Gene expression levels of heat shock proteins in the soleus and plantaris muscles of rats after hindlimb suspension or spaceflight.

PubMed

Ishihara, Akihiko; Fujino, Hidemi; Nagatomo, Fumiko; Takeda, Isao; Ohira, Yoshinobu

2008-12-01

Gene expression levels of heat shock proteins (HSPs) in the slow-twitch soleus and fast-twitch plantaris muscles of rats were determined after hindlimb suspension or spaceflight. Male rats were hindlimb-suspended for 14 d or exposed to microgravity for 9 d. The mRNA expression levels of HSP27, HSP70, and HSP84 in the hindlimb-suspended and microgravity-exposed groups were compared with those in the controls. The mRNA expression levels of the 3 HSPs in the soleus muscle under normal conditions were higher compared with those in the plantaris muscle. The mRNA expression levels of the 3 HSPs in the soleus muscle were inhibited by hindlimb suspension and spaceflight. The mRNA expression levels of the 3 HSPs in the plantaris muscle did not change after hindlimb suspension. It is suggested that the mRNA expression levels of the 3 HSPs are regulated by the mechanical and neural activity levels, and therefore the decreased mRNA expression levels of HSPs in the slow-twitch muscle following hindlimb suspension and spaceflight are related to a reduction in the mechanical and neural activity levels.

Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

PubMed Central

Pascal, Laura E; True, Lawrence D; Campbell, David S; Deutsch, Eric W; Risk, Michael; Coleman, Ilsa M; Eichner, Lillian J; Nelson, Peter S; Liu, Alvin Y

2008-01-01

Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers. PMID:18501003

Effects of a Transposable Element Insertion on Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Dunn, R. C.; Laurie, C. C.

1995-01-01

Variation in the DNA sequence and level of alcohol dehydrogenase (Adh) gene expression in Drosophila melanogaster have been studied to determine what types of DNA polymorphisms contribute to phenotypic variation in natural populations. The Adh gene, like many others, shows a high level of variability in both DNA sequence and quantitative level of expression. A number of transposable element insertions occur in the Adh region and one of these, a copia insertion in the 5' flanking region, is associated with unusually low Adh expression. To determine whether this insertion (called RI42) causes the low expression level, the insertion was excised from the cloned RI42 Adh gene and the effect was assessed by P-element transformation. Removal of this insertion causes a threefold increase in the level of ADH, clearly showing that it contributes to the naturally occurring variation in expression at this locus. Removal of all but one LTR also causes a threefold increase, indicating that the mechanism is not a simple sequence disruption. Furthermore, this copia insertion, which is located between the two Adh promoters and their upstream enhancer sequences, has differential effects on the levels of proximal and distal transcripts. Finally, a test for the possible modifying effects of two suppressor loci, su(w(a)) and su(f), on this insertional mutation was negative, in contrast to a previous report in the literature. PMID:7498745

4-Nitroquinoline-1-oxide effects human lung adenocarcinoma A549 cells by regulating the expression of POLD4

PubMed Central

HUANG, QIN-MIAO; ZENG, YI-MING; ZHANG, HUA-PING; LV, LIANG-CHAO; YANG, DONG-YONG; LIN, HUI-HUANG

2016-01-01

The aim of the present study was to explore the expression of POLD4 in human lung adenocarcinoma A549 cells under 4-nitroquinoline-1-oxide (4NQO) stimulation to investigate the role of POLD4 in smoking-induced lung cancer. The lung cancer A549 cell line was treated with 4NQO, with or without MG132 (an inhibitor of proteasome activity), and subsequently the POLD4 level was determined by western blot analysis. Secondly, the cell sensitivity to 4NQO and Taxol was determined when the POLD4 expression level was downregulated by siRNA. The POLD4 protein levels in the A549 cells decreased following treatment with 4NQO; however, MG132 could reverse this phenotype. Downregulation of the POLD4 expression by siRNA enhanced A549 cell sensitivity to 4NQO, but not to Taxol. In conclusion, 4NQO affects human lung adenocarcinoma A549 cells by regulating the expression of POLD4. PMID:26998273

Model-based Analysis of HER Activation in Cells Co-Expressing EGFR, HER2 and HER3.

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

Shankaran, Harish; Zhang, Yi; Tan, Yunbing

2013-08-22

The HER/ErbB family of receptor tyrosine kinases drive critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panelmore » of human mammary epithelial cells expressing varying levels of EGFR, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of epithelial cells lines with known HER expression levels. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1/1 and HER1/2 dimers, and not HER1/3 dimers, ii) HER1/2 and HER2/3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2/3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.« less

Quantifying HER-2 expression on circulating tumor cells by ACCEPT

PubMed Central

van Dalum, Guus; Decraene, Charles; Proudhon, Charlotte; Fehm, Tanja; Neubauer, Hans; Rack, Brigitte; Alunni-Fabbroni, Marianna; Terstappen, Leon W. M. M.; van Gils, Stephan A.; Brune, Christoph

2017-01-01

Circulating tumor cells (CTCs) isolated from blood can be probed for the expression of treatment targets. Immunofluorescence is often used for both the enumeration of CTC and the determination of protein expression levels related to treatment targets. Accurate and reproducible assessment of such treatment target expression levels is essential for their use in the clinic. To enable this, an open source image analysis program named ACCEPT was developed in the EU-FP7 CTCTrap and CANCER-ID programs. Here its application is shown on a retrospective cohort of 132 metastatic breast cancer patients from which blood samples were processed by CellSearch® and stained for HER-2 expression as additional marker. Images were digitally stored and reviewers identified a total of 4084 CTCs. CTC’s HER-2 expression was determined in the thumbnail images by ACCEPT. 150 of these images were selected and sent to six independent investigators to score the HER-2 expression with and without ACCEPT. Concordance rate of the operators’ scoring results for HER-2 on CTCs was 30% and could be increased using the ACCEPT tool to 51%. Automated assessment of HER-2 expression by ACCEPT on 4084 CTCs of 132 patients showed 8 (6.1%) patients with all CTCs expressing HER-2, 14 (10.6%) patients with no CTC expressing HER-2 and 110 (83.3%) patients with CTCs showing a varying HER-2 expression level. In total 1576 CTCs were determined HER-2 positive. We conclude that the use of image analysis enables a more reproducible quantification of treatment targets on CTCs and leads the way to fully automated and reproducible approaches. PMID:29084234

Expression and role of neuroglobin in rats with sepsis-associated encephalopathy.

PubMed

Zhang, Li-Na; Ai, Yu-Hang; Gong, Hua; Guo, Qu-Lian; Huang, Li; Liu, Zhi-Yong; Yao, Bo

2014-01-01

To determine the role of neuroglobin in the pathology of sepsis-associated encephalopathy and ascertain if neuroglobin has any protective effects against sepsis-associated encephalopathy. Randomized laboratory animal study. Research university animal laboratory. Two hundred and forty adult male Sprague-Dawley rats. Rats received cecal puncture and ligation (or sham) surgery to induce sepsis, then broken up into groups based on whether or not the rat developed sepsis-associated encephalopathy as determined by electroencephalograph and evoked potential recordings. The rats were then left untreated to examine the effect of sepsis-associated encephalopathy on neuroglobin, treated with a neuroglobin antisense nucleotide to block gene expression, or given hemin, a neuroglobin inducer. Following sepsis induction, diagnosis, and treatment, the brains were analyzed for both gross and ultrastructural morphology. Also, neuronal neuroglobin immunoreactivity and apoptosis (via terminal uridine nucleotide end-labeling) were examined. Blood serum levels were then analyzed for neuroglobin, superoxide dismutase, and malondialdehyde levels. We determined that sepsis-associated encephalopathy induces damage evident when examining both gross and ultrastructural morphology, as well as induces neuronal neuroglobin expression. Also, blockade of neuroglobin expression via antisense treatment will exacerbate these pathological effects, while increasing neuroglobin levels via hemin will ameliorate them. Blood analysis found that levels of superoxide dismutase and malondialdehyde mirrored the level of pathology found in the brain, while plasma neuroglobin levels reflected the amount of neuronal neuroglobin immunoreactivity. We conclude that neuroglobin is involved in the pathogenesis of sepsis-associated encephalopathy and has neuroprotective effects. We also determined that hemin has protective effects against sepsis-associated encephalopathy as well, most probably due to its effect on neuroglobin.

The effects of cyclosporin on the collagenolytic activity of gingival fibroblasts.

PubMed

Hyland, Paula L; Traynor, Patrick S; Myrillas, Theofilos T; Marley, John J; Linden, Gerard J; Winter, Paul; Leadbetter, Nicola; Cawston, Timothy E; Irwin, Chris R

2003-04-01

The immunosuppressive agent cyclosporin is associated with a number of major side-effects including the development of gingival overgrowth. Although the pathogenesis of cyclosporin-induced gingival overgrowth remains unclear, it has been suggested that the finely regulated balance between extracellular matrix synthesis and degradation may be disturbed, resulting in an accumulation of excess connective tissue components within the gingival tissue. The aim of this study was to investigate the effect of cyclosporin on matrix metalloproteinases (MMP)-1 and tissue inhibitors of MMP (TIMP)-1 expression at the mRNA, protein, and enzyme activity levels. Gingival fibroblasts were grown to confluence and then cultured in serum-free medium supplemented with cyclosporin over the concentration range of 0 to 2000 ng/ml. MMP-1 and TIMP-1 mRNA levels in cultures were determined by reverse transcription polymerase chain reaction (RT-PCR), protein levels in whole conditioned medium were assessed by enzyme-linked immunosorbent assay (ELISA), and collagenolytic activity determined using a 3H-acetylated type I collagen degradation assay. Tissue mRNA levels in normal and overgrown gingiva were also determined by RT-PCR. Results indicated that cyclosporin inhibited MMP-1 expression at both the mRNA and protein level in a dose- and time-dependent fashion. The effects on TIMP-1 expression were less clear, cyclosporin inhibiting mRNA expression, but having no effect on TIMP-1 protein levels at any concentration studied. Addition of the drug resulted in reduced levels of collagenolytic activity in the culture medium. MMP-1 mRNA expression was significantly reduced in overgrown compared to normal tissue. These results add support to the hypothesis that the accumulation of collagen seen in gingival overgrowth can be explained by a cyclosporin-induced inhibition of collagenolytic activity within the gingival tissues.

CCR5 Expression Levels in HIV-Uninfected Women Receiving Hormonal Contraception

PubMed Central

Sciaranghella, Gaia; Wang, Cuiwei; Hu, Haihong; Anastos, Kathryn; Merhi, Zaher; Nowicki, Marek; Stanczyk, Frank Z.; Greenblatt, Ruth M.; Cohen, Mardge; Golub, Elizabeth T.; Watts, D. Heather; Alter, Galit; Young, Mary A.; Tsibris, Athe M. N.

2015-01-01

Human immunodeficiency virus (HIV) infectivity increases as receptor/coreceptor expression levels increase. We determined peripheral CD4, CCR5, and CXCR4 expression levels in HIV-uninfected women who used depot medroxyprogesterone acetate (DMPA; n = 32), the levonorgestrel-releasing intrauterine device (LNG-IUD; n = 27), oral contraceptive pills (n = 32), or no hormonal contraception (n = 33). The use of LNG-IUD increased the proportion of CD4+ and CD8+ T cells that expressed CCR5; increases in the magnitude of T-cell subset CCR5 expression were observed with DMPA and LNG-IUD use (P < .01 for all comparisons). LNG-IUD and, to a lesser extent, DMPA use were associated with increased peripheral T-cell CCR5 expression. PMID:25895986

[The level of superoxide dismutase expression in primary and metastatic colorectal cancer cells in hypoxia and tissue normoxia].

PubMed

Skrzycki, Michał; Czeczot, Hanna; Chrzanowska, Alicja; Otto-Ślusarczyk, Dagmara

2015-11-01

Superoxide oxidase (SOD) is a key antioxidant enzyme protecting cells against oxidative stress, which might induce cancerogenesis. In tumor cells SOD influences the level of the reactive oxygen species (ROS) allowing for survival and proliferation. High rate of cells proliferation in tumor leads to their temporary hypoxia due to lower rate of angiogenesis. Therefore during tumor development, cancer cells function in conditions of hypoxia or tissue normoxia. The aim of study was to evaluate of SOD isoenzymes (SOD1 and SOD2) expression level in cell lines of primary (SW 480) and metastatic (SW 620) colorectal cancer, cultured in hypoxia (1% oxygen), tissue normoxia (10% oxygen), and atmospheric normoxia (21% oxygen). Cells were cultured in MEM medium in different oxygen concentrations (1%, 10%, 21%) in hypoxic chamber with oxygenation regulator. The number of living cells in lines SW 480 and 620 was determined by trypan blue method. Expression of SOD1 and SOD2 at the mRNA level was determined by RT-PCR and PCR. In both studied cell lines (SW 480 and SW 620), the number of living cells (viability) was increased in hypoxia and atmospheric normoxia. The expression level of SOD1 and SOD2 in studied cell lines was different. The lowest level of expression of both SOD isoenzymes was observed in hypoxia. In conditions of atmospheric normoxia the expression level of SOD1 in SW480 cell line was increased, and similar in SW620 cell line comparing to tissue normoxia. Whereas the SOD2 expression level in atmospheric normoxia conditions in both cell lines was significantly increased. Observed differences were statistically significant (p ≤ 0,05). The profile of expression of SOD1 and SOD2 in cell lines SW480 and SW620 indicates differentiated response of tumor cells depending on access to oxygen. Low level of SOD isoenzymes expression in SW480 and SW620 cells in hypoxia indicates decreased production of ROS. Differences of SOD isoenzymes expression level in tissue normoxia indicate their compensatory action, allowing to maintain the balance between O₂- removal and H₂O₂production in studied tumor cells. In atmospheric normoxia conditions increased expression level of SOD1 and SOD2 observed in studied cell lines points to oxidative stress. © 2015 MEDPRESS.

miR-379 Regulates Cyclin B1 Expression and Is Decreased in Breast Cancer

PubMed Central

Khan, Sonja; Brougham, Cathy L.; Ryan, James; Sahrudin, Arisha; O’Neill, Gregory; Wall, Deirdre; Curran, Catherine; Newell, John; Kerin, Michael J.; Dwyer, Roisin M.

2013-01-01

MicroRNAs are small non-coding RNA molecules that control gene expression post-transcriptionally, and are known to be altered in many diseases including breast cancer. The aim of this study was to determine the relevance of miR-379 in breast cancer. miR-379 expression was quantified in clinical samples including tissues from breast cancer patients (n=103), healthy controls (n=30) and patients with benign breast disease (n=35). The level of miR-379 and its putative target Cyclin B1 were investigated on all breast tissue specimens by RQ-PCR. Potential relationships with gene expression and patient clinicopathological details were also determined. The effect of miR-379 on Cyclin B1 protein expression and function was investigated using western blot, immunohistochemistry and proliferation assays respectively. Finally, the levels of circulating miR-379 were determined in whole blood from patients with breast cancer (n=40) and healthy controls (n=34). The level of miR-379 expression was significantly decreased in breast cancer (Mean(SEM) 1.9 (0.09) Log10 Relative Quantity (RQ)) compared to normal breast tissues (2.6 (0.16) Log10 RQ, p<0.01). miR-379 was also found to decrease significantly with increasing tumour stage. A significant negative correlation was determined between miR-379 and Cyclin B1 (r=-0.31, p<0.001). Functional assays revealed reduced proliferation (p<0.05) and decreased Cyclin B1 protein levels following transfection of breast cancer cells with miR-379. Circulating miR-379 was not significantly dysregulated in patients with breast cancer compared to healthy controls (p=0.42). This data presents miR-379 as a novel regulator of Cyclin B1 expression, with significant loss of the miRNA observed in breast tumours. PMID:23874748

Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

PubMed

Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

2014-05-01

To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and cultured without glutamine showed higher levels of ROS and IL-8 than those transfected with negative control siRNA; increased levels of ROS and IL-8 were suppressed by the treatment of glutamine. Glutamine deprivation induces ROS production, NF-κB activation, and IL-8 expression as well as a reduction in GSH in A-T fibroblasts, all of which are attenuated by glutamine supplementation.

The mRNA expression levels of uncoupling proteins 1 and 2 in mononuclear cells from patients with metabolic disorders: obesity and type 2 diabetes mellitus.

PubMed

Margaryan, Sona; Witkowicz, Agata; Partyka, Anna; Yepiskoposyan, Levon; Manukyan, Gayane; Karabon, Lidia

2017-10-19

Type 2 diabetes mellitus (T2DM) and obesity are metabolic disorders whose major hallmark is insulin resistance. Impaired mitochondrial activity, such as reduced ratio of energy production to respiration, has been implicated in the development of insulin resistance. Uncoupling proteins (UCPs) are proton carriers, expressed in the mitochondrial inner membrane, that uncouple oxygen consumption by the respiratory chain from ATP synthesis. The aim of the study was to determine transcriptional levels of UCP1 and UCP2 in peripheral blood mononuclear cells (PBMCs) from patients with metabolic disorders: T2DM, obesity and from healthy individuals. The mRNA levels of UCP1, UCP2 were determined by Real-Time PCR method using Applied Biosystems assays. The UCP1 mRNA expression level was not detectable in the majority of studied samples, while very low expression was found in PBMCs from 3 obese persons. UCP2 mRNA expression level was detectable in all samples. The median mRNA expression of UCP2 was lower in all patients with metabolic disorders as compared to the controls (0.20+0.14 vs. 0.010+0.009, p=0.05). When compared separately, the differences of medians UCP2 mRNA expression level between the obese individuals and the controls as well as between the T2DM patients and the controls did not reach statistical significance. Decreased UCP2 gene expression in mononuclear cells from obese and diabetic patients might contribute to the immunological abnormalities in these metabolic disorders and suggests its role as a candidate gene in future studies of obesity and diabetes.

Effects of Angiotensin Inhibitor Valsartan on the Expression of the Angiotensin II 1 Receptor, Matrix Metalloproteinases -2 and -9 in Human Bladder Cancer Cell Lines.

