury S.; Osterman, Ilya A.; Szal, Teresa; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Kusochek, Pavel; Bulkley, David; Malanicheva, Irina A.; Efimenko, Tatyana A.; Efremenkova, Olga V.; Konevega, Andrey L.; Shaw, Karen J.; Bogdanov, Alexey A.; Rodnina, Marina V.; Dontsova, Olga A.; Mankin, Alexander S.; Steitz, Thomas A.; Sergiev, Petr V.

2014-01-01

SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G. PMID:25306919

The extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity

PubMed Central

Pagliano, Cristina; La Rocca, Nicoletta; Andreucci, Flora; Deák, Zsuzsanna; Vass, Imre; Rascio, Nicoletta; Barbato, Roberto

2009-01-01

Background and Aims Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca2+ and Cl− as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl− and Ca2+, but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides. Methods Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT–PCR and time-resolved chlorophyll fluorescence were used. Key Results Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein. Conclusions In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII. PMID:19033288

First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

PubMed

Oh, Minyoung; Umasuthan, Navaneethaiyer; Elvitigala, Don Anushka Sandaruwan; Wan, Qiang; Jo, Eunyoung; Ko, Jiyeon; Noh, Gyeong Eon; Shin, Sangok; Rho, Sum; Lee, Jehee

2016-02-01

Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21 kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable in vivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

PubMed

Alejo, Jose L; Blanchard, Scott C

2017-10-10

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome

PubMed Central

Alejo, Jose L.; Blanchard, Scott C.

2017-01-01

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors. PMID:28973849

Molecular analysis of the von hippel-lindau disease gene.

PubMed

Chernoff, A; Kasparcova, V; Linehan, W M; Stolle, C A

2001-01-01

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that predisposes the affected individual to develop characteristic tumors. These include CNS hemangioblastoma, retinal angiomas, endolymphatic sac tumors, pancreatic cysts and tumors, epididymal cystadenomas, pheochromocytomas, renal cysts, and clear-cell renal carcinoma. The VHL gene was localized to 3p25 and then isolated by Latif et al. (1). The gene contains three exons with an open reading frame of 852 nucleotides, which encode a predicted protein of 284 amino acids. The VHL protein is believed to have several functions. It is involved in transcription regulation through its inhibition of elongation by binding to the B and C subunits of elongin. Mutations of VHL allow the B and C subunits to bind with the A subunit. This complex then overcomes "pausing" of RNA polymerase during mRNA transcription (2,3). Several studies suggest that the VHL protein is also involved in regulation of hypoxia-inducible transcripts, particularly vascular endothelial growth factor (VEGF), by altering mRNA stability (4,5). Therefore, VHL gene mutations permit the overexpression of VEGF under normoxic conditions, which leads to the angiogenesis believed to be required for tumor growth. The VHL-elongin BC complex (VBC) also binds two other proteins-CUL2 and Rbx1-in a complex that has structural similarity to other E3 ubiquitin ligase complexes (6). Such complexes mediate the degradation of cell-cycle regulatory proteins.

Evodiamine Inhibits Angiotensin II-Induced Rat Cardiomyocyte Hypertrophy.

PubMed

He, Na; Gong, Qi-Hai; Zhang, Feng; Zhang, Jing-Yi; Lin, Shu-Xian; Hou, Hua-Hua; Wu, Qin; Sun, An-Sheng

2018-05-01

To investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms. Cardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca 2+ ] i ) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis. Compared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca 2+ ] i ) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca 2+ ] i concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05). Evo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca 2+ ]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Fail-safe mechanism of GCN4 translational control—uORF2 promotes reinitiation by analogous mechanism to uORF1 and thus secures its key role in GCN4 expression

PubMed Central

Gunišová, Stanislava; Valášek, Leoš Shivaya

2014-01-01

One of the extensively studied mechanisms of gene-specific translational regulation is reinitiation. It takes place on messenger RNAs (mRNAs) where main ORF is preceded by upstream ORF (uORF). Even though uORFs generally down-regulate main ORF expression, specific uORFs exist that allow high level of downstream ORF expression. The key is their ability to retain 40S subunits on mRNA upon termination of their translation to resume scanning for the next AUG. Here, we took advantage of the exemplary model system of reinitiation, the mRNA of yeast transcriptional activator GCN4 containing four short uORFs, and show that contrary to previous reports, not only the first but the first two of its uORFs allow efficient reinitiation. Strikingly, we demonstrate that they utilize a similar molecular mechanism relying on several cis-acting 5′ reinitiation-promoting elements, one of which they share, and the interaction with the a/TIF32 subunit of translation initiation factor eIF3. Since a similar mechanism operates also on YAP1 uORF, our findings strongly suggest that basic principles of reinitiation are conserved. Furthermore, presence of two consecutive reinitiation-permissive uORFs followed by two reinitiation-non-permissive uORFs suggests that tightness of GCN4 translational control is ensured by a fail-safe mechanism that effectively prevents or triggers GCN4 expression under nutrient replete or deplete conditions, respectively. PMID:24623812

Constitutive Knockout of Kalirin-7 Leads to Increased Rates of Cocaine Self-Administration

PubMed Central

Kiraly, Drew D.; Nemirovsky, Natali E.; LaRese, Taylor P.; Tomek, Seven E.; Yahn, Stephanie L.; Olive, M. Foster; Eipper, Betty A.

2013-01-01

Kalirin-7 (Kal7) is a Rho-guanine nucleotide exchange factor that is localized in neuronal postsynaptic densities. Kal7 interacts with the NR2B subunit of the NMDA receptor and regulates aspects of dendritic spine dynamics both in vitro and in vivo. Chronic treatment with cocaine increases dendritic spine density in the nucleus accumbens (NAc) of rodents and primates. Kal7 mRNA and protein are upregulated in the NAc following cocaine treatment, and the presence of Kal7 is necessary for the normal proliferation of dendritic spines following cocaine use. Mice that constitutively lack Kal7 [Kalirin-7 knockout mice (Kal7KO)] demonstrate increased locomotor sensitization to cocaine and a decreased place preference for cocaine. Here, using an intravenous cocaine self-administration paradigm, Kal7KO mice exhibit increased administration of cocaine at lower doses as compared with wild-type (Wt) mice. Analyses of mRNA transcript levels from the NAc of mice that self-administered saline or cocaine reveal that larger splice variants of the Kalrn gene are increased by cocaine more dramatically in Kal7KO mice than in Wt mice. Additionally, transcripts encoding the NR2B subunit of the NMDA receptor increased in Wt mice that self-administered cocaine but were unchanged in similarly experienced Kal7KO mice. These findings suggest that Kal7 participates in the reinforcing effects of cocaine, and that Kal7 and cocaine interact to alter the expression of genes related to critical glutamatergic signaling pathways in the NAc. PMID:23894151

Ramipril restores PPARβ/δ and PPARγ expressions and reduces cardiac NADPH oxidase but fails to restore cardiac function and accompanied myosin heavy chain ratio shift in severe anthracycline-induced cardiomyopathy in rat.

PubMed

Cernecka, Hana; Doka, Gabriel; Srankova, Jasna; Pivackova, Lenka; Malikova, Eva; Galkova, Kristina; Kyselovic, Jan; Krenek, Peter; Klimas, Jan

2016-11-15

We hypothesized that peroxisome proliferator-activated receptors (PPARs) might be involved in a complex protective action of ACE inhibitors (ACEi) in anthracyclines-induced cardiomyopathy. For purpose of study, we compared effects of ramipril on cardiac dysfunction, cardiac failure markers and PPAR isoforms in moderate and severe chronic daunorubicin-induced cardiomyopathy. Male Wistar rats were administered with a single intravenous injection of daunorubicin: 5mg/kg (moderate cardiomyopathy), or 15mg/kg (severe cardiomyopathy) or co-administered with daunorubicin and ramipril (1mg/kg/d, orally) or vehicle for 8 weeks. Left ventricular function was measured invasively under anesthesia. Cardiac mRNA levels of heart failure markers (ANP, Myh6, Myh7, Myh7b) and PPARs (alpha, beta/delta and gama) were measured by qRT-PCR. Protein expression of NADPH subunit (gp91phox) was measured by Western blot. Moderate cardiomyopathy exhibited only minor cardiac dysfunction what was corrected by ramipril. In severe cardiomyopathy, hemodynamic dysfunction remained unaltered upon ramipril although it decreased the significantly up-regulated cardiac ANP mRNA expression. Simultaneously, while high-dose daunorubicin significantly decreased PPARbeta/delta and PPARgama mRNA, ramipril normalized these abnormalities. Similarly, ramipril reduced altered levels of oxidative stress-related gp91phox. On the other hand, ramipril was unable to correct both the significantly decreased relative abundance of Myh6 and increased Myh7 mRNA levels, respectively. In conclusion, ramipril had a protective effect on cardiac function exclusively in moderate chronic daunorubicin-induced cardiomyopathy. Although it normalized abnormal PPARs expression and exerted also additional protective effects also in severe cardiomyopathy, it was insufficient to influence impaired cardiac function probably because of a shift in myosin heavy chain isoform content. Copyright © 2016 Elsevier B.V. All rights reserved.

Differential expression and induction of two carbonic anhydrase isoforms in the gills of the euryhaline green crab, Carcinus maenas, in response to low salinity.

PubMed

Serrano, Laetitia; Henry, Raymond P

2008-06-01

Two isoforms of the enzyme carbonic anhydrase (CA) from the gills of the euryhaline green crab were sequenced and identified; these were found to match the cytoplasmic (CAc) and membrane-associated (CAg) isoforms known from other species. The mRNA of the membrane-associated isoform is present in significantly higher levels of abundance in gills of crabs acclimated to 32 ppt, at which the crab is an osmotic and ionic conformer. Upon transfer to low salinity (15 ppt), in which the crab is an osmoregulator, however, the cytoplasmic isoform undergoes a rapid 100-fold increase in abundance in the posterior gills, becoming the dominant isoform. CAg increases 3-fold initially and then remains elevated through 14 days of low salinity acclimation. The induction of CAc mRNA is believed to be the molecular basis for the 20 fold increase in CA protein-specific activity during low salinity acclimation. The initial increase in CAc mRNA takes place at 6 h, and maximal levels of expression are achieved by 24 h; this precedes the induction of CA activity and is within the time in which hemolymph osmotic and ionic concentrations stabilize at new acclimated levels. The increase in expression of the CAg isoform is believed to be more closely related to changes in the population of branchial chloride cells. Changes in the relative abundance of mRNA for the alpha-subunit of the Na(+)/K(+)-ATPase were smaller in magnitude than those for CAc, but the timing was similar. There were no changes in expression of a control gene, arginine kinase (AK) in posterior gills, and there were no significant changes in expression in anterior gills for any of the genes measured here. These results support the use of a control tissue (anterior gills) in addition to a control gene for expression studies.

Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease

PubMed Central

Molle, Céline; Zhang, Tong; Ysebrant de Lendonck, Laure; Gueydan, Cyril; Andrianne, Mathieu; Sherer, Félicie; Van Simaeys, Gaetan; Blackshear, Perry J.; Leo, Oberdan

2013-01-01

Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine–rich element (ARE)–binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow–derived dendritic cells (BMDCs) from TTP−/− mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3′ untranslated region. TTP−/− mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23–IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation. PMID:23940256

Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease.

PubMed

Molle, Céline; Zhang, Tong; Ysebrant de Lendonck, Laure; Gueydan, Cyril; Andrianne, Mathieu; Sherer, Félicie; Van Simaeys, Gaetan; Blackshear, Perry J; Leo, Oberdan; Goriely, Stanislas

2013-08-26

Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine-rich element (ARE)-binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow-derived dendritic cells (BMDCs) from TTP(-/-) mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3' untranslated region. TTP(-/-) mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23-IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation.

Lipofuscin accumulation and autophagy in glaucomatous human lamina cribrosa cells.

PubMed

McElnea, Elizabeth M; Hughes, Emily; McGoldrick, Aloysius; McCann, Amanda; Quill, Barry; Docherty, Neil; Irnaten, Mustapha; Farrell, Michael; Clark, Abbot F; O'Brien, Colm J; Wallace, Deborah M

2014-12-02

Disease associated alterations in the phenotype of lamina cribrosa (LC) cells are implicated in changes occurring at the optic nerve head (ONH) in glaucoma. Lipofuscin, the formation of which is driven by reactive oxygen species (ROS), is an intralysosomal, non-degradable, auto-fluorescent macromolecule which accumulates with age and can affect autophagy - the lysosomal degradation of a cell's constituents. We aimed to compare the content of lipofuscin-like material and markers of autophagy in LC cells from normal and glaucoma donor eyes. The number and size of peri-nuclear lysosomes were examined by transmission electron microscopy (TEM). Cellular auto-fluorescence was quantified by flow cytometry. Cathepsin K mRNA levels were assessed by PCR. Autophagy protein 5 (Atg5) mRNA and protein levels were analysed by PCR and Western blot. Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot. Immunohistochemical staining of LC3-II in ONH sections from normal and glaucomatous donor eyes was performed. A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells. Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein. Enhanced levels of LC3-II were found in both LC cells and optic nerve head sections from glaucoma donors. Increased lipofuscin formation is characteristic of LC cells from donors with glaucoma. This finding confirms the importance of oxidative stress in glaucoma pathogenesis. Intracellular lipofuscin accumulation may have important effects on autophagy the modification of which could form the basis for future novel glaucoma treatments.

BRCA1: A Novel Prognostic Factor in Resected Non-Small-Cell Lung Cancer

PubMed Central

Rosell, Rafael; Skrzypski, Marcin; Jassem, Ewa; Taron, Miquel; Bartolucci, Roberta; Sanchez, Jose Javier; Mendez, Pedro; Chaib, Imane; Perez-Roca, Laia; Szymanowska, Amelia; Rzyman, Witold; Puma, Francesco; Kobierska-Gulida, Grazyna; Farabi, Raffaele; Jassem, Jacek

2007-01-01

Background Although early-stage non-small-cell lung cancer (NSCLC) is considered a potentially curable disease following complete resection, patients have a wide spectrum of survival according to stage (IB, II, IIIA). Within each stage, gene expression profiles can identify patients with a higher risk of recurrence. We hypothesized that altered mRNA expression in nine genes could help to predict disease outcome: excision repair cross-complementing 1 (ERCC1), myeloid zinc finger 1 (MZF1) and Twist1 (which regulate N-cadherin expression), ribonucleotide reductase subunit M1 (RRM1), thioredoxin-1 (TRX1), tyrosyl-DNA phosphodiesterase (Tdp1), nuclear factor of activated T cells (NFAT), BRCA1, and the human homolog of yeast budding uninhibited by benzimidazole (BubR1). Methodology and Principal Findings We performed real-time quantitative polymerase chain reaction (RT-QPCR) in frozen lung cancer tissue specimens from 126 chemonaive NSCLC patients who had undergone surgical resection and evaluated the association between gene expression levels and survival. For validation, we used paraffin-embedded specimens from 58 other NSCLC patients. A strong inter-gene correlation was observed between expression levels of all genes except NFAT. A Cox proportional hazards model indicated that along with disease stage, BRCA1 mRNA expression significantly correlated with overall survival (hazard ratio [HR], 1.98 [95% confidence interval (CI), 1.11-6]; P = 0.02). In the independent cohort of 58 patients, BRCA1 mRNA expression also significantly correlated with survival (HR, 2.4 [95%CI, 1.01-5.92]; P = 0.04). Conclusions Overexpression of BRCA1 mRNA was strongly associated with poor survival in NSCLC patients, and the validation of this finding in an independent data set further strengthened this association. Since BRCA1 mRNA expression has previously been linked to differential sensitivity to cisplatin and antimicrotubule drugs, BRCA1 mRNA expression may provide additional information for customizing adjuvant antimicrotubule-based chemotherapy, especially in stage IB, where the role of adjuvant chemotherapy has not been clearly demonstrated. PMID:17987116

Role of Na+/K+-ATPase in Natriuretic Effect of Prolactin in a Model of Cholestasis of Pregnancy.

PubMed

Abramicheva, P A; Balakina, T A; Bulaeva, O A; Guseva, A A; Lopina, O D; Smirnova, O V

2017-05-01

Participation of Na+/K+-ATPase in the natriuretic effect of prolactin in a cholestasis of pregnancy model was investigated. The Na+/K+-ATPase activity in rat kidney medulla, where active sodium reabsorption occurs, decreased in the model of cholestasis of pregnancy and other hyperprolactinemia types compared with intact animals. This effect was not connected with the protein level of α1- and β-subunits of Na+/K+-ATPase measured by Western blotting in the kidney medulla. Decrease in Na+/K+-ATPase activity in the kidney cortex was not significant, as well as decrease in the quantity of mRNA and proteins of the α1- and β-subunits of Na+/K+-ATPase. There were no correlations between the Na+/K+-ATPase activity and sodium clearance, although sodium clearance increased significantly in the model of cholestasis of pregnancy and other hyperprolactinemia groups under conditions of stable glomerular filtration rate measured by creatinine clearance. We conclude that the Na+/K+-ATPase is not the only mediator of the natriuretic effect of prolactin in the model of cholestasis of pregnancy.

The control of lambda DNA terminase synthesis.

PubMed Central

Murialdo, H; Davidson, A; Chow, S; Gold, M

1987-01-01

Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques. Images PMID:3029667

Transcription elongation factors are involved in programming hormone production in pituitary neuroendocrine GH4C1 cells.

PubMed

Fujita, Toshitsugu; Piuz, Isabelle; Schlegel, Werner

2010-05-05

Transcription elongation of many eukaryotic genes is regulated. Two negative transcription elongation factors, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) are known to stall collaboratively RNA polymerase II promoter proximally. We discovered that DSIF and NELF are linked to hormone expression in rat pituitary GH4C1 cells. When NELF-E, a subunit of NELF or Spt5, a subunit of DSIF was stably knocked-down, prolactin (PRL) expression was increased both at the mRNA and protein levels. In contrast, stable knock-down of only Spt5 abolished growth hormone (GH) expression. Transient NELF-E knock-down increased coincidentally PRL expression and enhanced transcription of a PRL-promoter reporter gene. However, no direct interaction of NELF with the PRL gene could be demonstrated by chromatin immuno-precipitation. Thus, NELF suppressed PRL promoter activity indirectly. In conclusion, transcription regulation by NELF and DSIF is continuously involved in the control of hormone production and may contribute to neuroendocrine cell differentiation. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

The role of calsenilin/DREAM/KChIP3 in contextual fear conditioning.

PubMed

Alexander, Jon C; McDermott, Carmel M; Tunur, Tumay; Rands, Vicky; Stelly, Claire; Karhson, Debra; Bowlby, Mark R; An, W Frank; Sweatt, J David; Schrader, Laura A

2009-03-01

Potassium channel interacting proteins (KChIPs) are members of a family of calcium binding proteins that interact with Kv4 potassium (K(+)) channel primary subunits and also act as transcription factors. The Kv4 subunit is a primary K(+) channel pore-forming subunit, which contributes to the somatic and dendritic A-type currents throughout the nervous system. These A-type currents play a key role in the regulation of neuronal excitability and dendritic processing of incoming synaptic information. KChIP3 is also known as calsenilin and as the transcription factor, downstream regulatory element antagonist modulator (DREAM), which regulates a number of genes including prodynorphin. KChIP3 and Kv4 primary channel subunits are highly expressed in hippocampus, an area of the brain important for learning and memory. Through its various functions, KChIP3 may play a role in the regulation of synaptic plasticity and learning and memory. We evaluated the role of KChIP3 in a hippocampus-dependent memory task, contextual fear conditioning. Male KChIP3 knockout (KO) mice showed significantly enhanced memory 24 hours after training as measured by percent freezing. In addition, we found that membrane association and interaction with Kv4.2 of KChIP3 protein was significantly decreased and nuclear KChIP3 expression was increased six hours after the fear conditioning training paradigm with no significant change in KChIP3 mRNA. In addition, prodynorphin mRNA expression was significantly decreased six hours after fear conditioning training in wild-type (WT) but not in KO animals. These data suggest a role for regulation of gene expression by KChIP3/DREAM/calsenilin in consolidation of contextual fear conditioning memories.

Cadmium Induces Liver Cell Apoptosis through Caspase-3A Activation in Purse Red Common Carp (Cyprinus carpio)

PubMed Central

Qiao, Panpan; Liu, Shen; Zhang, Li; He, Penghui; Zhang, Xiaoyan; Wang, Yannan; Min, Weiping

2013-01-01

Caspase-3, the essential effector caspase, plays a pivotal role during caspase-dependent apoptosis. In this study, we isolated and characterized caspase-3A gene from common carp. The common carp caspase-3A comprising 273 amino acids showed 71.8% sequence similarity and 59.3% sequence identity to human caspase-3. It exhibited an evolutionarily conserved structure of mammalian caspase-3 genes, including a pro-domain, a large subunit, a small subunit and other motifs such as the pentapeptide active-site motif (QACRG) and the putative cleavage sites at the aspartic acids. Phylogenetic analysis demonstrated that common carp caspase-3A formed a clade with cyprinid fish caspase-3. To assess whether caspase-3A is involved in cadmium (Cd)-induced cell apoptosis in common carp, a Cd exposure experiment was performed. TUNEL analysis showed that Cd triggered liver cell apoptosis; caspase-3A activity was markedly increased; its proenzyme level was significantly decreased, and the levels of its cleaved forms were markedly increased. However, real-time quantitative PCR analysis revealed that the mRNA transcript level of caspase-3A was not significantly elevated. Immunoreactivities were observed in the cytoplasm of hepatocytes by immunohistochemical detection. The findings indicates that Cd can trigger liver cell apoptosis through the activation of caspase-3A. Caspase-3A may play an essential role in Cd-induced apoptosis. PMID:24349509

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

PubMed Central

Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

2014-01-01

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

Reduced Insulin/Insulin-like Growth Factor-1 Signaling and Dietary Restriction Inhibit Translation but Preserve Muscle Mass in Caenorhabditis elegans

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

Depuydt, Geert; Xie, Fang; Petyuk, Vladislav A.

Reduced signaling through the C. elegans insulin/IGF1 like tyrosine kinase receptor daf2 and dietary restriction via bacterial dilution are two well-characterized lifespan-extending interventions that operate in parallel or through (partially) independent mechanisms. Using accurate mass and time tag LCMS/MS quantitative proteomics we detected that the abundance of a large number of ribosomal subunits is decreased in response to dietary restriction as well as in the daf2(e1370) insulin/IGF1 receptor mutant. In addition, general protein synthesis levels in these long-lived worms are repressed. Surprisingly, ribosomal transcript levels were not correlated to actual protein abundance, suggesting that posttranscriptional regulation determines ribosome content. Proteomicsmore » also revealed increased presence of many structural muscle cell components in long-lived worms, which appears to result from prioritized preservation of muscle cell volume in nutrient-poor conditions or low insulin-like signaling. Activation of DAF16, but not diet-restriction, stimulates mRNA expression of muscle-related genes to prevent muscle atrophy. Important daf2 specific proteome changes include overexpression of aerobic metabolism enzymes and a general activation of stress responsive and immune defense systems, while increased abundance of many protein subunits of the proteasome core complex is a DR-specific characteristic.« less

Characteristics of ethanol-induced behavioral sensitization in rats: Molecular mediators and cross-sensitization between ethanol and cocaine.

PubMed

Xu, Shijie; Kang, Ung Gu

2017-09-01

Repeated exposure to drugs of abuse can induce a progressive increase in locomotor activity, known as behavioral sensitization. However, little is known about behavioral sensitization to ethanol. We examined whether ethanol could induce behavioral sensitization and investigated several molecular changes accompanying sensitization. We also assessed whether "cross-sensitization" occurred between ethanol and cocaine, another abused drug. Ethanol-induced sensitization was examined in rats after ethanol treatment (0.5 or 2g/kg) for 15days. The biochemical effects of low- or high-dose ethanol were examined in terms of N-methyl-d-aspartate (NMDA) receptor subunit phosphorylation or expression. Neuronal activity after ethanol treatment was assessed by measuring the level of early growth response (Egr-1) expression. Ethanol-induced behavioral sensitization was observed at the low dose (0.5g/kg) but not the high dose (2g/kg). Although acute treatment with the sensitizing dose of ethanol robustly increased Egr-1 protein and mRNA levels, the expression and phosphorylation of NMDA receptor subunits were not affected. The biochemical responses to ethanol seemed to be enhanced in ethanol-sensitized animals. Cross-sensitization between ethanol and cocaine was observed, which supports the hypothesis that there are commonalities among substances in the pathophysiology of substance dependence. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolation and Characterization of the PKAr Gene From a Plant Pathogen, Curvularia lunata.

PubMed

Liu, T; Ma, B C; Hou, J M; Zuo, Y H

2014-09-01

By using EST database from a full-length cDNA library of Curvularia lunata, we have isolated a 2.9 kb cDNA, termed PKAr. An ORF of 1,383 bp encoding a polypeptide of 460 amino acids with molecular weight 50.1 kDa, (GeneBank Acc. No. KF675744) was cloned. The deduced amino acid sequence of the PKAr shows 90 and 88 % identity with cAMP-dependent protein kinase A regulatory subunit from Alternaria alternate and Pyrenophora tritici-repentis Pt-1C-BFP, respectively. Database analysis revealed that the deduced amino acid sequence of PKAr shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. No introns were identified within the 1,383 bp of ORF compared with PKAr genomic DNA sequence. Southern blot indicated that PKAr existed as a single copy per genome. The mRNA expression level of PKAr in different development stages were demonstrated using real-time quantitative PCR. The results showed that the level of PKAr expression was highest in vegetative growth mycelium, which indicated it might play an important role in the vegetative growth of C. lunata. These results provided a fundamental supporting research on the function of PKAr in plant pathogen, C. lunata.

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

PubMed Central

Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

1988-01-01

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

Protective effects of 3-alkyl luteolin derivatives are mediated by Nrf2 transcriptional activity and decreased oxidative stress in Huntington's disease mouse striatal cells.

PubMed

Oliveira, Ana M; Cardoso, Susana M; Ribeiro, Márcio; Seixas, Raquel S G R; Silva, Artur M S; Rego, A Cristina

2015-12-01

Huntington's disease (HD) is a polyglutamine-expansion neurodegenerative disorder caused by increased number of CAG repeats in the HTT gene, encoding for the huntingtin protein. The mutation is linked to several intracellular mechanisms, including oxidative stress. Flavones are compounds with a protective role in neurodegenerative pathologies. In the present study we analyzed the protective effect of luteolin (Lut, 3',4',5,7-tetrahydroxyflavone) and four luteolin derivatives bearing 3-alkyl chains of 1, 4, 6 and 10 carbons (Lut-C1, Lut-C4, Lut-C6, Lut-C10) in striatal cells derived from HD knock-in mice expressing mutant Htt (STHdh(Q111/Q111)) versus wild-type striatal cells (STHdh(Q7/Q7)). HD cells showed increased caspase-3-like activity and intracellular reactive oxygen species (ROS), which were significantly decreased following treatment with Lut-C4 and Lut-C6 under concentrations that enhanced cell viability. Interestingly, Lut-C4 and Lut-C6 rose the nuclear levels of phospho(Ser40)-nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and Nrf2/ARE transcriptional activity. Concordantly with increased Nrf2/ARE transcription, Lut-C6 enhanced superoxide dismutase 1 (SOD1) mRNA and SOD activity and glutamate-cysteine ligase catalytic subunit (GCLc) mRNA and protein levels, while Lut-C4 induced mRNA levels of GCLc only in mutant striatal cells. Data suggest that Lut-C6 luteolin derivative (in particular) might be relevant for the development of antioxidant strategies in HD. Copyright © 2015 Elsevier Ltd. All rights reserved.

Goldfish neurokinin B: Cloning, tissue distribution, and potential role in regulating reproduction.

PubMed

Qi, Xin; Zhou, Wenyi; Li, Shuisheng; Liu, Yun; Ye, Gang; Liu, Xiaochun; Peng, Chun; Zhang, Yong; Lin, Haoran

2015-09-15

Neurokinin B (NKB) is a member of the tackykinin (TAC) family known to play a critical role in the neuroendocrine regulation of reproduction in mammals. However, its biological functions in teleosts are less clear. The aim of this study was to determine the role of NKB in fish reproduction using goldfish as a model. Two transcripts, TAC3a and TAC3b, which encode several NKBs, including NKBa-13, NKBa-10, NKBb-13, and NKBb-11, were cloned. Phylogenetic analysis revealed that NKBa-10 and NKBb-11 are closely related to mammalian NKB, while NKB-13s are more conserved in teleosts. Quantitative real-time PCR analyses in various tissues showed that TAC3a and TAC3b mRNAs were mainly expressed in the brain. In situ hybridization further detected TAC3a and TAC3b mRNAs in several regions of the brain known to be involved in the regulation of reproduction and metabolism, as well as in the neurohypophysis of the pituitary. To investigate the potential role of NKBs in reproduction, goldfish were injected intraperitoneally with synthetic NKBa-13, -10, NKBb-13, or -11 peptides and the mRNA levels of hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary gonadotropin subunits were measured. NKBa-13, -10, or NKBb-13, but not -11, significantly increased hypothalamic salmon GnRH and pituitary FSHβ and LHβ mRNA levels in both female and male goldfish. Finally, ovariectomy increased, while estradiol replacement reduced, TAC3a mRNA levels without affecting TAC3b expression in the hypothalamus. These data suggest that NKBa-13, -10, and NKBb-13 play a role in mediating the estrogen negative feedback regulation of gonadotropins. Copyright © 2014 Elsevier Inc. All rights reserved.

The mRNA expression of P450 aromatase, gonadotropin beta-subunits and FTZ-F1 in the orange-spotted grouper (Epinephelus Coioides) during 17alpha-methyltestosterone-induced precocious sex change.

PubMed

Zhang, Weimin; Zhang, Yong; Zhang, Lihong; Zhao, Huihong; Li, Xin; Huang, He; Lin, Haoran

2007-06-01

The orange-spotted grouper Epinephelus coioides is a protogynous hermaphroditic fish, but the physiological basis of its sex change remains largely unknown. In the present study, the 2-year-old orange-spotted grouper was induced to change sex precociously by oral administration of 17alpha-methyltestosterone (MT, 50 mg/Kg diet, twice a day at daily ration of 5% bodyweight) for 60 days. The serum testosterone levels were significantly elevated after MT treatment for 20 and 40 days as compared to control, but the levels of serum estradiol (E(2)) remained unchanged. The expression of P450aromA in the gonad significantly decreased after MT treatment for 20, 40, and 60 days. Accordingly, the enzyme activity of gonadal aromatase was also lower. The expression of FSHbeta subunit in the pituitary was significantly decreased after MT treatment for 20 days, but returned to the control levels after 40 and 60 days; however, the expression of LHbeta subunit was not altered significantly by MT treatment. The expression of FTZ-F1 in the gonad also decreased significantly in response to MT treatment for 40 and 60 days, but its expression in the pituitary was not altered significantly. Interestingly, when tested in vitro on ovarian fragments, MT had no direct effect on the expression of P450aromA and FTZ-F1 as well as the activity of gonadal aromatase, suggesting that the inhibition of gonadal P450aromatase and FTZ-F1 by MT may be mediated at upper levels of the brain-pituitary-gonadal axis. Taken together, these results indicated that FSH, P450aromA, FTZ-F1, and serum testosterone are associated with the MT-induced sex change of the orange-spotted grouper, but the cause-effect relationship between these factors and sex change in this species remains to be characterized. (c) 2006 Wiley-Liss, Inc.

Ammonia excretion in Caenorhabditis elegans: mechanism and evidence of ammonia transport of the Rhesus protein CeRhr-1

PubMed Central

Adlimoghaddam, Aida; Boeckstaens, Mélanie; Marini, Anna-Maria; Treberg, Jason R.; Brassinga, Ann-Karen C.; Weihrauch, Dirk

2015-01-01

ABSTRACT The soil-dwelling nematode Caenorhabditis elegans is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2 µmol gFW−1 day−1) and very little urea (0.21±0.004 µmol gFW−1 day−1). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA expression levels of the Rhesus protein CeRhr-2, V-type H+-ATPase (subunit A) and Na+/K+-ATPase (α-subunit) decreased correspondingly. Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H+-ATPase, carbonic anhydrase, Na+/K+-ATPase, and a functional microtubule network. These findings indicate that ammonia is excreted, not only by apical ammonia trapping, but also via vesicular transport and exocytosis. Exposure to 1 mmol l−1 NH4Cl caused a 10-fold increase in body ammonia and a tripling of ammonia excretion rates. Gene expression levels of CeRhr-1 and CeRhr-2, V-ATPase and Na+/K+-ATPase also increased significantly in response to 1 mmol l−1 NH4Cl. Importantly, a functional expression analysis showed, for the first time, ammonia transport capabilities for CeRhr-1 in a phylogenetically ancient invertebrate system, identifying these proteins as potential functional precursors to the vertebrate ammonia-transporting Rh-glycoproteins. PMID:25740900

Characterization of GABAA receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells

PubMed Central

Yuan, Nina Y.; Poe, Michael M.; Witzigmann, Christopher; Cook, James M.; Stafford, Douglas; Arnold, Leggy A.

2016-01-01

Introduction Automated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABAA receptor. Methods iCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABAA receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABAA receptor subunits were quantified by qRT-PCR. Results We have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15-25 MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC50 of 0.43 μM. Positive GABAA receptor modulators diazepam, HZ166, and CW-04-020 exhibited EC50 values of 0.42 μM, 1.56 μM, and 0.23 μM, respectively. The α2/α3/α5 selective compound HZ166-induced the highest potentiation (efficacy) of 810% of the current induced by 100 nM GABA. Quantification of GABAA receptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain. Discussion iCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABAA receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change. PMID:27544543

Involvement of PI3K, Akt, and RhoA in oestradiol regulation of cardiac iNOS expression.

PubMed

Zafirovic, Sonja; Sudar-Milovanovic, Emina; Obradovic, Milan; Djordjevic, Jelena; Jasnic, Nebojsa; Borovic, Milica Labudovic; Isenovic, Esma R

2018-02-12

Oestradiol is an important regulatory factor with several positive effects on the cardiovascular (CV) system. We evaluated the molecular mechanism of the in vivo effects of oestradiol on the regulation of cardiac inducible nitric oxide (NO) synthase (iNOS) expression and activity. Male Wistar rats were treated with oestradiol (40 mg/kg, intraperitoneally) and after 24 h the animals were sacrificed. The concentrations of NO and L-Arginine (L-Arg) were determined spectrophotometrically. For protein expressions of iNOS, p65 subunit of nuclear factor-κB (NFκB-p65), Ras homolog gene family-member A (RhoA), angiotensin II receptor type 1 (AT1R), insulin receptor substrate 1 (IRS-1), p85, p110 and protein kinase B (Akt), Western blot method was used. Co-immunoprecipitation was used for measuring the association of IRS-1 with the p85 subunit of phosphatidylinositol-3-kinase (PI3K). The expression of iNOS messenger ribonucleic acid (mRNA) was measured with the quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis of the tissue was used to detect localization and expression of iNOS in heart tissue. Oestradiol treatment reduced L-Arg concentration (p<0.01), iNOS mRNA (p<0.01) and protein (p<0.001) expression, level of RhoA (p<0.05) and AT1R (p<0.001) protein. In contrast, plasma NO (p<0.05), Akt phosphorylation at Thr308 (p<0.05) and protein level of p85 (p<0.001) increased after oestradiol treatment. Our results suggest that oestradiol in vivo regulates cardiac iNOS expression via the PI3K/Akt signaling pathway, through attenuation of RhoA and AT1R. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

17 beta-estradiol modifies nitric oxide-sensitive guanylyl cyclase expression and down-regulates its activity in rat anterior pituitary gland.

PubMed

Cabilla, Jimena P; Díaz, María del Carmen; Machiavelli, Leticia I; Poliandri, Ariel H; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

2006-09-01

Previous studies showed that 17 beta-estradiol (17 beta-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17 beta-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17 beta-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17 beta-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17 beta-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing alpha1 and decreasing beta1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6-9 h after 17 beta-E2 administration. 17beta-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17 beta-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17 beta-E2, because they were not observed with the administration of other steroids such as progesterone and 17 alpha-estradiol. This inhibitory action of 17beta-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17 beta-E2 action. 17 beta-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17 beta-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC beta 1 subunit and sGC activity in a specific, ER-dependent manner.

Effects of KCNQ2 gene truncation on M-type Kv7 potassium currents.

PubMed

Robbins, Jon; Passmore, Gayle M; Abogadie, Fe C; Reilly, Joanne M; Brown, David A

2013-01-01

The KCNQ2 gene product, Kv7.2, is a subunit of the M-channel, a low-threshold voltage-gated K(+) channel that regulates mammalian and human neuronal excitability. Spontaneous mutations one of the KCNQ2 genes cause disorders of neural excitability such as Benign Familial Neonatal Seizures. However there appear to be no reports in which both human KCNQ2 genes are mutated. We therefore asked what happens to M-channel function when both KCNQ2 genes are disrupted. We addressed this using sympathetic neurons isolated from mice in which the KCNQ2 gene was truncated at a position corresponding to the second transmembrane domain of the Kv7.2 protein. Since homozygote KCNQ2-/- mice die postnatally, experiments were largely restricted to neurons from late embryos. Quantitative PCR revealed an absence of KCNQ2 mRNA in ganglia from KCNQ2-/- embryos but 100-120% increase of KCNQ3 and KCNQ5 mRNAs; KCNQ2+/- ganglia showed ∼30% less KCNQ2 mRNA than wild-type (+/+) ganglia but 40-50% more KCNQ3 and KCNQ5 mRNA. Neurons from KCNQ2-/- embryos showed a complete absence of M-current, even after applying the Kv7 channel enhancer, retigabine. Neurons from heterozygote KCNQ2+/- embryos had ∼60% reduced M-current. In contrast, M-currents in neurons from adult KCNQ2+/- mice were no smaller than those in neurons from wild-type mice. Measurements of tetraethylammonium block did not indicate an increased expression of Kv7.5-containing subunits, implying a compensatory increase in Kv7.2 expression from the remaining KCNQ2 gene. We conclude that mouse embryonic M-channels have an absolute requirement for Kv7.2 subunits for functionality, that the reduced M-channel activity in heterozygote KCNQ2+/- mouse embryos results primarily from a gene-dosage effect, and that there is a compensatory increase in Kv7.2 expression in adult mice.

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

PubMed

Gallenberger, Martin; Meinel, Dominik M; Kroeber, Markus; Wegner, Michael; Milkereit, Philipp; Bösl, Michael R; Tamm, Ernst R

2011-02-01

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.

PubMed

Kumar, Parimal; Hellen, Christopher U T; Pestova, Tatyana V

2016-07-01

Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5'-terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5' end of mRNA, implying that Met-tRNAi (Met) inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAi (Met) can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m(7)G cap specifically interacts with eIF3l. © 2016 Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

PubMed Central

Aitken, Colin Echeverría; Beznosková, Petra; Vlčkova, Vladislava; Chiu, Wen-Ling; Zhou, Fujun; Valášek, Leoš Shivaya; Hinnebusch, Alan G; Lorsch, Jon R

2016-01-01

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA. DOI: http://dx.doi.org/10.7554/eLife.20934.001 PMID:27782884

Antepartal insulin-like growth factor concentrations indicating differences in the metabolic adaptive capacity of dairy cows

PubMed Central

Holzhausen, Lars; Araujo, Marcelo Gil; Heppelmann, Maike; Sipka, Anja; Pfarrer, Chistiane; Schuberth, Hans-Joachim; Bollwein, Heinrich

2014-01-01

Cows with different Insulin-like Growth Factor-I (IGF-I) concentrations showed comparable expression levels of hepatic growth hormone receptor (GHR). Suppressor of cytokine signaling 2 (SOCS2), could be responsible for additional inhibition of the GHR signal cascade. The aims were to monitor cows with high or low antepartal IGF-I concentrations (IGF-Ihigh or IGF-Ilow), evaluate the interrelationships of endocrine endpoints, and measure hepatic SOCS2 expression. Dairy cows (n = 20) were selected (240 to 254 days after artificial insemination (AI)). Blood samples were drawn daily (day -17 until calving) and IGF-I, GH, insulin, thyroid hormones, estradiol, and progesterone concentrations were measured. Liver biopsies were taken (day 264 ± 1 after AI and postpartum) to measure mRNA expression (IGF-I, IGFBP-2, IGFBP-3, IGFBP-4, acid labile subunit (ALS), SOCS2, deiodinase1, GHR1A). IGF-I concentrations in the two groups were different (p < 0.0001). However, GH concentrations and GHR1A mRNA expression were comparable (p > 0.05). Thyroxine levels and ALS expression were higher in the IGF-Ihigh cows compared to IGF-Ilow cows. Estradiol concentration tended to be greater in the IGF-Ilow group (p = 0.06). It was hypothesized that low IGF-I levels are associated with enhanced SOCS2 expression although this could not be decisively confirmed by the present study. PMID:24962413

Antepartal insulin-like growth factor concentrations indicating differences in the metabolic adaptive capacity of dairy cows.

PubMed

Piechotta, Marion; Holzhausen, Lars; Araujo, Marcelo Gil; Heppelmann, Maike; Sipka, Anja; Pfarrer, Chistiane; Schuberth, Hans-Joachim; Bollwein, Heinrich

2014-01-01

Cows with different Insulin-like Growth Factor-I (IGF-I) concentrations showed comparable expression levels of hepatic growth hormone receptor (GHR). Suppressor of cytokine signaling 2 (SOCS2), could be responsible for additional inhibition of the GHR signal cascade. The aims were to monitor cows with high or low antepartal IGF-I concentrations (IGF-I(high) or IGF-I(low)), evaluate the interrelationships of endocrine endpoints, and measure hepatic SOCS2 expression. Dairy cows (n = 20) were selected (240 to 254 days after artificial insemination (AI)). Blood samples were drawn daily (day -17 until calving) and IGF-I, GH, insulin, thyroid hormones, estradiol, and progesterone concentrations were measured. Liver biopsies were taken (day 264 ± 1 after AI and postpartum) to measure mRNA expression (IGF-I, IGFBP-2, IGFBP-3, IGFBP-4, acid labile subunit (ALS), SOCS2, deiodinase1, GHR1A). IGF-I concentrations in the two groups were different (p < 0.0001). However, GH concentrations and GHR1A mRNA expression were comparable (p > 0.05). Thyroxine levels and ALS expression were higher in the IGF-I(high) cows compared to IGF-I(low) cows. Estradiol concentration tended to be greater in the IGF-I(low) group (p = 0.06). It was hypothesized that low IGF-I levels are associated with enhanced SOCS2 expression although this could not be decisively confirmed by the present study.

Antiallergic effect of fisetin on IgE-mediated mast cell activation in vitro and on passive cutaneous anaphylaxis (PCA).

PubMed

Jo, Woo-Ri; Park, Hye-Jin

2017-10-01

Fisetin (3,7,3',4'-tetrahydroxyflavone), a naturally occurring bioactive flavonoid, has been shown to inhibit inflammation. However, little is known about the effect of fisetin on immunoglobulin E (IgE)-mediated allergic responses. In this study, the effect of fisetin on rat basophilic leukemia (RBL-2H3) cell-mediated allergic reactions was investigated. Fisetin inhibited β-hexosaminidase release and decreased the level of interleukin-4 and tumor necrosis factor-α mRNA in IgE/antigen (IgE/Ag)-stimulated RBL-2H3 cells. To elucidate the antiallergic mechanism, we examined the levels of signaling molecules responsible for degranulation and release of inflammatory cytokines. Fisetin decreased the levels of activated spleen tyrosine kinase, Gab2 proteins, linker of activated T cells, extracellular signal-related kinase 1/2 in the IgE/Ag-stimulated RBL2H3 cells, and NFκB and STAT3 proteins activated in the ear tissue of mice with passive cutaneous anaphylaxis (PCA). In addition, fisetin significantly lowered of FcɛRI α-subunit mRNA expression. Consistent with the cellular data, fisetin markedly suppressed RBL-2H3 cell-dependent PCA in IgE/Ag-sensitized mice. These results suggest that fisetin may have potential as a therapeutic agent for the treatment of allergic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Cbp80 is needed for the expression of piRNA components and piRNAs

PubMed Central

Colombo, Martino; Hernandez, Greco; Beuchle, Dirk; Berger, Fabienne; Peischl, Stephan; Bruggmann, Rémy

2017-01-01

Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts. PMID:28746365

Biochemical mechanisms of imidacloprid resistance in Nilaparvata lugens: over-expression of cytochrome P450 CYP6AY1.

PubMed

Ding, Zhiping; Wen, Yucong; Yang, Baojun; Zhang, Yixi; Liu, Shuhua; Liu, Zewen; Han, Zhaojun

2013-11-01

Imidacloprid is a key insecticide extensively used for control of Nilaparvata lugens, and its resistance had been reported both in the laboratory selected strains and field populations. A target site mutation Y151S in two nicotinic acetylcholine receptor subunits and enhanced oxidative detoxification have been identified in the laboratory resistant strain, contributing importantly to imidacloprid resistance in N. lugens. To date, however, imidacloprid resistance in field population is primarily attributable to enhanced oxidative detoxification by over-expressed P450 monooxygenases. A resistant strain (Res), originally collected from a field population and continuously selected in laboratory with imidacloprid for more than 40 generations, had 180.8-fold resistance to imidacloprid, compared to a susceptible strain (Sus). Expression of different putative P450 genes at mRNA levels was detected and compared between Res and Sus strains, and six genes were found expressed significantly higher in Res strain than in Sus strain. CYP6AY1 was found to be the most different expressed P450 gene and its mRNA level in Res strain was 17.9 times of that in Sus strain. By expressing in E. coli cells, CYP6AY1 was found to metabolize imidacloprid efficiently with initial velocity calculated of 0.851 ± 0.073 pmol/min/pmol P450. When CYP6AY1 mRNA levels in Res strain was reduced by RNA interference, imidacloprid susceptibility was recovered. In four field populations with different resistance levels, high levels of CYP6AY1 transcript were also found. In vitro and in vivo studies provided evidences that the over-expression of CYP6AY1 was one of the key factors contributing to imidacloprid resistance in the laboratory selected strain Res, which might also be the important mechanism for imidacloprid resistance in field populations, when the target site mutation was not prevalent at present. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ribosomal Protein Rps26 Influences 80S Ribosome Assembly in Saccharomyces cerevisiae

PubMed Central

Belyy, Alexander; Levanova, Nadezhda; Tabakova, Irina; Rospert, Sabine

2016-01-01

ABSTRACT The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5′ untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62–K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62–K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62–K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62–K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62–K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5′ untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62–K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast. PMID:27303706

Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

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

Ji, Cheng; Graduate University of Chinese Academy of Sciences, Beijing; Jin, Xia

Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in themore » pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.« less

Molecular characterization and mRNA expression of two key enzymes of hypoxia-sensing pathways in eastern oysters Crassostrea virginica (Gmelin): Hypoxia-inducible factor α (HIF-α) and HIF-prolyl hydroxylase (PHD)

PubMed Central

Piontkivska, Helen; Chung, J. Sook; Ivanina, Anna V.; Sokolov, Eugene P.; Techa, Sirinart; Sokolova, Inna M.

2010-01-01

Oxygen homeostasis is crucial for development, survival and normal function of all metazoans. A family of transcription factors called hypoxia-inducible factors (HIF) is critical in mediating the adaptive responses to reduced oxygen availability. The HIF transcription factor consists of a constitutively expressed β subunit and an oxygen-dependent α subunit; the abundance of the latter determines the activity of HIF and is regulated by a family of O2- and Fe2+-dependent enzymes prolyl hydroxylases (PHDs). Currently very little is known about the function of this important pathway and the molecular structure of its key players in hypoxia-tolerant intertidal mollusks including oysters, which are among the animal champions of anoxic and hypoxic tolerance and thus can serve as excellent models to study the role of HIF cascade in adaptations to oxygen deficiency. We have isolated transcripts of two key components of the oxygen sensing pathway - the oxygen-regulated HIF-α subunit and PHD - from an intertidal mollusk, the eastern oyster Crassostrea virginica, and determined the transcriptional responses of these two genes to anoxia, hypoxia and cadmium (Cd) stress. HIF-α and PHD homologs from eastern oysters C. virginica show significant sequence similarity and share key functional domains with the earlier described isoforms from vertebrates and invertebrates. Phylogenetic analysis shows that genetic diversification of HIF and PHD isoforms occurred within the vertebrate lineage indicating functional diversification and specialization of the oxygen-sensing pathways in this group, which parallels situation observed for many other important genes. HIF-α and PHD homologs are broadly expressed at the mRNA level in different oyster tissues and show transcriptional responses to prolonged hypoxia in the gills consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. Similarity in amino acid sequence, domain structure and transcriptional responses between HIF-α and PHD homologs from oysters and other invertebrate and vertebrate species implies the highly conserved functions of these genes throughout the evolutionary history of animals, in accordance with their critical role in oxygen sensing and homeostasis. PMID:21106446

Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

PubMed Central

Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

2011-01-01

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

Ablation of the auditory cortex results in changes in the expression of neurotransmission-related mRNAs in the cochlea.

PubMed

Lamas, Verónica; Juiz, José M; Merchán, Miguel A

2017-03-01

The auditory cortex (AC) dynamically regulates responses of the Organ of Corti to sound through descending connections to both the medial (MOC) and lateral (LOC) olivocochlear efferent systems. We have recently provided evidence that AC has a reinforcement role in the responses to sound of the auditory brainstem nuclei. In a molecular level, we have shown that descending inputs from AC are needed to regulate the expression of molecules involved in outer hair cell (OHC) electromotility control, such as prestin and the α10 nicotinic acetylcholine receptor (nAchR). In this report, we show that descending connections from AC to olivocochlear neurons are necessary to regulate the expression of molecules involved in cochlear afferent signaling. RT-qPCR was performed in rats at 1, 7 and 15 days after unilateral ablation of the AC, and analyzed the time course changes in gene transcripts involved in neurotransmission at the first auditory synapse. This included the glutamate metabolism enzyme glutamate decarboxylase 1 (glud1) and AMPA glutamate receptor subunits GluA2-4. In addition, gene transcripts involved in efferent regulation of type I spiral ganglion neuron (SGN) excitability mediated by LOC, such as the α7 nAchR, the D2 dopamine receptor, and the α1, and γ2 GABAA receptor subunits, were also investigated. Unilateral AC ablation induced up-regulation of GluA3 receptor subunit transcripts, whereas both GluA2 and GluA4 mRNA receptors were down-regulated already at 1 day after the ablation. Unilateral removal of the AC also resulted in up-regulation of the transcripts for α7 nAchR subunit, D2 dopamine receptor, and α1 GABAA receptor subunit at 1 day after the ablation. Fifteen days after the injury, AC ablations induced an up-regulation of glud1 transcripts. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Separate functional properties of NMDARs regulate distinct aspects of spatial cognition.