PubMed

Yang, Delin; Huo, Qian; Luan, Ting; Wang, Jiansong; Tang, Zhaoran; Wang, Haifeng

2016-08-01

In order to investigate how valsartan-the angiotensin II 1 receptor (AT1R) antagonist-affects the expressions of AT1R antigen, matrix metalloproteinases (MMPs) -2 and -9 in carcinoma of urinary bladder (CUB) cell lines with different invasive abilities. Three cell lines, EJ-M3, EJ, and BIU-87, with different invasive abilities were cultured and treated with valsartan. Cell proliferation states were determined by the methyl thiazolyl tetrazolium (MTT) method. The expressions at protein level and gene level were determined by Western blot and real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR), respectively. The invasive abilities and migratory abilities of the three cell lines were determined by Transwell in vitro cell invasion assay and wound healing assay, respectively. MTT results show that valsartan can inhibit the proliferation of CUB cells, and the inhibition effect is enhanced with the increase of concentration. AngII promotes the MMP2 and MMP9 expressions (both protein and gene levels) in CUB cells through AT1R, but their expressions can be effectively inhibited by valsartan, the AngII inhibitor. AngII inhibitor may become a novel drug that can inhibit CUB metastasis and prolong the survival of CUB patients.

Expression of classical components of the renin-angiotensin system in the human eye.

PubMed

White, Andrew J R; Cheruvu, Sarat C; Sarris, Maria; Liyanage, Surabhi S; Lumbers, Eugenie; Chui, Jeanie; Wakefield, Denis; McCluskey, Peter J

2015-03-01

The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions. © The Author(s) 2014.

Elevated levels of serum-soluble triggering receptor expressed on myeloid cells-1 in patients with IBD do not correlate with intestinal TREM-1 mRNA expression and endoscopic disease activity.

PubMed

Saurer, Leslie; Rihs, Silvia; Birrer, Michèle; Saxer-Seculic, Nikolina; Radsak, Markus; Mueller, Christoph

2012-10-01

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory responses. We have previously demonstrated a substantial increase in TREM-1-expressing macrophages in the inflamed intestinal mucosa of patients with inflammatory bowel diseases (IBD). TREM-1 is also produced as a soluble receptor (sTREM-1). Here, we aimed to determine whether serum sTREM-1 could be used as a surrogate marker of disease activity in patients with IBD. Intestinal biopsies and concurrently collected sera from patients with Crohn's disease (CD) and Ulcerative colitis (UC) enrolled in the Swiss IBD cohort study were analyzed for intestinal TREM-1 mRNA and serum sTREM-1 expression. TREM-1 mRNA and sTREM-1 were correlated with the endoscopically determined disease activity. Serum sTREM-1 and TREM-1 mRNA expression levels were further determined in sera and colonic tissues collected at various time-points post disease induction in an experimental mouse model of colitis and correlated with disease activity. Expression of TREM-1 mRNA was upregulated in intestinal biopsies from patients with active disease but not in patients with quiescent disease. Serum sTREM-1 was elevated in IBD patients compared to normal controls. No substantial differences in sTREM-1 expression levels were found in patients with active versus quiescent disease. In colitic mice, colonic TREM-1 mRNA and serum sTREM-1 were also upregulated. While colonic TREM-1 mRNA expression levels correlated with disease activity, augmented serum sTREM-1 in fact associated with a milder course of disease. Analysis of sTREM-1 as a surrogate marker of disease activity in patients with IBD warrants caution. Copyright © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

Transient expression and cellular localization of recombinant proteins in cultured insect cells

USDA-ARS?s Scientific Manuscript database

Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

The influence of social networks on patients' attitudes toward type II diabetes.

PubMed

Mani, Nandini; Caiola, Enrico; Fortuna, Robert J

2011-10-01

Social networks are increasingly recognized as important determinants of many chronic diseases, yet little data exist regarding the influence of social networks on diabetes. We surveyed diabetic patients to determine how social networks affect their overall level of concern regarding diabetes and its complications. We adapted a previously published instrument and surveyed 240 diabetic patients at two primary care practices. Patients recorded the number of family and friends who had diabetes and rated their level of concern about diabetes on a scale of 0% (no concern) to 100% (extremely concerned). Our primary outcome variable was patients' level of concern (<75% or ≥75%). We developed logistic regression models to determine the effect of disease burden in patients' social networks on expressed level of concern about diabetes. We received 154 surveys (64% response rate). We found that for each additional family member with diabetes, patients expressed a greater level of concern about diabetes (AOR 1.5; 95% CI 1.2-2.0) and its potential complications (AOR 1.4; 95% CI 1.1-1.7). Similarly, patients with an increased number of friends with diabetes expressed greater concern about diabetes (AOR 1.5; 95% CI 1.2-1.9) and its complications (AOR 1.3; 95% CI 1.1-1.7). Patients with a higher prevalence of diabetes within their social networks expressed greater concern about diabetes and diabetic complications. Determining disease burden within patients' social networks may allow physicians to better understand patients' perspectives on their disease and ultimately help them achieve meaningful behavioral change.

Epithelial Membrane Protein-2 in Human Proliferative Vitreoretinopathy and Epiretinal Membranes.

PubMed

Telander, David G; Yu, Alfred K; Forward, Krisztina I; Morales, Shawn A; Morse, Lawrence S; Park, Susanna S; Gordon, Lynn K

2016-06-01

To determine the level of epithelial membrane protein-2 (EMP2) expression in preretinal membranes from surgical patients with proliferative vitreoretinopathy (PVR) or epiretinal membranes (ERMs). EMP2, an integrin regulator, is expressed in the retinal pigment epithelium and understanding EMP2 expression in human retinal disease may help determine whether EMP2 is a potential therapeutic target. Preretinal membranes were collected during surgical vitrectomies after obtaining consents. The membranes were fixed, processed, sectioned, and protein expression of EMP2 was evaluated by immunohistochemistry. The staining intensity (SI) and percentage of positive cells (PP) in membranes were compared by masked observers. Membranes were categorized by their cause and type including inflammatory and traumatic. All of the membranes stained positive for EMP2. Proliferative vitreoretinopathy-induced membranes (all causes) showed greater expression of EMP2 than ERMs with higher SI (1.81 vs. 1.38; P = 0.07) and PP (2.08 vs. 1.54; P = 0.09). However all the PVR subgroups had similar levels of EMP2 expression without statistically significant differences by Kruskal-Wallis test. Inflammatory PVR had higher expression of EMP2 than ERMs (SI of 2.58 vs. 1.38); however, this was not statistically significant. No correlation was found between duration of PVR membrane and EMP2 expression. EMP2 was detected by RT-PCR in all samples (n = 6) tested. All studied ERMs and PVR membranes express EMP2. Levels of EMP2 trended higher in all PVR subgroups than in ERMs, especially in inflammatory and traumatic PVR. Future studies are needed to determine the role of EMP2 in the pathogenesis and treatment of various retinal conditions including PVR.

Differential gene expression in queen–worker caste determination in bumble-bees

PubMed Central

Pereboom, Jeffrey J. M; Jordan, William C; Sumner, Seirian; Hammond, Robert L; Bourke, Andrew F. G

2005-01-01

Investigating how differential gene expression underlies caste determination in the social Hymenoptera is central to understanding how variation in gene expression underlies adaptive phenotypic diversity. We investigated for the first time the association between differential gene expression and queen–worker caste determination in the bumble-bee Bombus terrestris. Using suppression subtractive hybridization we isolated 12 genes that were differentially expressed in queen- and worker-destined larvae. We found that the sets of genes underlying caste differences in larvae and adults failed to overlap greatly. We also found that B. terrestris shares some of the genes whose differential expression is associated with caste determination in the honeybee, Apis mellifera, but their expression patterns were not identical. Instead, we found B. terrestris to exhibit a novel pattern, whereby most genes upregulated (i.e. showing relatively higher levels of expression) in queen-destined larvae early in development were upregulated in worker-destined larvae late in development. Overall, our results suggest that caste determination in B. terrestris involves a difference not so much in the identity of genes expressed by queen- and worker-destined larvae, but primarily in the relative timing of their expression. This conclusion is of potential importance in the further study of phenotypic diversification via differential gene expression. PMID:16024376

Environmental sex determination mechanisms in reptiles.

PubMed

Merchant-Larios, H; Díaz-Hernández, V

2013-01-01

Temperature-dependent sex determination (TSD) was first discovered in reptiles. Since then, a great diversity of sex-determining responses to temperature has been reported. Higher temperatures can produce either males or females, and the temperature ranges and lengths of exposure that influence TSD are remarkably variable among species. In addition, transitory gene regulatory networks leading to gonadal TSD have evolved. Although most genes involved in gonadal development are conserved in vertebrates, including TSD species, temporal and spatial gene expression patterns vary among species. Despite variation in TSD pattern and gene expression heterochrony, the structural framework, the medullary cords, and cortex of the bipotential gonad have been strongly conserved. Aromatase (CYP19), which regulates gonadal estrogen levels, is proposed to be the main target of a putative thermosensitive factor for TSD. However, manipulation of estrogen levels rarely mimics the precise timing of temperature effects on expression of gonadal genes, as occurs with TSD. Estrogen levels may influence sex determination or gonad differentiation depending on the species. Furthermore, the process leading to sex determination under the influence of temperature poses problems that are not encountered by species with genetic sex determination. Yolk steroids of maternal origin and steroids produced by the embryonic nervous system should also be considered as sources of hormones that may play a role in TSD. Copyright © 2012 S. Karger AG, Basel.

Environmental effects on resistance gene expression in milk stage popcorn kernels and associations with mycotoxin production

USDA-ARS?s Scientific Manuscript database

Like other forms of maize, popcorn is subject to increased levels of contamination by a variety of different mycotoxins under stress conditions, although levels generally are less than dent maize under comparable stress. Gene array analysis was used to determine expression differences of disease res...

24 CFR 51.103 - Criteria and standards.

Code of Federal Regulations, 2010 CFR

2010-04-01

... decibels to sound levels in the night from 10 p.m. to 7 a.m. Mathematical expressions for average sound..., as indicated in § 51.106(a)(3). Methods for assessing the contribution of loud impulsive sounds to day-night average sound level at a site and mathematical expressions for determining whether a sound...

BMP-2 up-regulates PTEN expression and induces apoptosis of pulmonary artery smooth muscle cells under hypoxia.

PubMed

Pi, Weifeng; Guo, Xuejun; Su, Liping; Xu, Weiguo

2012-01-01

To investigate the role of bone morphogenetic protein 2 (BMP-2) in regulation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and apoptosis of pulmonary artery smooth muscle cells (PASMCs) under hypoxia. Normal human PASMCs were cultured in growth medium (GM) and treated with BMP-2 from 5-80 ng/ml under hypoxia (5% CO(2)+94% N(2)+1% O(2)) for 72 hours. Gene expression of PTEN, AKT-1 and AKT-2 were determined by quantitative RT-PCR (QRT-PCR). Protein expression levels of PTEN, AKT and phosph-AKT (pAKT) were determined. Apoptosis of PASMCs were determined by measuring activities of caspases-3, -8 and -9. siRNA-smad-4, bpV(HOpic) (PTEN inhibitor) and GW9662 (PPARγ antagonist) were used to determine the signalling pathways. Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferation rate was achieved at 60 ng/ml concentration under hypoxia (82.2±2.8%). BMP-2 increased PTEN gene expression level, while AKT-1 and AKT-2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. AKT activity significantly reduced in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2. PTEN expression remained unchanged when Smad-4 expression was inhibited by siRNA-Smad-4. bpV(HOpic) and GW9662 (PPARγ inhibitor) inhibited PTEN protein expression and recovered PASMCs proliferation rate. BMP-2 increased PTEN expression under hypoxia in a dose dependent pattern. BMP-2 reduced AKT activity and increased caspase activity of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway.

Regulatory Divergence between Parental Alleles Determines Gene Expression Patterns in Hybrids

PubMed Central

Combes, Marie-Christine; Hueber, Yann; Dereeper, Alexis; Rialle, Stéphanie; Herrera, Juan-Carlos; Lashermes, Philippe

2015-01-01

Both hybridization and allopolyploidization generate novel phenotypes by conciliating divergent genomes and regulatory networks in the same cellular context. To understand the rewiring of gene expression in hybrids, the total expression of 21,025 genes and the allele-specific expression of over 11,000 genes were quantified in interspecific hybrids and their parental species, Coffea canephora and Coffea eugenioides using RNA-seq technology. Between parental species, cis- and trans-regulatory divergences affected around 32% and 35% of analyzed genes, respectively, with nearly 17% of them showing both. The relative importance of trans-regulatory divergences between both species could be related to their low genetic divergence and perennial habit. In hybrids, among divergently expressed genes between parental species and hybrids, 77% was expressed like one parent (expression level dominance), including 65% like C. eugenioides. Gene expression was shown to result from the expression of both alleles affected by intertwined parental trans-regulatory factors. A strong impact of C. eugenioides trans-regulatory factors on the upregulation of C. canephora alleles was revealed. The gene expression patterns appeared determined by complex combinations of cis- and trans-regulatory divergences. In particular, the observed biased expression level dominance seemed to be derived from the asymmetric effects of trans-regulatory parental factors on regulation of alleles. More generally, this study illustrates the effects of divergent trans-regulatory parental factors on the gene expression pattern in hybrids. The characteristics of the transcriptional response to hybridization appear to be determined by the compatibility of gene regulatory networks and therefore depend on genetic divergences between the parental species and their evolutionary history. PMID:25819221

Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

PubMed Central

Hahnvajanawong, Chariya; Chaiyagool, Jariya; Seubwai, Wunchana; Bhudhisawasdi, Vajarabhongsa; Namwat, Nisana; Khuntikeo, Narong; Sripa, Banchob; Pugkhem, Ake; Tassaneeyakul, Wichittra

2012-01-01

AIM: To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA). METHODS: The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues. Tumor cell viability was determined morphologically with hematoxylin and eosin- and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues. The mRNA expression of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) was determined with real-time reverse transcriptase-polymerase chain reaction. The levels of gene expression and the sensitivity to 5-FU were evaluated. RESULTS: Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital, Khon Kaen University from 2007 to 2009. HDRA was used to determine the response of these CCA tissues to 5-FU. Based on the dose-response curve, 200 μg/mL 5-FU was selected as the test concentration. The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU. When the relationship between TP, OPRT, TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined, only OPRT mRNA expression was significantly correlated with the response to 5-FU. The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12, P < 0.05). CONCLUSION: OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA. Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients. PMID:22912546

Decoding Facial Expressions: A New Test with Decoding Norms.

ERIC Educational Resources Information Center

Leathers, Dale G.; Emigh, Ted H.

1980-01-01

Describes the development and testing of a new facial meaning sensitivity test designed to determine how specialized are the meanings that can be decoded from facial expressions. Demonstrates the use of the test to measure a receiver's current level of skill in decoding facial expressions. (JMF)

Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes.

PubMed

Ansari, M Y; Khan, N M; Ahmad, I; Haqqi, T M

2017-08-08

Mitochondrial dysfunction, oxidative stress and chondrocyte death are important contributors to the development and pathogenesis of osteoarthritis (OA). In this study, we determined the expression and role of Parkin in the clearance of damaged/dysfunctional mitochondria, regulation of reactive oxygen species (ROS) levels and chondrocyte survival under pathological conditions. Human chondrocytes were from the unaffected area of knee OA cartilage (n = 12) and were stimulated with IL-1β to mimic pathological conditions. Mitochondrial membrane depolarization and ROS levels were determined using specific dyes and flow cytometry. Autophagy was determined by Western blotting for ATG5, Beclin1, immunofluorescence staining and confocal microscopy. Gene expression was determined by RT-qPCR. siRNA, wild-type and mutant Parkin plasmids were transfected using Amaxa system. Apoptosis was determined by PI staining of chondrocytes and TUNEL assay. IL-1β-stimulated OA chondrocytes showed high levels of ROS generation, mitochondrial membrane damage, accumulation of damaged mitochondria and higher incidence of apoptosis. IL-1β stimulation of chondrocytes with depleted Parkin expression resulted in sustained high levels of ROS, accumulation of damaged/dysfunctional mitochondria and enhanced apoptosis. Parkin translocation to depolarized/damaged mitochondria and recruitment of p62/SQSTM1 was required for the elimination of damaged/dysfunctional mitochondria in IL-1β-stimulated OA chondrocytes. Importantly we demonstrate that Parkin elimination of depolarized/damaged mitochondria required the Parkin ubiquitin ligase activity and resulted in reduced ROS levels and inhibition of apoptosis in OA chondrocytes under pathological conditions. Our data demonstrates that Parkin functions to eliminate depolarized/damaged mitochondria in chondrocytes which is necessary for mitochondrial quality control, regulation of ROS levels and chondrocyte survival under pathological conditions. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

The Association between Expressive Grammar Intervention and Social and Emergent Literacy Outcomes for Preschoolers with SLI

ERIC Educational Resources Information Center

Washington, Karla N.

2013-01-01

Purpose: To determine whether (a) expressive grammar intervention facilitated social and emergent literacy outcomes better than no intervention and (b) expressive grammar gains and/or initial expressive grammar level predicted social and emergent literacy outcomes. Method: This investigation was a follow-up to a recently published study exploring…

The Janus kinase inhibitor tofacitinib inhibits TNF-α-induced gliostatin expression in rheumatoid fibroblast-like synoviocytes.

PubMed

Kawaguchi, Yohei; Waguri-Nagaya, Yuko; Tatematsu, Naoe; Oguri, Yusuke; Kobayashi, Masaaki; Nozaki, Masahiro; Asai, Kiyofumi; Aoyama, Mineyoshi; Otsuka, Takanobu

2018-01-15

Gliostatin (GLS) is known to have angiogenic and arthritogenic activity, and GLS expression levels in serum from patients with rheumatoid arthritis (RA) are significantly correlated with the disease activity. Tofacitinib is a novel oral Janus kinase (JAK) inhibitor and is effective in treating RA. However, the mechanism of action of tofacitinib in fibroblast-like synoviocytes (FLSs) has not been elucidated. The purpose of this study was to investigate the modulatory effects of tofacitinib on serum GLS levels in patients with RA and GLS production in FLSs derived from patients with RA. Six patients with RA who had failed therapy with at least one TNF inhibitor and were receiving tofacitinib therapy were included in the study. Serum samples were collected to measure CRP, MMP-3 and GLS expression. FLSs derived from patients with RA were cultured and stimulated by TNFα with or without tofacitinib. GLS expression levels were determined using reverse transcription-polymerase chain reaction (RT-PCR), EIA and immunocytochemistry, and signal transducer and activator of transcription (STAT) protein phosphorylation levels were determined by western blotting. Treatment with tofacitinib decreased serum GLS levels in all patients. GLS mRNA and protein expression levels were significantly increased by treatment with TNF-α alone, and these increases were suppressed by treatment with tofacitinib, which also inhibited TNF-α-induced STAT1 phosphorylation. JAK/STAT activation plays a pivotal role in TNF-α-mediated GLS up-regulation in RA. Suppression of GLS expression in FLSs has been suggested to be one of the mechanisms through which tofacitinib exerts its anti-inflammatory effects.