PubMed

Sanders, Erin M; Nyarko-Odoom, Akua O; Zhao, Kevin; Nguyen, Michael; Liao, Hong Hong; Keith, Matthew; Pyon, Jane; Kozma, Alyssa; Sanyal, Mohima; McHail, Daniel G; Dumas, Theodore C

2018-06-01

N -methyl-d-aspartate receptors (NMDARs) at excitatory synapses are central to activity-dependent synaptic plasticity and learning and memory. NMDARs act as ionotropic and metabotropic receptors by elevating postsynaptic calcium concentrations and by direct intracellular protein signaling. In the forebrain, these properties are controlled largely by the auxiliary GluN2 subunits, GluN2A and GluN2B. While calcium conductance through NMDAR channels and intracellular protein signaling make separate contributions to synaptic plasticity, it is not known if these properties individually influence learning and memory. To address this issue, we created chimeric GluN2 subunits containing the amino-terminal domain and transmembrane domains from GluN2A or GluN2B fused to the carboxy-terminal domain of GluN2B (termed ABc) or GluN2A ATD (termed BAc), respectively, and expressed these mutated GluN2 subunits in transgenic mice. Expression was confirmed at the mRNA level and protein subunit translation and translocation into dendrites were observed in forebrain neurons. In the spatial version of the Morris water maze, BAc mice displayed signs of a learning deficit. In contrast, ABc animals performed similarly to wild-types during training, but showed a more direct approach to the goal location during a long-term memory test. There was no effect of ABc or BAc expression in a nonspatial water escape task. Since background expression is predominantly GluN2A in mature animals, the results suggest that spatial learning is more sensitive to manipulations of the amino-terminal domain and transmembrane domains (calcium conductance) and long-term memory is regulated more by the carboxy-terminal domain (intracellular protein signaling). © 2018 Sanders et al.; Published by Cold Spring Harbor Laboratory Press.

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160.

PubMed

Perrin, Arnaud; Rousseau, Joël; Tremblay, Jacques P

2017-03-17

Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR) and proteins (immunohistochemistry and western blot) were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Identification and characterization of a gibberellin-regulated protein, which is ASR5, in the basal region of rice leaf sheaths.

PubMed

Takasaki, Hironori; Mahmood, Tariq; Matsuoka, Makoto; Matsumoto, Hiroshi; Komatsu, Setsuko

2008-04-01

Gibberellins (GAs) regulate growth and development in higher plants. To identify GA-regulated proteins during rice leaf sheath elongation, a proteomic approach was used. Proteins from the basal region of leaf sheath in rice seedling treated with GA(3) were analyzed by fluorescence two-dimensional difference gel electrophoresis. The levels of abscisic acid-stress-ripening-inducible 5 protein (ASR5), elongation factor-1 beta, translationally controlled tumor protein, fructose-bisphosphate aldolase and a novel protein increased; whereas the level of RuBisCO subunit binding-protein decreased by GA(3) treatment. ASR5 out of these six proteins was significantly regulated by GA(3) at the protein level but not at the mRNA level in the basal region of leaf sheaths. Since this protein is regulated not only by abscisic acid but also by GA(3), these results indicate that ASR5 might be involved in plant growth in addition to stress in the basal regions of leaf sheaths.

Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

PubMed

De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

2016-02-01

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Increased IL-27/IL-27R expression in association with the immunopathology of murine ocular toxoplasmosis.

PubMed

Tong, Xinxin; Chen, Shengjie; Zheng, Huanqin; Huang, Shiguang; Lu, Fangli

2018-05-19

Interleukin 27 (IL-27) is a member of the IL-6/IL-12 family, and IL-27 receptor (IL-27R) consists of WSX-1 (the IL-27Rα subunit) and the signal-transducing subunit gp130. Human and mouse mast cells (MCs) express the IL-27R. To explore the expressions of IL-27/IL-27R subunits (WSX-1 and gp130) during acute ocular toxoplasmosis (OT), we established mouse model by intraocular injection of 500 Toxoplasma gondii RH strain tachyzoites. Histopathological changes were analyzed, MCs were counted by toluidine blue staining, and tryptase + /IL-27 + MCs were examined by immunofluorescence double-staining in the eyes and cervical lymph nodes (CLNs) of T. gondii-infected mice. The mRNA expressions of IL-27p28, WSX-1, gp130, and tachyzoite specific surface antigen 1 (SAG1) in the eyes and CLNs of T. gondii-infected mice, and the expressions of WSX-1 and gp130 in the murine mastocytoma cell line P815 infected with T. gondii tachyzoites in vitro were examined by using quantitative real-time reverse transcription-polymerase chain reaction. Our results showed that, after T. gondii infection, severe histopathological changes, increased numbers of total MCs and degranulated MCs, elevated expressions of IL-27p28, WSX-1, and gp130 were found in the eyes and CLNs, and significant correlations between the levels of IL-27 and SAG1 existed in the eyes and CLNs of T. gondii-infected mice. In addition, increased levels of WSX-1 and gp130 were examined in T. gondii-infected P815 cells. Our data suggested that IL-27/IL-27R expression induced by T. gondii infection may regulate MC-mediated immune response during acute OT in mouse model.

Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia

PubMed Central

Catts, Vibeke Sørensen; Derminio, Dominique Suzanne; Hahn, Chang-Gyu; Weickert, Cynthia Shannon

2015-01-01

Background: There is converging evidence of involvement of N-methyl-d-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Our group recently identified a decrease in total NR1 mRNA and protein expression in the dorsolateral prefrontal cortex in a case-control study of individuals with schizophrenia (n=37/group). The NR1 subunit is critical to NMDA receptor function at the postsynaptic density, a cellular structure rich in the scaffolding protein, PSD-95. The extent to which the NMDA receptor NR1 subunit is altered at the site of action, in the postsynaptic density, is not clear. Aims: To extend our previous results by measuring levels of NR1 and PSD-95 protein in postsynaptic density-enriched fractions of prefrontal cortex from the same individuals in the case-control study noted above. Methods: Postsynaptic density-enriched fractions were isolated from fresh-frozen prefrontal cortex (BA10) and subjected to western blot analysis for NR1 and PSD-95. Results: We found a 20% decrease in NR1 protein (t(66)=−2.874, P=0.006) and a 30% decrease in PSD-95 protein (t(63)=−2.668, P=0.010) in postsynaptic density-enriched fractions from individuals with schizophrenia relative to unaffected controls. Conclusions: Individuals with schizophrenia have less NR1 protein, and therefore potentially fewer functional NMDA receptors, at the postsynaptic density. The associated decrease in PSD-95 protein at the postsynaptic density suggests that not only are glutamate receptors compromised in individuals with schizophrenia, but the overall spine architecture and downstream signaling supported by PSD-95 may also be deficient. PMID:27336043

Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia.

PubMed

Catts, Vibeke Sørensen; Derminio, Dominique Suzanne; Hahn, Chang-Gyu; Weickert, Cynthia Shannon

2015-01-01

There is converging evidence of involvement of N-methyl-d-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Our group recently identified a decrease in total NR1 mRNA and protein expression in the dorsolateral prefrontal cortex in a case-control study of individuals with schizophrenia (n=37/group). The NR1 subunit is critical to NMDA receptor function at the postsynaptic density, a cellular structure rich in the scaffolding protein, PSD-95. The extent to which the NMDA receptor NR1 subunit is altered at the site of action, in the postsynaptic density, is not clear. To extend our previous results by measuring levels of NR1 and PSD-95 protein in postsynaptic density-enriched fractions of prefrontal cortex from the same individuals in the case-control study noted above. Postsynaptic density-enriched fractions were isolated from fresh-frozen prefrontal cortex (BA10) and subjected to western blot analysis for NR1 and PSD-95. We found a 20% decrease in NR1 protein (t(66)=-2.874, P=0.006) and a 30% decrease in PSD-95 protein (t(63)=-2.668, P=0.010) in postsynaptic density-enriched fractions from individuals with schizophrenia relative to unaffected controls. Individuals with schizophrenia have less NR1 protein, and therefore potentially fewer functional NMDA receptors, at the postsynaptic density. The associated decrease in PSD-95 protein at the postsynaptic density suggests that not only are glutamate receptors compromised in individuals with schizophrenia, but the overall spine architecture and downstream signaling supported by PSD-95 may also be deficient.

SWI/SNF Associates with Nascent Pre-mRNPs and Regulates Alternative Pre-mRNA Processing

PubMed Central

Tyagi, Anu; Ryme, Jessica; Brodin, David; Östlund Farrants, Ann Kristin; Visa, Neus

2009-01-01

The SWI/SNF chromatin remodeling complexes regulate the transcription of many genes by remodeling nucleosomes at promoter regions. In Drosophila, SWI/SNF plays an important role in ecdysone-dependent transcription regulation. Studies in human cells suggest that Brahma (Brm), the ATPase subunit of SWI/SNF, regulates alternative pre-mRNA splicing by modulating transcription elongation rates. We describe, here, experiments that study the association of Brm with transcribed genes in Chironomus tentans and Drosophila melanogaster, the purpose of which was to further elucidate the mechanisms by which Brm regulates pre-mRNA processing. We show that Brm becomes incorporated into nascent Balbiani ring pre-mRNPs co-transcriptionally and that the human Brm and Brg1 proteins are associated with RNPs. We have analyzed the expression profiles of D. melanogaster S2 cells in which the levels of individual SWI/SNF subunits have been reduced by RNA interference, and we show that depletion of SWI/SNF core subunits changes the relative abundance of alternative transcripts from a subset of genes. This observation, and the fact that a fraction of Brm is not associated with chromatin but with nascent pre-mRNPs, suggest that SWI/SNF affects pre-mRNA processing by acting at the RNA level. Ontology enrichment tests indicate that the genes that are regulated post-transcriptionally by SWI/SNF are mostly enzymes and transcription factors that regulate postembryonic developmental processes. In summary, the data suggest that SWI/SNF becomes incorporated into nascent pre-mRNPs and acts post-transcriptionally to regulate not only the amount of mRNA synthesized from a given promoter but also the type of alternative transcript produced. PMID:19424417

Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2010-01-01

Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

Quantitative analysis of a deeply sequenced marine microbial metatranscriptome.

PubMed

Gifford, Scott M; Sharma, Shalabh; Rinta-Kanto, Johanna M; Moran, Mary Ann

2011-03-01

The potential of metatranscriptomic sequencing to provide insights into the environmental factors that regulate microbial activities depends on how fully the sequence libraries capture community expression (that is, sample-sequencing depth and coverage depth), and the sensitivity with which expression differences between communities can be detected (that is, statistical power for hypothesis testing). In this study, we use an internal standard approach to make absolute (per liter) estimates of transcript numbers, a significant advantage over proportional estimates that can be biased by expression changes in unrelated genes. Coastal waters of the southeastern United States contain 1 × 10(12) bacterioplankton mRNA molecules per liter of seawater (~200 mRNA molecules per bacterial cell). Even for the large bacterioplankton libraries obtained in this study (~500,000 possible protein-encoding sequences in each of two libraries after discarding rRNAs and small RNAs from >1 million 454 FLX pyrosequencing reads), sample-sequencing depth was only 0.00001%. Expression levels of 82 genes diagnostic for transformations in the marine nitrogen, phosphorus and sulfur cycles ranged from below detection (<1 × 10(6) transcripts per liter) for 36 genes (for example, phosphonate metabolism gene phnH, dissimilatory nitrate reductase subunit napA) to >2.7 × 10(9) transcripts per liter (ammonia transporter amt and ammonia monooxygenase subunit amoC). Half of the categories for which expression was detected, however, had too few copy numbers for robust statistical resolution, as would be required for comparative (experimental or time-series) expression studies. By representing whole community gene abundance and expression in absolute units (per volume or mass of environment), 'omics' data can be better leveraged to improve understanding of microbially mediated processes in the ocean.

The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

PubMed

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

2012-02-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

PubMed Central

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc

2012-01-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism. PMID:22156529

Estrogen Responsiveness of the TFIID Subunit TAF4B in the Normal Mouse Ovary and in Ovarian Tumors1

PubMed Central

Wardell, Jennifer R.; Hodgkinson, Kendra M.; Binder, April K.; Seymour, Kimberly A.; Korach, Kenneth S.; Vanderhyden, Barbara C.; Freiman, Richard N.

2013-01-01

ABSTRACT Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation. PMID:24068106

Estrogen responsiveness of the TFIID subunit TAF4B in the normal mouse ovary and in ovarian tumors.

PubMed

Wardell, Jennifer R; Hodgkinson, Kendra M; Binder, April K; Seymour, Kimberly A; Korach, Kenneth S; Vanderhyden, Barbara C; Freiman, Richard N

2013-11-01

Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation.

Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

PubMed

Vela, J; Vitorica, J; Ruano, D

2001-12-01

We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

Effect of caffeic acid phenethyl ester on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

PubMed

Choi, E-Y; Choe, S-H; Hyeon, J-Y; Choi, J-I; Choi, I S; Kim, S-J

2015-12-01

Caffeic acid phenethyl ester (CAPE) has numerous potentially beneficial properties, including antioxidant, immunomodulatory and anti-inflammatory activities. However, the effect of CAPE on periodontal disease has not been studied before. This study was designed to investigate the efficacy of CAPE in ameliorating the production of proinflammatory mediators in macrophages activated by lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease. LPS from P. intermedia ATCC 25611 was isolated by using the standard hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO), interleukin (IL)-1β and IL-6. We used real-time polymerase chain reaction to quantify inducible NO synthase, IL-1β, IL-6, heme oxygenase (HO)-1 and suppressors of cytokine signaling (SOCS) 1 mRNA expression. HO-1 protein expression and levels of signaling proteins were assessed by immunoblot analysis. DNA-binding activities of NF-κB subunits were analyzed by using the enzyme-linked immunosorbent assay-based kits. CAPE exerted significant inhibitory effects on P. intermedia LPS-induced production of NO, IL-1β and IL-6 as well as their mRNA expression in RAW264.7 cells. CAPE-induced HO-1 expression in cells activated with P. intermedia LPS, and selective inhibition of HO-1 activity by tin protoporphyrin IX attenuated the inhibitory effect of CAPE on LPS-induced NO production. CAPE did not interfere with IκB-α degradation induced by P. intermedia LPS. Instead, CAPE decreased nuclear translocation of NF-κB p65 and p50 subunits induced with LPS, and lessened LPS-induced p50 binding activity. Further, CAPE showed strong inhibitory effects on LPS-induced signal transducer and activator of transcription 1 and 3 phosphorylation. Besides, CAPE significantly elevated SOCS1 mRNA expression in P. intermedia LPS-stimulated cells. Modulation of host response by CAPE may represent an attractive strategy towards the treatment of periodontal disease. In vivo studies are required to appraise the potential of CAPE further as an immunomodulator in the treatment of periodontal disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In situ hybridization analysis of anterior pituitary hormone gene expression during fetal mouse development.

PubMed

Japón, M A; Rubinstein, M; Low, M J

1994-08-01

We used 35S-labeled oligonucleotides and cRNAs (riboprobes) to detect the temporal order and spatial pattern of anterior pituitary hormone gene expression in (B6CBF1 x B6CBF1)F2 fetal mice from embryonic Day 9.5 (E9.5) to postnatal Day 1 (P1). Pro-opiomelanocortin (POMC) mRNA was expressed in the basal diencephalon on Day E10.5, in the ventromedial zone of the pars distalis on Day E12.5, and in the pars intermedia on Day E14.5. The common alpha-glycoprotein subunit (alpha-GSU) mRNA first appeared in the anterior wall of Rathke's pouch on Day E11.5 and extended to the pars tuberalis and ventromedial zone of the pars distalis on Day E12.5. Thyroid-stimulating hormone-beta (TSH beta) subunit mRNA was expressed initially in both the pas tuberalis and ventromedial pars distalis on Day E14.5, with an identical spatial distribution to alpha-GSU at the time. In contrast, luteinizing hormone-beta (LH beta) subunit and follicle-stimulating hormone beta (FSH beta) subunit mRNAs were detected initially only in the ventromedial pars distalis on Days E16.5 and E17.5, respectively, in an identical distribution to each other. POMC-, alpha-GSU-, TSH beta, LH beta-, and FSH beta-positive cells within the pars distalis all increased in number and autoradiographic signal with differing degrees of spatial expansion posteriorly, laterally, and dorsally up to Day P1. POMC expression was typically the most intense and extended circumferentially to include the entire lateral and dorsal surfaces of the pars distalis. The expression of both growth hormone (GH) and prolactin (PRL) started coincidentally on Day E15.5. However PRL cells localized in the ventromedial area similarly to POMC and the glycoprotein hormone subunits, whereas GH cells were found initially in a more lateral and central distribution within the lobes of the pars distalis. Somatotrophs increased dramatically in number and autoradiographic signal, extending throughout the pars distalis except for the most peripheral layer of cells on Day E17.5. Mammotrophs also increased in number but less abundantly than somatotrophs, and PRL expression remained more confined to central-medial and ventrolateral areas of the pars distalis up to Day P1. These data demonstrate distinctive patterns of expression for each of the major anterior pituitary hormone genes during development of the mouse pituitary gland and suggest that different groups of committed cells are the immediate precursors to the terminally differentiated hormone-secreting cell types.

Toward a Whole-Cell Model of Ribosome Biogenesis: Kinetic Modeling of SSU Assembly

PubMed Central

Earnest, Tyler M.; Lai, Jonathan; Chen, Ke; Hallock, Michael J.; Williamson, James R.; Luthey-Schulten, Zaida

2015-01-01

Central to all life is the assembly of the ribosome: a coordinated process involving the hierarchical association of ribosomal proteins to the RNAs forming the small and large ribosomal subunits. The process is further complicated by effects arising from the intracellular heterogeneous environment and the location of ribosomal operons within the cell. We provide a simplified model of ribosome biogenesis in slow-growing Escherichia coli. Kinetic models of in vitro small-subunit reconstitution at the level of individual protein/ribosomal RNA interactions are developed for two temperature regimes. The model at low temperatures predicts the existence of a novel 5′→3′→central assembly pathway, which we investigate further using molecular dynamics. The high-temperature assembly network is incorporated into a model of in vivo ribosome biogenesis in slow-growing E. coli. The model, described in terms of reaction-diffusion master equations, contains 1336 reactions and 251 species that dynamically couple transcription and translation to ribosome assembly. We use the Lattice Microbes software package to simulate the stochastic production of mRNA, proteins, and ribosome intermediates over a full cell cycle of 120 min. The whole-cell model captures the correct growth rate of ribosomes, predicts the localization of early assembly intermediates to the nucleoid region, and reproduces the known assembly timescales for the small subunit with no modifications made to the embedded in vitro assembly network. PMID:26333594

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).

[Aging-related ionic remodeling of L-type voltage dependent calcium channel in left atria of canine].

PubMed

Zhou, Xian-hui; Zhang, Jian; Gan, Tian-yi; Xu, Guo-jun; Tang, Bao-peng

2012-04-01

To investigate aging-related ionic remodeling of L-type voltage dependent calcium channel (LVDCC) in left atria of canine. Seven adult (2.0 - 2.5 years) and 10 aged (> 8 years) dogs were used. The current of LVDCC was recorded by patch clamp technique in the whole cell mode. The action potential duration (APD(90)), amplitude of action potential plateau (APA), I(Ca-L) peak current density of LVDCC were recorded. The mRNA and protein expressions of α1c subunit (Ca(V1.2)), sarcoplasmic reticulum Ca(2+)-ATPase (SECRA(2)), Calpain-I, ryanodine receptor (RYR(2)) were detected by quantitative RT-PCR and Western blot, respectively. I(Ca-L) peak current density [(-8.11 ± 0.54) pA/pF vs. (-14.04 ± 0.82) pA/pF, P < 0.05] was significantly reduced and action potential duration to 90% repolarization (APD(90)) significantly prolonged [(340.5 ± 10.1) ms vs. (320.0 ± 7.9) ms, P < 0.05] in aged group than in adult group. The mRNA gene expression level of Ca(V1.2) was significantly lower (0.90 ± 0.35 vs. 2.38 ± 0.40, P < 0.05) while mRNA expression of RYR(2) was significantly higher (4.39 ± 4.68 vs. 1.49 ± 1.69, P < 0.05) in the aged dogs than in the adult dogs. mRNA expression of SECRA(2) and Calpain-I was similar between the two groups. Similarly, the protein expression level of Ca(V1.2) was significantly lower (0.13 ± 0.10 vs. 0.29 ± 0.12, P < 0.05) while the protein expression level of RYR(2) was significantly higher (0.18 ± 0.21 vs. 0.08 ± 0.36, P < 0.05) in the aged dogs than in the adult dogs. Again, protein expression of SECRA(2), PLN(1) and Calpain-I was similar between the two groups. These data suggest that aging could induce mRNA and protein expression changes of Ca(V1.2) and RYR(2) of LVDCC which might serve as the molecular basis of I(Ca-L) remodeling in aged dogs and might be linked to the increased likelihood of developing atrial fibrillation (AF) in aged dogs.

Inhalation of Roman chamomile essential oil attenuates depressive-like behaviors in Wistar Kyoto rats.

PubMed

Kong, Yingying; Wang, Ting; Wang, Rong; Ma, Yichuan; Song, Shanshan; Liu, Juan; Hu, Weiwei; Li, Shengtian

2017-06-01

The idea of aromatherapy, using essential oils, has been considered as an alternative antidepressant treatment. In the present study, we investigated the effect of Roman chamomile essential oil inhalation for two weeks on depressive-like behaviors in Wistar-Kyoto (WKY) rats. We found that inhalation of either Roman chamomile or one of its main components α-pinene, attenuated depressive-like behavior in WKY rats in the forced swim test. Using isobaric tags for relative and absolute quantitation analysis (iTRAQ), we found that inhalation of α-pinene increased expression of proteins that are involved in oxidative phosphorylation, such as cytochrome c oxidase subunit 6C-2, cytochrome c oxidase subunit 7A2, ATPase inhibitor in the hippocampus, and cytochrome c oxidase subunit 6C-2, ATP synthase subunit e, Acyl carrier protein, and Cytochrome b-c1 complex subunit 6 in the PFC (prefrontal cortex). In addition, using the quantitative real-time polymerase chain reaction technique, we confirmed an increase of parvalbumin mRNA expression in the hippocampus, which was shown to be upregulated by 2.8-fold in iTRAQ analysis, in α-pinene treated WKY rats. These findings collectively suggest the involvement of mitochondrial functions and parvalbumin-related signaling in the antidepressant effect of α-pinene inhalation.

Molecular Characterization and Expression of α-Globin and β-Globin Genes in the Euryhaline Flounder (Platichthys flesus)

PubMed Central

Lu, Weiqun; Mayolle, Aurelie; Cui, Guoqiang; Luo, Lei; Balment, Richard J.

2011-01-01

In order to understand the possible role of globin genes in fish salinity adaptation, we report the molecular characterization and expression of all four subunits of haemoglobin, and their response to salinity challenge in flounder. The entire open reading frames of α1-globin and α2-globin genes were 432 and 435 bp long, respectively, whereas the β1-globin and β2-globin genes were both 447 bp. Although the head kidney (pronephros) is the predicted major site of haematopoiesis, real-time PCR revealed that expression of α-globin and β-globin in kidney (mesonephros) was 1.5 times higher than in head kidney. Notably, the α1-globin and β1-globin mRNA expression was higher than α2-globin and β2-globin in kidney. Expression levels of all four globin subunits were higher in freshwater- (FW-) than in seawater- (SW-)adapted fish kidney. If globins do play a role in salinity adaptation, this is likely to be more important in combating the hemodilution faced by fish in FW than the dehydration and salt loading which occur in SW. PMID:21969841

Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis

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

Lee, Boo-Ja; Kwon, Sun Jae; Kim, Sung-Kyu

Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P{sub 0} interaction, but not during compatible TMV-P{sub 1.2} interaction. When CaRPN7::GFP fusion protein was targeted in onionmore » cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.« less

ATP6V1H regulates the growth and differentiation of bone marrow stromal cells.

PubMed

Li, Lin; Yang, Shaoqing; Zhang, Yanli; Ji, Dongrui; Jin, Zuolin; Duan, Xiaohong

2018-05-18

ATP6V1H encodes subunit H of vacuolar ATPase (V-ATPase) and may regulate osteoclastic function. The deficiency of ATP6V1H caused bone loss in human, mouse and zebrafish. In this report, we identified the mechanisms by which ATP6V1H regulates proliferation and differentiation of bone marrow stromal cells (BMSCs). We found that ATP6V1H was expressed in BMSCs, andAtp6v1h +/- BMSCs exhibited the lower proliferation rate, cell cycle arrest and reduced osteogenic differentiation capacity, as well as the increased adipogenic potentials. Histologic analysis confirmed less bone formation and more fatty degeneration in Atp6v1h +/- mice in the different age groups. Q-PCR analysis revealed that loss of ATP6V1H function downregulated the mRNA level of TGF-β1 receptor, and its binding molecule, subunit β of adaptor protein complex 2 (AP-2), suggesting ATP6V1H regulates the proliferation and differentiation of BMSCs by interacting with TGF-β receptor I and AP-2 complex. Copyright © 2018. Published by Elsevier Inc.

The Exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins

PubMed Central

Hessle, Viktoria; Björk, Petra; Sokolowski, Marcus; de Valdivia, Ernesto González; Silverstein, Rebecca; Artemenko, Konstantin; Tyagi, Anu; Maddalo, Gianluca; Ilag, Leopold; Helbig, Roger; Zubarev, Roman A.

2009-01-01

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins. PMID:19494042

Mutated cancer autoantigen implicated cause of paraneoplastic myasthenia gravis.

PubMed

Zekeridou, Anastasia; Griesmann, Guy E; Lennon, Vanda A

2018-05-09

Anti-tumor immune responses are postulated to initiate paraneoplastic neurological disorders when proteins normally restricted to neural cells are expressed as oncoproteins. Mutated oncopeptides could bypass self-tolerant T-cells to activate cytotoxic effector T-lymphocytes and requisite helper T-lymphocytes to stimulate autoantibody production by B-lymphocytes. We investigated muscle-type nicotinic acetylcholine receptor (AChR) antigen expression at transcriptional and protein levels in a small-cell lung cancer line (SCLC) established from a patient with AChR-IgG-positive myasthenia gravis. I We identified mRNA transcripts encoding the two AChR α1-subunit isoforms, and seven alternative-splicing products, three yielding premature stop codons. Despite detecting native muscle-type AChR pentamers in the tumor, we did not identify mutant α1-peptides. However, we found α1-subunit-derived peptides bound to tumor MHC1-protein. In a control SCLC from an ANNA-1(anti-Hu)-IgG-positive patient, we identified MHC1-complexed Hu protein-derived peptides, but not AChR peptides. Our findings support onconeural protein products as pertinent immunogens initiating paraneoplastic neurological autoimmunity. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Effect of gel re-organization and tensional forces on alpha2beta1 integrin levels in dermal fibroblasts.

PubMed

Jenkins, G; Redwood, K L; Meadows, L; Green, M R

1999-07-01

Mechanical forces are known to play an important role in regulating cell function in a wide range of biological systems. This is of particular relevance to dermal fibroblast function, given that the skin is known to be held under an intrinsic natural tension. To understand more about the generation of force by dermal fibroblasts and their ability to respond to changes in it, we have studied the role of the beta1 integrin receptors expressed by dermal fibroblasts in their ability to generate tensional forces within a collagen type I matrix and the effect of altered tensional force on integrin expression by dermal fibroblasts. Using a purpose-built culture force monitor, function-blocking antibodies directed towards the beta1 receptors dramatically reduced the tensional forces generated by dermal fibroblasts in a 3D collagen I matrix. However, the specific involvement of alpha1 or alpha2 subunits could not be demonstrated. Analysis of cellular response demonstrated that cells isolated from contracting collagen gels expressed fourfold higher levels of alpha2 mRNA than cells isolated from fully restrained gels. The levels of beta1 messenger RNA were relatively unaffected by reductions in force. Cells exposed to single reductions in force, however, did not exhibit alterations in either alpha1 or beta1 mRNA levels. We propose, therefore that alpha2beta1 integrin receptor levels in dermal fibroblasts are not altered in response to single reductions of gel tension, but do change following a continual change in force and associated matrix re-organization

Enhanced Delivery and Potency of Self-Amplifying mRNA Vaccines by Electroporation in Situ

PubMed Central

Cu, Yen; Broderick, Kate E.; Banerjee, Kaustuv; Hickman, Julie; Otten, Gillis; Barnett, Susan; Kichaev, Gleb; Sardesai, Niranjan Y.; Ulmer, Jeffrey B.; Geall, Andrew

2013-01-01

Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA), and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA. PMID:26344119

Identification and transcription profiling of NDUFS8 in Aedes taeniorhynchus (Diptera:Culididae): developmental regulation and environmental response

USDA-ARS?s Scientific Manuscript database

The cDNA of a NADH dehydrogenase -ubiquinone Fe-S protein 8 subunit (NDUFS8) gene from Aedes (Ochlerotatus) taeniorhynchus Wiedemann has been cloned and sequenced. The full-length mRNA sequence (824 bp) of AetNDUFS8 encodes an open reading region of 651 bp (i.e., 217 amino acids). To detect whether ...

Yeast eIF4A enhances recruitment of mRNAs regardless of their structural complexity

PubMed Central

Yourik, Paul; Aitken, Colin Echeverría; Zhou, Fujun; Gupta, Neha

2017-01-01

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5'-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that eIF4A's ATPase activity is markedly stimulated in the presence of the PIC, independently of eIF4E•eIF4G, but dependent on subunits i and g of the heteromeric eIF3 complex. Surprisingly, eIF4A accelerated the rate of recruitment of all mRNAs tested, regardless of their degree of structural complexity. Structures in the 5'-UTR and 3' of the start codon synergistically inhibit mRNA recruitment in a manner relieved by eIF4A, indicating that the factor does not act solely to melt hairpins in 5'-UTRs. Our findings that eIF4A functionally interacts with the PIC and plays important roles beyond unwinding 5'-UTR structure is consistent with a recent proposal that eIF4A modulates the conformation of the 40S ribosomal subunit to promote mRNA recruitment. PMID:29192585

The Rtr1p CTD phosphatase autoregulates its mRNA through a degradation pathway involving the REX exonucleases

PubMed Central

Hodko, Domagoj; Ward, Taylor; Chanfreau, Guillaume

2016-01-01

Rtr1p is a phosphatase that impacts gene expression by modulating the phosphorylation status of the C-terminal domain of the large subunit of RNA polymerase II. Here, we show that Rtr1p is a component of a novel mRNA degradation pathway that promotes its autoregulation through turnover of its own mRNA. We show that the 3′UTR of the RTR1 mRNA contains a cis element that destabilizes this mRNA. RTR1 mRNA turnover is achieved through binding of Rtr1p to the RTR1 mRNP in a manner that is dependent on this cis element. Genetic evidence shows that Rtr1p-mediated decay of the RTR1 mRNA involves the 5′-3′ DExD/H-box RNA helicase Dhh1p and the 3′-5′ exonucleases Rex2p and Rex3p. Rtr1p and Rex3p are found associated with Dhh1p, suggesting a model for recruiting the REX exonucleases to the RTR1 mRNA for degradation. Rtr1p-mediated decay potentially impacts additional transcripts, including the unspliced BMH2 pre-mRNA. We propose that Rtr1p may imprint its RNA targets cotranscriptionally and determine their downstream degradation mechanism by directing these transcripts to a novel turnover pathway that involves Rtr1p, Dhh1p, and the REX family of exonucleases. PMID:26843527

Regulated lysosomal trafficking as a mechanism for regulating GABAA receptor abundance at synapses in Caenorhabditis elegans.

PubMed

Davis, Kathleen M; Sturt, Brianne L; Friedmann, Andrew J; Richmond, Janet E; Bessereau, Jean-Louis; Grant, Barth D; Bamber, Bruce A

2010-08-01

GABA(A) receptor plasticity is important for both normal brain function and disease progression. We are studying GABA(A) receptor plasticity in Caenorhabditis elegans using a genetic approach. Acute exposure of worms to the GABA(A) agonist muscimol hyperpolarizes postsynaptic cells, causing paralysis. Worms adapt after several hours, but show uncoordinated locomotion consistent with decreased GABA signaling. Using patch-clamp and immunofluorescence approaches, we show that GABA(A) receptors are selectively removed from synapses during adaptation. Subunit mRNA levels were unchanged, suggesting a post-transcriptional mechanism. Mutants with defective lysosome function (cup-5) show elevated GABA(A) receptor levels at synapses prior to muscimol exposure. During adaptation, these receptors are removed more slowly, and accumulate in intracellular organelles positive for the late endosome marker GFP-RAB-7. These findings suggest that chronic agonist exposure increases endocytosis and lysosomal trafficking of GABA(A) receptors, leading to reduced levels of synaptic GABA(A) receptors and reduced postsynaptic GABA sensitivity.

The effect of soybean isoflavone on the dysregulation of NMDA receptor signaling pathway induced by β-amyloid peptides 1-42 in rats.

PubMed

Xi, Yuan-Di; Ding, Juan; Han, Jing; Zhang, Dan-Di; Liu, Jin-Meng; Feng, Ling-Li; Xiao, Rong

2015-05-01

Synaptic damage is the key factor of cognitive impairment. The purpose of this study was to understand the effect of soybean isoflavone (SIF) on synaptic damage induced by β-amyloid peptide 1-42 (Aβ1-42) in rats. Adult male Wistar rats were randomly divided into control, Aβ1-42, SIF, and SIF + Aβ1-42 (SIF pretreatment) groups according to body weight. SIF was treated orally by gavage in SIF and SIF + Aβ1-42 groups. After 14 days pretreatment with SIF or vehicle, Aβ1-42 was injected into the lateral cerebral ventricle of rats in Aβ1-42 and SIF + Aβ1-42 groups using miniosmotic pump. The level of Aβ1-42 and the expression of N-methyl-D-aspartic-acid receptor (NMDAR) were observed by immunohistochemistry. Reverse transcriptase polymerase chain reaction was used to detect the mRNA levels of NMDAR, calmodulin (CaM), calcium/CaM-dependent protein kinase II (CaMKII), cAMP-response element binding protein (CREB), and brain-derived neurotrophic factor (BDNF). The results showed that Aβ1-42 down-regulated mRNA and protein expression of the NR1 and NR2B subunits of NMDAR, SIF pretreatment could reverse these changes. The mRNA expression of CaM, CaMKII, CREB, and BDNF were down-regulated by Aβ1-42, but they were all regulated by SIF pretreatment. These results suggest that SIF pretreatment could antagonize the neuron damage in rats induced by Aβ1-42, and its mechanism might be associated with the NMDA receptor and CaM/CaMKII/CREB/BDNF signaling pathway, which are the synaptic plasticity-related molecules.

Tissue specific expression of the retinoic acid receptor-beta 2: regulation by short open reading frames in the 5'-noncoding region

PubMed Central

1994-01-01

The 40-S subunit of eukaryotic ribosomes binds to the capped 5'-end of mRNA and scans for the first AUG in a favorable sequence context to initiate translation. Most eukaryotic mRNAs therefore have a short 5'- untranslated region (5'-UTR) and no AUGs upstream of the translational start site; features that seem to assure efficient translation. However, approximately 5-10% of all eukaryotic mRNAs, particularly those encoding for regulatory proteins, have complex leader sequences that seem to compromise translational initiation. The retinoic-acid- receptor-beta 2 (RAR beta 2) mRNA is such a transcript with a long (461 nucleotides) 5'-UTR that contains five, partially overlapping, upstream open reading frames (uORFs) that precede the major ORF. We have begun to investigate the function of this complex 5'-UTR in transgenic mice, by introducing mutations in the start/stop codons of the uORFs in RAR beta 2-lacZ reporter constructs. When we compared the expression patterns of mutant and wild-type constructs we found that these mutations affected expression of the downstream RAR beta 2-ORF, resulting in an altered regulation of RAR beta 2-lacZ expression in heart and brain. Other tissues were unaffected. RNA analysis of adult tissues demonstrated that the uORFs act at the level of translation; adult brains and hearts of transgenic mice carrying a construct with either the wild-type or a mutant UTR, had the same levels of mRNA, but only the mutant produced protein. Our study outlines an unexpected role for uORFs: control of tissue-specific and developmentally regulated gene expression. PMID:7962071

Conserved regional patterns of GABA-related transcript expression in the neocortex of subjects with schizophrenia.

PubMed

Hashimoto, Takanori; Bazmi, H Holly; Mirnics, Karoly; Wu, Qiang; Sampson, Allan R; Lewis, David A

2008-04-01

Individuals with schizophrenia exhibit disturbances in a number of cognitive, affective, sensory, and motor functions that depend on the circuitry of different cortical areas. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related genes. Consequently, the authors sought to determine whether this pattern of altered gene expression is restricted to the dorsolateral prefrontal cortex or could also contribute to the dysfunction of other cortical areas in subjects with schizophrenia. Real-time quantitative polymerase chain reaction was used to assess the levels of eight GABA-related transcripts in four cortical areas (dorsolateral prefrontal cortex, anterior cingulate cortex, and primary motor and primary visual cortices) of subjects (N=12) with schizophrenia and matched normal comparison subjects. Expression levels of seven transcripts were lower in subjects with schizophrenia, with the magnitude of reduction for each transcript comparable across the four areas. The largest reductions were detected for mRNA encoding somatostatin and parvalbumin, followed by moderate decreases in mRNA expression for the 67-kilodalton isoform of glutamic acid decarboxylase, the GABA membrane transporter GAT-1, and the alpha 1 and delta subunits of GABA(A) receptors. In contrast, the expression of calretinin mRNA did not differ between the subject groups in any of the four areas. Because the areas examined represent the major functional domains (e.g., association, limbic, motor, and sensory) of the cerebral cortex, our findings suggest that a conserved set of molecular alterations affecting GABA neurotransmission contribute to the pathophysiology of different clinical features of schizophrenia.

mRNA Regulation of Cardiac Iron Transporters and Ferritin Subunits in a Mouse Model of Iron Overload

PubMed Central

Brewer, Casey J.; Wood, Ruth I.; Wood, John C.

2014-01-01

Iron cardiomyopathy is the leading cause of death in iron overload. Men have twice the mortality rate of women, though the cause is unknown. In hemojuvelin-knockout mice, a model of the disease, males load more cardiac iron than females. We postulated that sex differences in cardiac iron import cause differences in cardiac iron concentration. RT-PCR was used to measure mRNA of cardiac iron transporters in hemojuvelin-knockout mice. No sex differences were discovered among putative importers of non-transferrin bound iron (L-type and T-type calcium channels, ZRT/IRT-like protein 14 zinc channels). Transferrin-bound iron transporters were also analyzed; these are controlled by the iron regulatory element/iron regulatory protein (IRE/IRP) system. There was a positive relationship between cardiac iron and ferroportin mRNA in both sexes, but it was significantly steeper in females (p<0.05). Transferrin receptor 1 and divalent metal transporter 1 were more highly expressed in females than males (p<0.01 and p<0.0001, respectively), consistent with their lower cardiac iron levels, as predicted by IRE/IRP regulatory pathways. Light-chain (L) ferritin showed a positive correlation with cardiac iron that was nearly identical in males and females (R2=0.41, p<0.01 and R2=0.56, p<0.05, respectively), while heavy-chain (H) ferritin was constitutively expressed in both sexes. This represents the first report of IRE/IRP regulatory pathways in the heart. Transcriptional regulation of ferroportin was suggested in both sexes, creating a potential mechanism for differential set points for iron export. Constitutive H-ferritin expression suggests a logical limit to cardiac iron buffering capacity at levels known to produce heart failure in humans. PMID:25220979

Interactive effects of cortisol treatment and ambient seawater challenge on gill Na+,K+-ATPase and CFTR expression in two strains of Atlantic salmon smolts

USGS Publications Warehouse

Singer, T.D.; Finstad, B.; McCormick, S.D.; Wiseman, S.B.; Schulte, P.M.; McKinley, R.S.

2003-01-01

During peak smoltification, the interactive effects of cortisol and ambient seawater challenge were compared in two strains of Atlantic salmon (Salmo salar) smolts: a domesticated strain, AquaGen and a native River Imsa strain. Tissue and blood samples were taken from untreated fish on 20 May. Fish were then transferred to experimental tanks, allowed to recover for 24 h and cortisol (50 mg kg body mass-1), dissolved in vegetable oil, or vegetable oil alone (sham) was implanted. Samples were taken 5 days post implantation. Fish were then exposed to 24 h ambient seawater challenge (FW-SW) or freshwater to freshwater replacement (FW-FW) and sampled as before. Sham implantation had no significant impact on any of the measured parameters. Cortisol implantation significantly elevated plasma cortisol in FW-FW Imsa smolts, while no effect was observed in FW-SW Imsa smolts. Cortisol implantation had no effect on the plasma cortisol levels of AquaGen smolts regardless of FW-SW challenge. Increased plasma cortisol corresponded with significantly higher plasma glucose levels in FW-FW Imsa smolts. Plasma Na+ and Cl- levels were not affected by cortisol implantation but were significantly increased in FW-SW smolts of both strains. Gill Na+,K+-ATPase activity increased in response to cortisol implantation in only FW-FW smolts but not FW-SW smolts. Gill Na+,K+-ATPase ??-subunit mRNA levels were not affected by strain, cortisol injection or transfer protocol, while both CFTR I and CFTR II mRNA levels were significantly higher in AquaGen versus Imsa smolts regardless of treatment. CFTR I mRNA was elevated following cortisol implantation in FW-FW smolts from both strains suggesting CFTR I expression is under the control of cortisol. These findings also suggest that plasma cortisol levels are regulated differently between strains and that cortisol implantation and ambient FW-SW challenge interact, interfering with the individual effects of each of these factors. ?? 2003 Published by Elsevier Science B.V.

Enriched environment influences hormonal status and hippocampal brain derived neurotrophic factor in a sex dependent manner.

PubMed

Bakos, J; Hlavacova, N; Rajman, M; Ondicova, K; Koros, C; Kitraki, E; Steinbusch, H W M; Jezova, D

2009-12-01

The present study is aimed at testing the hypothesis that an enriched environment (EE) induces sex-dependent changes in stress hormone release and in markers of increased brain plasticity. The focus was on hypothalamic-pituitary-adrenocortical (HPA) axis activity, plasma levels of stress hormones, gene expression of glutamate receptor subunits and concentrations of brain-derived neurotrophic factor (BDNF) in selected brain regions. Rats exposed to EE were housed in groups of 12 in large cages with various objects, which were frequently changed, for 6 weeks. Control animals were housed four per cage under standard conditions. In females the EE-induced rise in hippocampal BDNF, a neurotrophic factor associated with increased neural plasticity, was more pronounced than in males. Similar sex-specific changes were observed in BDNF concentrations in the hypothalamus. EE also significantly attenuated oxytocin and aldosterone levels only in female but not male rats. Plasma testosterone positively correlated with hippocampal BDNF in female but not male rats housed in EE. In male rats housing in EE led to enhanced levels of testosterone and adrenocorticotropic hormone (ACTH), this was not seen in females. Hippocampal glucocorticoid but not mineralocorticoid receptor levels decreased in rats housed in EE irrespective of sex. Housing conditions failed to modify mRNA levels of glutamate receptor type 1 (Glur1) and metabotropic glutamate receptor subtype 5 (mGlur5) subunits of glutamate receptors in the forebrain. Moreover, a negative association between corticosterone and BDNF was observed in both sexes. The results demonstrate that the association between hormones and changes in brain plasticity is sex related. In particular, testosterone seems to be involved in the regulatory processes related to neuroplasticity in females.

The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation

PubMed Central

Olivares, Eduardo; Landry, Dori M.; Cáceres, C. Joaquín; Pino, Karla; Rossi, Federico; Navarrete, Camilo; Huidobro-Toro, Juan Pablo; Thompson, Sunnie R.

2014-01-01

ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5′-untranslated region (5′UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5′UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCE The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene expression. PMID:24623421

Carbonic anhydrase 2-like and Na⁺-K⁺-ATPase α gene expression in medaka (Oryzias latipes) under carbonate alkalinity stress.

PubMed

Yao, Zongli; Lai, Qifang; Hao, Zhuoran; Chen, Ling; Lin, Tingting; Zhou, Kai; Wang, Hui

2015-12-01

High carbonate alkalinity is one of the major stress factors for living organisms in saline-alkaline water areas. Acute and chronic effects of carbonate alkalinity on expression of two genes, carbonic anhydrase 2-like (CA2-like) and Na(+)-K(+)-ATPase α subunit (NKA-α) mRNA in medaka (Oryzias latipes) were evaluated to better understand the responses important for coping with a carbonate alkalinity stress. In the acute exposure experiment, the expression of CA2-like and NKA-α mRNA in the gill and kidney of medaka were examined from 0 h to 7 days exposed to 30.4 mM carbonate alkalinity water. Exposure to high carbonate alkalinity resulted in a transitory alkalosis, followed by a transient increase in gill and kidney CA2-like and NKA-α mRNA expression. In the chronic exposure experiment, the expression of these two genes was examined in the gill and kidney at 50 days post-exposure to six different carbonate alkalinity concentrations ranging from 1.5 to 30.4 mM. Gill and kidney CA2-like mRNA levels in 30.4 mM were approximately 10 and 30 times higher than that of the control (1.5 mM), respectively. Less differences were found in NKA-α expression in the 50-days exposure. The results indicate that when transferred to high carbonate alkalinity water, a transitory alkalosis may occur in medaka, followed by compensatory acid-base and ion regulatory responses. Thus, CA2-like and NKA-α are at least two of the important factors that contribute to the regulation of alkalinity stress.

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

A microfluidic method for synthesis of transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia

PubMed Central

Yang, Zhaogang; Yu, Bo; Zhu, Jing; Huang, Xiaomeng; Xie, Jing; Xu, Songlin; Yang, Xiaojuan; Wang, Xinmei; Yung, Bryant C.; Lee, L. James; Lee, Robert J.; Teng, Lesheng

2015-01-01

siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for targeted delivery of LOR-1284 siRNA to acute myeloid leukemia (AML) cells. In vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4–11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than free LOR-1284 in reducing the R2 mRNA and protein levels. Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific thereapeutic delivery of LOR-1284 to AML. PMID:25003978

Rrp6p controls mRNA polyA tail length and its decoration with polyA binding proteins

PubMed Central

Schmid, Manfred; Poulsen, Mathias Bach; Olszewski, Pawel; Pelechano, Vicent; Saguez, Cyril; Gupta, Ishaan; Steinmetz, Lars M.; Moore, Claire; Jensen, Torben Heick

2012-01-01

PolyA (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here we find that the nuclear exosome subunit Rrp6p counteracts the in vitro and in vivo extension of mature pA tails by the non-canonical pA polymerase Trf4p. Moreover, PABP loading onto nascent pA tails is controlled by Rrp6p; while Pab1p is the major PABP, Nab2p only associates in the absence of Rrp6p. This is because Rrp6p can interact with Nab2p and displace it from pA tails, potentially leading to RNA turnover as evidenced for certain pre-mRNAs. We suggest that a nuclear mRNP surveillance step involves targeting of Rrp6p by Nab2p-bound pA-tailed RNPs and that pre-mRNA abundance is regulated at this level. PMID:22683267

Changes in Protein Synthesis in Rapeseed (Brassica napus) Seedlings during a Low Temperature Treatment 1

PubMed Central

Meza-Basso, Luis; Alberdi, Miren; Raynal, Monique; Ferrero-Cadinanos, Maria-Luz; Delseny, Michel

1986-01-01

Changes induced by cold treatment in young rapeseed (Brassica napus) seedlings were investigated at the molecular level. Following germination at 18°C for 48 hours, one half of the seedlings was transferred to 0°C for another 48 hour period, the other half being kept at 18°C as a control. Newly synthesized proteins were labeled for the last 6 hours of incubation with [35S]methionine. The different polypeptides were separated by two-dimensional electrophoresis in polyacrylamide gels. Newly synthesized proteins were revealed by fluorography. Protein synthesis clearly continues at 0°C and some polypeptides preferentially accumulate at this temperature. On the other hand, synthesis of several others is repressed while many are insensitive to cold treatment. Similar changes are also observed when mRNA is prepared from cold treated seedlings, translated in vitro in a reticulocyte cell free system and compared with the products of mRNA extracted from control samples. Among the genes which are repressed we identified the small subunit of ribulose 1,6-bisphosphate carboxylase. These changes are also detectable after shorter treatments. Images Fig. 1 Fig. 2 Fig. 3 PMID:16665102

Discrimination of heterogenous mRNAs encoding strychnine-sensitive glycine receptors in Xenopus oocytes by antisense oligonucleotides.

PubMed Central

Akagi, H; Patton, D E; Miledi, R

1989-01-01

Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors. Images PMID:2479016

Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

PubMed

Kaydamov, C; Tewes, A; Adler, K; Manteuffel, R

2000-04-25

We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

PubMed Central

Shen, Peter S.; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H.; Cox, James; Cheng, Yifan; Lambowitz, Alan M.; Weissman, Jonathan S.; Brandman, Onn; Frost, Adam

2015-01-01

In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”). PMID:25554787

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

PubMed

Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

2008-12-01

Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

PubMed Central

Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P.; Romby, Pascale; Springer, Mathias; Marzi, Stefano

2013-01-01

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

[A study on the construction, expression and immunosterility of Lagurus laguru zona pellucida 3 DNA vaccine pVAX1-sig-LTB-lZP3-C3d3].

PubMed

Li, Chen-Chen; Yu, Ji-Yun; Jiang, Min; Tu, Yi-Xian; Ma, Xiao-Lin; Zhang, Fu-Chun

2011-09-01

To enhance the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 DNA vaccine, and to achieve the prospect of application through the pVAX1-sig-LTB-lZP3-C3d3 different immunity pathway. Two adjuvant molecules were constructed into the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 as DNA vaccine which contains Escherichia coli heat-labile enterotoxin B subunit and the molecular adjuvant 3 copies of C3d. The results of RT-PCR and western blot showed that the DNA vaccine was expressed in mRNA and protein level. The female C57BL/6 mice were immunized by three ways: intramuscular injection, intranasal or oral route.Antibody levels and types were detected by ELISA. ELISA results showed that recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 immunization induced specific IgG, IgA levels were significantly different comparing with control (P<0.01). Antifertility experiment showed that the experimental group reduced the average fertility significantly different compared with the control group (P<0.01). Restriction analysis, RT-PCR and Western blot showed that the recombinant plasmid constructed correctly and can be the expression of mRNA and protein levels.It resulted that the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 can induce the specific immune response efficiently and enhance the immunocontraceptive effects.

Anti-neuroinflammatory effects of grossamide from hemp seed via suppression of TLR-4-mediated NF-κB signaling pathways in lipopolysaccharide-stimulated BV2 microglia cells.