Osthole suppresses the proliferation and accelerates the apoptosis of human glioma cells via the upregulation of microRNA-16 and downregulation of MMP-9.

PubMed

Lin, Kai; Gao, Zhiyu; Shang, Bin; Sui, Shaohua; Fu, Qiang

2015-09-01

Osthole (7-methoxy-8-isoamyl alkenyl coumarin) has been reported to exhibit marked anticancer effects on several types of cancer. The expression levels of matrix metalloproteinase-9 (MMP-9) are closely associated with the pathogenesis of glioma. Furthermore, it is reported that the upregulation of microRNA‑16 (miR‑16) by the MMP‑9 signaling pathway can restrain the proliferation of cancer cells. To examine whether osthole increases the anticancer effect on human glioma cells in the present study, the common glioma cell line, U87, was treated with osthole at concentrations of 0, 50, 100 and 200 µΜ. The effects of osthole on cell viability were determined using a 3‑(4,5‑dimethylthiazol‑2‑thiazolyl)‑2,5‑diphenyl‑tetrazolium bromide assay. The rate of cellular apoptosis was analyzed by measuring the activity of caspase‑3 and using flow cytometry. The expression of MMP‑9 was determined using gelatin zymography assays and the expression of miR‑16 was determined using reverse transcription‑quantitative polymerase chain reaction. The results demonstrated that osthole significantly suppressed the proliferation and accelerated the apoptosis of the U87 cells. Furthermore, increased expression levels of miR‑16 and reduced protein expression levels of MMP‑9 were found in the U87 cells. In addition, miR‑16 was found to regulate the expression of MMP‑9 in the U87 cells through transfection of miR‑16 precursor and anti‑miR‑16 into the U87 cells. In conclusion, these observations indicated that osthole suppressed the proliferation and accelerated the apoptosis of human glioma cells through upregulation of the expression of miR‑16 and downregulation of the expression of MMP-9.

Determinants in the β and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.

PubMed

Rudell, Jolene Chang; Borges, Lucia S; Rudell, John B; Beck, Kenneth A; Ferns, Michael J

2014-01-03

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.

Fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of LncRNA MEG3

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

Hu, Duanmin; Su, Cunjin; Jiang, Min

There is still no suitable drug for pancreatic cancer treatment, which is one of the most aggressive human tumors. Maternally expressed gene 3 (MEG3), a LncRNA, has been suggested as a tumor suppressor in a range of human tumors. Studies found fenofibrate exerted anti-tumor roles in various human cancer cell lines. However, its role in pancreatic cancer remains unknown. The present study aimed to explore the impacts of fenofibrate on pancreatic cancer cell lines, and to investigate MEG3 role in its anti-tumor mechanisms. We used MTT assay to determine cells proliferation, genome-wide LncRNA microarray analysis to identify differently expressed LncRNAs,more » siRNA or pCDNA-MEG3 transfection to interfere or upregulate MEG3 expression, western blot to detect protein levels, real-time PCR to determine MEG3 level. Fenofibrate significantly inhibited proliferation of pancreatic cancer cells, increased MEG3 expression and p53 levels. Moreover, knockdown of MEG3 attenuated cytotoxicity induced by fenofibrate. Furthermore, overexpression of MEG3 induced cells death and increased p53 expression. Our results indicated fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of MEG3. - Highlights: • We found that fenofibrate suppressed proliferation of pancreatic cancer cells. • We found fenofibrate increased LncRNA-MEG3 expression and p53 level in PANC-1 cells. • Inhibition of MEG3 expression attenuated anti-tumor effects of fenofibrate.« less

SIRT6 expression and oxidative DNA damage in individuals with prediabetes and type 2 diabetes mellitus.

PubMed

Çalışkan, Z; Mutlu, T; Güven, M; Tunçdemir, M; Niyazioğlu, M; Hacioglu, Y; Dincer, Y

2018-02-05

Sirtuins (SIRTs) is a family of NAD + dependent histone deacetylases. SIRT6 takes play in glucose homeostasis, genomic stability and DNA repair. Although increased oxidative DNA damage and decreased DNA repair activity were determined in diabetes mellitus, the possible relation between level of oxidative DNA damage and SIRT6 expression has not been investigated so far. We determined SIRT6 expression and urinary 8-hydroxy deoxyguanosine (8-OHdG) levels, marker of oxidative DNA damage, in cases with prediabetes (PreDM) and type 2 diabetes mellitus (T2DM). SIRT6 gene expression was determined in peripheral blood leukocytes of 70 patients with type 2 diabetes, 50 cases in prediabetic stage and 40 healthy subjects. SIRT6 mRNA levels were determined by quantitive real time- polymerase chain reaction. SIRT6 protein was detected by immunocytochemical staining. Urinary 8-hydroxy deoxyguanosine (8-OHdG) levels were measured by ELISA. There was no significant difference between groups for SIRT6 mRNA level. SIRT6 immunopositivity in T2DM group was lower when compared to those in preDM group (P<0.05). SIRT6 positive cell number in T2DM and preDM groups were lower in comparison to control group (P<0.01 for both), however, when study groups were subdivided into two groups according to their age, the difference between preDM and control groups disappeared in both mid-aged and old-aged groups. The urinary 8-OHdG level was found to be higher in the T2DM group in comparison to preDM group (P<0.05). When age is taken into consideration, urinary 8-OHdG level in the T2DM group was found to be higher than those in both preDM and control groups in the old-aged cases but no significant difference was determined between groups in the mid-aged cases. There was no relation between SIRT6 expression and urinary 8-OHDG excretion. It was concluded that SIRT6 may take play in development of T2DM but this effect seems to be independent from repair of oxidative DNA damage. Copyright © 2017 Elsevier B.V. All rights reserved.

Oncogene GAEC1 regulates CAPN10 expression which predicts survival in esophageal squamous cell carcinoma

PubMed Central

Chan, Dessy; Tsoi, Miriam Yuen-Tung; Liu, Christina Di; Chan, Sau-Hing; Law, Simon Ying-Kit; Chan, Kwok-Wah; Chan, Yuen-Piu; Gopalan, Vinod; Lam, Alfred King-Yin; Tang, Johnny Cheuk-On

2013-01-01

AIM: To identify the downstream regulated genes of GAEC1 oncogene in esophageal squamous cell carcinoma and their clinicopathological significance. METHODS: The anti-proliferative effect of knocking down the expression of GAEC1 oncogene was studied by using the RNA interference (RNAi) approach through transfecting the GAEC1-overexpressed esophageal carcinoma cell line KYSE150 with the pSilencer vector cloned with a GAEC1-targeted sequence, followed by MTS cell proliferation assay and cell cycle analysis using flow cytometry. RNA was then extracted from the parental, pSilencer-GAEC1-targeted sequence transfected and pSilencer negative control vector transfected KYSE150 cells for further analysis of different patterns in gene expression. Genes differentially expressed with suppressed GAEC1 expression were then determined using Human Genome U133 Plus 2.0 cDNA microarray analysis by comparing with the parental cells and normalized with the pSilencer negative control vector transfected cells. The most prominently regulated genes were then studied by immunohistochemical staining using tissue microarrays to determine their clinicopathological correlations in esophageal squamous cell carcinoma by statistical analyses. RESULTS: The RNAi approach of knocking down gene expression showed the effective suppression of GAEC1 expression in esophageal squamous cell carcinoma cell line KYSE150 that resulted in the inhibition of cell proliferation and increase of apoptotic population. cDNA microarray analysis for identifying differentially expressed genes detected the greatest levels of downregulation of calpain 10 (CAPN10) and upregulation of trinucleotide repeat containing 6C (TNRC6C) transcripts when GAEC1 expression was suppressed. At the tissue level, the high level expression of calpain 10 protein was significantly associated with longer patient survival (month) of esophageal squamous cell carcinoma compared to the patients with low level of calpain 10 expression (37.73 ± 16.33 vs 12.62 ± 12.44, P = 0.032). No significant correction was observed among the TNRC6C protein expression level and the clinocopathologcial features of esophageal squamous cell carcinoma. CONCLUSION: GAEC1 regulates the expression of CAPN10 and TNRC6C downstream. Calpain 10 expression is a potential prognostic marker in patients with esophageal squamous cell carcinoma. PMID:23687414

Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy.

PubMed

Walz, Jenna A; Mace, Charles R

2018-06-05

Immunophenotyping is typically achieved using flow cytometry, but any influence a biomarker may have on adhesion or surface recognition cannot be determined concurrently. In this manuscript, we demonstrate the utility of lateral microscopy for correlating cell surface biomarker expression levels with quantitative descriptions of cell morphology. With our imaging system, we observed single cells from two T cell lines and two B cell lines adhere to antibody-coated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angles on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations.

C-reactive protein expression is up-regulated in apical lesions of endodontic origin in association with interleukin-6.

PubMed

Garrido, Mauricio; Dezerega, Andrea; Bordagaray, María José; Reyes, Montserrat; Vernal, Rolando; Melgar-Rodríguez, Samantha; Ciuchi, Pía; Paredes, Rodolfo; García-Sesnich, Jocelyn; Ahumada-Montalva, Pablo; Hernández, Marcela

2015-04-01

C-reactive protein (CRP) is the prototype component of acute-phase proteins induced ultimately by interleukin (IL)-6 in the liver, but it is unknown whether periradicular tissues locally express CRP. The present study aimed to identify whether CRP messenger RNA synthesis occurs in situ within apical lesions of endodontic origin (ALEOs) and healthy periodontal ligament and its association with IL-6 and to determine their protein levels and tissue localization. Patients with asymptomatic apical periodontitis and healthy volunteers presenting at the School of Dentistry, University of Chile, Santiago, Chile, were enrolled. ALEOs and healthy teeth were obtained and processed for either immunohistochemistry and double immunofluorescence to assess IL-6 and CRP tissue localization, whereas healthy periodontal ligaments were processed as controls for real-time reverse-transcription polymerase chain reaction for their RNA expression levels and multiplex assay to determine their protein levels. Statistic analysis was performed using the unpaired t test or Mann-Whitney test according to data distribution and Pearson correlation. IL-6 and CRP were synthesized in ALEOs, whereas their RNA expression and protein levels were significantly higher when compared with healthy periodontal ligament. IL-6 and CRP immunolocalized to the inflammatory cells, vascular endothelial cells, and mesenchymal cells. Both, IL-6 and CRP colocalized in ALEOs, and a positive correlation was found between their expression levels (P < .05). IL-6 and CRP messenger RNA are constitutively expressed in periodontal ligament and up-regulated in ALEOs along with higher protein levels. Given their pleiotropic effects, IL-6 and CRP protein levels in apical tissues might partially explain the development and progression of ALEOs as well as potentially asymptomatic apical periodontitis-associated systemic low-grade inflammation. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Identification of prostaglandin receptors in human ureters.

PubMed

Oll, Matthias; Baumann, Claudia; Behbahani, Turang E; von Ruecker, Alexander; Müller, Stefan C; Ellinger, Jörg

2012-12-10

Prostaglandins play an important role in ureteral obstruction, but the detailed expression profiles of the prostaglandin receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR) remain unknown in the different parts of the human ureter. The expression pattern of PTGER1, PTGER2, PTGER3, PTGER4 and PTGFR was determined in human distal, mid and proximal ureter and renal pelvis samples using immunohistochemistry (protein levels) and quantitative real-time PCR (mRNA). PTGER1 was highly expressed in most samples irrespective of the ureteral localization; however, urothelial cells had higher levels of PTGER1 than smooth muscle cells. PTGFR was also moderately to strongly expressed in urothelial and smooth muscle cells. In comparison, PTGER2-4 expression was mostly unexpressed or weakly expressed in urothelial and smooth cells in all regions. Our data indicate high levels of PTGER1 in ureters.

The Expression of Dectin-1, Irak1 and Rip2 During the Host Response to Aspergillus fumigatus.

PubMed

Liu, Jinguo; Yu, Lin; Chen, Cuicui; Zhou, Jian; Gong, Xin; Li, Dandan; Hou, Dongni; Song, Yuanlin; Shao, Changzhou

2018-04-01

C-type lectin receptors (CLRs), Toll-like receptors (TLRs), and Nod-like receptors (NLRs) have the ability to recognize Aspergillus fumigatus (A. fumigates) and induce innate immune response. Dectin-1 is a well-described CLR, while interleukin-1 receptor-associated kinase 1 (Irak1) and receptor-interacting protein 2 (Rip2) are pivotal adaptor proteins of TLRs and NLRs signaling pathways, respectively. Our primary aim is to elucidate whether Dectin-1 regulates the expression of Irak1 and Rip2, and confirm that CLRs, TLRs, and NLRs pathways act synergistically in response to A. fumigatus infection. Pulmonary infection mouse models were established. Myeloid cells were differentiated in cell culture and examined by inverted microscopy, flow cytometry, and scanning electron microscopy. The relative mRNA levels were determined by qRT-PCR. The protein expression levels were determined by immunohistochemistry and Western blot. The expression of Dectin-1, Irak1, Rip2, and phosphorylation level of nuclear factor (NF)-κB p65 were induced by conidia in immunocompetent mice, while their expression and phosphorylation level were inhibited in immunocompromised mice after the administration of conidia. Conidia increased the expression of Dectin-1, Irak1, and Rip2 in myeloid cells, while Dectin-1 silencing significantly reduced their expression. Our findings demonstrate that Dectin-1, Irak1, and Rip2 are involved in response to A. fumigatus infection. Dectin-1 modulates the expression of Irak1 and Rip2. Additionally, these three signaling pathways are interconnected, and CLRs pathway plays a dominant role against A. fumigatus invasion.

Expression Divergence Is Correlated with Sequence Evolution but Not Positive Selection in Conifers.

PubMed

Hodgins, Kathryn A; Yeaman, Sam; Nurkowski, Kristin A; Rieseberg, Loren H; Aitken, Sally N

2016-06-01

The evolutionary and genomic determinants of sequence evolution in conifers are poorly understood, and previous studies have found only limited evidence for positive selection. Using RNAseq data, we compared gene expression profiles to patterns of divergence and polymorphism in 44 seedlings of lodgepole pine (Pinus contorta) and 39 seedlings of interior spruce (Picea glauca × engelmannii) to elucidate the evolutionary forces that shape their genomes and their plastic responses to abiotic stress. We found that rapidly diverging genes tend to have greater expression divergence, lower expression levels, reduced levels of synonymous site diversity, and longer proteins than slowly diverging genes. Similar patterns were identified for the untranslated regions, but with some exceptions. We found evidence that genes with low expression levels had a larger fraction of nearly neutral sites, suggesting a primary role for negative selection in determining the association between evolutionary rate and expression level. There was limited evidence for differences in the rate of positive selection among genes with divergent versus conserved expression profiles and some evidence supporting relaxed selection in genes diverging in expression between the species. Finally, we identified a small number of genes that showed evidence of site-specific positive selection using divergence data alone. However, estimates of the proportion of sites fixed by positive selection (α) were in the range of other plant species with large effective population sizes suggesting relatively high rates of adaptive divergence among conifers. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Gender Difference of Hepatic and Intestinal CYP3A4 in CYP3AHumanized Mice Generated by a Human Chromosome-engineering Technique.

PubMed

Kobayashi, Kaoru; Abe, Chihiro; Endo, Mika; Kazuki, Yasuhiro; Oshimura, Mitsuo; Chiba, Kan

2017-11-17

Cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme that is expressed in the liver and small intestine of humans. Various factors influence the expression of CYP3A4, but gender difference in CYP3A4 expression remains debatable. To clarify gender difference of hepatic and intestinal CYP3A4 in CYP3A-humanized mice generated by a human artificial chromosome (HAC) vector system. The CYP3A-humanized (CYP3AHAC) mice have essential regulatory regions, including promoters and enhancers, and unknown elements affecting the expression of CYP3A4. We examined the expression and activity of hepatic and intestinal CYP3A4 in male and female CYP3A-HAC mice. CYP3A activity was determined as α- and 4-hydroxylation activity of triazolam in liver and intestinal microsomes. Expression level of CYP3A protein was determined by Western blot analysis. Expression level of CYP3A4 mRNA was measured by quantitative real-time PCR. The results showed that triazolam hydroxylation activities and protein levels of CYP3A in the liver were significantly higher in female than in male CYP3A-HAC mice, whereas those in the intestine were not significantly different between the genders. In addition, the expression of CYP3A4 mRNA showed a tendency similar to that found for the activity and expression of CYP3A protein in the liver and intestine of CYP3A-HAC mice. These findings suggest that the expression and activity levels of CYP3A4 in the liver are higher in females than in males, whereas there is no gender difference in the levels in the intestine of CYP3A-HAC mice. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Relative expression of proprotein convertases in rat ovaries during pregnancy.

PubMed

Kwok, Simon Cm; Chakraborty, Damayanti; Soares, Michael J; Dai, Guoli

2013-12-11

Proprotein convertases are a family of serine proteinases that are related to bacterial subtilisin and yeast kexin. They are involved in posttranslational processing of the precursors of a vast number of cellular proteins. With the exception of PC1/3, the relative expression levels of the proprotein convertases in the ovary during pregnancy have not been reported. The purpose of this study is to determine by real-time PCR the relative expression levels of all nine proprotein convertases in rat ovaries during pregnancy and at 3 days postpartum. RNA was extracted from ovaries at Day 0, 4, 9, 11, 13, 15, 18, and 20 of pregnancy as well as 3 days postpartum. Relative expression levels of Pcsk1, Pcsk2, Furin, Pcsk4, Pcsk5, Pcsk6, Pcsk7, Mbtps1 and Pcsk9 were determined with real-time PCR. Results were reported as fold-change over the level at Day 0 of pregnancy. Results showed that Pcsk1 and Pcsk6 were upregulated as gestation advanced, in parallel with an observed increase in relaxin transcript. Pcsk2 showed downregulation as gestation advanced, while Pcsk5 showed relatively higher levels in early pregnancy and postpartum, but lower level in mid-pregnancy. On the other hand, Furin, Pcsk4, Pcsk7, Mbtps1 and Pcsk9 showed little change of expression throughout gestation. PC1/3 (PCSK1) and PACE4 (PCSK6) may play an important role in proprotein processing in the ovary during late pregnancy.

Multi-level Expression Design Language: Requirement level (MEDL-R) system evaluation

NASA Technical Reports Server (NTRS)

1980-01-01

An evaluation of the Multi-Level Expression Design Language Requirements Level (MEDL-R) system was conducted to determine whether it would be of use in the Goddard Space Flight Center Code 580 software development environment. The evaluation is based upon a study of the MEDL-R concept of requirement languages, the functions performed by MEDL-R, and the MEDL-R language syntax. Recommendations are made for changes to MEDL-R that would make it useful in the Code 580 environment.

Expression analysis of Drosophila doublesex, transformer-2, intersex, fruitless-like, and vitellogenin homologs in the parahaploid predator Metaseiulus occidentalis (Chelicerata: Acari: Phytoseiidae).

PubMed

Pomerantz, Aaron F; Hoy, Marjorie A

2015-01-01

Characterization and expression analyses are essential to gain insight into sex-determination pathways in members of the Acari. Little is known about sex determination at the molecular level in the western orchard predatory mite Metaseiulus occidentalis (Arthropoda: Chelicerata: Arachnida: Acari: Phytoseiidae), a parahaploid species. In this study, eight genes previously identified as putative homologs to genes involved in the sex-determination pathway in Drosophila melanogaster were evaluated for sex-specific alternative splicing and sex-biased expression using reverse-transcriptase PCR and quantitative real-time PCR techniques, respectively. The homologs evaluated in M. occidentalis included two doublesex-like genes (Moccdsx1 and Moccdsx2), transformer-2 (Mocctra-2), intersex (Moccix), two fruitless-like genes (MoccBTB1 and MoccBTB2), as well as two vitellogenin-like genes (Moccvg1 and Moccvg2). Single transcripts of equal size were detected in males and females for Moccdsx1, Moccdsx2, Mocctra-2, Moccix, and MoccBTB2, suggesting that their pre-mRNAs do not undergo alternative splicing in a sex-specific manner. Three genes, Moccdsx1, Moccdsx2 and MoccBTB2, displayed male-biased expression relative to females. One gene, Moccix, displayed female-biased expression relative to males. Two genes, Mocctra-2 and MoccBTB1, did not display detectable differences in transcript abundance in males and females. Expression of Moccvg1 and Moccvg2 were detected in females only, and transcript levels were up-regulated in mated females relative to unmated females. To our knowledge, this represents the first attempt to elucidate expression patterns of putative sex-determination genes in an acarine. This study is an initial step towards understanding the sex-determination pathway in the parahaploid M. occidentalis.

Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation

PubMed Central

Soto, Lilian; Ferrier, Ashley; Aravena, Octavio; Fonseca, Elianet; Berendsen, Jorge; Biere, Andrea; Bueno, Daniel; Ramos, Verónica; Aguillón, Juan Carlos; Catalán, Diego

2015-01-01

The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis. PMID:26483788

Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses.

PubMed

Tay, Szun Szun; Wong, Yik Chun; McDonald, David M; Wood, Nicole A W; Roediger, Ben; Sierro, Frederic; Mcguffog, Claire; Alexander, Ian E; Bishop, G Alex; Gamble, Jennifer R; Weninger, Wolfgang; McCaughan, Geoffrey W; Bertolino, Patrick; Bowen, David G

2014-06-24

CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

[Correlation between EGLN1 gene, protein express in lung tissue of rats and pulmonary artery pressure at different altitude].

PubMed

Li, S H; Li, S; Sun, L; Bai, Z Z; Yang, Q Y; Ga, Q; Jin, G E

2016-08-23

To investigate the correlation between pulmonary artery pressure (PAP) and the expression level of Egl nine homologue 1 (EGLN1) gene or its protein in lung tissue of rats at different altitudes. Totally 121 male Wistar rats were randomly divided into low altitude group (n=11), moderate altitude group and high altitude group, the rats in moderate altitude and high altitude group were further divided into 1(st) day, 3(rd) days, 7(th) days, 15(th) day and 30(th) day group according to the exposure time to hypoxic environment, each group 11 rats. The low altitude group, the PAP of rats were determined by physiological signal acquisition system, and tissue samples were collected in liquid nitrogen container for storage at an altitude of 498 m area. Moderate altitude group rats were placed in altitude of 2 260 meters of natural environment, 5 high altitude groups rats were placed in the hypobaric hypoxic chamber, simulating altitude of 4 500 meters. The PAP of rats in moderate altitude group and high altitude group were also determined by physiological signal acquisition system, and tissue samples were collected when rats were exposed to hypoxia at 1(st), 3(rd), 7(th), 15(th) and 30(th) day; Western blot was used to determine expression levels of EGLN1 protein, and person correlation analysis was used to analyze whether the protein was related to the formation of pulmonary arterial hypertension (PH) under hypoxia. Real-time quantitive PCR method determined expression levels of EGLN1 mRNA in lung tissues, and the relative expression method was used to analyze PCR data, and finally assess whether the EGLN1 gene was the initial cause of the formation of PH during hypoxia. The mean PAP of rats was (20.0±3.2) mmHg (1 mmHg=0.133 kPa) in low altitude group; in moderate altitude group, mean PAP began to increase slightly when rats were exposed to hypoxia on the 15(th) day and reached at (22.7±4.1) mmHg on hypoxic 30(th) day, but compared with the low altitude group, there was no statistical difference (P> 0.05); the mean PAP of rats in high altitude group began to rise on the 7(th) day (28.7±7.7) mmHg, which was higher than that in low altitude group (P<0.05), and significantly increased to (42.3±9.1) mmHg (P<0.001) on hypoxic 30(th) day; it was significantly proportional with exposure to hypoxic time, and compared to low altitude group and moderate altitude group, there was significant difference (P<0.05). EGLN1 protein expression in the lung tissue of rats had no significant difference between the low altitude group and moderate altitude group, and its expression level in the high altitude group were significantly decreased, furthermore, the expression level decreased with the increase of hypoxia exposure time (P<0.05); PAP and EGLN1 protein expression levels showed a negative correlation (r=-0.662). The transcription level of mRNA EGLN1 in high altitude group was significantly increased under hypobaric hypoxia, it was 72 times more than that of the moderate altitude group, and nearly 300 times than that of the low altitude group, respectively (both P<0.001=. EGLN1 gene expression in lung tissue of rat is affected by hypoxia, the expression level increases with the increase of the altitude; but the protein expression level, in contrast with gene expression level, is decreased with the increase of altitude and is significantly negatively correlated with mean PAP.

Omega-3 Fatty Acid Deficiency Increases Stearoyl-CoA Desaturase Expression and Activity Indices in Rat Liver: Positive Association with Non-Fasting Plasma Triglyceride Levels

PubMed Central

Hofacer, Rylon; Magrisso, I. Jack; Jandacek, Ronald; Rider, Therese; Tso, Patrick; Benoit, Stephen C.; McNamara, Robert K.

2011-01-01

Although omega-3 (n-3) fatty acids negatively regulate triglyceride biosynthesis, the mechanisms mediating this effect are poorly understood, and emerging evidence suggests that stearoyl-CoA desaturase (Scd1) is required for de novo triglyceride biosynthesis. To investigate this mechanism, we determined the effects of perinatal n-3 deficiency and postnatal repletion on rat liver Scd1 mRNA expression and activity indices (liver 16:1/16:0 & 18:1/18:0 ratios), and determined relationships with postprandial (non-fasting) plasma triglyceride levels. Rats were fed conventional diets with or without the n-3 fatty acid precursor α-linolenic acid (ALA, 18:3n-3) during perinatal development (E0-P100), and a subset of rats fed the ALA− diet were switched to the ALA+ diet post-weaning (P21-P100, repletion). Compared with controls, rats fed the ALA− diet exhibited significantly lower liver long-chain n-3 fatty acid compositions and elevations in monounsaturated fatty acid composition, both of which were normalized in repleted rats. Liver Scd1 mRNA expression and activity indices (16:1/16:0 & 18:1/18:0 ratios) were significantly greater in n-3 deficient rats compared with controls and repleted rats. Among all rats, liver Scd1 mRNA expression was positively correlated with liver 18:1/18:0 and 16:1/16:0 ratios. Plasma triglyceride levels, but not glucose or insulin levels, were significantly greater in n-3 deficient rats compared with controls and repleted rats. Liver Scd1 mRNA expression and activity indices were positively correlated with plasma triglyceride levels. These preclinical findings demonstrate that n-3 fatty acid status is an important determinant of liver Scd1 mRNA expression and activity, and suggest that down-regulation of Scd1 is a mechanism by which n-3 fatty acids repress constitutive triglyceride biosynthesis. PMID:22047910

Determination of transport wind speed in the gaussian plume diffusion equation for low-lying point sources

NASA Astrophysics Data System (ADS)

Wang, I. T.

A general method for determining the effective transport wind speed, overlineu, in the Gaussian plume equation is discussed. Physical arguments are given for using the generalized overlineu instead of the often adopted release-level wind speed with the plume diffusion equation. Simple analytical expressions for overlineu applicable to low-level point releases and a wide range of atmospheric conditions are developed. A non-linear plume kinematic equation is derived using these expressions. Crosswind-integrated SF 6 concentration data from the 1983 PNL tracer experiment are used to evaluate the proposed analytical procedures along with the usual approach of using the release-level wind speed. Results of the evaluation are briefly discussed.

Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales

PubMed Central

Margres, Mark J.; Wray, Kenneth P.; Seavy, Margaret; McGivern, James J.; Herrera, Nathanael D.; Rokyta, Darin R.

2016-01-01

Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression proteins, enabling low-expression proteins to evolve and potentially lead to more rapid adaptation. PMID:26546003

Mucin gene expression is not regulated by estrogen and/or progesterone in the ocular surface epithelia of mice.

PubMed

Lange, Christine; Fernandez, Jolene; Shim, David; Spurr-Michaud, Sandra; Tisdale, Ann; Gipson, Ilene K

2003-07-01

Dry eye syndrome is prevalent in post-menopausal women, and post-menopausal women secrete less mucus in their reproductive tracts. Using a mouse model, the purpose of this study was to determine if estrogen and/or progesterone regulates Muc4 and Muc5AC gene expression in the ocular surface epithelia, as the hormones do in reproductive tract epithelia. Adult C57BL/6 mice were ovariectomized, and 19 days later, pellets containing estrogen, progesterone, or a combination were inserted subcutaneously. Ocular surface and reproductive tract tissues were harvested following seven days of hormone treatment. A control group consisted of ovariectomized mice that received no hormone treatment. Real-time reverse transcription-polymerase chain reaction was used to determine the tissue expression levels of mucin mRNA of each treatment group relative to the control. Muc4 mRNA expression levels were determined for the reproductive tract, and both Muc4 and Muc5AC expression levels were determined for the ocular surface epithelia. Muc4 and Muc5AC gene expression in ocular surface and Muc4 in reproductive tract epithelia was demonstrated by In Situ hybridization, and Muc4 and Muc5AC protein was demonstrated in the epithelia of animals in the experimental groups. The mRNA expression levels of Muc4 and Muc5AC and the immunofluorescence localization pattern in the ocular surface epithelia were not significantly different in any hormone treatment group when compared to the control ovariectomized group. By comparison, mice that were administered estrogen had a significant increase of Muc4 mRNA in the reproductive tract epithelia, progesterone given in combination with estrogen antagonized the upregulatory effects of estrogen in the reproductive tract, and the amount of Muc4 mRNA in the reproductive tract of progesterone-treated animals was not different from ovariectomized controls. Immunofluorescence localization of Muc4 in the reproductive tract epithelia of the experimental groups correlated to message levels, with lack of Muc4 protein detected in the control and progesterone groups. In comparison to reproductive tract epithelia, Muc4 and Muc5AC are not hormonally regulated by estrogen or progesterone in the ocular surface epithelia of mice. These data demonstrate that regulation of epithelial mucin genes is tissue specific.

An Analysis of First Amendment Protection for Student Expression, Mid-1900s-2011

ERIC Educational Resources Information Center

Conaway, Anne F.

2012-01-01

This dissertation sought to determine if federal-level, post-secondary student freedom of expression case law was developing in a similar path to that at the K-12 level of education. It also investigated the ways in which a K-12, highly speech-restrictive legal standard arising from the K-12 case "Hazelwood v. Kuhlmeier" has been…

Examination of Anxiety Levels and Anger Expression Manners of Undergraduate Table Tennis Players

ERIC Educational Resources Information Center

Karademir, Tamer; Türkçapar, Ünal

2016-01-01

This research was done for the determination of how their anxiety levels' and anger expressions' get shaped according to some variances. For this reason there were 76 female 125 male totally 201 sportsmen, who participated to the table tennis championship between universities in 2016 and ages differ from 18 to 28, were included the research group.…

Effect of folate deficiency on promoter methylation and gene expression of Esr1, Cav1, and Elavl1, and its influence on spermatogenesis.

PubMed

Yuan, Hong-Fang; Zhao, Kai; Zang, Yu; Liu, Chun-Yan; Hu, Zhi-Yong; Wei, Jia-Jing; Zhou, Ting; Li, Ying; Zhang, Hui-Ping

2017-04-11

This study aims to investigate the effect of folate deficiency on the male reproductive function and the underlying mechanism. A total of 269 screened participants from 421 recruitments were enrolled in this study. An animal model of folate deficiency was constructed. Folate concentration was measured in the ejaculate, and its association with semen parameters was then determined. The expression and promoter methylation status of ESR1, CAV1, and ELAVL1 were also evaluated. Results showed that seminal plasma folate level was significantly lower among subjects with azoospermia than those with normozoospermia. Low folate level was significantly correlated with low sperm concentration in men with normozoospermia. Folate deficiency significantly reduced the expression of ESR1, CAV1, and ELAVL1, which are critical to spermatogenesis. However, low folate levels did not increase the methylation levels of the promoter regions of ESR1, CAV1, and ELAVL1 in human sperm DNA. Thus, folate deficiency impairs spermatogenesis may partly due to inhibiting the expression of these genes. Thus future research should determine the significance of sufficient folate status in male fertilization and subsequent pregnancy outcomes.

Regulatory divergence between parental alleles determines gene expression patterns in hybrids.

PubMed

Combes, Marie-Christine; Hueber, Yann; Dereeper, Alexis; Rialle, Stéphanie; Herrera, Juan-Carlos; Lashermes, Philippe

2015-03-29

Both hybridization and allopolyploidization generate novel phenotypes by conciliating divergent genomes and regulatory networks in the same cellular context. To understand the rewiring of gene expression in hybrids, the total expression of 21,025 genes and the allele-specific expression of over 11,000 genes were quantified in interspecific hybrids and their parental species, Coffea canephora and Coffea eugenioides using RNA-seq technology. Between parental species, cis- and trans-regulatory divergences affected around 32% and 35% of analyzed genes, respectively, with nearly 17% of them showing both. The relative importance of trans-regulatory divergences between both species could be related to their low genetic divergence and perennial habit. In hybrids, among divergently expressed genes between parental species and hybrids, 77% was expressed like one parent (expression level dominance), including 65% like C. eugenioides. Gene expression was shown to result from the expression of both alleles affected by intertwined parental trans-regulatory factors. A strong impact of C. eugenioides trans-regulatory factors on the upregulation of C. canephora alleles was revealed. The gene expression patterns appeared determined by complex combinations of cis- and trans-regulatory divergences. In particular, the observed biased expression level dominance seemed to be derived from the asymmetric effects of trans-regulatory parental factors on regulation of alleles. More generally, this study illustrates the effects of divergent trans-regulatory parental factors on the gene expression pattern in hybrids. The characteristics of the transcriptional response to hybridization appear to be determined by the compatibility of gene regulatory networks and therefore depend on genetic divergences between the parental species and their evolutionary history. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The MNS glycophorin variant GP.Mur affects differential erythroid expression of Rh/RhAG transcripts.

PubMed

Hsu, K; Kuo, M-S; Yao, C-C; Cheng, H-C; Lin, H-J; Chan, Y-S; Lin, M

2017-10-01

The band 3 macrocomplex (also known as the ankyrin-associated complex) on the red cell membrane comprises two interacting subcomplexes: a band 3/glycophorin A subcomplex, and a Rh/RhAG subcomplex. Glycophorin B (GPB) is a component of the Rh/RhAG subcomplex that is also structurally associated with glycophorin A (GPA). Expression of glycophorin B-A-B hybrid GP.Mur enhances band 3 expression and is associated with lower levels of Rh-associated glycoprotein (RhAG) and Rh polypeptides. The goal of this study was to determine whether GP.Mur influenced erythroid Rh/RhAG expression at the transcript level. GP.Mur was serologically determined in healthy participants from Taitung County, Taiwan. RNA was extracted from the reticulocyte-enriched fraction of peripheral blood, followed by reverse transcription and quantitative PCR for RhAG, RhD and RhCcEe. Quantification by real-time PCR revealed significantly fewer RhAG and RhCcEe transcripts in the reticulocytes from subjects with homozygous GYP*Mur. Independent from GYP.Mur, both RhAG and RhD transcript levels were threefold or higher than that of RhCcEe. Also, in GYP.Mur and the control samples alike, direct quantitative associations were observed between the transcript levels of RhAG and RhD, but not between that of RhAG and RhCcEe. Erythroid RhD and RhCcEe were differentially expressed at the transcript levels, which could be related to their different degrees of interaction or sensitivity to RhAG. Further, the reduction or absence of glycophorin B in GYP.Mur erythroid cells affected transcript expressions of RhAG and RhCcEe. Thus, GPB and GP.Mur differentially influenced Rh/RhAG expressions prior to protein translation. © 2017 International Society of Blood Transfusion.