PubMed

Luo, Qian; Yan, Xiaoli; Bobrovskaya, Larisa; Ji, Mei; Yuan, Huiqing; Lou, Hongxiang; Fan, Peihong

2017-04-01

Grossamide, a representative lignanamide in hemp seed, has been reported to possess potential anti-inflammatory effects. However, the potential anti-neuroinflammatory effects and underlying mechanisms of action of grossamide are still unclear. Therefore, the present study investigated the possible effects and underlying mechanisms of grossamide against lipopolysaccharide (LPS)-induced inflammatory response in BV2 microglia cells. BV2 microglia cells were pre-treated with various concentrations of grossamide before being stimulated with LPS to induce inflammation. The levels of pro-inflammatory cytokines were determined using the enzyme-linked immunoassay (ELISA) and mRNA expression levels were measured by real-time PCR. The translocation of nuclear factor-kappa B (NF-κB) and contribution of TLR4-mediated NF-κB activation on inflammatory effects were evaluated by immunostaining and Western blot analysis. This study demonstrated that grossamide significantly inhibited the secretion of pro-inflammatory mediators such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), and decreased the level of LPS-mediated IL-6 and TNF-α mRNA. In addition, it significantly reduced the phosphorylation levels of NF-κB subunit p65 in a concentration-dependent manner and suppressed translocation of NF-κB p65 into the nucleus. Furthermore, grossamide markedly attenuated the LPS-induced expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). Taken together, these data suggest that grossamide could be a potential therapeutic candidate for inhibiting neuroinflammation in neurodegenerative diseases.

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

Eckardt, N.A.; Pell, E.J.

Leaf senescence is characterized by loss of the major photosynthetic enzyme, Ribulose bisphosphate carboxylase (Rubisco). Exposure to ozone (O[sub 3]) is often associated with a premature decline in the quantity of this enzyme. Declines in Rubisco quantity could arise through inhibition of synthesis or enhancement of degradation. Several experiments were conducted to investigate the effect of O[sub 3] on these events in immature and mature leaves of potato. The effect of O[sub 3] on Rubisco synthesis was investigated indirectly by measuring the relative quantities of mRNA for the rubisco large (rbcL) and small (rbcS) subunits following a 5 hour exposuremore » to 0.309 [mu]L L[sup [minus]1] O[sup 3] or charcoal-filtered air. O[sup 3] treatment was associated with a significant loss in rbcS mRNA in immature and mature potato leaves sampled immediately following the exposure. After the O[sup 3] exposure, a set of plants was placed in the dark at 30 C for two days. Levels of rbcS mRNA declined rapidly during the first twelve hours of dark incubation, thus declines in Rubisco quantity following two days of dark incubation were ascribed to degradation. Enhanced degradation due to O[sub 3] during the dark incubation was observed in the mature leaves, but not in the immature leaves. We conclude that O[sub 3] can cause both inhibited synthesis and enhanced degradation of Rubisco, and the response in dependent on leaf age.« less

Alteration of glutamate/GABA balance during acute alcohol intoxication in rats: effect of Xingnaojing injection.

PubMed

Wei, Jingjing; Yao, Limei; Yang, Lei; Zhao, Wei; Shi, Si; Cai, Qingyan; Chen, Dingsheng; Li, Weirong; Wang, Qi

2015-05-26

Xingnaojing Injection (XNJI) is a modern Chinese formula came from famous Chinese medicine An Gong Niu Huang Pill. XNJI has been used for treatment of cerebral diseases and stroke in China, and is approved by the State Food and Drug Administration of China for the treatment of acute alcohol intoxication (AAI). XNJI belongs to the ethnopharmacological family of medicines. In this study, we investigated the mechanisms of the XNJI effect on AAI. To investigate the effects of XNJI on glutamate, gamma-aminobutyric acid (GABA) and related receptor in lateral hypothalamic area (LHA) of AAI rat. Adult male Sprague-Dawley rats were implanted with microdialysis probes in LHA. Rats were randomly divided into control, model, 1.36mg/kg XNJI, 0.68mg/kg XNJI and 0.34mg/kg XNJI groups. During microdialysis, baseline samples were collected from 1h to 2.5h; thereafter, the rats were given an intraperitoneal injection of 52% ethanol, 5.2g/kg, or saline for control group. Twenty minutes later, three doses of XNJI was given by unilateral injection respectively, while saline for control and model groups, and samples were collected for the next 4h. The extracellular glutamate and GABA levels were measured in the LHA by a high performance liquid chromatography coupled with fluorescence detector (HPLC-FLU). The expression levels of related receptors N-methyl-d-aspartate receptor (NR) subunit NR2A, NR2B and GABAA were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Ethanol (5.2g/kg) significantly decreased the extracellular levels of glutamate and increased extracellular GABA in LHA. On the other hand ethanol significantly decreased NR2A and NR2B mRNAs expression, and increase GABAA mRNA expression. XNJI could increase the extracellular level of glutamate and decrease that of GABA; moreover, induced an increase in NR2A and NR2B mRNA expression, and a decrease in GABAA mRNA expression in LHA. The current changes in glutamate, GABA and mRNA expressions of related receptors in LHA after injection of XNJI suggest that changes in these neurotransmitters and receptors as a potential mechanism of action for AAI. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mouse Mammary Tumor Virus c-rel Transgenic Mice Develop Mammary Tumors

PubMed Central

Romieu-Mourez, Raphaëlle; Kim, Dong W.; Min Shin, Sang; Demicco, Elizabeth G.; Landesman-Bollag, Esther; Seldin, David C.; Cardiff, Robert D.; Sonenshein, Gail E.

2003-01-01

Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-κB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-κB. Analysis of the composition of NF-κB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-κB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-κB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary tumorigenesis. PMID:12897145

Heteromeric Slick/Slack K+ channels show graded sensitivity to cell volume changes

PubMed Central

Hashem, Nadia; Calloe, Kirstine; Klaerke, Dan A.

2017-01-01

Slick and Slack high-conductance K+ channels are found in the CNS, kidneys, pancreas, among other organs, where they play an important role in cell excitability as well as in ion transport processes. They are both activated by Na+ and Cl- but show a differential regulation by cell volume changes. Slick has been shown to be regulated by cell volume changes, whereas Slack is insensitive. α-subunits of these channels form homomeric as well as heteromeric channels. It is the aim of this work to explore whether the subunit composition of the Slick/Slack heteromeric channel affects the response to osmotic challenges. In order to provide with the adequate water permeability to the cell membrane of Xenopus laevis oocytes, mRNA of aquaporin 1 was co-expressed with homomeric or heteromeric Slick and Slack α-subunits. Oocytes were superfused with hypotonic or hypertonic buffers and changes in currents were measured by two-electrode voltage clamp. This work presents the first heteromeric K+ channel with a characteristic graded sensitivity to small and fast changes in cell volume. Our results show that the cell volume sensitivity of Slick/Slack heteromeric channels is dependent on the number of volume sensitive Slick α-subunits in the tetrameric channels, giving rise to graded cell volume sensitivity. Regulation of the subunit composition of a channel may constitute a novel mechanism to determine volume sensitivity of cells. PMID:28222129

Heteromeric Slick/Slack K+ channels show graded sensitivity to cell volume changes.

PubMed

Tejada, Maria A; Hashem, Nadia; Calloe, Kirstine; Klaerke, Dan A

2017-01-01

Slick and Slack high-conductance K+ channels are found in the CNS, kidneys, pancreas, among other organs, where they play an important role in cell excitability as well as in ion transport processes. They are both activated by Na+ and Cl- but show a differential regulation by cell volume changes. Slick has been shown to be regulated by cell volume changes, whereas Slack is insensitive. α-subunits of these channels form homomeric as well as heteromeric channels. It is the aim of this work to explore whether the subunit composition of the Slick/Slack heteromeric channel affects the response to osmotic challenges. In order to provide with the adequate water permeability to the cell membrane of Xenopus laevis oocytes, mRNA of aquaporin 1 was co-expressed with homomeric or heteromeric Slick and Slack α-subunits. Oocytes were superfused with hypotonic or hypertonic buffers and changes in currents were measured by two-electrode voltage clamp. This work presents the first heteromeric K+ channel with a characteristic graded sensitivity to small and fast changes in cell volume. Our results show that the cell volume sensitivity of Slick/Slack heteromeric channels is dependent on the number of volume sensitive Slick α-subunits in the tetrameric channels, giving rise to graded cell volume sensitivity. Regulation of the subunit composition of a channel may constitute a novel mechanism to determine volume sensitivity of cells.

Ferritin gene transcription is regulated by iron in soybean cell cultures.

PubMed

Lescure, A M; Proudhon, D; Pesey, H; Ragland, M; Theil, E C; Briat, J F

1991-09-15

Iron-regulated ferritin synthesis in animals is dominated by translational control of stored mRNA; iron-induced transcription of ferritin genes, when it occurs, changes the subunit composition of ferritin mRNA and protein and is coupled to translational control. Ferritins in plants and animals have evolved from a common progenitor, based on the similarity of protein sequence; however, sequence divergence occurs in the C termini; structure prediction suggests that plant ferritin has the E-helix, which, in horse ferritin, forms a large channel at the tetrameric interface. In contemporary plants, a transit peptide is encoded by ferritin mRNA to target the protein to plastids. Iron-regulated synthesis of ferritin in plants and animals appears to be very different since the 50- to 60-fold increases of ferritin protein, previously observed to be induced by iron in cultured soybean cells, is accompanied by an equivalent accumulation of hybridizable ferritin mRNA and by increased transcription of ferritin genes. Ferritin mRNA from iron-induced cells and the constitutive ferritin mRNA from soybean hypocotyls are identical. The iron-induced protein is translocated normally to plastids. Differences in animal ferritin structure coincide with the various iron storage functions (reserve for iron proteins and detoxification). In contrast, the constancy of structure of soybean ferritin, iron-induced and constitutive, coupled with the potential for vacuolar storage of excess iron in plants suggest that rapid synthesis of ferritin from a stored ferritin mRNA may not be needed in plants for detoxification of iron.

GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism

PubMed Central

Fatemi, S. Hossein; Folsom, Timothy D.

2016-01-01

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in Fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. PMID:25432637

Maturation profile of inferior olivary neurons expressing ionotropic glutamate receptors in rats: role in coding linear accelerations.

PubMed

Li, Chuan; Han, Lei; Ma, Chun-Wai; Lai, Suk-King; Lai, Chun-Hong; Shum, Daisy Kwok Yan; Chan, Ying-Shing

2013-07-01

Using sinusoidal oscillations of linear acceleration along both the horizontal and vertical planes to stimulate otolith organs in the inner ear, we charted the postnatal time at which responsive neurons in the rat inferior olive (IO) first showed Fos expression, an indicator of neuronal recruitment into the otolith circuit. Neurons in subnucleus dorsomedial cell column (DMCC) were activated by vertical stimulation as early as P9 and by horizontal (interaural) stimulation as early as P11. By P13, neurons in the β subnucleus of IO (IOβ) became responsive to horizontal stimulation along the interaural and antero-posterior directions. By P21, neurons in the rostral IOβ became also responsive to vertical stimulation, but those in the caudal IOβ remained responsive only to horizontal stimulation. Nearly all functionally activated neurons in DMCC and IOβ were immunopositive for the NR1 subunit of the NMDA receptor and the GluR2/3 subunit of the AMPA receptor. In situ hybridization studies further indicated abundant mRNA signals of the glutamate receptor subunits by the end of the second postnatal week. This is reinforced by whole-cell patch-clamp data in which glutamate receptor-mediated miniature excitatory postsynaptic currents of rostral IOβ neurons showed postnatal increase in amplitude, reaching the adult level by P14. Further, these neurons exhibited subthreshold oscillations in membrane potential as from P14. Taken together, our results support that ionotropic glutamate receptors in the IO enable postnatal coding of gravity-related information and that the rostral IOβ is the only IO subnucleus that encodes spatial orientations in 3-D.

Curcumin suppresses the production of interleukin-6 in Prevotella intermedia lipopolysaccharide-activated RAW 264.7 cells

PubMed Central

2011-01-01

Purpose Curcumin is known to exert numerous biological effects including anti-inflammatory activity. In this study, we investigated the effects of curcumin on the production of interleukin-6 (IL-6) by murine macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory periodontal disease, and sought to determine the underlying mechanisms of action. Methods LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time polymerase chain reaction to detect IL-6 mRNA expression. IκB-α degradation, nuclear translocation of NF-κB subunits, and STAT1 phosphorylation were characterized via immunoblotting. DNA-binding of NF-κB was also analyzed. Results Curcumin strongly suppressed the production of IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW 264.7 cells. Curcumin did not inhibit the degradation of IκB-α induced by P. intermedia LPS. Curcumin blocked NF-κB signaling through the inhibition of nuclear translocation of NF-κB p50 subunit. Curcumin also attenuated DNA binding activity of p50 and p65 subunits and suppressed STAT1 phosphorylation. Conclusions Although further study is required to explore the detailed mechanism of action, curcumin may contribute to blockade of the host-destructive processes mediated by IL-6 and appears to have potential therapeutic values in the treatment of inflammatory periodontal disease. PMID:21811692

Modulation of the Proteasome Pathway by Nano-Curcumin and Curcumin in Retinal Pigment Epithelial Cells.

PubMed

Ramos de Carvalho, J Emanuel; Verwoert, Milan T; Vogels, Ilse M C; Schipper-Krom, Sabine; Van Noorden, Cornelis J F; Reits, Eric A; Klaassen, Ingeborg; Schlingemann, Reinier O

2018-01-01

Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome regulation in retinal pigment epithelial (RPE) cells. Viability, cell cycle progression, and reactive oxygen species (ROS) production were determined after treatment with nano-curcumin or curcumin. Subsequently, the effects of nano-curcumin and curcumin on proteasome activity and the gene and protein expression of proteasome subunits PA28α, α7, β5, and β5i were assessed. Nano-curcumin (5-100 μM) did not show significant cytotoxicity or anti-oxidative effects against H2O2-induced oxidative stress, whereas curcumin (≥10 μM) was cytotoxic and a potent inducer of ROS production. Both nano-curcumin and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased activity of the proteasome subunits β2 and β5i/β1 and reduced activity of β5/β1i. Likewise, nano-curcumin and curcumin affected mRNA and protein levels of household and immunoproteasome subunits. Nano-curcumin is less toxic to RPE cells and less prone to induce ROS production than curcumin. Both nano-curcumin and curcumin increase proteasome-mediated proteolytic activity. These results suggest that nano-curcumin may be regarded as a proteasome-modulating agent of limited cytotoxicity for RPE cells. The Author(s). Published by S. Karger AG, Basel.

Arsenic responsive microRNAs in vivo and their potential involvement in arsenic-induced oxidative stress

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

Ren, Xuefeng, E-mail: xuefengr@buffalo.edu; Department of Pharmacology and Toxicology, School of Biomedical Sciences, The State University of New York, Buffalo, NY 14214; Gaile, Daniel P.

Arsenic exposure is postulated to modify microRNA (miRNA) expression, leading to changes of gene expression and toxicities, but studies relating the responses of miRNAs to arsenic exposure are lacking, especially with respect to in vivo studies. We utilized high-throughput sequencing technology and generated miRNA expression profiles of liver tissues from Sprague Dawley (SD) rats exposed to various concentrations of sodium arsenite (0, 0.1, 1, 10 and 100 mg/L) for 60 days. Unsupervised hierarchical clustering analysis of the miRNA expression profiles clustered the SD rats into different groups based on the arsenic exposure status, indicating a highly significant association between arsenicmore » exposure and cluster membership (p-value of 0.0012). Multiple miRNA expressions were altered by arsenic in an exposure concentration-dependent manner. Among the identified arsenic-responsive miRNAs, several are predicted to target Nfe2l2-regulated antioxidant genes, including glutamate–cysteine ligase (GCL) catalytic subunit (GCLC) and modifier subunit (GCLM) which are involved in glutathione (GSH) synthesis. Exposure to low concentrations of arsenic increased mRNA expression for Gclc and Gclm, while high concentrations significantly reduced their expression, which were correlated to changes in hepatic GCL activity and GSH level. Moreover, our data suggested that other mechanisms, e.g., miRNAs, rather than Nfe2l2-signaling pathway, could be involved in the regulation of mRNA expression of Gclc and Gclm post-arsenic exposure in vivo. Together, our findings show that arsenic exposure disrupts the genome-wide expression of miRNAs in vivo, which could lead to the biological consequence, such as an altered balance of antioxidant defense and oxidative stress. - Highlights: • Chronic arsenic exposure induces changes of hepatic miRNA expression profiles. • Hepatic GCL activity and GSH level in rats are altered following arsenic exposure. • Arsenic induced GCL expression change is independent to Nfe2l2-signaling pathway. • Expression of several miRNAs predicted to target GCL changed after arsenic exposure.« less

Changes in cortical N-methyl-D-aspartate receptors and post-synaptic density protein 95 in schizophrenia, mood disorders and suicide.

PubMed

Dean, Brian; Gibbons, Andrew S; Boer, Simone; Uezato, Akihito; Meador-Woodruff, James; Scarr, Elizabeth; McCullumsmith, Robert E

2016-03-01

In humans, depending on dose, blocking the N-methyl-D-aspartate receptor (NMDAR) with ketamine can cause psychomimetic or antidepressant effects. The overall outcome for drugs such as ketamine depends on dose and the number of its available binding sites in the central nervous system, and to understand something of the latter variable we measure NMDAR in the frontal pole, dorsolateral prefrontal, anterior cingulate and parietal cortices from people with schizophrenia, bipolar disorder, major depressive disorders and age/sex matched controls. We measured levels of NMDARs (using [(3)H]MK-801 binding) and NMDAR sub-unit mRNAs (GRINs: using in situ hybridisation) as well as post-synaptic density protein 95 (anterior cingulate cortex only; not major depressive disorders: an NMDAR post-synaptic associated protein) in bipolar disorder, schizophrenia and controls. Compared to controls, levels of NMDAR were lower in the outer laminae of the dorsolateral prefrontal cortex (-17%, p = 0.01) in people with schizophrenia. In bipolar disorder, levels of NMDAR binding (laminae IV-VI; -19%, p < 0.01) and GRIN2C mRNA (laminae I-VI; -27%, p < 0.05) were lower in the anterior cingulate cortex and NMDAR binding was lower in the outer lamina IV of the dorsolateral prefrontal cortex (-19%, p < 0.01). In major depressive disorders, levels of GRIN2D mRNA were higher in frontal pole (+22%, p < 0.05). In suicide completers, levels of GRIN2B mRNA were higher in parietal cortex (+20%, p < 0.01) but lower (-35%, p = 0.02) in dorsolateral prefrontal cortex while post-synaptic density protein 95 was higher (+26%, p < 0.05) in anterior cingulate cortex. These data suggest that differences in cortical NMDAR expression and post-synaptic density protein 95 are present in psychiatric disorders and suicide completion and may contribute to different responses to ketamine. © The Royal Australian and New Zealand College of Psychiatrists 2015.

A thyroid hormone receptor mutation that dissociates thyroid hormone regulation of gene expression in vivo

PubMed Central

Machado, Danielle S.; Sabet, Amin; Santiago, Leticia A.; Sidhaye, Aniket R.; Chiamolera, Maria I.; Ortiga-Carvalho, Tania M.; Wondisford, Fredric E.

2009-01-01

Resistance to thyroid hormone (RTH) is most often due to point mutations in the β-isoform of the thyroid hormone (TH) receptor (TR-β). The majority of mutations involve the ligand-binding domain, where they block TH binding and receptor function on both stimulatory and inhibitory TH response elements. In contrast, a few mutations in the ligand-binding domain are reported to maintain TH binding and yet cause RTH in certain tissues. We introduced one such naturally occurring human RTH mutation (R429Q) into the germline of mice at the TR-β locus. R429Q knock-in (KI) mice demonstrated elevated serum TH and inappropriately normal thyroid-stimulating hormone (TSH) levels, consistent with hypothalamic–pituitary RTH. In contrast, 3 hepatic genes positively regulated by TH (Dio1, Gpd1, and Thrsp) were increased in R429Q KI animals. Mice were then rendered hypothyroid, followed by graded T3 replacement. Hypothyroid R429Q KI mice displayed elevated TSH subunit mRNA levels, and T3 treatment failed to normally suppress these levels. T3 treatment, however, stimulated pituitary Gh levels to a greater degree in R429Q KI than in control mice. Gsta, a hepatic gene negatively regulated by TH, was not suppressed in R429Q KI mice after T3 treatment, but hepatic Dio1 and Thrsp mRNA levels increased in response to TH. Cardiac myosin heavy chain isoform gene expression also showed a specific defect in TH inhibition. In summary, the R429Q mutation is associated with selective impairment of TH-mediated gene repression, suggesting that the affected domain, necessary for TR homodimerization and corepressor binding, has a critical role in negative gene regulation by TH. PMID:19439650

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

mRNA 3' of the A site bound codon is located close to protein S3 on the human 80S ribosome.

PubMed

Molotkov, Maxim V; Graifer, Dmitri M; Popugaeva, Elena A; Bulygin, Konstantin N; Meschaninova, Maria I; Ven'yaminova, Aliya G; Karpova, Galina G

2006-07-01

Ribosomal proteins neighboring the mRNA downstream of the codon bound at the decoding site of human 80S ribosomes were identified using three sets of mRNA analogues that contained a UUU triplet at the 5' terminus and a perfluorophenylazide cross-linker at guanosine, adenosine or uridine residues placed at various locations 3' of this triplet. The positions of modified mRNA nucleotides on the ribosome were governed by tRNA(Phe) cognate to the UUU triplet targeted to the P site. Upon mild UV-irradiation, the mRNA analogues cross-linked preferentially to the 40S subunit, to the proteins and to a lesser extent to the 18S rRNA. Cross-linked nucleotides of 18S rRNA were identified previously. In the present study, it is shown that among the proteins the main target for cross-linking with all the mRNA analogues tested was protein S3 (homologous to prokaryotic S3, S3p); minor cross-linking to protein S2 (S5p) was also detected. Both proteins cross-linked to mRNA analogues in the ternary complexes as well as in the binary complexes (without tRNA). In the ternary complexes protein S15 (S19p) also cross-linked, the yield of the cross-link decreased significantly when the modified nucleotide moved from position +5 to position +12 with respect to the first nucleotide of the P site bound codon. In several ternary complexes minor cross-linking to protein S30 was likewise detected. The results of this study indicate that S3 is a key protein at the mRNA binding site neighboring mRNA downstream of the codon at the decoding site in the human ribosome.

GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth; Johnson, David; Borghese, Cecilia M.; Hanrahan, Jane R.; Johnston, Graham A. R.; Chebib, Mary; Harris, R. Adron

2014-01-01

GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo. PMID:24454882

Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant

PubMed Central

Zheng, Zhaoqing; Sabirzhanov, Boris

2012-01-01

Previously, we proposed a two-stage model for an in vitro neural correlate of eyeblink classical conditioning involving the initial synaptic incorporation of glutamate receptor A1 (GluA1)-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type receptors (AMPARs) followed by delivery of GluA4-containing AMPARs that support acquisition of conditioned responses. To test specific elements of our model for conditioning, selective knockdown of GluA4 AMPAR subunits was used using small-interfering RNAs (siRNAs). Recently, we sequenced and characterized the GluA4 subunit and its splice variants from pond turtles, Trachemys scripta elegans (tGluA4). Analysis of the relative abundance of mRNA expression by real-time RT-PCR showed that the flip/flop variants of tGluA4, tGluA4c, and a novel truncated variant tGluA4trc1 are major isoforms in the turtle brain. Here, transfection of in vitro brain stem preparations with anti-tGluA4 siRNA suppressed conditioning, tGluA4 mRNA and protein expression, and synaptic delivery of tGluA4-containing AMPARs but not tGluA1 subunits. Significantly, transfection of abducens motor neurons by nerve injections of tGluA4 flop rescue plasmid prior to anti-tGluA4 siRNA application restored conditioning and synaptic incorporation of tGluA4-containing AMPARs. In contrast, treatment with rescue plasmids for tGluA4 flip or tGluA4trc1 failed to rescue conditioning. Finally, treatment with a siRNA directed against GluA1 subunits inhibited conditioning and synaptic delivery of tGluA1-containing AMPARs and importantly, those containing tGluA4. These data strongly support our two-stage model of conditioning and our hypothesis that synaptic incorporation of tGluA4-containing AMPARs underlies the acquisition of in vitro classical conditioning. Furthermore, they suggest that tGluA4 flop may have a critical role in conditioning mechanisms compared with the other tGluA4 splice variants. PMID:22490558

Functional analyses of the interaction of chicken interleukin 23 subunit p19 with IL-12 subunit p40 to form the IL-23 complex.

PubMed

Truong, Anh Duc; Hoang, Cong Thanh; Hong, Yeojin; Lee, Janggeun; Lee, Kyungbaek; Lillehoj, Hyun S; Hong, Yeong Ho

2017-12-01

This study represents the first description of the cloning of chicken IL-23p19 (ChIL-23α) and the function of the IL-23 complex in birds. Multiple alignment of ChIL-23α with other known IL-23α amino acid sequences revealed regions of amino acid conservation. The homologies of ChIL-23α, IL-12p35, and similar mammalian subunits ranged between 26% and 42%. ChIL-23α consisted of four exons and three introns; similar to those in humans and mice, and limited conservation of synteny between the human and chicken genomes was observed. Using bioinformatics tools, we identified the NF-κB, C/EBPα-β, c-Jun, c-Rel, AP-1, GATA-1, and ER promoter sites in ChIL-23α. Moreover, IL-23α mRNA was more highly expressed than IL-12p40 and IL-12p35 mRNA in several organs of chickens infected with Salmonella. In addition, ChIL-23 complex are associated with IL-23R, IL-12Rβ1 receptors; activate the JAK2/TYK2, STAT1/3, SOCS1 genes, and induced proinflammatory cytokines in immune cells. Collectively, these results indicate that ChIL-23 is a member of the IL-12 family, has proinflammatory properties related to IL-23R and IL-12Rβ1 receptor expression, and activates the JAK/STAT signaling pathway that results in the interaction of ChIL-23α with ChIL-12p40 to form the novel ChIL-23 complex. Our results provide novel insights into the regulation of immunity, inflammation, and immunopathology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Shiga toxin 2 subtypes of enterohemorrhagic E. coli O157:H- E32511 analyzed by RT-qPCR and top-down proteomics using MALDI-TOF-TOF-MS

USDA-ARS?s Scientific Manuscript database

We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-M...