Metallothionein-1 and nitric oxide expression are inversely correlated in a murine model of Chagas disease

PubMed Central

Gonzalez-Mejia, Martha Elba; Torres-Rasgado, Enrique; Porchia, Leonardo M; Salgado, Hilda Rosas; Totolhua, José-Luis; Ortega, Arturo; Hernández-Kelly, Luisa Clara Regina; Ruiz-Vivanco, Guadalupe; Báez-Duarte, Blanca G; Pérez-Fuentes, Ricardo

2014-01-01

Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of Chagas disease using endemic T. cruzi RyCH1 in BALB/c mice, which were divided into four groups: infected non-treated (Inf), infected N-monomethyl-L-arginine treated (Inf L-NAME), non-infected L-NAME treated and non-infected vehicle-treated. We determined blood parasitaemia and NO levels, the extent of parasite nests in tissues and liver MT-I expression levels. It was observed that NO levels were increasing in Inf mice in a time-dependent manner. Inf L-NAME mice had fewer T. cruzi nests in cardiac and skeletal muscle with decreased blood NO levels at day 135 post infection. This affect was negatively correlated with an increase of MT-I expression (r = -0.8462, p < 0.0001). In conclusion, we determined that in Chagas disease, an unknown inhibitory mechanism reduces MT-I expression, allowing augmented NO levels. PMID:24676665

Expression analysis of several antiviral related genes to BmNPV in different resistant strains of silkworm, Bombyx mori

PubMed Central

Cheng, Yang; Wang, Xue-yang; Du, Chang; Gao, Juan; Xu, Jia-ping

2014-01-01

Abstract Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a highly pathogenic virus in the sericultural industry, often causing severe damage leading to large economic losses. The immune mechanisms of B. mori against this virus remain obscure. Previous studies had demonstrated Bmlipase-1, BmNox and Bmserine protease-2 showing antiviral activity in vitro , but data on the transcription levels of these proteins in different resistant strains were not reported. In order to determine the resistance level of the four different strains (P50, A35, A40, A53) and gain a better understanding of the mechanism of resistance to BmNPV in B. mori , the relative expression level of the genes coding the three antiviral proteins in larval haemolymph and midgut of different B. mori strains resistant to BmNPV was determined. The results showed that these genes expressed significantly higher in the resistant strains compared to the susceptible strain, and the differential expression levels were consistent with the LC50 values in different strains. The transcription level of the target genes almost all up-regulated in the larvae midgut and down-regulated in the haemolymph. The results indicate the correlation of these genes to BmNPV resistance in B. mori. PMID:25373223

Claudin gene expression patterns do not associate with interspecific differences in paracellular nutrient absorption.

PubMed

Price, Edwin R; Rott, Katherine H; Caviedes-Vidal, Enrique; Karasov, William H

2016-01-01

Bats exhibit higher paracellular absorption of glucose-sized molecules than non-flying mammals, a phenomenon that may be driven by higher permeability of the intestinal tight junctions. The various claudins, occludin, and other proteins making up the tight junctions are thought to determine their permeability properties. Here we show that absorption of the paracellular probe l-arabinose is higher in a bat (Eptesicus fuscus) than in a vole (Microtus pennsylvanicus) or a hedgehog (Atelerix albiventris). Furthermore, histological measurements demonstrated that hedgehogs have many more enterocytes in their intestines, suggesting that bats cannot have higher absorption of arabinose simply by having more tight junctions. We therefore investigated the mRNA levels of several claudins and occludin, because these proteins may affect permeability of tight junctions to macronutrients. To assess the expression levels of claudins per tight junction, we normalized the mRNA levels of the claudins to the constitutively expressed tight junction protein ZO-1, and combined these with measurements previously made in a bat and a rodent to determine if there were among-species differences. Although expression ratios of several genes varied among species, there was not a consistent difference between bats and non-flyers in the expression ratio of any particular gene. Protein expression patterns may differ from mRNA expression patterns, and might better explain differences among species in arabinose absorption. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantification of three steroid hormone receptors of the leopard gecko (Eublepharis macularius), a lizard with temperature-dependent sex determination: their tissue distributions and the effect of environmental change on their expressions.

PubMed

Endo, Daisuke; Park, Min Kyun

2003-12-01

Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones function through their specific receptors, so the expression levels of the receptors may reflect the responsibility of target organs. However, there was no effective method to quantify the expression levels of these receptors in reptilian species. In this study, we established the competitive-PCR assay systems for the quantification of the mRNA expression levels of three sex steroid hormone receptors in the leopard gecko. These assay systems were successfully able to detect the mRNA expression level of each receptor in various organs of male adult leopard geckoes. The expression levels of mRNA of these receptors were highly various depending on the organs assayed. This is the first report regarding the tissue distributions of sex steroid hormone receptor expressions in reptile. The effects of environmental conditions on these hormone receptor expressions were also examined. After the low temperature and short photoperiod treatment for 6 weeks, only the androgen receptor expression was significantly increased in the testes. The competitive-PCR assay systems established in this report should be applicable for various studies of the molecular mechanism underlying the reproductive activity of the leopard gecko.

Opposing roles of the aldo-keto reductases AKR1B1 and AKR1B10 in colorectal cancer.

PubMed

Taskoparan, Betul; Seza, Esin Gulce; Demirkol, Secil; Tuncer, Sinem; Stefek, Milan; Gure, Ali Osmay; Banerjee, Sreeparna

2017-12-01

Aldo-keto reductases (including AKR1B1 and AKR1B10) constitute a family of oxidoreductases that have been implicated in the pathophysiology of diabetes and cancer, including colorectal cancer (CRC). Available data indicate that, despite their similarities in structure and enzymatic functions, their roles in CRC may be divergent. Here, we aimed to determine the expression and functional implications of AKR1B1 and AKR1B10 in CRC. AKR1B1 and AKR1B10 gene expression levels were analyzed using publicly available microarray data and ex vivo CRC-derived cDNA samples. Gene Set Enrichment Analysis (GSEA), The Cancer Genome Atlas (TCGA) RNA-seq data and The Cancer Proteome Atlas (TCPA) proteome data were analyzed to determine the effect of high and low AKR1B1 and AKR1B10 expression levels in CRC patients. Proliferation, cell cycle progression, cellular motility, adhesion and inflammation were determined in CRC-derived cell lines in which these genes were either exogenously overexpressed or silenced. We found that the expression of AKR1B1 was unaltered, whereas that of AKR1B10 was decreased in primary CRCs. GSEA revealed that, while high AKR1B1 expression was associated with increased cell cycle progression, cellular motility and inflammation, high AKR1B10 expression was associated with a weak inflammatory phenotype. Functional studies carried out in CRC-derived cell lines confirmed these data. Microarray data analysis indicated that high expression levels of AKR1B1 and AKR1B10 were significantly associated with shorter and longer disease-free survival rates, respectively. A combined gene expression signature of AKR1B10 (low) and AKR1B1 (high) showed a better prognostic stratification of CRC patients independent of confounding factors. Despite their similarities, the expression levels and functions of AKR1B1 and AKR1B10 are highly divergent in CRC, and they may have prognostic implications.

Growth differentiation factor‑5 induces tenomodulin expression via phosphorylation of p38 and promotes viability of murine mesenchymal stem cells from compact bone.

PubMed

Qu, Yanlong; Zhou, Li; Lv, Bing; Wang, Chunlei; Li, Pengwei

2018-03-01

Growth differentiation factor (GDF)‑5 serves a role in tissue development and tenomodulin serves an important role in the development of tendons. The effects of GDF‑5 on mesenchymal stem cells (MSCs), particularly with regards to tendon bioengineering, are poorly understood. The present study aimed to investigate the effects of GDF‑5 on cell viability and tenomodulin expression in MSCs from murine compact bone. MSCs were isolated from murine compact bones and confirmed by flow cytometric analysis. In addition, the adipogenic, osteoblastic and chondrocyte differentiation capabilities of the MSCs were determined. MSCs were treated with GDF‑5 and the effects of GDF‑5 on MSC viability were determined. The mRNA and protein expression levels of tenomodulin were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. MSCs from murine compact bone were successfully isolated. GDF‑5 had optimal effects on cell viability at 100 ng/ml (+36.9% of control group without GDF‑5 treatment, P<0.01) and its effects peaked after 6 days of treatment (+56.6% of control group, P<0.001). Compared with the control group, treatment with 100 ng/ml GDF‑5 for 4 days enhanced the mRNA expression levels of tenomodulin (3.56±0.94 vs. 1.02±0.25; P<0.05). In addition, p38 was activated by GDF‑5, as determined by enhanced expression levels of phosphorylated p38 (p‑p38). The GDF‑5‑induced protein expression levels of p‑p38 and tenomodulin were markedly inhibited following treatment with SB203580, an inhibitor of p38 mitogen‑activated protein kinase. These results suggested that GDF‑5 treatment may increase tenomodulin protein expression via phosphorylation of p38 in MSCs from murine compact bone. These findings may aid the future development of tendon bioengineering.

Adipose tissue IL-8 is increased in normal weight women after menopause and reduced after gastric bypass surgery in obese women.

PubMed

Alvehus, Malin; Simonyte, Kotryna; Andersson, Therése; Söderström, Ingegerd; Burén, Jonas; Rask, Eva; Mattsson, Cecilia; Olsson, Tommy

2012-11-01

The menopausal transition is characterized by increased body fat accumulation, including redistribution from peripheral to central fat depots. This distribution is associated with an increased risk of type 2 diabetes and cardiovascular disease that are linked to low-grade inflammation. We determined whether postmenopausal women have higher levels of inflammatory markers, compared with premenopausal women. We also wanted to determine whether these markers are reduced by stable weight loss in obese women. Anthropometric data, blood samples and subcutaneous adipose tissue biopsies were collected from normal weight premenopausal and postmenopausal women and obese women before and 2 years after gastric bypass (GBP) surgery. Serum protein levels and adipose tissue gene expression of inflammatory markers were investigated. IL-8 expression in adipose tissue and circulating levels were higher in postmenopausal vs premenopausal women. IL-8 expression was associated with waist circumference, independent of menopausal status. IL-6 expression and serum levels of monocyte chemoattractant protein (MCP)-1 were higher in postmenopausal vs premenopausal women. Two years after GBP surgery, adipose expression of IL-8, tumour necrosis factor-α and MCP-1 decreased significantly. Serum insulin levels were associated with inflammation-related gene expression before GBP surgery, but these associations disappeared after surgery. Postmenopausal women have an increased inflammatory response in the subcutaneous fat and circulation. Inflammatory markers in adipose tissue decreased significantly after surgery-induced weight loss. This effect may be beneficial for metabolic control and reduced cardiovascular risk after weight loss. © 2011 Blackwell Publishing Ltd.

Analysis of TP53 gene expression and p53 level of human hypopharyngeal FaDu (HTB-43) head and neck cancer cell line after microRNA-181a inhibition.

PubMed

Cheah, Y K; Cheng, R W; Yeap, S K; Khoo, C H; See, H S

2014-03-17

The identification of new biomarkers for early detection of highly recurrent head and neck cancer is urgently needed. MicroRNAs (miRNAs) are small and non-coding RNAs that regulate cancer-related gene expression, such as tumor protein 53 (TP53) gene expression. This study was carried out to analyze TP53 gene expression using real-time PCR and to determine changes in intracellular p53 level by flow cytometry after downregulation of miRNA-181a miRNA inhibitor in the FaDu cell line. TP53 gene expression showed a 3-fold increment and the p53 protein level was also increased in the miRNA-181a-treated cells. In conclusion, miRNA-181a binds to the TP53 gene and inhibits its expression, decreasing the synthesis of p53.

Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

PubMed

Jiao, Wei; Liu, Fei; Tang, Feng-Zhu; Lan, Jiao; Xiao, Rui-Ping; Chen, Xing-Zhou; Ye, Hui-Lan; Cai, Yong-Lin

2013-01-01

To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

Effective Hepatocyte Transplantation Using Rat Hepatocytes with Low Asialoglycoprotein Receptor Expression

PubMed Central

Ise, Hirohiko; Nikaido, Toshio; Negishi, Naoki; Sugihara, Nobuhiro; Suzuki, Fumitaka; Akaike, Toshihiro; Ikeda, Uichi

2004-01-01

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 × 105 cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied ∼76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for ∼12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes. PMID:15277224

Effective hepatocyte transplantation using rat hepatocytes with low asialoglycoprotein receptor expression.

PubMed

Ise, Hirohiko; Nikaido, Toshio; Negishi, Naoki; Sugihara, Nobuhiro; Suzuki, Fumitaka; Akaike, Toshihiro; Ikeda, Uichi

2004-08-01

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 x 10(5) cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied approximately 76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for approximately 12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes.

Expression of lysozyme in the life history of the house fly (Musca domestica l.).

PubMed

Nayduch, Dana; Joyner, Chester

2013-07-01

From egg to adult, all life history stages of house flies associate with septic environments teeming with bacteria. House fly lysozyme was first identified in the larval midgut, where it is used for digestion of microbe-rich meals because of its broad-spectrum activity against gram-positive and gram-negative bacteria as well as fungi. This study aimed to determine the temporal expression of lysozyme in the life history of house flies (from egg through adults) on both the mRNA and protein level, and to determine the tissue-specific expression of lysozyme in adult flies induced by feeding Staphylococcus aureus. From 30-min postoviposition through adulthood, all life history stages of the house fly express lysozyme on the mRNA level. In adult flies, lysozyme is expressed both locally in the alimentary canal and systemically in the fat body. Interestingly, we found that during the normal life history of flies, lysozyme protein was only detected in larval stages and older adults, likely because of ingestion of immune-stimulating levels of bacteria, not experienced during egg, pupa, and teneral adult stages. Constitutive expression on the mRNA level implies that this effector is a primary defense molecule in all stages of the house fly life history, and that a mechanism for posttranscriptional control of mature lysozyme enzyme expression may be present. Lysozyme active enzyme primarily serves both a digestive and defensive function in larval and adult flies, and may be a key player in the ability of Musca domestica L. to thrive in microbe-rich environments.

Characterization, expression and function analysis of DAX1 gene of scallop ( Chlamys farreri jones and preston 1904) during its gametogenesis

NASA Astrophysics Data System (ADS)

Li, Hailong; Liu, Jianguo; Huang, Xiaoting; Wang, Dan; Zhang, Zhifeng

2014-08-01

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop ( Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at mRNA and protein levels. The cDNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame (ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher ( P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.

Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

PubMed Central

2010-01-01

Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this one could aid in overcoming the technical bottlenecks in working with TM proteins and could potentially aid in increasing the rate of structure determination. PMID:21114855

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression.

PubMed

Shannon, Diane B; McKeown, Scott T W; Lundy, Fionnuala T; Irwin, Chris R

2006-01-01

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.

Expression and function of methylthioadenosine phosphorylase in chronic liver disease.

PubMed

Czech, Barbara; Dettmer, Katja; Valletta, Daniela; Saugspier, Michael; Koch, Andreas; Stevens, Axel P; Thasler, Wolfgang E; Müller, Martina; Oefner, Peter J; Bosserhoff, Anja-Katrin; Hellerbrand, Claus

2013-01-01

To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease. MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chromatography-tandem mass spectrometry. MTAP was downregulated in hepatocytes in murine fibrosis models and in patients with chronic liver disease, leading to a concomitant increase in MTA levels. In contrast, activated hepatic stellate cells (HSCs) showed strong MTAP expression in cirrhotic livers. However, also MTA levels in activated HSCs were significantly higher than in hepatocytes, and there was a significant correlation between MTA levels and collagen expression in diseased human liver tissue indicating that activated HSCs significantly contribute to elevated MTA in diseased livers. MTAP suppression by siRNA resulted in increased MTA levels, NFκB activation and apoptosis resistance, while overexpression of MTAP caused the opposite effects in HSCs. The anti-apoptotic effect of low MTAP expression and high MTA levels, respectively, was mediated by induced expression of survivin, while inhibition of survivin abolished the anti-apoptotic effect of MTA on HSCs. Treatment with a DNA demethylating agent induced MTAP and reduced survivin expression, while oxidative stress reduced MTAP levels but enhanced survivin expression in HSCs. MTAP mediated regulation of MTA links polyamine metabolism with NFκB activation and apoptosis in HSCs. MTAP and MTAP modulating mechanisms appear as promising prognostic markers and therapeutic targets for hepatic fibrosis.

Expression and Function of Methylthioadenosine Phosphorylase in Chronic Liver Disease

PubMed Central

Czech, Barbara; Dettmer, Katja; Valletta, Daniela; Saugspier, Michael; Koch, Andreas; Stevens, Axel P.; Thasler, Wolfgang E.; Müller, Martina; Oefner, Peter J.; Bosserhoff, Anja-Katrin; Hellerbrand, Claus

2013-01-01

To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease. Design MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chromatography-tandem mass spectrometry. Results MTAP was downregulated in hepatocytes in murine fibrosis models and in patients with chronic liver disease, leading to a concomitant increase in MTA levels. In contrast, activated hepatic stellate cells (HSCs) showed strong MTAP expression in cirrhotic livers. However, also MTA levels in activated HSCs were significantly higher than in hepatocytes, and there was a significant correlation between MTA levels and collagen expression in diseased human liver tissue indicating that activated HSCs significantly contribute to elevated MTA in diseased livers. MTAP suppression by siRNA resulted in increased MTA levels, NFκB activation and apoptosis resistance, while overexpression of MTAP caused the opposite effects in HSCs. The anti-apoptotic effect of low MTAP expression and high MTA levels, respectively, was mediated by induced expression of survivin, while inhibition of survivin abolished the anti-apoptotic effect of MTA on HSCs. Treatment with a DNA demethylating agent induced MTAP and reduced survivin expression, while oxidative stress reduced MTAP levels but enhanced survivin expression in HSCs. Conclusion MTAP mediated regulation of MTA links polyamine metabolism with NFκB activation and apoptosis in HSCs. MTAP and MTAP modulating mechanisms appear as promising prognostic markers and therapeutic targets for hepatic fibrosis. PMID:24324622

Antisense oligodeoxynucleotide inhibits vascular endothelial growth factor expression in U937 foam cells.

PubMed

Yang, Peng-Yuan; Rui, Yao-Cheng; Jin, You-Xin; Li, Tie-Jun; Qiu, Yan; Zhang, Li; Wang, Jie-Song

2003-06-01

To study the expression of vascular endothelial growth factor (VEGF) induced by oxidized low density liporotein (ox-LDL) and the inhibitory effects of antisense oligodeoxynucleotide (asODN) on the levels of VEGF protein and mRNA in the U937 foam cells. U937 cells were incubated with ox-LDL 80 mg/L for 48 h, then, the foam cells were treated with asODN (0, 5, 10, and 20 micromol/L). The VEGF concentration in the media was determined by ELISA. The VEGF protein expression level in cells was measured by immuohistochemistry; the positive ratio detected by a morphometrical analysis system was used as the amount of the VEGF expression level. The VEGF mRNA level was examined by Northern blotting. After U937 cells were incubated with ox-LDL, VEGF expression level increased greatly both in the cells and in the media. asODN markedly inhibited the increase of VEGF. After treatment with asODN 20 micromol/L, the VEGF protein concentration in the media decreased by 45.0%, the VEGF positive ratio detected by immuohistochemistry in cells decreased by 64.9%, and the VEGF mRNA level decreased by 47.1%. The expression of VEGF in U937 foam cells was strong. asODN inhibited VEGF expression significantly in U937 foam cells in vitro.

Relative expression of proprotein convertases in rat ovaries during pregnancy

PubMed Central

2013-01-01

Background Proprotein convertases are a family of serine proteinases that are related to bacterial subtilisin and yeast kexin. They are involved in posttranslational processing of the precursors of a vast number of cellular proteins. With the exception of PC1/3, the relative expression levels of the proprotein convertases in the ovary during pregnancy have not been reported. The purpose of this study is to determine by real-time PCR the relative expression levels of all nine proprotein convertases in rat ovaries during pregnancy and at 3 days postpartum. Methods RNA was extracted from ovaries at Day 0, 4, 9, 11, 13, 15, 18, and 20 of pregnancy as well as 3 days postpartum. Relative expression levels of Pcsk1, Pcsk2, Furin, Pcsk4, Pcsk5, Pcsk6, Pcsk7, Mbtps1 and Pcsk9 were determined with real-time PCR. Results were reported as fold-change over the level at Day 0 of pregnancy. Results Results showed that Pcsk1 and Pcsk6 were upregulated as gestation advanced, in parallel with an observed increase in relaxin transcript. Pcsk2 showed downregulation as gestation advanced, while Pcsk5 showed relatively higher levels in early pregnancy and postpartum, but lower level in mid-pregnancy. On the other hand, Furin, Pcsk4, Pcsk7, Mbtps1 and Pcsk9 showed little change of expression throughout gestation. Conclusion PC1/3 (PCSK1) and PACE4 (PCSK6) may play an important role in proprotein processing in the ovary during late pregnancy. PMID:24330629

Angiopoietin-like protein 8/betatrophin as a new determinant of type 2 diabetes remission after bariatric surgery.

PubMed

Ejarque, Miriam; Borlaug, Marianne; Vilarrasa, Nuria; Martinez-Perez, Bruno; Llauradó, Gemma; Megía, Ana; Helland, Thomas; Gutierrez, Cristina; Serena, Carolina; Folkestad, Oddry; Nuñez-Roa, Catalina; Roche, Kelly; Casajoana, Ana; Fradera, Rosa; González-Clemente, José Miguel; López, Miguel; Mohn, Arne C; Nedrebø, Bjørn G; Nogueiras, Ruben; Mellgren, Gunnar; Fernø, Johan; Fernández-Veledo, Sonia; Vendrell, Joan

2017-06-01

This work aimed to explore the link between angiopoietin-like protein 8 (ANGPTL8) and weight loss after metabolic surgery. In the cross-sectional study (n = 100), circulating ANGPTL8 concentrations were significantly lower in morbidly obese than in lean subjects, and strikingly lower in morbidly obese patients with type 2 diabetes mellitus (T2DM). Conversely, ANGPTL8 expression in subcutaneous adipose tissue (SAT) was higher in morbidly obese patients, particularly in those with T2DM, whereas its expression in visceral adipose tissue was unchanged. The main predictors for circulating levels of ANGPTL8 were BMI and T2DM, whereas ANGPTL8 expression in SAT was determined by the presence of T2DM. The prospective cohort studies before and 1 year after bariatric surgery in morbidly obese patients with (n = 45) and without (n = 30) T2DM, revealed a significant increase of circulating ANGPTL8 levels 1 year after the bariatric surgery. Intriguingly, this increment, which was predicted by basal ANGPTL8 concentrations, appeared as a determinant of T2DM remission. In conclusion, circulating ANGPTL8 levels have an inverse relationship with SAT expression. Low basal levels of ANGPTL8 rebound after bariatric surgery. The increment in ANGPTL8 concentrations at 1 month of follow-up after weight loss emerged as a significant predictor of the T2DM remission at 1 year of follow-up. Copyright © 2017 Elsevier Inc. All rights reserved.

Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

PubMed

Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C

2016-06-22

In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.

HDAC1 and HDAC2 are Differentially Expressed in Endometriosis

PubMed Central

Colón-Díaz, Maricarmen; Báez-Vega, Perla; García, Miosotis; Ruiz, Abigail; Monteiro, Janice B.; Fourquet, Jessica; Bayona, Manuel; Alvarez-Garriga, Carolina; Achille, Alexandra; Seto, Edward; Flores, Idhaliz

2012-01-01

Epigenetic mechanisms have been ascribed important roles in endometriosis. Covalent histone modifications at lysine residues have been shown to regulate gene expression and thus contribute to pathological states in many diseases. In endometriosis, histone deacetylase inhibition (HDACi) resulted in reactivation of E-cadherin, attenuation of invasion, decreased proliferation of endometriotic cells, and caused lesion regression in an animal model. This study was conducted to assess basal and hormone-regulated gene expression levels of HDAC1 and HDAC2 (HDAC1/2) in cell lines and protein expression levels in tissues. Basal and steroid hormone-regulated HDAC1/2 gene expression levels were determined by quantitative polymerase chain reaction in cell lines and tissues. Protein levels were measured by immunohistochemistry (IHC) in tissues on an endometriosis tissue microarray (TMA). Basal HDAC1/2 gene expression levels were significantly higher in endometriotic versus endometrial stromal cells, which was confirmed by Western blot analysis. Estradiol (E2) and progesterone (P4) significantly downregulated HDAC1 expression in endometrial epithelial cells. Levels of HDAC2 were upregulated by E2 and downregulated by E2 + P4 in endometrial stromal cells. Hormone modulation of HDAC1/2 gene expression was lost in the endometriotic cell line. Immunohistochemistry showed that HDAC1/2 proteins were expressed in a substantial proportion of lesions and endometrium from patients, and their expression levels varied according to lesion localization. The highest proportion of strong HDAC1 immunostaining was seen in ovarian, skin, and gastrointestinal lesions, and of HDAC2 in skin lesions and endometrium from patients with endometriosis. These studies suggest that endometriosis etiology may be partially explained by epigenetic regulation of gene expression due to dysregulations in the expression of HDACs. PMID:22344732

Accumulation of Flavonoid Glycosides and UFGT Gene Expression in Mulberry Leaves (Morus alba L.) before and after Frost.

PubMed

Yu, Xiaofeng; Zhu, Yiling; Fan, Jingyi; Wang, Dujun; Gong, Xiaohui; Ouyang, Zhen

2017-08-01

In order to determine the molecular mechanism underlying the influence of frost on chemical changes in mulberry leaves, the UFGT activity, expression level, and accumulation of flavonoid glycosides in mulberry leaves (Morus alba L.) were studied. The expression of UFGT gene was investigated by quantitative real-time PCR (qRT-PCR) and the UFGT activity, accumulation of flavonoid glycosides was studied by high performance liquid chromatography. Then, the correlation between the expression level of UFGT, the UFGT activity, and the flavonoid glycosides accumulation with temperature was explored. The accumulation of isoquercitrin and astragalin is significantly positively correlated with UFGT gene expression and UFGT activity. On the contrary, the average temperature was significantly negatively correlated with the level of UFGT gene expression and UFGT activity. The results show that after frost, low temperature can induce the expression of UFGT gene in mulberry leaves, resulting in the accumulation of flavonoid glycosides. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

PubMed Central

Thiel, Cora S.; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E.

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. PMID:25654098

Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

PubMed

Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

HOXC6 promotes gastric cancer cell invasion by upregulating the expression of MMP9.

PubMed

Chen, Shi-Wei; Zhang, Qing; Xu, Zhi-Feng; Wang, Hai-Ping; Shi, Yi; Xu, Feng; Zhang, Wen-Jian; Wang, Ping; Li, Yong

2016-10-01

Previous studies have demonstrated that the homoebox C6 (HOXC6) gene is highly expressed in gastric cancer tissues and is associated with the depth of tumor invasion, and is associated with poor prognosis of gastric cancer patients expressing HOXC6. The present study investigated the effect and underlying mechanism of HOXC6 on the proliferation and metastasis of gastric cancer cells in vitro. Reverse transcription‑quantitative polymerase chain (PCR) reaction was used to investigate the expression levels of HOXC6 in different gastric cancer cell lines and the effect of different levels of expression on the proliferation of gastric cancer cells was determined by cell growth curve and plate colony formation. The effect of HOXC6 on the anchorage‑independent proliferation of gastric cancer cells was determined by soft agar colony formation assay while the Transwell invasion assay was used to investigate the effect of different levels of HOXC6 expression on the invasive and metastatic abilities of gastric cancer cells. Semi‑quantitative PCR was used to detect the effect of different levels of HOXC6 expression on the expression of matrix metalloproteinase (MMP)2 and MMP9 in gastric cancer cells. Immunoblotting was used to assess MMP9 signaling in the gastric cancer cells. The HOXC6 gene is highly expressed in the majority of the gastric cancer cell lines. Overexpression of HOXC6 promoted gastric cancer cell proliferation and colony formation ability while HOXC6 downregulation inhibited cell proliferation and clone forming ability. HOXC6 overexpression also enhanced the soft agar colony formation ability of gastric cancer cells while HOXC6 downregulation decreased the colony formation ability. Upregulated HOXC6 increased the migration and invasion abilities of gastric cancer cells while interfering with HOXC6 expression inhibited the migration and invasion of the gastric cancer cells. The expression of MMP9 was enhanced with an upregulation of HOXC6 expression while HOXC6 downregulation lowered MMP9 gene expression levels. Increased expression of HOXC6 in gastric cancer cell lines significantly activated extracellular signal‑regulated kinase signaling and upregulated MMP9. The HOXC6 gene promotes the proliferation of gastric cancer cells while upregulation of MMP9 promotes migration and invasion of gastric cancer cells.

pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis.

PubMed

Sheen, Patricia; Lozano, Katherine; Gilman, Robert H; Valencia, Hugo J; Loli, Sebastian; Fuentes, Patricia; Grandjean, Louis; Zimic, Mirko

2013-09-01

Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance. Copyright © 2013 Elsevier Ltd. All rights reserved.

pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis

PubMed Central

Sheen, Patricia; Lozano, Katherine; Gilman, Robert H.; Valencia, Hugo J.; Loli, Sebastian; Fuentes, Patricia; Grandjean, Louis; Zimic, Mirko

2013-01-01

Summary Background Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. Objective This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Methods Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Findings Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance. PMID:23867321

Cytotype Control of Drosophila Melanogaster P Element Transposition: Genomic Position Determines Maternal Repression

PubMed Central

Misra, S.; Buratowski, R. M.; Ohkawa, T.; Rio, D. C.

1993-01-01

P element transposition in Drosophila is controlled by the cytotype regulatory state: in P cytotype, transposition is repressed, whereas in M cytotype, transposition can occur. P cytotype is determined by a combination of maternally inherited factors and chromosomal P elements in the zygote. Transformant strains containing single elements that encoded the 66-kD P element protein zygotically repressed transposition, but did not display the maternal repression characteristic of P cytotype. Upon mobilization to new genomic positions, some of these repressor elements showed significant maternal repression of transposition in genetic assays, involving a true maternal effect. Thus, the genomic position of repressor elements can determine the maternal vs. zygotic inheritance of P cytotype. Immunoblotting experiments indicate that this genomic position effect does not operate solely by controlling the expression level of the 66-kD repressor protein during oogenesis. Likewise, P element derivatives containing the hsp26 maternal regulator sequence expressed high levels of the 66-kD protein during oogenesis, but showed no detectable maternal repression. These data suggest that the location of a repressor element in the genome may determine maternal inheritance of P cytotype by a mechanism involving more than the overall level of expression of the 66-kD protein in the ovary. PMID:8293979

The plateau zokors' learning and memory ability is related to the high expression levels of foxP2 in the brain.

PubMed

Ma, Ben-Yuan; Wei, Lian; Sun, Sheng-Zhen; Wang, Duo-Wei; Wei, Deng-Bang

2014-04-25

Plateau zokor (Myospalax baileyi) is a subterranean mammal. Plateau zokor has high learning and memory ability, and can determine the location of blocking obstacles in their tunnels. Forkhead box p2 (FOXP2) is a transcription factor implicated in the neural control of orofacial coordination and sensory-motor integration, particularly with respect to learning, memory and vocalization. To explore the association of foxP2 with the high learning and memory ability of plateau zokor, the cDNA of foxP2 of plateau zokor was sequenced; by using plateau pika as control, the expression levels of foxP2 mRNA and FOXP2 protein in brain of plateau zokor were determined by real-time PCR and Western blot, respectively; and the location of FOXP2 protein in the brain of plateau zokor was determined by immunohistochemistry. The result showed that the cDNA sequence of plateau zokor foxP2 was similar to that of other mammals and the amino acid sequences showed a relatively high degree of conservation, with the exception of two particular amino acid substitutions [a Gln (Q)-to-His (H) change at position 231 and a Ser (S)-to-Ile (I) change at position 235]. Higher expression levels of foxP2 mRNA (3-fold higher) and FOXP2 protein (>2-fold higher) were detected in plateau zokor brain relative to plateau pika brain. In plateau zokor brain, FOXP2 protein was highly expressed in the cerebral cortex, thalamus and the striatum (a basal ganglia brain region). The results suggest that the high learning and memory ability of plateau zokor is related to the high expression levels of foxP2 in the brain.

ERCC1 and ERCC2 as predictive biomarkers to oxaliplatin-based chemotherapy in colorectal cancer patients from Egypt.

PubMed

Kassem, Amira B; Salem, Salem Eid; Abdelrahim, Mohamed E; Said, Amira S A; Salahuddin, Ahmad; Hussein, Marwa Mahmoud; Bahnassy, Abeer A

2017-02-01

The impact of Excision repair cross-complementation group 1 (ERCC1) and group 2 (ERCC2) expression levels on the efficacy of oxaliplatin-based chemotherapy is still controversial. The present study was conducted to determine the predictive value of these molecular biomarkers in stage III and IV colorectal cancer (CRC) patients receiving oxaliplatin (OX)-based chemotherapy as first-line treatment. The study included 80 CRC patients who received first line oxaliplatin based chemotherapy The expression levels of ERCC1 and ERCC2-mRNA and proteins were determined in the primary tumors by quantitative real time reverse transcription polymerase chain reaction(RT-qPCR) and immunohistochemistry (IHC); respectively. The results of mRNA expression were correlated with patients' characteristics, response to treatment, overall- and event free survival (OS & EFS). Sixty four out of the 80 patients were legible for assessment of ERCC1 and ERCC2 expression. The cut-off levels of ERCC1and ERCC2-RNA were 3.8×10 -3 & 4.6×10 -3 ; respectively. Reduced ERCC1 and ERCC2 RNA expressions were detected in 50 (78.1%) and 48 (75%) cases, respectively whereas reduced proteins were detected in 48 cases (75%) for ERCC1 and ERCC2. After The median follow up period was 30.5months (range: 7-104months), Patients with low mRNAERCC1levels showed significantly longer OS (p=0.011) and EFS (p˂0.001). However, no significant relation was found between ERCC2 levels and OS or EFS. In multivariate analysis performance status (PS), stage of the disease and ERCC1-mRNA expression were independent prognostic factors for EFS whereas tumor histology and stage of the disease were independent factors for OS. ERCC1 expression levels may help in selecting patients who benefit from oxaliplatin chemotherapy in stage III & IV CRC. Further large trials are needed to validate these data. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.

PubMed

Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N; Marfurt, Jutta

2016-01-01

Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. Copyright © 2015 Pava et al.

Hematopoietic lineage cell-specific protein 1 immunoreactivity indicates an increased risk of poor overall survival in patients with ovarian carcinoma.

PubMed

Liu, Wenting; Kajiyama, Hiroaki; Shibata, Kiyosumi; Koya, Yoshihiro; Senga, Takeshi; Kikkawa, Fumitaka

2018-06-01

Hematopoietic lineage cell-specific protein 1 (HS1) is a 75-kDa intracellular protein that is expressed primarily in hematopoietic cells. Several previous studies have demonstrated the association between HS1 expression and a poor prognosis in hematopoietic malignancies; however, in solid tumors, no studies not been reported. The present study examined the distribution and expression of HS1 in human epithelial ovarian carcinoma (EOC) to determine its clinical significance. Paraffin sections were obtained from EOC tissues and immunostained with HS1 antibody, and then the staining intensities were evaluated. Overall survival (OS) was determined using the Kaplan-Meier estimator method, and multivariate analysis was performed using the Cox proportional hazards analysis. In total, 195 patients with EOC (median age, 56 years) were enrolled into the present study. HS1 immunoreactivity was categorized based on expression levels: Low (89/195; 45.6%) and high (106/195; 54.4%). Results demonstrated no association between expression level(s) and any clinicopathological parameter including age, International Federation of Gynecology and Obstetrics (FIGO) staging, type of chemotherapy or type of surgery received. The 5-year OS rates of patients who demonstrated low (n=89) and high (n=106) HS1 expression were 90.4 and 66.7%, respectively. The OS times for patients with high HS1 expression were significantly shorter compared with those for patients exhibiting low HS1 expression (P=0.0065). Results obtained from the multivariate analysis demonstrated that the FIGO stage and the amount of HS1 expressed were significant independent prognostic markers for poorer OS (hazard ratio, 3.539; 95% confidence interval, 1.221-12.811; P=0.0187). High HS1 expression levels may serve as a useful biomarker in patients with EOC who are likely to exhibit an unfavorable clinical outcome.

Prostaglandin E2 Induces IL-6 and IL-8 Production by the EP Receptors/Akt/NF-κB Pathways in Nasal Polyp-Derived Fibroblasts.