Molecular mechanics of 30S subunit head rotation.

PubMed

Mohan, Srividya; Donohue, John Paul; Noller, Harry F

2014-09-16

During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2.

Molecular mechanics of 30S subunit head rotation

PubMed Central

Mohan, Srividya; Donohue, John Paul; Noller, Harry F.

2014-01-01

During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2. PMID:25187561

Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis

PubMed Central

Komarova, Anastassia V.; Real, Eléonore; Borman, Andrew M.; Brocard, Michèle; England, Patrick; Tordo, Noël; Hershey, John W.B.; Jacob, Yves

2007-01-01

Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5′-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein–protein interaction with the cellular translation machinery. PMID:17287294

Pharmacological and immunochemical characterization of α2* nicotinic acetylcholine receptors (nAChRs) in mouse brain

PubMed Central

Whiteaker, Paul; Wilking, Jennifer A; Brown, Robert WB; Brennan, Robert J; Collins, Allan C; Lindstrom, Jon M; Boulter, Jim

2009-01-01

Aim: α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2* nAChRs in these mouse brain regions. Methods: α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing (α2*) nAChRs in these two regions. Results: [125I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8±1.7 and 92±26 fmol̇mg-1 protein, respectively). Pharmacological characterization using the β2-selective drug A85380 to inhibit [125I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2* nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good (>90% of β2* and >80% of β4* nAChRs were routinely recovered). Conclusion: Immunoprecipitation experiments indicated that interpeduncular nucleus α2* nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits. PMID:19498420

Transcriptional and post-translational control of chlorophyll biosynthesis by dark-operative protochlorophyllide oxidoreductase in Norway spruce.

PubMed

Stolárik, Tibor; Hedtke, Boris; Šantrůček, Jiří; Ilík, Petr; Grimm, Bernhard; Pavlovič, Andrej

2017-05-01

Unlike angiosperms, gymnosperms use two different enzymes for the reduction of protochlorophyllide to chlorophyllide: the light-dependent protochlorophyllide oxidoreductase (LPOR) and the dark-operative protochlorophyllide oxidoreductase (DPOR). In this study, we examined the specific role of both enzymes for chlorophyll synthesis in response to different light/dark and temperature conditions at different developmental stages (cotyledons and needles) of Norway spruce (Picea abies Karst.). The accumulation of chlorophyll and chlorophyll-binding proteins strongly decreased during dark growth in secondary needles at room temperature as well as in cotyledons at low temperature (7 °C) indicating suppression of DPOR activity. The levels of the three DPOR subunits ChlL, ChlN, and ChlB and the transcripts of their encoding genes were diminished in dark-grown secondary needles. The low temperature had minor effects on the transcription and translation of these genes in cotyledons, which is suggestive for post-translational control in chlorophyll biosynthesis. Taking into account the higher solubility of oxygen at low temperature and oxygen sensitivity of DPOR, we mimicked low-temperature condition by the exposure of seedlings to higher oxygen content (33%). The treatment resulted in an etiolated phenotype of dark-grown seedlings, confirming an oxygen-dependent control of DPOR activity in spruce cotyledons. Moreover, light-dependent suppression of mRNA and protein level of DPOR subunits indicates that more efficiently operating LPOR takes over the DPOR function under light conditions, especially in secondary needles.

Cytosolic increased labile Zn2+ contributes to arrhythmogenic action potentials in left ventricular cardiomyocytes through protein thiol oxidation and cellular ATP depletion.

PubMed

Degirmenci, Sinan; Olgar, Yusuf; Durak, Aysegul; Tuncay, Erkan; Turan, Belma

2018-07-01

Intracellular labile (free) Zn 2+ -level ([Zn 2+ ] i ) is low and increases markedly under pathophysiological conditions in cardiomyocytes. High [Zn 2+ ] i is associated with alterations in excitability and ionic-conductances while exact mechanisms are not clarified yet. Therefore, we examined the elevated-[Zn 2+ ] i on some sarcolemmal ionic-mechanisms, which can mediate cardiomyocyte dysfunction. High-[Zn 2+ ] i induced significant changes in action potential (AP) parameters, including depolarization in resting membrane-potential and prolongations in AP-repolarizing phases. We detected also the time-dependent effects such as induction of spontaneous APs at the time of ≥ 3 min following [Zn 2+ ] i increases, a manner of cellular ATP dependent and reversible with disulfide-reducing agent dithiothreitol, DTT. High-[Zn 2+ ] i induced inhibitions in voltage-dependent K + -channel currents, such as transient outward K + -currents, I to , steady-state currents, I ss and inward-rectifier K + -currents, I K1 , reversible with DTT seemed to be responsible from the prolongations in APs. We, for the first time, demonstrated that lowering cellular ATP level induced significant decreaeses in both I ss and I K1 , while no effect on I to . However, the increased-[Zn 2+ ] i could induce marked activation in ATP-sensitive K + -channel currents, I KATP , depending on low cellular ATP and thiol-oxidation levels of these channels. The mRNA levels of Kv4.3, Kv1.4 and Kv2.1 were depressed markedly with increased-[Zn 2+ ] i with no change in mRNA level of Kv4.2, while the mRNA level of I KATP subunit, SUR2A was increased significantly with increased-[Zn 2+ ] i , being reversible with DTT. Overall we demonstrated that high-[Zn 2+ ] i, even if nanomolar levels, alters cardiac function via prolonged APs of cardiomyocytes, at most, due to inhibitions in voltage-dependent K + -currents, although activation of I KATP is playing cardioprotective role, through some biochemical changes in cellular ATP- and thiol-oxidation levels. It seems, a well-controlled [Zn 2+ ] i can be novel therapeutic target for cardiac complications under pathological conditions including oxidative stress. Copyright © 2018 Elsevier GmbH. All rights reserved.

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency.

PubMed

Greene, Dina N; Whitney, Spencer M; Matsumura, Ichiro

2007-06-15

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.

Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis.

PubMed

Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Ferrando, Sara; Gallus, Lorenzo; Giovine, Marco

2015-08-01

Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and β. Here, we report the cDNA cloning, characterization, and expression analysis of the α and β subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the β subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and βP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2β2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.

Trefoil factor 2 (TFF2) deficiency in murine digestive tract influences the immune system.

PubMed

Baus-Loncar, Mirela; Schmid, Janinne; Lalani, El-Nasir; Rosewell, Ian; Goodlad, Robert A; Stamp, Gordon W H; Blin, Nikolaus; Kayademir, Tuncay

2005-01-01

The gastrointestinal trefoil factor family (TFF1, TFF2, TFF3) peptides are considered to play an important role in maintaining the integrity of the mucosa. The physiological role of TFF2 in the protection of the GI tract was investigated in TFF2 deficiency. TFF2-/- mice were generated and differential expression of various genes was assessed by using a mouse expression microarray, quantitative real time PCR, Northern blots or immunohistochemistry. On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach. Copyright (c) 2005 S. Karger AG, Basel.

The pan-Kv7 (KCNQ) Channel Opener Retigabine Inhibits Striatal Excitability by Direct Action on Striatal Neurons In Vivo.

PubMed

Hansen, Henrik H; Weikop, Pia; Mikkelsen, Maria D; Rode, Frederik; Mikkelsen, Jens D

2017-01-01

Central Kv7 (KCNQ) channels are voltage-dependent potassium channels composed of different combinations of four Kv7 subunits, being differently expressed in the brain. Notably, striatal dopaminergic neurotransmission is strongly suppressed by systemic administration of the pan-Kv7 channel opener retigabine. The effect of retigabine likely involves the inhibition of the activity in mesencephalic dopaminergic neurons projecting to the striatum, but whether Kv7 channels expressed in the striatum may also play a role is not resolved. We therefore assessed the effect of intrastriatal retigabine administration on striatal neuronal excitability in the rat determined by c-Fos immunoreactivity, a marker of neuronal activation. When retigabine was applied locally in the striatum, this resulted in a marked reduction in the number of c-Fos-positive neurons after a strong excitatory striatal stimulus induced by acute systemic haloperidol administration in the rat. The relative mRNA levels of Kv7 subunits in the rat striatum were found to be Kv7.2 = Kv7.3 = Kv7.5 > >Kv7.4. These data suggest that intrastriatal Kv7 channels play a direct role in regulating striatal excitability in vivo. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway.

PubMed

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Verma, Deepika; Priyanka, Kumari; Bal, Amanjit; Gill, Kiran Dip

2015-12-01

The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6h before aluminium (10mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits - ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Activation of RNA Polymerase III Transcription in Cells Transformed by Simian Virus 40

PubMed Central

Larminie, Christopher G. C.; Sutcliffe, Josephine E.; Tosh, Kerrie; Winter, Andrew G.; Felton-Edkins, Zoe A.; White, Robert J.

1999-01-01

RNA polymerase (Pol) III transcription is abnormally active in fibroblasts that have been transformed by simian virus 40 (SV40). This report presents evidence that two separate components of the general Pol III transcription apparatus, TFIIIB and TFIIIC2, are deregulated following SV40 transformation. TFIIIC2 subunits are expressed at abnormally high levels in SV40-transformed cells, an effect which is observed at both protein and mRNA levels. In untransformed fibroblasts, TFIIIB is subject to repression through association with the retinoblastoma protein RB. The interaction between RB and TFIIIB is compromised following SV40 transformation. Furthermore, the large T antigen of SV40 is shown to relieve repression by RB. The E7 oncoprotein of human papillomavirus can also activate Pol III transcription, an effect that is dependent on its ability to bind to RB. The data provide evidence that both TFIIIB and TFIIIC2 are targets for activation by DNA tumor viruses. PMID:10373542

Effects of Ibuprofen on Cognition and NMDA Receptor Subunit Expression Across Aging

PubMed Central

Loza, Alejandra Márquez; Elias, Valerie; Wong, Carmen P.; Ho, Emily; Bermudez, Michelle; Magnusson, Kathy R.

2017-01-01

Age-related declines in long- and short-term memory show relationships to decreases in N-methyl-D-aspartate (NMDA) receptor expression, which may involve inflammation. This study was designed to determine effects of an anti-inflammatory drug, ibuprofen, on cognitive function and NMDA receptor expression across aging. Male C57BL/6 mice (ages 5, 14, 20, and 26 months) were fed ibuprofen (375 ppm) in NIH31 diet or diet alone for 6 weeks prior to testing. Behavioral testing using the Morris water maze showed that older mice performed significantly worse than younger in spatial long-term memory, reversal, and short-term memory tasks. Ibuprofen enhanced overall performance in the short-term memory task, but this appeared to be more related to improved executive function than memory. Ibuprofen induced significant decreases over all ages in the mRNA densities for GluN2B subunit, all GluN1 splice variants, and GluN1-1 splice forms in the frontal cortex and in protein expression of GluN2A, GluN2B and GluN1 C2′ cassettes in the hippocampus. GluN1-3 splice form mRNA and C2′ cassette protein were significantly increased across ages in frontal lobes of ibuprofen-treated mice. Ibuprofen did not alter expression of pro-inflammatory cytokines IL-1β and TNFα, but did reduce the area of reactive astrocyte immunostaining in frontal cortex of aged mice. Enhancement in executive function showed a relationship to increased GluN1-3 mRNA and decreased gliosis. These findings suggest that inflammation may play a role in executive function declines in aged animals, but other effects of ibuprofen on NMDA receptors appeared to be unrelated to aging or inflammation. PMID:28057539

Angiotensin II AT1 receptor blocker candesartan prevents the fast up-regulation of cerebrocortical benzodiazepine-1 receptors induced by acute inflammatory and restraint stress

PubMed Central

Sánchez-Lemus, Enrique; Honda, Masaru; Saavedra, Juan M.

2012-01-01

Centrally acting Angiotensin II AT1 receptor blockers (ARBs) protect from stress-induced disorders and decrease anxiety in a model of inflammatory stress, the systemic injection of bacterial endotoxin lipopolysaccharide (LPS). In order to better understand the anxiolytic effect of ARBs, we treated rats with LPS (50 µg/kg) with or without three days of pretreatment with the ARB candesartan (1 mg/kg/day), and studied cortical benzodiazepine (BZ) and corticotrophin-releasing factor (CRF) receptors. We compared the cortical BZ and CRF receptors expression pattern induced by LPS with that produced in restraint stress. Inflammation stress produced a generalized increase in cortical BZ1 receptors and reduced mRNA expression of the GABAA receptor γ2 subunit in cingulate cortex; changes were prevented by candesartan pretreatment. Moreover, restraint stress produced similar increases in cortical BZ1 receptor binding, and candesartan prevented these changes. Treatment with candesartan alone increased cortical BZ1 binding, and decreased γ2 subunit mRNA expression in the cingulate cortex. Conversely, we did not find changes in CRF1 receptor expression in any of the cortical areas studied, either after inflammation or restraint stress. Cortical CRF2 receptor binding was undetectable, but CRF2 mRNA expression was decreased by inflammation stress, a change prevented by candesartan. We conclude that stress promotes rapid and widespread changes in cortical BZ1 receptor expression; and that the stress-induced BZ1 receptor expression is under the control of AT1 receptor activity. The results suggest that the anti-anxiety effect of ARBs may be associated with their capacity to regulate stress-induced alterations in cortical BZ1 receptors. PMID:22503782

Molecular characterization and expression analysis of AMPK α subunit isoform genes from Scophthalmus maximus responding to salinity stress.

PubMed

Zeng, Lin; Liu, Bin; Wu, Chang-Wen; Lei, Ji-Lin; Xu, Mei-Ying; Zhu, Ai-Yi; Zhang, Jian-She; Hong, Wan-Shu

2016-12-01

AMP-activated protein kinase (AMPK) is a highly conserved and multi-functional protein kinase that plays important roles in both intracellular energy balance and cellular stress response. In the present study, molecular characterization, tissue distribution and gene expression levels of the AMPK α1 and α2 genes from turbot (Scophthalmus maximus) under salinity stress are described. The complete coding regions of the AMPK α1 and α2 genes were isolated from turbot through degenerate primers in combination with RACE using muscle cDNA. The complete coding regions of AMPK α1 (1722 bp) and α2 (1674 bp) encoded 573 and 557 amino acids peptides, respectively. Multiple alignments, structural analysis and phylogenetic tree construction indicated that S. maximus AMPK α1 and α2 shared a high amino acid identity with other species, especially fish. AMPK α1 and α2 genes could be detected in all tested tissues, indicating that they are constitutively expressed. Salinity challenges significantly altered the gene expression levels of AMPK α1 and α2 mRNA in a salinity- and time-dependent manners in S. maximus gill tissues, suggesting that AMPK α1 and α2 played important roles in mediating the salinity stress in S. maximus. The expression levels of AMPK α1 and α2 mRNA were a positive correlation with gill Na + , K + -ATPase activities. These findings will aid our understanding of the molecular mechanism of juvenile turbot in response to environmental salinity changes.

Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P

PubMed Central

Jarrous, Nayef; Wolenski, Joseph S.; Wesolowski, Donna; Lee, Christopher; Altman, Sidney

1999-01-01

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors. PMID:10444065

An Alternative Splice Product of IκB Kinase (IKKγ), IKKγ-Δ, Differentially Mediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-κB Activation

PubMed Central

Hai, Tao; Yeung, Man-Lung; Wood, Thomas G.; Wei, Yuanfen; Yamaoka, Shoji; Gatalica, Zoran; Jeang, Kuan-Teh; Brasier, Allan R.

2006-01-01

NF-κB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IκB kinase (IKK) “signalsome”. The core IKK consists of two catalytic serine kinases, IKKα and IKKβ, and a noncatalytic subunit, IKKγ. IKKγ is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKγ mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKγ-Δ. Using a specific reverse transcription-PCR assay, we find that IKKγ-Δ is widely expressed in cultured human cells and normal human tissues. Because IKKγ-Δ protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-κB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKγ-Δ has strong homo- and heterotypic association with wild-type (WT) IKKγ and, like IKKγ WT, associates with the IKKβ kinase. Similarly, IKKγ-Δ mediates IKK kinase activity and downstream NF-κB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-κB-inducing kinase-IKKα signaling pathway. Surprisingly, however, in contrast to IKKγ WT, IKKγ-Δ is not able to mediate HTLV-1 Tax-induced NF-κB-dependent transcription, even though IKKγ-Δ binds and colocalizes with Tax. These observations suggest that IKKγ-Δ is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKγ-Δ expression, therefore, may affect signal transduction cascades coupling to IKK. PMID:16611882

Ferritin gene transcription is regulated by iron in soybean cell cultures.

PubMed Central

Lescure, A M; Proudhon, D; Pesey, H; Ragland, M; Theil, E C; Briat, J F

1991-01-01

Iron-regulated ferritin synthesis in animals is dominated by translational control of stored mRNA; iron-induced transcription of ferritin genes, when it occurs, changes the subunit composition of ferritin mRNA and protein and is coupled to translational control. Ferritins in plants and animals have evolved from a common progenitor, based on the similarity of protein sequence; however, sequence divergence occurs in the C termini; structure prediction suggests that plant ferritin has the E-helix, which, in horse ferritin, forms a large channel at the tetrameric interface. In contemporary plants, a transit peptide is encoded by ferritin mRNA to target the protein to plastids. Iron-regulated synthesis of ferritin in plants and animals appears to be very different since the 50- to 60-fold increases of ferritin protein, previously observed to be induced by iron in cultured soybean cells, is accompanied by an equivalent accumulation of hybridizable ferritin mRNA and by increased transcription of ferritin genes. Ferritin mRNA from iron-induced cells and the constitutive ferritin mRNA from soybean hypocotyls are identical. The iron-induced protein is translocated normally to plastids. Differences in animal ferritin structure coincide with the various iron storage functions (reserve for iron proteins and detoxification). In contrast, the constancy of structure of soybean ferritin, iron-induced and constitutive, coupled with the potential for vacuolar storage of excess iron in plants suggest that rapid synthesis of ferritin from a stored ferritin mRNA may not be needed in plants for detoxification of iron. Images PMID:1896472

Copper ions stimulate the proliferation of hepatic stellate cells via oxygen stress in vitro.

PubMed

Xu, San-qing; Zhu, Hui-yun; Lin, Jian-guo; Su, Tang-feng; Liu, Yan; Luo, Xiao-ping

2013-02-01

This study examined the effect of copper ions on the proliferation of hepatic stellate cells (HSCs) and the role of oxidative stress in this process in order to gain insight into the mechanism of hepatic fibrosis in Wilson's disease. LX-2 cells, a cell line of human HSCs, were cultured in vitro and treated with different agents including copper sulfate, N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO) for different time. The proliferation of LX-2 cells was measured by non-radioactive cell proliferation assay. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of platelet-derived growth factor receptor β subunit (PDGFβR), ELISA to determine the level of glutathione (GSH) and oxidized glutathione (GSSG), dichlorofluorescein assay to measure the level of reactive oxygen species (ROS), and lipid hydroperoxide assay to quantify the level of lipid peroxide (LPO). The results showed that copper sulfate over a certain concentration range could promote the proliferation of LX-2 cells in a time- and dose-dependent manner. The effect was most manifest when LX-2 cells were treated with copper sulfate at a concentration of 100 μmol/L for 24 h. Additionally, copper sulfate could dose-dependently increase the levels of ROS and LPO, and decrease the ratio of GSH/GSSG in LX-2 cells. The copper-induced increase in mRNA and protein expression of PDGFβR was significantly inhibited in LX-2 cells pre-treated with NAC, a precursor of GSH, and this phenomenon could be reversed by the intervention of BSO, an inhibitor of NAC. It was concluded that copper ions may directly stimulate the proliferation of HSCs via oxidative stress. Anti-oxidative stress therapies may help suppress the copper-induced activation and proliferation of HSCs.

CD25 is expressed by canine cutaneous mast cell tumors but not by cutaneous connective tissue mast cells.

PubMed

Meyer, A; Gruber, A D; Klopfleisch, R

2012-11-01

Canine cutaneous mast cell tumors (MCT) of different histological grades have distinct biological behaviors. However, little is known about underlying molecular mechanisms that lead to tumor development and increasing malignancy with higher tumor grade. Recent studies have identified the interleukin-2 receptor (IL-2R) subunits CD25 and CD2 as markers that distinguish nonneoplastic from neoplastic mast cells in human systemic mastocytosis. In this study, their potential as a marker for canine MCT and their possible impact on MCT carcinogenesis were evaluated. mRNA expression levels of both genes were compared between grade 1 (n = 12) and grade 3 (n = 8) MCT, and protein expression levels of CD25 were compared in 90 MCT of different tumor grades. mRNA expression levels of both CD25 and CD2 were upregulated in grade 3 MCT. In contrast, CD25 protein was expressed by fewer tumor cells and at decreased levels in grade 3 tumors, while most grade 1 MCT had strong CD25 protein expression. Moreover, CD25 was not expressed by nonneoplastic, resting cutaneous mast cells, while few presumably activated mast cells in tissue samples from dogs with allergic dermatitis had weak CD25 expression. Taken together, these findings suggest that CD25 may play a critical role in early MCT development and may be a stimulatory factor in grade 1 MCT, while grade 3 MCT seem to be less dependent on CD25. Because of the low number of CD25-positive tumor cells in high-grade tumors, the usefulness of CD25 as a tumor marker is, however, questionable.

Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes.

PubMed

Xiao, Wusheng; Sarsour, Ehab H; Wagner, Brett A; Doskey, Claire M; Buettner, Garry R; Domann, Frederick E; Goswami, Prabhat C

2016-02-01

Polychlorinated biphenyls (PCBs) and their metabolites are environmental pollutants that are known to have adverse health effects. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of 4-monochlorobiphenyl (PCB3, present in the environment and human blood) is toxic to human skin keratinocytes, and breast and prostate epithelial cells. This study investigates the hypothesis that 4-ClBQ-induced metabolic oxidative stress regulates toxicity in human keratinocytes. Results from Seahorse XF96 Analyzer showed that the 4-ClBQ treatment increased extracellular acidification rate, proton production rate, oxygen consumption rate and ATP content, indicative of metabolic oxidative stress. Results from a q-RT-PCR assay showed significant increases in the mRNA levels of hexokinase 2 (hk2), pyruvate kinase M2 (pkm2) and glucose-6-phosphate dehydrogenase (g6pd), and decreases in the mRNA levels of succinate dehydrogenase (complex II) subunit C and D (sdhc and sdhd). Pharmacological inhibition of G6PD-activity enhanced the toxicity of 4-ClBQ, suggesting that the protective function of the pentose phosphate pathway is functional in 4-ClBQ-treated cells. The decrease in sdhc and sdhd expression was associated with a significant decrease in complex II activity and increase in mitochondrial levels of ROS. Overexpression of sdhc and sdhd suppressed 4-ClBQ-induced inhibition of complex II activity, increase in mitochondrial levels of ROS, and toxicity. These results suggest that the 4-ClBQ treatment induces metabolic oxidative stress in HaCaT cells, and while the protective function of the pentose phosphate pathway is active, inhibition of complex II activity sensitizes HaCaT cells to 4-ClBQ-induced toxicity.

Temperature Modulates the Effects of Ocean Acidification on Intestinal Ion Transport in Atlantic Cod, Gadus morhua

PubMed Central

Hu, Marian Y.; Michael, Katharina; Kreiss, Cornelia M.; Stumpp, Meike; Dupont, Sam; Tseng, Yung-Che; Lucassen, Magnus

2016-01-01

CO2-driven seawater acidification has been demonstrated to enhance intestinal bicarbonate secretion rates in teleosts, leading to an increased release of CaCO3 under simulated ocean acidification scenarios. In this study, we investigated if increasing CO2 levels stimulate the intestinal acid–base regulatory machinery of Atlantic cod (Gadus morhua) and whether temperatures at the upper limit of thermal tolerance stimulate or counteract ion regulatory capacities. Juvenile G. morhua were acclimated for 4 weeks to three CO2 levels (550, 1200, and 2200 μatm) covering present and near-future natural variability, at optimum (10°C) and summer maximum temperature (18°C), respectively. Immunohistochemical analyses revealed the subcellular localization of ion transporters, including Na+/K+-ATPase (NKA), Na+/H+-exchanger 3 (NHE3), Na+/HCO3− cotransporter (NBC1), pendrin-like Cl−/HCO3− exchanger (SLC26a6), V-type H+-ATPase subunit a (VHA), and Cl− channel 3 (CLC3) in epithelial cells of the anterior intestine. At 10°C, proteins and mRNA were generally up-regulated for most transporters in the intestinal epithelium after acclimation to higher CO2 levels. This supports recent findings demonstrating increased intestinal HCO3− secretion rates in response to CO2 induced seawater acidification. At 18°C, mRNA expression and protein concentrations of most ion transporters remained unchanged or were even decreased, suggesting thermal compensation. This response may be energetically favorable to retain blood HCO3− levels to stabilize pHe, but may negatively affect intestinal salt and water resorption of marine teleosts in future oceans. PMID:27313538

Temperature Modulates the Effects of Ocean Acidification on Intestinal Ion Transport in Atlantic Cod, Gadus morhua.