PubMed

Cho, Jung-Sun; Han, In-Hye; Lee, Hye Rim; Lee, Heung-Man

2014-09-01

Interleukin 6 (IL-6) and IL-8 participate in the pathogenesis of chronic rhinosinusitis with nasal polyps, and their levels are increased by prostaglandin E2 (PGE2) in different cell types. The purposes of this study were to determine whether PGE2 has any effect on the increase in the levels of IL-6 and IL-8 in nasal polyp-derived fibroblasts (NPDFs) and subsequently investigate the possible mechanism of this effect. Different concentrations of PGE2 were used to stimulate NPDFs at different time intervals. NPDFs were treated with agonists and antagonists of E prostanoid (EP) receptors. To determine the signaling pathway for the expression of PGE2-induced IL-6 and IL-8, PGE2 was treated with Akt and NF-κB inhibitors in NPDFs. Reverse transcription-polymerase chain reaction for IL-6 and IL-8 mRNAs was performed. IL-6 and IL-8 levels were measured byenzyme-linked immunosorbent assay (ELISA). The activation of Akt and NF-κB was evaluated by western blot analysis. PGE2 significantly increased the mRNA and protein expression levels of IL-6 and IL-8 in NPDFs. The EP2 and EP4 agonists and antagonists induced and inhibited IL-6 expression. However, the EP4 agonist and antagonist were only observed to induce and inhibit IL-8 expression level. The Akt and NF-κB inhibitors significantly blocked PGE2-induced expression of IL-6 and IL-8. PGE2 increases IL-6 expression via EP2 and EP4 receptors, and IL-8 expression via the EP4 receptor in NPDFs. It also activates the Akt and NF-κB signal pathways for the production of IL-6 and IL-8 in NPDFs. These results suggest that signaling pathway for IL-6 and IL-8 expression induced by PGE2 might be a useful therapeutic target for the treatment of nasal polyposis.

L-dopa decarboxylase (DDC) gene expression is related to outcome in patients with prostate cancer.

PubMed

Koutalellis, Georgios; Stravodimos, Konstantinos; Avgeris, Margaritis; Mavridis, Konstantinos; Scorilas, Andreas; Lazaris, Andreas; Constantinides, Constantinos

2012-09-01

What's known on the subject? and What does the study add? L-dopa decarboxylase (DDC) has been documented as a novel co-activator of androgen receptor transcriptional activity. Recently, it was shown that DDC gene expression is significantly higher in patients with PCa than in those with BPH. In the present study, there was a significant association between the DDC gene expression levels and the pathological stage and Gleason score of patients with prostate cancer (PCa). Moreover, DDC expression was shown to be an unfavourable prognostic marker of biochemical recurrence and disease-free survival in patients with PCa treated by radical prostatectomy. To determine whether L-dopa decarboxylase gene (DDC) expression levels in patients with prostate cancer (PCa) correlate to biochemical recurrence and disease prognosis after radical prostatectomy (RP). The present study consisted of 56 samples with confirmed malignancy from patients with PCa who had undergone RP at a single tertiary academic centre. Total RNA was isolated from tissue specimens and a SYBR Green fluorescence-based quantitative real-time polymerase chain reaction methodology was developed for the determination of DDC mRNA expression levels of the tested tissues. Follow-up time ranged between 1.0 and 62.0 months (mean ± SE, 28.6 ± 2.1 month; median, 31.5 months). Time to biochemical recurrence was defined as the interval between the surgery and the measurement of two consecutive values of prostate-specific antigen (PSA) ≥0.2 ng/mL. DDC expression levels were found to be positively correlated with the tumour-node-metastasis stage (P = 0.021) and Gleason score (P = 0.036) of the patients with PCa. Patients with PCa with raised DDC expression levels run a significantly higher risk of biochemical recurrence after RP, as indicated by Cox proportional regression analysis (P = 0.021). Multivariate Cox proportional regression models revealed the preoperative PSA-, age- and digital rectal examination-independent prognostic value of DDC expression for the prediction of disease-free survival (DFS) among patients with PCa (P = 0.036). Kaplan-Meier survival analysis confirms the significantly shorter DFS after RP of PCa with higher DDC expression levels (P = 0.015). This is the first study indicating the potential of DDC expression as a novel prognostic biomarker in patients with PCa who have undergone RP. For further evaluation and clinical application of the findings of the present study, a direct analysis of mRNA and/or its protein expression level in preoperative biopsy, blood serum and urine should be conducted. © 2012 BJU INTERNATIONAL.

Impact of a high-cholesterol diet on expression levels of Niemann-Pick C1-like 1 and intestinal transporters in rats and mice.

PubMed

Kawase, Atsushi; Araki, Yasuha; Ueda, Yukiko; Nakazaki, Sayaka; Iwaki, Masahiro

2016-08-01

Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 are all involved in intestinal cholesterol absorption. It is unclear whether a high-cholesterol (HC) diet affects the expression of these transporters in rats and mice as well as humans. We examined the effects of an HC diet on their expression in small intestine and the differences between rats and mice in the responsive of this expression to an HC diet. In addition to these transporters, alterations in six representative drug and nutrient transporters (multidrug resistance-associated protein, breast cancer resistance protein, peptide transporter, sodium-glucose linked transporter, glucose transporter, and L-type amino acid transporter) and transcriptional factors such as hepatocyte nuclear factor (HNF)4α, sterol regulatory element-binding protein (SREBP)2, and liver X receptor (LXR)α were determined. In rats and mice fed an HC diet for 7 days, the mRNA and protein levels of NPC1L1 in the small intestine were determined by real-time reverse transcription polymerase chain reaction and western blotting, respectively. The mRNA levels of ABCG5 and ABCG8, six representative transporters, and transcriptional factors such as HNF4α, SREBP2, and LXR were examined. Significant decreases in the expression levels of NPC1L1 were observed in mice, but not rats, fed the HC diet. The mRNA levels of ABCG5 and ABCG8 were significantly increased in HC rats but not in mice. Only minor changes in the mRNA levels of the other transporters were seen in HC rats and mice. Decreased mRNA levels of HNF4α and SREBP2 in mice could be involved in the reduction in NPC1L1 expression observed upon the introduction of an HC diet. These results indicate that the effects of an HC diet on the expression levels of NPC1L1, ABCG5, and ABCG8 differ between mice and rats.

Isoform-specific regulation of cytochrome P450 expression and activity by estradiol in female rats

PubMed Central

Choi, Su-Young; Fischer, Liam; Yang, Kyunghee; Chung, Hyejin; Jeong, Hyunyoung

2011-01-01

Estradiol (E2) is the major endogenous estrogen, and its plasma concentration increases up to 100-fold during pregnancy in humans. Accumulating evidence suggests that an elevated level of E2 may influence hepatic drug metabolism, potentially being responsible for altered drug metabolism during pregnancy. We characterized effects of E2 on expression and activities of cytochrome P450 enzymes (CYPs) in an in vivo system using rats. To this end, female rats were treated with estradiol benzoate (EB) or known CYP inducers. Liver tissues were obtained after 5 days of treatment, and mRNA and protein expression levels as well as activities of major hepatic CYPs were determined by qRT-PCR, immunoblot, and microsomal assay. E2 increased CYP1A2 expression and activity to a smaller extent than β-naphthoflavone did. E2 also enhanced CYP2C expression (CYP2C6, CYP2C7, and CYP2C12) to levels comparable to those observed by phenobarbital. E2 upregulated CYP3A9 expression, while expression of CYP3A1 was downregulated. Expression of hepatic nuclear receptors (PXR and CAR) and the obligate redox partner of CYPs (POR) was downregulated in EB-treated rats, suggesting their potential involvement in regulation of CYP expression and activity by E2. In summary, in female rats E2 regulates expression of hepatic CYPs in a CYP isoform-specific manner although the directional changes are different from those clinically observed during human pregnancy. Further study is warranted to determine whether the changes in drug metabolism during human pregnancy are attributable to involvement of hormones other than E2. PMID:21219883

Expression of the cytoskeleton regulatory protein Mena in human gastric carcinoma and its prognostic significance

PubMed Central

Xu, Lihua; Tan, Huo; Liu, Ruiming; Huang, Qungai; Zhang, Nana; Li, Xi; Wang, Jiani

2017-01-01

The cytoskeleton regulatory protein Mena is reportedly overexpressed in breast cancer; however, data regarding its expression level and clinical significance in gastric carcinoma (GC) is limited. The aim of the present study was to investigate Mena expression levels and prognostic significance in GC. Mena mRNA expression level was determined by reverse transcription-quantitative polymerase chain reaction in 10 paired GC and adjacent normal tissues. The Mena protein expression level was analyzed in paraffin-embedded GC samples and adjacent normal tissues by immunohistochemistry. Statistical analyses were also performed to evaluate the clinicopathological significance of Mena. The results revealed that the mRNA expression level of Mena was significantly higher in G Ct issues compared with in adjacent normal tissues from10 paired samples. In the paraffin-embedded tissue samples, the protein expression level of Mena was higher in G Ct issues compared with in adjacent normal tissues. Compared with adjacent normal tissues, Mena overexpression was observed in 52.83% (56/106) of patients. The overexpression of Mena was significantly associated with the T stage (P=0.033), tumor-node-metastasis (TNM) stage (P<0.001) and decreased overall survival (P<0.001). Based on a multivariate analysis, Mena expression level was an independent prognostic factor for overall survival time. In conclusion, Mena wasoverexpressed in G C tissues and significantly associated with the T stage, TNM stage and overall survival time. Mena may therefore be suitable as a prognostic indicator for patients with GC. PMID:29113241

Expression of the cytoskeleton regulatory protein Mena in human gastric carcinoma and its prognostic significance.

PubMed

Xu, Lihua; Tan, Huo; Liu, Ruiming; Huang, Qungai; Zhang, Nana; Li, Xi; Wang, Jiani

2017-11-01

The cytoskeleton regulatory protein Mena is reportedly overexpressed in breast cancer; however, data regarding its expression level and clinical significance in gastric carcinoma (GC) is limited. The aim of the present study was to investigate Mena expression levels and prognostic significance in GC. Mena mRNA expression level was determined by reverse transcription-quantitative polymerase chain reaction in 10 paired GC and adjacent normal tissues. The Mena protein expression level was analyzed in paraffin-embedded GC samples and adjacent normal tissues by immunohistochemistry. Statistical analyses were also performed to evaluate the clinicopathological significance of Mena. The results revealed that the mRNA expression level of Mena was significantly higher in G Ct issues compared with in adjacent normal tissues from10 paired samples. In the paraffin-embedded tissue samples, the protein expression level of Mena was higher in G Ct issues compared with in adjacent normal tissues. Compared with adjacent normal tissues, Mena overexpression was observed in 52.83% (56/106) of patients. The overexpression of Mena was significantly associated with the T stage (P=0.033), tumor-node-metastasis (TNM) stage (P<0.001) and decreased overall survival (P<0.001). Based on a multivariate analysis, Mena expression level was an independent prognostic factor for overall survival time. In conclusion, Mena wasoverexpressed in G C tissues and significantly associated with the T stage, TNM stage and overall survival time. Mena may therefore be suitable as a prognostic indicator for patients with GC.

Elevated HLA-A expression impairs HIV control through inhibition of NKG2A-expressing cells.

PubMed

Ramsuran, Veron; Naranbhai, Vivek; Horowitz, Amir; Qi, Ying; Martin, Maureen P; Yuki, Yuko; Gao, Xiaojiang; Walker-Sperling, Victoria; Del Prete, Gregory Q; Schneider, Douglas K; Lifson, Jeffrey D; Fellay, Jacques; Deeks, Steven G; Martin, Jeffrey N; Goedert, James J; Wolinsky, Steven M; Michael, Nelson L; Kirk, Gregory D; Buchbinder, Susan; Haas, David; Ndung'u, Thumbi; Goulder, Philip; Parham, Peter; Walker, Bruce D; Carlson, Jonathan M; Carrington, Mary

2018-01-05

The highly polymorphic human leukocyte antigen ( HLA ) locus encodes cell surface proteins that are critical for immunity. HLA-A expression levels vary in an allele-dependent manner, diversifying allele-specific effects beyond peptide-binding preference. Analysis of 9763 HIV-infected individuals from 21 cohorts shows that higher HLA-A levels confer poorer control of HIV. Elevated HLA-A expression provides enhanced levels of an HLA-A-derived signal peptide that specifically binds and determines expression levels of HLA-E, the ligand for the inhibitory NKG2A natural killer (NK) cell receptor. HLA-B haplotypes that favor NKG2A-mediated NK cell licensing (i.e., education) exacerbate the deleterious effect of high HLA-A on HIV control, consistent with NKG2A-mediated inhibition impairing NK cell clearance of HIV-infected targets. Therapeutic blockade of HLA-E:NKG2A interaction may yield benefit in HIV disease. Copyright © 2017, American Association for the Advancement of Science.

High-level mRNA quantification of proliferation marker pKi-67 is correlated with favorable prognosis in colorectal carcinoma.

PubMed

Ihmann, Thomas; Liu, Jian; Schwabe, Wolfgang; Häusler, Peter; Behnke, Detlev; Bruch, Hans-Peter; Broll, Rainer; Windhövel, Ute; Duchrow, Michael

2004-12-01

The present study retrospectively examines the expression of pKi-67 mRNA and protein in colorectal carcinoma and their correlation to the outcome of patients. Immunohistochemistry and quantitative RT-PCR were used to analyze the expression of pKi-67 in 43 archival specimens of patients with curatively resected primary colorectal carcinoma, who were not treated with neo-adjuvant therapy. We determined a median pKi-67 (MIB-1) labeling index of 31.3% (range 10.3-66.4%), and a mean mRNA level of 0.1769 (DeltaC(T): range 0.01-0.69); indices and levels did not correlate. High pKi-67 mRNA DeltaC(T) values were associated with a significantly favorable prognosis, while pKi-67 labeling indices were not correlated to prognostic outcome. A multivariate analysis of clinical and biological factors indicated that tumor stage (UICC) and pKi-67 mRNA expression level were independent prognostic factors. Quantitatively determined pKi-67 mRNA can be a good and new prognostic indicator for primary resected colorectal carcinoma.

Regulation of alternative splicing at the single-cell level.

PubMed

Faigenbloom, Lior; Rubinstein, Nimrod D; Kloog, Yoel; Mayrose, Itay; Pupko, Tal; Stein, Reuven

2015-12-28

Alternative splicing is a key cellular mechanism for generating distinct isoforms, whose relative abundances regulate critical cellular processes. It is therefore essential that inclusion levels of alternative exons be tightly regulated. However, how the precision of inclusion levels among individual cells is governed is poorly understood. Using single-cell gene expression, we show that the precision of inclusion levels of alternative exons is determined by the degree of evolutionary conservation at their flanking intronic regions. Moreover, the inclusion levels of alternative exons, as well as the expression levels of the transcripts harboring them, also contribute to this precision. We further show that alternative exons whose inclusion levels are considerably changed during stem cell differentiation are also subject to this regulation. Our results imply that alternative splicing is coordinately regulated to achieve accuracy in relative isoform abundances and that such accuracy may be important in determining cell fate. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Studying the MicroRNA role as a survival predictor and revealing its part in malignancy level determination in patients with supratentorial gliomas of brain

NASA Astrophysics Data System (ADS)

Stupak, E. V.; Veryaskina, Yu. A.; Titov, S. E.; Achmerova, L. G.; Stupak, V. V.; Dolzhenko, D. A.; Rabinovich, S. S.; Narodov, A. A.; Ivanov, M. K.; Zhimulev, I. F.; Kolesnikov, N. N.

2017-09-01

The numerous data show, that microRNA (miRNA) are direct participants of carcinogenesis. Also miRNA plays the role of a diagnostic and prognostic marker for different types of cancer, including gliomas. The aim of this research is to make the comparative analysis of 10 micro RNA (miR-124, -125b, -16, -181b, -191, -21, -221, -223, -31 and -451) expression profiles. The analysis was made for gliomas with different malignancy degree, then compared with the samples of the adjacent not changed tissues (n = 90). During the study the specific profiles of miRNA expression for various histotypes of tumors were revealed. It was determined, that miRNA acts as a predictor of patient survival in the cases with malignant supratentorial brain tumors. The diagnostic approaches based on miRNA expression profile were designed. It will help to determine the malignancy level and to predict the course of the disease.

Chemotherapy-Induced Monoamine Oxidase Expression in Prostate Carcinoma Functions as a Cytoprotective Resistance Enzyme and Associates with Clinical Outcomes

PubMed Central

Huang, Chung-Ying; Harris, William P.; Sim, Hong Gee; Lucas, Jared M.; Coleman, Ilsa; Higano, Celestia S.; Gulati, Roman; True, Lawrence D.; Vessella, Robert; Lange, Paul H.; Garzotto, Mark; Beer, Tomasz M.; Nelson, Peter S.

2014-01-01

To identify molecular alterations in prostate cancers associating with relapse following neoadjuvant chemotherapy and radical prostatectomy patients with high-risk localized prostate cancer were enrolled into a phase I-II clinical trial of neoadjuvant chemotherapy with docetaxel and mitoxantrone followed by prostatectomy. Pre-treatment prostate tissue was acquired by needle biopsy and post-treatment tissue was acquired by prostatectomy. Prostate cancer gene expression measurements were determined in 31 patients who completed 4 cycles of neoadjuvant chemotherapy. We identified 141 genes with significant transcript level alterations following chemotherapy that associated with subsequent biochemical relapse. This group included the transcript encoding monoamine oxidase A (MAOA). In vitro, cytotoxic chemotherapy induced the expression of MAOA and elevated MAOA levels enhanced cell survival following docetaxel exposure. MAOA activity increased the levels of reactive oxygen species and increased the expression and nuclear translocation of HIF1α. The suppression of MAOA activity using the irreversible inhibitor clorgyline augmented the apoptotic responses induced by docetaxel. In summary, we determined that the expression of MAOA is induced by exposure to cytotoxic chemotherapy, increases HIF1α, and contributes to docetaxel resistance. As MAOA inhibitors have been approved for human use, regimens combining MAOA inhibitors with docetaxel may improve clinical outcomes. PMID:25198178

Chemotherapy-induced monoamine oxidase expression in prostate carcinoma functions as a cytoprotective resistance enzyme and associates with clinical outcomes.