PubMed

Hu, Marian Y; Michael, Katharina; Kreiss, Cornelia M; Stumpp, Meike; Dupont, Sam; Tseng, Yung-Che; Lucassen, Magnus

2016-01-01

CO2-driven seawater acidification has been demonstrated to enhance intestinal bicarbonate secretion rates in teleosts, leading to an increased release of CaCO3 under simulated ocean acidification scenarios. In this study, we investigated if increasing CO2 levels stimulate the intestinal acid-base regulatory machinery of Atlantic cod (Gadus morhua) and whether temperatures at the upper limit of thermal tolerance stimulate or counteract ion regulatory capacities. Juvenile G. morhua were acclimated for 4 weeks to three CO2 levels (550, 1200, and 2200 μatm) covering present and near-future natural variability, at optimum (10°C) and summer maximum temperature (18°C), respectively. Immunohistochemical analyses revealed the subcellular localization of ion transporters, including Na(+)/K(+)-ATPase (NKA), Na(+)/H(+)-exchanger 3 (NHE3), Na(+)/[Formula: see text] cotransporter (NBC1), pendrin-like Cl(-)/[Formula: see text] exchanger (SLC26a6), V-type H(+)-ATPase subunit a (VHA), and Cl(-) channel 3 (CLC3) in epithelial cells of the anterior intestine. At 10°C, proteins and mRNA were generally up-regulated for most transporters in the intestinal epithelium after acclimation to higher CO2 levels. This supports recent findings demonstrating increased intestinal [Formula: see text] secretion rates in response to CO2 induced seawater acidification. At 18°C, mRNA expression and protein concentrations of most ion transporters remained unchanged or were even decreased, suggesting thermal compensation. This response may be energetically favorable to retain blood [Formula: see text] levels to stabilize pHe, but may negatively affect intestinal salt and water resorption of marine teleosts in future oceans.

Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer.

PubMed

Sand, Michael; Skrygan, Marina; Georgas, Dimitrios; Arenz, Christoph; Gambichler, Thilo; Sand, Daniel; Altmeyer, Peter; Bechara, Falk G

2012-11-01

The microprocessor complex mediates intranuclear biogenesis of precursor microRNAs from the primary microRNA transcript. Extranuclear, mature microRNAs are incorporated into the RNA-induced silencing complex (RISC) before interaction with complementary target mRNA leads to transcriptional repression or cleavage. In this study, we investigated the expression profiles of the microprocessor complex subunit DiGeorge syndrome critical region gene 8 (DGCR8) and the RISC components argonaute-1 (AGO1), argonaute-2 (AGO2), as well as double-stranded RNA-binding proteins PACT, TARBP1, and TARBP2 in epithelial skin cancer and its premalignant stage. Patients with premalignant actinic keratoses (AK, n = 6), basal cell carcinomas (BCC, n = 15), and squamous cell carcinomas (SCC, n = 7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional), from healthy skin sites (intraindividual controls), and from healthy skin sites in a healthy control group (n = 16; interindividual control). The DGCR8, AGO1, AGO2, PACT, TARBP1, and TARBP2 mRNA expression levels were detected by quantitative real-time reverse transcriptase polymerase chain reaction. The DGCR8, AGO1, AGO2, PACT, and TARBP1 expression levels were significantly higher in the AK, BCC, and SCC groups than the healthy controls (P < 0.05). There was no significant difference in the TARBP2 expression levels between groups (P > 0.05). This study indicates that major components of the miRNA pathway, such as the microprocessor complex and RISC, are dysregulated in epithelial skin cancer. Copyright © 2011 Wiley Periodicals, Inc.

Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype

PubMed Central

Gilbert, Merle L.; Yang, Linghai; Su, Thomas

2015-01-01

PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus. PMID:25587115

Oxaliplatin Alters Expression of T1R2 Receptor and Sensitivity to Sweet Taste in Rats.

PubMed

Ohishi, Akihiro; Nishida, Kentaro; Yamanaka, Yuri; Miyata, Ai; Ikukawa, Akiko; Yabu, Miharu; Miyamoto, Karin; Bansho, Saho; Nagasawa, Kazuki

2016-01-01

As one of the adverse effects of oxaliplatin, a key agent in colon cancer chemotherapy, a taste disorder is a severe issue in a clinical situation because it decreases the quality of life of patients. However, there is little information on the mechanism underlying the oxaliplatin-induced taste disorder. Here, we examined the molecular and behavioral characteristics of the oxaliplatin-induced taste disorder in rats. Oxaliplatin (4-16 mg/kg) was administered to Sprague-Dawley (SD) rats intraperitoneally for 2 d. Expression levels of mRNA and protein of taste receptors in circumvallate papillae (CP) were measured by real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry, respectively. Taste sensitivity was assessed by their behavioral change using a brief-access test. Morphological change of the taste buds in CP was evaluated by hematoxyline-eosin (HE) staining, and the number of taste cells in taste buds was counted by immunohistochemical analysis. Among taste receptors, the expression levels of mRNA and protein of T1R2, a sweet taste receptor subunit, were increased transiently in CP of oxaliplatin-administered rats on day 7. In a brief-access test, the lick ratio was decreased in oxaliplatin-administered rats on day 7 and the alteration was recovered to the control level on day 14. There was no detectable alteration in the morphology of taste buds, number of taste cells or plasma zinc level in oxaliplatin-administered rats. These results suggest that decreased sensitivity to sweet taste in oxaliplatin-administered rats is due, at least in part, to increased expression of T1R2, while these alterations are reversible.

Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

PubMed Central

LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

2012-01-01

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation. PMID:23044750

Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.

PubMed

Lacruz, Rodrigo S; Brookes, Steven J; Wen, Xin; Jimenez, Jaime M; Vikman, Susanna; Hu, Ping; White, Shane N; Lyngstadaas, S Petter; Okamoto, Curtis T; Smith, Charles E; Paine, Michael L

2013-03-01

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation. Copyright © 2013 American Society for Bone and Mineral Research.

Regulation of leaf organ size by the Arabidopsis RPT2a 19S proteasome subunit.

PubMed

Sonoda, Yutaka; Sako, Kaori; Maki, Yuko; Yamazaki, Naoko; Yamamoto, Hiroko; Ikeda, Akira; Yamaguchi, Junji

2009-10-01

The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins, to control many cellular events. To further understand this pathway, we focused on the RPT2 subunit of the 26S proteasome regulatory particle. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit, with a difference of only three amino acids in the protein sequences. Both genes showed similar mRNA accumulation patterns. However, the rpt2a mutant showed a specific phenotype of enlarged leaves caused by increased cell size, in correlation with increased ploidy. Detailed analyses revealed that cell expansion is increased in the rpt2a mutant by extended endoreduplication early in leaf development. The transcription of genes encoding cell cycle-related components, for DNA replication licensing and the G2/M phase, was also promoted in the rpt2a mutant, suggesting that extended endoreduplication was caused by increased DNA replication, and disrupted regulation of the G2/M checkpoint, at the proliferation stage of leaf development.

Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

PubMed Central

Erzberger, Jan P.; Stengel, Florian; Pellarin, Riccardo; Zhang, Suyang; Schaefer, Tanja; Aylett, Christopher H.S.; Cimermančič, Peter; Boehringer, Daniel; Sali, Andrej; Aebersold, Ruedi; Ban, Nenad

2014-01-01

Summary Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA. PMID:25171412

Ensemble cryo-EM elucidates the mechanism of translation fidelity

PubMed Central

Loveland, Anna B.; Demo, Gabriel; Grigorieff, Nikolaus; Korostelev, Andrei A.

2017-01-01

SUMMARY Faithful gene translation depends on accurate decoding, whose structural mechanism remains a matter of debate. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by EF-Tu. We present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by EF-Tu. Both cognate and near-cognate tRNA anticodons explore the A site of an open 30S subunit, while inactive EF-Tu is separated from the 50S subunit. A transient conformation of decoding-center nucleotide G530 stabilizes the cognate codon-anticodon helix, initiating step-wise “latching” of the decoding center. The resulting 30S domain closure docks EF-Tu at the sarcin-ricin loop of the 50S subunit, activating EF-Tu for GTP hydrolysis and ensuing aminoacyl-tRNA accommodation. By contrast, near-cognate complexes fail to induce the G530 latch, thus favoring open 30S pre-accommodation intermediates with inactive EF-Tu. This work unveils long-sought structural differences between the pre-accommodation of cognate and near-cognate tRNA that elucidate the mechanism of accurate decoding. PMID:28538735

Construction of nanometer cisplatin core-ferritin (NCC-F) and proteomic analysis of gastric cancer cell apoptosis induced with cisplatin released from the NCC-F.

PubMed

Ji, Xue-Tao; Huang, Lin; Huang, He-Qing

2012-06-18

Both transmission electron microscopy (TEM) and fluorescence spectrometry were used to reveal the characteristics of both subunit disassociation and recombination in apo-pig pancreas ferritin (apoPPF) in an alkaline medium ranging reversibly from pH 7.0 to 13.0. The experimental results indicated that apoPPF could be completely disassociated into 24 free subunits at pH 13.0, and then these subunits could be quickly reassembled into the original apoPPF once the pH of the reactive medium was returned to pH7.0. This novel and simple method could be used to effectively construct a novel nanometer cisplatin core-PPF (NCC-PPF). The major characteristics of NCC-PPF were investigated using various analytical methods such as ultraviolet-spectrometry, circular dichroism spectrometry and TEM, which indicated that its molecular structure was still similar to that of the original apoPPF. Results from the inductively coupled plasma mass spectrometer (ICP-MS) method showed that 11.26 cisplatin (CDDP) molecules were successfully packaged within the NCC-PPF shell, indicating that each molecule of apoPPF had the ability to enwrap 11.26 CDDP molecules for constructing the NCC-PPF. Flow cytometry showed that NCC-PPF also had the ability to release CDDP for inducing the apoptosis of gastric cancer cells BGC823 (GCC), but this phenomenon could scarcely be observed using apoPPF. A differential proteomic technique using two-dimensional electrophoresis (2-DE) gels selected and identified the differential proteins from cell apoptosis in order to reveal the molecular pathway of GCC apoptosis by both NCC-PPF and free CDDP, giving 13 differential expression proteins. These differential proteins could be further classified into six groups, which were described as being involved in the regulation of apoptosis, RNA transcription, oxidative stress response, signal transduction, cell metabolism, and cytoskeleton changes. In addition, a real-time PCR method was used to prove the expression level of mRNA and to identify the reliability of the protein expression according to these differential proteins, which indicated that the mRNA level changes of six differential proteins corresponded to those of its differential protein expression in 2-DE gels. These studies played an important role in reasonably revealing the different pathways of GCC apoptosis induced with both the CDDP released by NCC-PPF and the free CDDP. We thus suggest that apoPPF has great potential for constructing a nanometer carrier filled with various drugs for application in clinical work. Copyright © 2012 Elsevier B.V. All rights reserved.

Impaired insulin signaling pathway in ovarian follicles of cows with cystic ovarian disease.

PubMed

Hein, G J; Panzani, C G; Rodríguez, F M; Salvetti, N R; Díaz, P U; Gareis, N C; Benítez, G A; Ortega, H H; Rey, F

2015-05-01

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Follicular cell steroidogenesis and proliferation in ovulatory follicles is stimulated by hormones such as insulin and its necessary post-receptor response. The aim of this study was to determine the expression of insulin receptor (IR), IR substrate-1 (IRS1) and phosphatidylinositol 3-kinase (PI3K), key intermediates in the insulin pathway, in control cows and cows with spontaneous COD and ACTH-induced COD. IR and IRS1 mRNA levels were greater in granulosa cells and lower in follicular cysts than in control tertiary follicles. PI3K mRNA levels were similar in all follicles evaluated, whereas the expression of IR, IRS1 and PI3K was similar in theca cells. Protein expression of IR was higher in control tertiary follicles than in the same structures in animals with COD and with cysts. IRS1 and PI3K protein expression showed the same pattern in tertiary and cystic follicles. However, the protein expression of subunit alpha p85 of PI3K was greater in theca cells from tertiary follicles than in cystic follicles. These results provide new insights into the insulin response in cows with COD. The lower gene and protein expressions of some insulin downstream effectors at an early stage of the signaling pathway could negatively influence the functionality of ovaries and contribute to follicle persistence. Copyright © 2015 Elsevier B.V. All rights reserved.

Mangiferin suppressed advanced glycation end products (AGEs) through NF-κB deactivation and displayed anti-inflammatory effects in streptozotocin and high fat diet-diabetic cardiomyopathy rats.

PubMed

Hou, Jun; Zheng, Dezhi; Fung, Gabriel; Deng, Haoyu; Chen, Lin; Liang, Jiali; Jiang, Yan; Hu, Yonghe

2016-03-01

Given the importance of the aggregation of advanced glycation end products (AGEs) and cardiac inflammation in the onset and progression of diabetic cardiomyopathy (DCM), our objective in this study was to demonstrate the cardioprotective effect of mangiferin, an antidiabetic and anti-inflammatory agent, on diabetic rat model. The DCM model was established by a high-fat diet and a low dose of streptozotocin. DCM rats were treated orally with mangiferin (20 mg/kg) for 16 weeks. Serum and left ventricular myocardium were collected for determination of inflammatory cytokines. AGEs mRNA and protein expression of nuclear factor kappa B (NF-κB) and receptor for AGEs (RAGE) in myocardium were assayed by real-time PCR and Western blot. ROS levels were measured by dihydroethidium fluorescence staining. NF-κB binding activity was assayed by TransAM NF-κB p65 ELISA kit. Chronic treatment with mangiferin decreased the levels of myocardial enzymes (CK-MB, LDH) and inflammatory mediators (TNF-α, IL-1β). Meanwhile, NF-κB is inhibited by the reduction of nuclear translocation of p65 subunit, and mangiferin reduced AGE production and decreased the mRNA and protein expression of RAGE in DCM rats. Our data indicated that mangiferin could significantly ameliorate DCM by preventing the release of inflammatory cytokines, and inhibiting ROS accumulation, AGE/RAGE production, and NF-κB nuclear translocation, suggesting that mangiferin treatment might be beneficial in DCM.

Expression of tumor necrosis factor-α and interleukin-1β genes in the cochlea and inferior colliculus in salicylate-induced tinnitus

PubMed Central

2011-01-01

Background Changes in the gene expressions for tumor necrosis factor-α (TNF-α) and/or interleukin-1β (IL-1β) during tinnitus have not been previously reported. We evaluated tinnitus and mRNA expression levels of TNF-α, IL-1β, and N-methyl D-aspartate receptor subunit 2B (NR2B) genes in cochlea and inferior colliculus (IC) of mice after intraperitoneal injections of salicylate. Methods Forty-eight 3-month-old male SAMP8 mice were randomly and equally divided into two groups: salicylate-treated and saline-treated. All mice were trained to perform an active avoidance task for 5 days. Once conditioned, an active avoidance task was performed 2 hours after daily intraperitoneal injections of saline, either alone or containing 300 mg/kg sodium salicylate. Total numbers of times (tinnitus score) the mice climbed during the inter-trial silent period for 10 trials were recorded daily for 4 days (days 7 to 10), and then mice were euthanized for determination of mRNA expression levels of TNF-α, IL-1β, and NR2B genes in cochlea and IC at day 10. Results Tinnitus scores increased in response to daily salicylate treatments. The mRNA expression levels of TNF-α increased significantly for the salicylate-treated group compared to the control group in both cochlea (1.89 ± 0.22 vs. 0.87 ± 0.07, P < 0.0001) and IC (2.12 ± 0.23 vs. 1.73 ± 0.22, p = 0.0040). mRNA expression levels for the IL-1β gene also increased significantly in the salicylate group compared to the control group in both cochlea (3.50 ± 1.05 vs. 2.80 ± 0.28, p < 0.0001) and IC (2.94 ± 0.51 versus 1.24 ± 0.52, p = 0.0013). Linear regression analysis revealed a significant positive association between tinnitus scores and expression levels of TNF-α, IL-1β, and NR2B genes in cochlea and IC. In addition, expression levels of the TNF-α gene were positively correlated with those of the NR2Bgene in both cochlea and IC; whereas, the expression levels of the IL-1β gene was positively correlated with that of the NR2B gene in IC, but not in cochlea. Conclusion We conclude that salicylate treatment resulting in tinnitus augments expression of the TNF-α and IL-1β genes in cochlea and IC of mice, and we suggest that these proinflammatory cytokines might lead to tinnitus directly or via modulating the NMDA receptor. PMID:21477330

A microfluidic method to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia

NASA Astrophysics Data System (ADS)

Yang, Zhaogang; Yu, Bo; Zhu, Jing; Huang, Xiaomeng; Xie, Jing; Xu, Songlin; Yang, Xiaojuan; Wang, Xinmei; Yung, Bryant C.; Lee, L. James; Lee, Robert J.; Teng, Lesheng

2014-07-01

The siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for the targeted delivery of LOR-1284 siRNA into acute myeloid leukemia (AML) cells. The in vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4-11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than the free LOR-1284 in reducing the R2 mRNA and protein levels. The Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to the free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with the decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific therapeutic delivery of LOR-1284 into AML cells.

Development, food intake, and ethinylestradiol influence hepatic triglyceride lipase and LDL-receptor mRNA levels in rats.

PubMed

Staels, B; Jansen, H; van Tol, A; Stahnke, G; Will, H; Verhoeven, G; Auwerx, J

1990-07-01

The influence of development and ethinylestradiol on low density lipoprotein (LDL)-receptor mRNA and hepatic triglyceride lipase (HTGL) activity and mRNA levels was studied in rat liver and intestine. Intestinal LDL-receptor mRNA levels are maximal in the perinatal period, whereas liver LDL-receptor and HTGL mRNA levels are highest after weaning in adult life. All mRNA levels reach a maximum between day 15 and 20 when rats still consume a lipid-rich diet, and increase twofold during weaning. Liver and intestinal LDL-receptor mRNA levels are not influenced by ovariectomy, but increase after ethinylestradiol treatment. Liver LDL-receptor mRNA shows a dose-dependent increase after ethinylestradiol and a sevenfold rise in liver LDL-receptor mRNA is attained with a dose of 2000 micrograms/day. Intestinal LDL-receptor mRNA increases slightly more than twofold after ethinylestradiol and this increase is not dose-dependent. Changes in LDL-receptor mRNA are independent of changes in food intake induced by ethinylestradiol treatment, since they are still observed after pair-feeding. The ethinylestradiol-induced increases in LDL-receptor mRNA levels are reflected by decreased serum apoB levels. HTGL mRNA levels increase after ovariectomy and show a dose-dependent decrease after ethinylestradiol. Pair-feeding abolishes the increase seen after ovariectomy, while the estrogen-mediated decrease is attenuated. These alterations in HTGL mRNA are reflected by similar changes in liver HTGL activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyanidin-3-O-glucoside ameliorates diabetic nephropathy through regulation of glutathione pool.

PubMed

Qin, Yan; Zhai, Qianqian; Li, Yan; Cao, Meng; Xu, Yun; Zhao, Kelei; Wang, Tao

2018-07-01

Diabetic nephropathy (DN) is a common complication of diabetes and the major cause of chronic kidney disease. Cyanidin 3-glucoside (C3G) is the most widespread anthocyanin in nature. In the present study, we aimed to investigate the possible effects of C3G on DN in db/db mice. We found that body weights and high levels of fasting blood glucose, serum insulin, C-peptide, glycosylated hemoglobin A1c, and systolic blood pressure in diabetic mice were significantly reduced by C3G. C3G also reduced the ratio of kidney to body weight and the levels of blood urea nitrogen (BUN), serum creatinine, urinary albumin content and albumin/creatinine ratio (ACR), ameliorated the pathological changes of kidneys, reduced the surface area of Bowman's capsule, glomerular tuft, Bowman's space, and decreased renal expression of collagen IV, fibronectin, transforming growth factor β 1 (TGFβ1), matrix metalloprotein 9 (MMP9) and α-smooth muscle actin (α-SMA) in db/db mice. The Lee's index, perirenal white adipose tissue weight, and high levels of blood and renal triglyceride and cholesterol were decreased by C3G. Moreover, C3G reduced systemic levels and renal expression of tumor necrosis factor ɑ (TNFɑ), IL-1ɑ, and monocyte chemotactic protein-1 (MCP-1), indicating the inhibition of inflammation. Furthermore, C3G increased glutathione (GSH) level and decreased GSSG level in kidneys of diabetic mice. The renal mRNA expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) was increased by C3G in diabetic mice. Buthionine sulphoximine (BSO), an inhibitor of GSH synthesis, inhibited the effects of C3G on glucose metabolic dysfunction and DN. The data demonstrates that enhancement of GSH pool is involved in the renal-protective effects of C3G. Overall, C3G could be a promising therapeutic option for attenuation of diabetes and DN. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Coexpression of the KCNA3B gene product with Kv1.5 leads to a novel A-type potassium channel.

PubMed

Leicher, T; Bähring, R; Isbrandt, D; Pongs, O

1998-12-25

Shaker-related voltage-gated potassium (Kv) channels may be heterooligomers consisting of membrane-integral alpha-subunits associated with auxiliary cytoplasmic beta-subunits. In this study we have cloned the human Kvbeta3.1 subunit and the corresponding KCNA3B gene. Identification of sequence-tagged sites in the gene mapped KCNA3B to band p13.1 of human chromosome 17. Comparison of the KCNA1B, KCNA2B, and KCNA3B gene structures showed that the three Kvbeta genes have very disparate lengths varying from >/=350 kb (KCNA1B) to approximately 7 kb (KCNA3B). Yet, the exon patterns of the three genes, which code for the seven known mammalian Kvbeta subunits, are very similar. The Kvbeta1 and Kvbeta2 splice variants are generated by alternative use of 5'-exons. Mouse Kvbeta4, a potential splice variant of Kvbeta3, is a read-through product where the open reading frame starts within the sequence intervening between Kvbeta3 exons 7 and 8. The human KCNA3B sequence does not contain a mouse Kvbeta4-like open reading frame. Human Kvbeta3 mRNA is specifically expressed in the brain, where it is predominantly detected in the cerebellum. The heterologous coexpression of human Kv1.5 and Kvbeta3.1 subunits in Chinese hamster ovary cells yielded a novel Kv channel mediating very fast inactivating (A-type) outward currents upon depolarization. Thus, the expression of Kvbeta3.1 subunits potentially extends the possibilities to express diverse A-type Kv channels in the human brain.

Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

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

Watkins, D.C.; Northup, J.K.; Malbon, C.C.

1987-05-01

Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased themore » amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.« less

A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes.

PubMed

Montes de Oca Balderas, Pavel; Aguilera, Penélope

2015-01-01

Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

PubMed

Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Mice lacking the G protein γ3-subunit show resistance to opioids and diet induced obesity

PubMed Central

Schwindinger, William F.; Borrell, Brandon M.; Waldman, Lora C.

2009-01-01

Contributing to the obesity epidemic, there is increasing evidence that overconsumption of high-fat foods may be analogous to drug addiction in that the palatability of these foods is associated with activation of specific reward pathways in the brain. With this perspective, we report that mice lacking the G protein γ3-subunit (Gng3−/− mice) show resistance to high-fat diet-induced weight gain over the course of a 12-wk study. Compared with Gng3+/+ controls, female Gng3−/− mice exhibit a 40% reduction in weight gain and a 53% decrease in fat pad mass, whereas male Gng3−/− mice display an 18% reduction in weight gain and no significant decrease in fat pad mass. The basis for the lowered weight gain is related to reduced food consumption for female and male Gng3−/− mice of 13% and 14%, respectively. Female Gng3−/− mice also show a lesser preference for high-fat chow than their female Gng3+/+ littermates, suggesting an attenuated effect on a reward pathway associated with overconsumption of fat. One possible candidate is the μ-opioid receptor (Oprm1) signaling cascade. Supporting a defect in this signaling pathway, Gng3−/− mice show marked reductions in both acute and chronic morphine responsiveness, as well as increases in endogenous opioid mRNA levels in reward-related regions of the brain. Taken together, these data suggest that the decreased weight gain of Gng3−/− mice may be related to a reduced rewarding effect of the high-fat diet resulting from a defect in Oprm1 signaling and loss of the G protein γ3-subunit. PMID:19759336

GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.

PubMed

Fatemi, S Hossein; Folsom, Timothy D

2015-09-01

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland.

PubMed

Inoue, Makiko; Shiina, Tomoya; Aizawa, Sayaka; Sakata, Ichiro; Takagi, Hiroyasu; Sakai, Takafumi

2012-06-01

Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.

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

Takahashi, Akinori; Kikuguchi, Chisato; Morita, Masahiro

Highlights: Black-Right-Pointing-Pointer CNOT3 depletion increases the mitotic index. Black-Right-Pointing-Pointer CNOT3 inhibits the expression of MAD1. Black-Right-Pointing-Pointer CNOT3 destabilizes the MAD1 mRNA. Black-Right-Pointing-Pointer MAD1 knockdown attenuates the CNOT3 depletion-induced mitotic arrest. -- Abstract: The stability of mRNA influences the dynamics of gene expression. The CCR4-NOT complex, the major deadenylase in mammalian cells, shortens the mRNA poly(A) tail and contributes to the destabilization of mRNAs. The CCR4-NOT complex plays pivotal roles in various physiological functions, including cell proliferation, apoptosis, and metabolism. Here, we show that CNOT3, a subunit of the CCR4-NOT complex, is involved in the regulation of the spindle assembly checkpoint,more » suggesting that the CCR4-NOT complex also plays a part in the regulation of mitosis. CNOT3 depletion increases the population of mitotic-arrested cells and specifically increases the expression of MAD1 mRNA and its protein product that plays a part in the spindle assembly checkpoint. We showed that CNOT3 depletion stabilizes the MAD1 mRNA, and that MAD1 knockdown attenuates the CNOT3 depletion-induced increase of the mitotic index. Basing on these observations, we propose that CNOT3 is involved in the regulation of the spindle assembly checkpoint through its ability to regulate the stability of MAD1 mRNA.« less

Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.

PubMed

Han, L Q; Zhou, Z; Ma, Y; Batistel, F; Osorio, J S; Loor, J J

2018-04-18

Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at -30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from -30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice

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

Li, Yuan-Yuan; Yu, Li-Fang; Zhang, Li-Na

2013-12-01

AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently withoutmore » changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome. - Highlights: • C24 activates AMPK through antagonizing autoinhibition within α subunit. • C24 activates AMPK in hepatocytes and decreases glucose output via AMPK. • C24 exerts beneficial effects on diabetic db/db mice. • C24 represents a novel therapeutic for treatment of metabolic syndrome.« less

Schizophrenia-like GABAergic gene expression deficits in cerebellar Golgi cells from rats chronically exposed to low-dose phencyclidine

PubMed Central

Bullock, W. Michael; Bolognani, Federico; Botta, Paolo; Valenzuela, C. Fernando; Perrone-Bizzozero, Nora I.

2009-01-01

One of the most consistent findings in schizophrenia is the decreased expression of the GABA synthesizing enzymes GAD67 and GAD65 in specific interneuron populations. This dysfunction is observed in distributed brain regions including the prefrontal cortex, hippocampus, and cerebellum. In an effort to understand the mechanisms for this GABA deficit, we investigated the effect of the N-methyl-D-aspartate receptor (NMDAR) antagonist phencyclidine (PCP), which elicits schizophrenia-like symptoms in both humans and animal models, in a chronic, low-dose exposure paradigm. Adult rats were given PCP at a dose of 2.58 mg/kg/day i.p. for a month, after which levels of various GABAergic cell mRNAs and other neuromodulators were examined in the cerebellum by RT-qPCR. Administration of PCP decreased the expression of GAD67, GAD65, and the presynaptic GABA transporter GAT-1, and increased GABAA receptor subunits similar to those seen in patients with schizophrenia. Additionally, we found that the mRNA levels of two Golgi cell selective NMDAR subunits, NR2B and NR2D, were decreased in PCP treated rats. Furthermore, we localized the deficits in GAD67 expression solely to these interneurons. Slice electrophysiological studies showed that spontaneous firing of Golgi cells was reduced by acute exposure to low dose PCP, suggesting that these neurons are particularly vulnerable to NMDA receptor antagonism. In conclusion, our results demonstrate that chronic exposure to low levels of PCP in rats mimics the GABAergic alterations reported in the cerebellum of patients with schizophrenia (Bullock et al., Am J Psychiatry 165: 1594-1603, 2008), further supporting the validity of this animal model. PMID:19651169

Kv7 channels are upregulated during striatal neuron development and promote maturation of human iPSC-derived neurons.

PubMed

Telezhkin, Vsevolod; Straccia, Marco; Yarova, Polina; Pardo, Monica; Yung, Sun; Vinh, Ngoc-Nga; Hancock, Jane M; Barriga, Gerardo Garcia-Diaz; Brown, David A; Rosser, Anne E; Brown, Jonathan T; Canals, Josep M; Randall, Andrew D; Allen, Nicholas D; Kemp, Paul J

2018-05-24

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

Properties and Expression of Na+/K+-ATPase α-Subunit Isoforms in the Brain of the Swamp Eel, Monopterus albus, Which Has Unusually High Brain Ammonia Tolerance

PubMed Central

Chen, Xiu L.; Wee, Nicklaus L. J. E.; Hiong, Kum C.; Ong, Jasmine L. Y.; Chng, You R.; Ching, Biyun; Wong, Wai P.; Chew, Shit F.; Ip, Yuen K.

2013-01-01

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l−1) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g−1). Na+/K+-ATPase (Nka) is an essential transporter in brain cells, and since NH4 + can substitute for K+ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K+ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4 + to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na+/K+-ATPase and Na+/NH4 +-ATPase activities over a range of K+/NH4 + concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l−1 NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na+/NH4 +-ATPase activities were significantly lower than the Na+/K+-ATPase activities assayed at various NH4 +/K+ concentrations. Furthermore, the effectiveness of NH4 + to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K+ specificity of K+ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus. PMID:24391932

Mus musculus-microRNA-449a ameliorates neuropathic pain by decreasing the level of KCNMA1 and TRPA1, and increasing the level of TPTE.

PubMed

Lu, Shan; Ma, Sichao; Wang, Yunyun; Huang, Tao; Zhu, Zhihua; Zhao, Guoqing

2017-07-01

Neuropathic pain is a nerve disorder characterized by the dysregulation of ion channels in dorsal root ganglion (DRG) neurons. MicroRNAs (miRs) may be associated with the molecular mechanisms underlying the altered levels of ion channels; however, the molecular mechanisms remain widely unknown. To investigate these mechanisms, the present study conducted a genomic analysis of miR between a unilateral spared nerve injury (SNI) model and sham control. Differentially expressed miRs between the SNI and sham groups were selected for transfection of DRG cells, a polymerase chain reaction (PCR) array analysis was subsequently performed. A total of three significantly differently expressed genes were selected from the results of the PCR array and further analyzed by reverse transcription‑quantitative PCR. Genomic analysis revealed that Mus musculus miR‑449a (mmu‑miR‑449a) was reduced in the SNI groups compared with the sham controls. The PCR array indicated that mmu‑miR‑449a‑transfection reduced the mRNA expression levels of transient receptor potential cation channel subfamily A member 1 (TRPA1), and calcium‑activated potassium channel subunit α‑1 (KCNMA1) and increased the level of transmembrane phosphatase with tension homology (TPTE) in the DRG cells (P<0.05). qRT‑PCR analysis further indicated that mmu‑miR‑449a transfection caused similar alterations in the mRNA expression levels of TRPA1, KCNMA1 and TPTE in DRG cells, respectively (P<0.05). Therefore, mmu‑miR‑449a may ameliorate neuropathic pain by decreasing the activity of the channel proteins TRPA1 and KCNMA1 and increasing the levels of TPTE. mmu‑miR‑449a may be a potential therapeutic molecule for the alleviation of neuropathic pain.

Modulation of Excitability of Stellate Neurons in the Ventral Cochlear Nucleus of Mice by ATP-Sensitive Potassium Channels.

PubMed

Bal, Ramazan; Ozturk, Gurkan; Etem, Ebru Onalan; Him, Aydin; Cengiz, Nurattin; Kuloglu, Tuncay; Tuzcu, Mehmet; Yildirim, Caner; Tektemur, Ahmet

2018-02-01

Major voltage-activated ionic channels of stellate cells in the ventral part of cochlear nucleus (CN) were largely characterized previously. However, it is not known if these cells are equipped with other ion channels apart from the voltage-sensitive ones. In the current study, it was aimed to study subunit composition and function of ATP-sensitive potassium channels (K ATP ) in stellate cells of the ventral cochlear nucleus. Subunits of K ATP channels, Kir6.1, Kir6.2, SUR1, and SUR2, were expressed at the mRNA level and at the protein level in the mouse VCN tissue. The specific and clearly visible bands for all subunits but that for Kir6.1 were seen in Western blot. Using immunohistochemical staining technique, stellate cells were strongly labeled with SUR1 and Kir6.2 antibodies and moderately labeled with SUR2 antibody, whereas the labeling signals for Kir6.1 were too weak. In patch clamp recordings, K ATP agonists including cromakalim (50 µM), diazoxide (0.2 mM), 3-Amino-1,2,4-triazole (ATZ) (1 mM), 2,2-Dithiobis (5-nitro pyridine) (DTNP) (330 µM), 6-Chloro-3-isopropylamino- 4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC 55-0118) (1 µM), 6-chloro-3-(methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414) (1 µM), and H 2 O 2 (0.88 mM) induced marked responses in stellate cells, characterized by membrane hyperpolarization which were blocked by K ATP antagonists. Blockers of K ATP channels, glibenclamide (0.2 mM), tolbutamide (0.1 mM) as well as 5-hydroxydecanoic acid (1 mM), and catalase (500 IU/ml) caused depolarization of stellate cells, increasing spontaneous action potential firing. In conclusion, K ATP channels seemed to be composed dominantly of Kir 6.2 subunit and SUR1 and SUR2 and activation or inhibition of K ATP channels regulates firing properties of stellate cells by means of influencing resting membrane potential and input resistance.

All-trans retinoic acid increases the expression of oxidative myosin heavy chain through the PPARδ pathway in bovine muscle cells derived from satellite cells.

PubMed

Kim, Jongkyoo; Wellmann, Kimberly B; Smith, Zachary K; Johnson, Bradley J

2018-04-24

All-trans retinoic acid (ATRA) has been associated with various physiological phenomenon in mammalian adipose tissue and skeletal muscle. We hypothesized that ATRA may affect skeletal muscle fiber type in bovine satellite cell culture through various transcriptional processes. Bovine primary satellite cell (BSC) culture experiments were conducted to determine dose effects of ATRA on expression of genes and protein levels related to skeletal muscle fiber type and metabolism. The semimembranosus from crossbred steers (n = 2 steers), aged approximately 24 months, were used to isolate BSC for 3 separate assays. Myogenic differentiation was induced using 3% horse serum upon cultured BSC with increasing doses (0, 1, 10, 100, 1000 nM) of ATRA. After 96 h of incubation, cells were harvested and used to measure the gene expression of protein kinase B (Akt), AMP-activated protein kinase alpha (AMPK), glucose transporter 4 (GLUT4), myogenin, lipoprotein lipase (LPL), myosin heavy chain (MHC) I, MHC IIA,MHC IIX, insulin like growth factor -1 (IGF-1), Peroxisome proliferator activated receptor gamma (PPARγ), PPARδ, and Smad transcription factor 3 (SMAD3) mRNA relative to ribosomal protein subunit 9 (RPS9). The mRNA expression of LPL was increased (P < 0.05) with 100 and 1000nM of ATRA. Expression of GLUT4 was altered (P < 0.05) by ATRA. The treatment of ATRA (1000nM) also increased (P < 0.05) mRNA gene expression of SMAD3. The gene expression of both PPARδ and PPARγ were increased (P < 0.05) with 1000nM of ATRA. Protein level of PPARδ was also affected (P < 0.05) by 1000nM of ATRA and resulted in a greater (P < 0.05) protein level of PPARδ compared to CON. All-trans retinoic acid (10nM) increased gene expression of MHC I (P < 0.05) compared to CON. Expression of MHC IIA was also influenced (P < 0.05) by ATRA. The mRNA expression of MHC IIX was decreased (P < 0.05) with 100 and 1000nM of ATRA.In muscle cells, ATRA may cause muscle fibers to transition towards the MHC isoform that prefers oxidative metabolism, as evidenced by increased expression of genes associated with the MHC I isoform. These changes in MHC isoforms appeared to be brought about by changing PPARδ gene expression and protein levels.

Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells.

PubMed

Qian, Jie; Mummalaneni, Shobha K; Alkahtani, Reem M; Mahavadi, Sunila; Murthy, Karnam S; Grider, John R; Lyall, Vijay

2016-01-01

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and β nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-β-erythroidine, a α4β2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and β4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

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.

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury

PubMed Central

Drexel, Meinrad; Puhakka, Noora; Kirchmair, Elke; Hörtnagl, Heide; Pitkänen, Asla; Sperk, Günther

2015-01-01

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei. This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’. PMID:25229716

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury.

PubMed

Drexel, Meinrad; Puhakka, Noora; Kirchmair, Elke; Hörtnagl, Heide; Pitkänen, Asla; Sperk, Günther

2015-01-01

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Transcriptome and Proteome Analyses of TNFAIP8 Knockdown Cancer Cells Reveal New Insights into Molecular Determinants of Cell Survival and Tumor Progression.

PubMed

Day, Timothy F; Mewani, Rajshree R; Starr, Joshua; Li, Xin; Chakravarty, Debyani; Ressom, Habtom; Zou, Xiaojun; Eidelman, Ofer; Pollard, Harvey B; Srivastava, Meera; Kasid, Usha N

2017-01-01

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

AMPKα, hs-CRP and FcγR in diabetic nephropathy and drug intervention.

PubMed

Sun, Ping; Lu, Lei; Chen, Jun; Liu, Xiao Dan; Zhang, Qing; Wang, Xu

2018-06-01

The aim of this study was to investigate the roles of AMP-activated protein kinase α subunit (AMPKα), hypersensitive C-reactive protein (hs-CRP) and Fcγ receptor (FcγR) in diabetic nephropathy and drug intervention effects. Sixty Sprague Dawley male rats were randomly divided into the control (n=30) and observation (n=30) groups. The model of type 2 diabetic nephropathy was established by high-fat and high-glucose diet and streptozotocin injection. The rats in the observation group were treated with baicalein and the rats in control group did not receive any drug intervention. The pathological changes of kidneys were observed by hematoxylin and eosin (H&E) staining. The expression of AMPKα mRNA in renal tissue was detected by reverse transcription-polymerase chain reaction (RT-PCR). The levels of hs-CRP and FcγR were measured by enzyme-linked immunosorbent assay (ELISA) at 1, 4, 6 and 8 weeks after drug intervention and blood urea nitrogen (BUN) and the 24 h urinary micro-albumin (U-ALB) levels were compared at 1, 4, 6 and 8 weeks after intervention. After 8 weeks of drug intervention, the pathological changes of kidneys in the observation group were significantly lower than those in the control group (p<0.05), while the relative expression levels of AMPKα mRNA and protein in the control group were higher than those in the observation group (p<0.05). The levels of hs-CRP, BUN and 24 h U-ALB in the control group were significantly higher than those in the observation group at different time-points after drug intervention and the level of FcγR in the control group was significantly lower than that in the observation group (p<0.05). Baicalein may protect renal function by inhibiting the expression of AMPKα and inflammatory reaction, and can also decrease BUN and 24 h U-ALB levels and improve the pathological changes of the kidney.

High dosage of cannabidiol (CBD) alleviates pentylenetetrazole-induced epilepsy in rats by exerting an anticonvulsive effect

PubMed Central

Mao, Ke; You, Chao; Lei, Ding; Zhang, Heng

2015-01-01

The study was designed to investigate the effect of various concentrations of cannabidiol (CBD) in rats with chronic epilepsy. The chronic epilepsy rat model was prepared by intraperitoneally injecting pentylenetetrazole to the rats pre-treated with CBD (10, 20 and 50 mg/kg) for 28 consecutive days. Behavioral measurements of convulsion following pentylenetetrazole treatment and morphological changes of the hippocampal neurons with hematoxylin and eosin staining were used to observe the epileptic behaviour. Immunohistochemistry was used to detect the expression levels of glial fibrillary acidic protein and inducible nitric oxide synthase (iNOS) in the hippocampus. The mRNA expression of N-methyl-D-aspartic acid (NMDA) receptor subunits (NR1 and NR2B) was detected by reverse transcription polymerase chain reaction. The results revealed a significant decrease in the daily average grade of epileptic seizures on treatment with CBD (50 mg/kg). The neuronal loss and astrocyte hyperplasia in the hippocampal area were also decreased. CBD treatment did not affect the expression of iNOS in the hippocampus; however, the expression of NR1 was decreased significantly. Thus, CBD administration inhibited the effect of pentylenetetrazole in rats, decreased the astrocytic hyperplasia, decreased neuronal damage in the hippocampus caused by seizures and selectively reduced the expression of the NR1 subunit of NMDA. Therefore, CBD exhibits an anticonvulsive effect in the rats with chronic epilepsy. PMID:26309534

High dosage of cannabidiol (CBD) alleviates pentylenetetrazole-induced epilepsy in rats by exerting an anticonvulsive effect.

PubMed

Mao, Ke; You, Chao; Lei, Ding; Zhang, Heng

2015-01-01

The study was designed to investigate the effect of various concentrations of cannabidiol (CBD) in rats with chronic epilepsy. The chronic epilepsy rat model was prepared by intraperitoneally injecting pentylenetetrazole to the rats pre-treated with CBD (10, 20 and 50 mg/kg) for 28 consecutive days. Behavioral measurements of convulsion following pentylenetetrazole treatment and morphological changes of the hippocampal neurons with hematoxylin and eosin staining were used to observe the epileptic behaviour. Immunohistochemistry was used to detect the expression levels of glial fibrillary acidic protein and inducible nitric oxide synthase (iNOS) in the hippocampus. The mRNA expression of N-methyl-D-aspartic acid (NMDA) receptor subunits (NR1 and NR2B) was detected by reverse transcription polymerase chain reaction. The results revealed a significant decrease in the daily average grade of epileptic seizures on treatment with CBD (50 mg/kg). The neuronal loss and astrocyte hyperplasia in the hippocampal area were also decreased. CBD treatment did not affect the expression of iNOS in the hippocampus; however, the expression of NR1 was decreased significantly. Thus, CBD administration inhibited the effect of pentylenetetrazole in rats, decreased the astrocytic hyperplasia, decreased neuronal damage in the hippocampus caused by seizures and selectively reduced the expression of the NR1 subunit of NMDA. Therefore, CBD exhibits an anticonvulsive effect in the rats with chronic epilepsy.

The Mediator complex and transcription regulation

PubMed Central

Poss, Zachary C.; Ebmeier, Christopher C.

2013-01-01

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein.

PubMed

Islas-Osuna, María A; Silva-Moreno, Begonia; Caceres-Carrizosa, Nidia; García-Robles, Jesús M; Sotelo-Mundo, Rogerio R; Yepiz-Plascencia, Gloria M

2006-05-01

Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.

Acetylcholine affects osteocytic MLO-Y4 cells via acetylcholine receptors.

PubMed

Ma, Yuanyuan; Li, Xianxian; Fu, Jing; Li, Yue; Gao, Li; Yang, Ling; Zhang, Ping; Shen, Jiefei; Wang, Hang

2014-03-25

The identification of the neuronal control of bone remodeling has become one of the many significant recent advances in bone biology. Cholinergic activity has recently been shown to favor bone mass accrual by complex cellular regulatory networks. Here, we identified the gene expression of the muscarinic and nicotinic acetylcholine receptors (m- and nAChRs) in mice tibia tissue and in osteocytic MLO-Y4 cells. Acetylcholine, which is a classical neurotransmitter and an osteo-neuromediator, not only influences the mRNA expression of the AChR subunits but also significantly induces the proliferation and viability of osteocytes. Moreover, acetylcholine treatment caused the reciprocal regulation of RANKL and OPG mRNA expression, which resulted in a significant increase in the mRNA ratio of RANKL:OPG in osteocytes via acetylcholine receptors. The expression of neuropeptide Y and reelin, which are two neurogenic markers, was also modulated by acetylcholine via m- and nAChRs in MLO-Y4 cells. These results indicated that osteocytic acetylcholine receptors might be a new valuable mediator for cell functions and even for bone remodeling. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Sirtuin 1 (Sirt1) Overexpression in BaF3 Cells Contributes to Cell Proliferation Promotion, Apoptosis Resistance and Pro-Inflammatory Cytokine Production.

PubMed

Wang, Qian; Yan, Chao; Xin, Miaomiao; Han, Li; Zhang, Yunqing; Sun, Mingshu

2017-03-27

BACKGROUND B lymphocyte hyperactivity is a main characteristic of systemic lupus erythematosus (SLE), and B lymphocytes play a prominent pathogenic role in the development and progression of SLE. The aim of this study was to investigate the role of Sirtuin 1 (Sirt1) in B lymphocytes. MATERIAL AND METHODS Mouse B lymphocytes BaF3 was transfected with Sirt1 vector or shRNA against Sirt1. Then the transfected cells viability and apoptosis were respectively determined by MTT assay and flow cytometry. In addition, the mRNA levels of three pro-inflammatory cytokines and p53 were detected by RT-PCR. Furthermore, the expression levels of nuclear factor-kappa B (NF-κB) pathway proteins were measured by Western blot. RESULTS Overexpression of Sirt1 significantly increased cell proliferation (p<0.05 or p<0.01) and significantly suppressed apoptosis (p<0.05). The mRNA level expressions of interleukin 1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α) were significantly upregulated (p<0.05 or p<0.01), whereas p53 was significantly downregulated (p<0.05) by Sirt1 overexpression. In addition, the inhibitory subunit of NF-κB (IκBα) and p65 were significantly activated and phosphorylated (p<0.01 or p<0.001), and B-Cell CLL/Lymphoma 3 (Bcl-3) was significantly upregulated (p<0.05) by Sirt1 overexpression. CONCLUSIONS These results suggested that Sirt1 overexpression could promote BaF3 cell proliferation, inhibit apoptosis, and upregulate pro-inflammatory cytokines. The NF-κB pathway might be involved in these effects of Sirt1 on BaF3 cells, and Sirt1 might be a potential risk factor of SLE.

TRPV1: Contribution to Retinal Ganglion Cell Apoptosis and Increased Intracellular Ca2+ with Exposure to Hydrostatic Pressure

PubMed Central

Sappington, Rebecca M.; Sidorova, Tatiana; Long, Daniel J.; Calkins, David J.

2013-01-01

Purpose Elevated hydrostatic pressure induces retinal ganglion cell (RGC) apoptosis in culture. The authors investigated whether the transient receptor potential vanilloid 1 (TRPV1) channel, which contributes to pressure sensing and Ca2+-dependent cell death in other systems, also contributes to pressure-induced RGC death and whether this contribution involves Ca2+. Methods trpv1 mRNA expression in RGCs was probed with the use of PCR and TRPV1 protein localization through immunocytochemistry. Subunit-specific antagonism (iodo-resiniferatoxin) and agonism (capsaicin) were used to probe how TRPV1 activation affects the survival of isolated RGCs at ambient and elevated hydrostatic pressure (+70 mm Hg). Finally, for RGCs under pressure, the authors tested whether EGTA chelation of Ca2+ improves survival and whether, with the Ca2+ dye Fluo-4 AM, TRPV1 contributes to increased intracellular Ca2+. Results RGCs express trpv1 mRNA, with robust TRPV1 protein localization to the cell body and axon. For isolated RGCs under pressure, TRPV1 antagonism increased cell density and reduced apoptosis to ambient levels (P ≤ 0.05), whereas for RGCs at ambient pressure, TRPV1 agonism reduced density and increased apoptosis to levels for elevated pressure (P ≤ 0.01). Chelation of extracellular Ca2+ reduced RGC apoptosis at elevated pressure by nearly twofold (P ≤ 0.01). Exposure to elevated hydrostatic pressure induced a fourfold increase in RGC intracellular Ca2+ that was reduced by half with TRPV1 antagonism. Finally, in the DBA/2 mouse model of glaucoma, levels of TRPV1 in RGCs increased with elevated IOP. Conclusions RGC apoptosis induced by elevated hydrostatic pressure arises substantially through TRPV1, likely through the influx of extracellular Ca2+. PMID:18952924

Docosahexaenoic acid prevents paraquat-induced reactive oxygen species production in dopaminergic neurons via enhancement of glutathione homeostasis

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

Lee, Hyoung Jun; Han, Jeongsu; Jang, Yunseon

Highlights: • DHA prevents PQ-induced dopaminergic neuronal loss via decreasing of excessive ROS. • DHA increases GR and GCLm derivate GSH pool by enhancement of Nrf2 expression. • Protective mechanism is removal of PQ-induced ROS via DHA-dependent GSH pool. • DHA may be a good preventive strategy for Parkinson’s disease (PD) therapy. - Abstract: Omega-3 polyunsaturated fatty acid levels are reduced in the substantia nigra area in Parkinson’s disease patients and animal models, implicating docosahexaenoic acid (DHA) as a potential treatment for preventing Parkinson’s disease and suggesting the need for investigations into how DHA might protect against neurotoxin-induced dopaminergic neuronmore » loss. The herbicide paraquat (PQ) induces dopaminergic neuron loss through the excessive production of reactive oxygen species (ROS). We found that treatment of dopaminergic SN4741 cells with PQ reduced cell viability in a dose-dependent manner, but pretreatment with DHA ameliorated the toxic effect of PQ. To determine the toxic mechanism of PQ, we measured intracellular ROS content in different organelles with specific dyes. As expected, all types of ROS were increased by PQ treatment, but DHA pretreatment selectively decreased cytosolic hydrogen peroxide content. Furthermore, DHA treatment-induced increases in glutathione reductase and glutamate cysteine ligase modifier subunit (GCLm) mRNA expression were positively correlated with glutathione (GSH) content. Consistent with this increase in GCLm mRNA levels, Western blot analysis revealed that DHA pretreatment increased nuclear factor-erythroid 2 related factor 2 (Nrf2) protein levels. These findings indicate that DHA prevents PQ-induced neuronal cell loss by enhancing Nrf2-regulated GSH homeostasis.« less

GABAA receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

PubMed Central

Chandra, Dev; Korpi, Esa R; Miralles, Celia P; De Blas, Angel L; Homanics, Gregg E

2005-01-01

Background Gamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. Results Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. Conclusion We conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably reduced the amount of γ2, and that 2) attenuated expression of γ2 increased anxiety-like behaviors but did not lead to differences in the hypnotic response to benzodiazepine site ligands. This suggests that reduced synaptic inhibition can lead to a phenotype of increased anxiety-like behavior. In contrast, normal drug effects can be maintained despite a dramatic reduction in GABAA-R targets. PMID:15850489

E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

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

Fang, Zejun; Gong, Chaoju; Liu, Hong

2015-08-21

As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression ofmore » E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and invasion of CRC cells by activating RRM2. • E2F1 is upregulated in CRC tissues and positively associated with RRM2 level. • E2F1-mediated RRM2 transcription will provide a new strategy in CRC.« less

Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression

PubMed Central

2013-01-01

Background Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). Results Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 μg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. Conclusions Avermectins are believed to exert their toxicity to invertebrates through interaction with glutamate-gated and GABA-gated chloride channels. Further potential drug targets include other Cys-loop ion channels such as nAChR. The present study demonstrates decreased transcript abundances of GABA-Cl and nAChR subunits in EMB-resistant salmon lice, suggesting their involvement in avermectin toxicity in caligids. PMID:23773482