PubMed

Gordon, Ryan R; Wu, Mengchu; Huang, Chung-Ying; Harris, William P; Sim, Hong Gee; Lucas, Jared M; Coleman, Ilsa; Higano, Celestia S; Gulati, Roman; True, Lawrence D; Vessella, Robert; Lange, Paul H; Garzotto, Mark; Beer, Tomasz M; Nelson, Peter S

2014-01-01

To identify molecular alterations in prostate cancers associating with relapse following neoadjuvant chemotherapy and radical prostatectomy patients with high-risk localized prostate cancer were enrolled into a phase I-II clinical trial of neoadjuvant chemotherapy with docetaxel and mitoxantrone followed by prostatectomy. Pre-treatment prostate tissue was acquired by needle biopsy and post-treatment tissue was acquired by prostatectomy. Prostate cancer gene expression measurements were determined in 31 patients who completed 4 cycles of neoadjuvant chemotherapy. We identified 141 genes with significant transcript level alterations following chemotherapy that associated with subsequent biochemical relapse. This group included the transcript encoding monoamine oxidase A (MAOA). In vitro, cytotoxic chemotherapy induced the expression of MAOA and elevated MAOA levels enhanced cell survival following docetaxel exposure. MAOA activity increased the levels of reactive oxygen species and increased the expression and nuclear translocation of HIF1α. The suppression of MAOA activity using the irreversible inhibitor clorgyline augmented the apoptotic responses induced by docetaxel. In summary, we determined that the expression of MAOA is induced by exposure to cytotoxic chemotherapy, increases HIF1α, and contributes to docetaxel resistance. As MAOA inhibitors have been approved for human use, regimens combining MAOA inhibitors with docetaxel may improve clinical outcomes.

Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP).

PubMed

Habibi, Narjeskhatoon; Norouzi, Alireza; Mohd Hashim, Siti Z; Shamsir, Mohd Shahir; Samian, Razip

2015-11-01

Recombinant protein overexpression, an important biotechnological process, is ruled by complex biological rules which are mostly unknown, is in need of an intelligent algorithm so as to avoid resource-intensive lab-based trial and error experiments in order to determine the expression level of the recombinant protein. The purpose of this study is to propose a predictive model to estimate the level of recombinant protein overexpression for the first time in the literature using a machine learning approach based on the sequence, expression vector, and expression host. The expression host was confined to Escherichia coli which is the most popular bacterial host to overexpress recombinant proteins. To provide a handle to the problem, the overexpression level was categorized as low, medium and high. A set of features which were likely to affect the overexpression level was generated based on the known facts (e.g. gene length) and knowledge gathered from related literature. Then, a representative sub-set of features generated in the previous objective was determined using feature selection techniques. Finally a predictive model was developed using random forest classifier which was able to adequately classify the multi-class imbalanced small dataset constructed. The result showed that the predictive model provided a promising accuracy of 80% on average, in estimating the overexpression level of a recombinant protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression analysis of several antiviral related genes to BmNPV in different resistant strains of silkworm, Bombyx mori.

PubMed

Cheng, Yang; Wang, Xue-yang; Du, Chang; Gao, Juan; Xu, Jia-ping

2014-05-30

Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a highly pathogenic virus in the sericultural industry, often causing severe damage leading to large economic losses. The immune mechanisms of B. mori against this virus remain obscure. Previous studies had demonstrated Bmlipase-1, BmNox and Bmserine protease-2 showing antiviral activity in vitro, but data on the transcription levels of these proteins in different resistant strains were not reported. In order to determine the resistance level of the four different strains (P50, A35, A40, A53) and gain a better understanding of the mechanism of resistance to BmNPV in B. mori, the relative expression level of the genes coding the three antiviral proteins in larval haemolymph and midgut of different B. mori strains resistant to BmNPV was determined. The results showed that these genes expressed significantly higher in the resistant strains compared to the susceptible strain, and the differential expression levels were consistent with the LC50 values in different strains. The transcription level of the target genes almost all up-regulated in the larvae midgut and down-regulated in the haemolymph. The results indicate the correlation of these genes to BmNPV resistance in B. mori. This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.

[Influence of physiologic 17 beta-estradiol concentrations on gene E6 expression in HVP type 18 in vitro].

PubMed

Dziubińska-Parol, Izabella; Gasowska, Urszula; Rzymowska, Jolanta; Kwaśniewska, Anna

2003-09-01

Many recent studies indicate that long term use of contraceptives is a strong risk factor in the development of cervical cancer. Steroid hormones, in persistent papilloma virus infection act on various levels, one of them is enhancing transforming activity of the virus. The aim of the study was to estimate if physiological concentrations of 17 beta-estradiol could influence expression of viral transforming genes. HeLa cell lines were incubated with three different physiological concentrations and and on the third day of incubation the level of E6 gene expression was determined. Results show no differences in expression between the control culter, and cultures incubated with physiological concentrations. It indicates that normal levels of 17 beta-estradiol don't play role in transforming process but it also shows need to analyse higher levels of hormones by quantitative analyses in prospective studies.

[Research of expression of L-DOPA decarboxylase in laryngeal cancer].

PubMed

Lai, Shisheng; Wan, Zhili

2014-12-01

This study aimed to investigate the expression levels of L-DOPA decarboxylase (DDC) mRNA and protein in laryngeal cancer, and to determine the clinical significance of DDC in diagnosis and prognosis of laryngeal cancer. Total RNA was isolated from 106 tissue samples surgically removed from 53 laryngeal cancer patients. A quantitative real-time polymerase chain reaction (RT-PCR) methodology based on SYBR Green I fluorescent dye was developed for the quantification of mRNA levels. In addition, Western Blot analysis was performed to detect the expression level of DDC protein. DDC mRNA expression in both primary (P= 0. 000) and recurrent (P=0. 033) laryngeal cancer samples downregulated significantly compared with their nonmalignant counterparts. Moreover, expression of DDC mRNA was not associated with age and histologic grade, but the significantly decreased mRNA were correlated with early TMN stage (P=0. 021). Additionally, DDC protein was detected in both cancerous and noncancerous tissues. Expression levels of DDC may play a vital role in the progression of laryngeal cancer, which can be served as a promising biomarker for the future clinical management of laryngeal cancer patients.

Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions-benign adenomas and inflammatory bowel disease.

PubMed

Dziaman, Tomasz; Gackowski, Daniel; Guz, Jolanta; Linowiecka, Kinga; Bodnar, Magdalena; Starczak, Marta; Zarakowska, Ewelina; Modrzejewska, Martyna; Szpila, Anna; Szpotan, Justyna; Gawronski, Maciej; Labejszo, Anna; Liebert, Ariel; Banaszkiewicz, Zbigniew; Klopocka, Maria; Foksinski, Marek; Marszalek, Andrzej; Olinski, Ryszard

2018-01-01

Active demethylation of 5-methyl-2'-deoxycytidine (5-mdC) in DNA occurs by oxidation to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further oxidation to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC), and is carried out by enzymes of the ten-eleven translocation family (TETs 1, 2, 3). Decreased level of epigenetic DNA modifications in cancer tissue may be a consequence of reduced activity/expression of TET proteins. To determine the role of epigenetic DNA modifications in colon cancer development, we analyzed their levels in normal colon and various colonic pathologies. Moreover, we determined the expressions of TETs at mRNA and protein level.The study included material from patients with inflammatory bowel disease (IBD), benign polyps (AD), and colorectal cancer (CRC). The levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in examined tissues were determined by means of isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). The expressions of TET mRNA were measured with RT-qPCR, and the expressions of TET proteins were determined immunohistochemically. IBD was characterized by the highest level of 8-oxodG among all analyzed tissues, as well as by a decrease in 5-hmdC and 5-mdC levels (at a midrange between normal colon and CRC). AD had the lowest levels of 5-hmdC and 5-mdC of all examined tissues and showed an increase in 8-oxodG and 5-(hydroxymethyl)-2'-deoxyuridine (5-hmdU) levels. CRC was characterized by lower levels of 5-hmdC and 5-mdC, the lowest level of 5-fdC among all analyzed tissues, and relatively high content of 5-cadC. The expression of TET1 mRNA in CRC and AD was significantly weaker than in IBD and normal colon. Furthermore, CRC and AD showed significantly lower levels of TET2 and AID mRNA than normal colonic tissue. Our findings suggest that a complex relationship between aberrant pattern of DNA epigenetic modification and cancer development does not depend solely on the transcriptional status of TET proteins, but also on the characteristics of premalignant/malignant cells. This study showed for the first time that the examined colonic pathologies had their unique epigenetic marks, distinguishing them from each other, as well as from normal colonic tissue. A decrease in 5-fdC level may be a characteristic feature of largely undifferentiated cancer cells.

Evaluation of the miRNA-146a and miRNA-155 Expression Levels in Patients with Oral Lichen Planus.

PubMed

Ahmadi-Motamayel, Fatemeh; Bayat, Zeynab; Hajilooi, Mehrdad; Shahryar-Hesami, Soroosh; Mahdavinezhad, Ali; Samie, Lida; Solgi, Ghasem

2017-12-01

Oral Lichen Planus (OLP) is a chronic autoimmune disease that could be considered as a potential premalignant status. To evaluate the miRNA-146a and miRNA-155 expression levels in patients with oral Lichen planus lesions compared to healthy subjects with normal oral mucosa. Forty patients with oral lichen planus and 18 healthy age and gender-matched controls were recruited in this case-control study. Oral lichen planus was diagnosed clinically and pathologically. The expression levels of two miRNAs in peripheral blood samples were determined using commercial TaqMan MicroRNA Assays. Relative quantification of gene expression was calculated by the 2-ΔΔct method. The expression levels of miRNA-146a and miRNA-155 in patients with oral Lichen planus were significantly higher than those of healthy controls. Also, a direct but insignificant correlation was found between miRNA-155 and miRNA-146a expression levels among the patient group. Our findings indicate that miRNA-146a and miRNA-155 could be potential biomarkers for the immunopathogenesis of oral lichen planus.

miR-23b as a potential tumor suppressor and its regulation by DNA methylation in cervical cancer.

PubMed

Campos-Viguri, Gabriela Elizabeth; Jiménez-Wences, Hilda; Peralta-Zaragoza, Oscar; Torres-Altamirano, Gricenda; Soto-Flores, Diana Guillermina; Hernández-Sotelo, Daniel; Alarcón-Romero, Luz Del Carmen; Jiménez-López, Marco Antonio; Illades-Aguiar, Berenice; Fernández-Tilapa, Gloria

2015-01-01

The aberrant expression of miR-23b is involved in the development and progression of cancer. The aim of this study was to evaluate the potential role of methylation in the silencing of miR-23b in cervical cancer cell lines and to determine its expression in stages of malignant progression and in cervical cancer tissues HPV16-positive. The methylation of the miR-23b promoter was determined in HeLa, SiHa, CaSki and C33A cells using a Human Cancer miRNA EpiTectMethyl II Signature PCR Array®. The cells were treated with 5-Aza-2'-deoxycytidine, and the expression of miR-23b, uPa, c-Met and Zeb1 was determined by qRT-PCR. miR-92a and GAPDH were used as controls. The expression of miR-23b was determined in cervical scrapes and biopsies of women without squamous intraepithelial lesions, with precursor lesions and with cervical cancer, all were HPV16-positive. The Fisher exact and Mann-Whitney tests were used to compare the differences of the expression of miR-23b, uPa, c-Met and Zeb1 among cell groups, and the difference among patients, respectively. The association between the expression of miR-23b and cervical cancer was determined by logistic regression with a confidence level of 95 %. A value of p < 0.05 was considered statistically significant. In C33A, HeLa and CaSki cells, methylation was associated with decreased expression of miR-23b. After treatment with 5-Aza-CdR, the expression of miR-23b increased in all cell lines and the expression of c-Met decreased in HeLa cells, while uPa and Zeb1 decreased in C33A and CaSki cells. In SiHa cells the expression of uPa, c-Met and Zeb1 increased. The expression of miR-23b decreased in relation to the increase in the severity of the lesion and was significantly lower in cervical cancer. In women with premalignant lesions HPV16-positive, decreased levels of miR-23b increased the risk of cervical cancer (OR = 36, 95 % CI = 6.7-192.6, p < 0.05). The results suggest that the expression of miR-23b is regulated by the methylation of its promoter and is possible that this microRNA influence the expression of uPa, c-Met and Zeb1 in cervical cancer cells lines. In women with premalignant lesions and cervical cancer infected with HPV16, the expression level of miR-23b agree with a tumor suppressor gene.

Preliminary Study on Gene Expression of Chitinase-Like Cytokines in Human Airway Epithelial Cell Under Chitin and Chitosan Microparticles Treatment.

PubMed

Alimohammadi, Masumeh; Yeganeh, Farshid; Haji Molla Hoseini, Mostafa

2016-06-01

Small-sized chitin and chitosan microparticles (MPs) reduce allergic inflammation. We examined the capacity of these glycans to stimulate A549 human airway epithelial cells to determine the feasibility of using of these glycans as allergic therapeutic modality. A549 cells were treated with MPs and then expressions levels of chitinase domain-containing 1 (CHID1) and chitinase 3-like 1 (CHI3L1) genes were determined by quantitative real-time PCR. IL-6 production was measured by ELISA. Chitin MPs resulted in upregulation of CHI3L1 expression by 35.7-fold while mRNA expression did not change with chitosan MPs. Compared to the untreated group, production of IL-6 was significantly decreased in the chitosan MPs-treated group, but chitin MPs treatment cause elevation of IL-6 level. This study demonstrates that chitin potently induces CHI3L1 expression, but chitosan is relatively inert. This effect and inhibition of pro-inflammatory cytokine (IL-6) suggest that chitosan MPs may possess more potential for therapeutic uses in human airway allergic inflammation.

Reduced SIRT1 expression correlates with enhanced oxidative stress in compensated and decompensated heart failure

PubMed Central

Akkafa, Feridun; Halil Altiparmak, Ibrahim; Erkus, Musluhittin Emre; Aksoy, Nurten; Kaya, Caner; Ozer, Ahmet; Sezen, Hatice; Oztuzcu, Serdar; Koyuncu, Ismail; Umurhan, Berrin

2015-01-01

Sirtuin-1 (SIRT1) is a longevity factor in mammals initiating the cell survival mechanisms, and preventing ischemic injury in heart. In the etiopathogenesis of heart failure (HF), impairment in cardiomyocyte survival is a notable factor. Oxidative stress comprises a critical impact on cardiomyocyte lifespan in HF. The aim of the present study was to investigate SIRT1 expression in patients with compensated (cHF) and decompensated HF (dHF), and its correlation with oxidative stress. SIRT1 expression in peripheral leukocytes was examined using quantitative RT-PCR in 163 HF patients and 84 controls. Serum total oxidant status (TOS) and total antioxidant status (TAS) were measured via colorimetric assays, and oxidative stress index (OSI) was calculated. Lipid parameters were also determined by routine laboratory methods. SIRT1 mRNA expression was significantly downregulated in HF with more robust decrease in dHF (p=0.002, control vs cHF; p<0.001, control vs dHF). Markedly increased oxidative stress defined as elevated TOS, OSI and low TAS levels were detected in HF patients comparing with the controls (TAS; p=0.010, control vs cHF, p=0.045 control vs dHF, TOS; p=0.004 control vs cHF; p<0.001 control vs dHF, OSI; p<0.001 for both comparisons, respectively). With SIRT1 expression levels, TAS, TOS, OSI, and high density lipoprotein levels in cHF and dHF were determined correlated. SIRT1 expression were significantly reduced in both HF subtypes, particularly in dHF. SIRT1 expression was correlated with the oxidant levels and antioxidant capacity. Data suggest that SIRT1 may have a significant contribution in regulation of oxidant/antioxidant balance in HF etiology and compensation status. PMID:26233702

beta(2)microglobulin mRNA expression levels are prognostic for lymph node metastasis in colorectal cancer patients.

PubMed

Shrout, J; Yousefzadeh, M; Dodd, A; Kirven, K; Blum, C; Graham, A; Benjamin, K; Hoda, R; Krishna, M; Romano, M; Wallace, M; Garrett-Mayer, E; Mitas, M

2008-06-17

Colorectal cancer (CRC) is the fourth most common non-cutaneous malignancy in the United States and the second most frequent cause of cancer-related death. One of the most important determinants of CRC survival is lymph node metastasis. To determine whether molecular markers might be prognostic for lymph node metastases, we measured by quantitative real-time RT-PCR the expression levels of 15 cancer-associated genes in formalin-fixed paraffin-embedded primary tissues derived from stage I-IV CRC patients with (n=20) and without (n=18) nodal metastases. Using the mean of the 15 genes as an internal reference control, we observed that low expression of beta(2)microglobulin (B2M) was a strong prognostic indicator of lymph node metastases (area under the curve (AUC)=0.85; 95% confidence interval (CI)=0.69-0.94). We also observed that the expression ratio of B2M/Spint2 had the highest prognostic accuracy (AUC=0.87; 95% CI=0.71-0.96) of all potential two-gene combinations. Expression values of Spint2 correlated with the mean of the entire gene set at an R(2) value of 0.97, providing evidence that Spint2 serves not as an independent prognostic gene, but rather as a reliable reference control gene. These studies are the first to demonstrate a prognostic role of B2M at the mRNA level and suggest that low B2M expression levels might be useful for identifying patients with lymph node metastasis and/or poor survival.

Differential retention of alpha-vitamin E is correlated with its transporter gene expression and growth inhibition efficacy in prostate cancer cells.

PubMed

Ni, Jing; Pang, See-Too; Yeh, Shuyuan

2007-04-01

Epidemiological studies showed Vit E has protective effects against prostate cancer (PCa). Interestingly, different prostate cancer cells have different sensitivity to alpha-Vit E or VES treatment. The goal of this study is to determine whether cellular Vit E bioavailability and its transport proteins are important contributing factors. alpha-Vit E and its ester form, VES, were used to treat prostate cancer LNCaP, PC3, and DU145 cells, and their growth rates were determined by MTT assay. Cellular levels of Vit E were quantified using HPLC as the index of bioavailability. The expression levels of Vit E transport proteins were determined by real-time PCR. Among these PCa cells, only LNCaP cells were sensitive to 20 microM alpha-Vit E treatment, while both LNCaP and PC3 cells were sensitive to 20 microM VES treatment. Coordinately, cellular levels of alpha-Vit E and VES positively correlated to their inhibitory effects. Further study found expression levels of Vit E transport proteins, including tocopherol associated protein (TAP), scavenger receptor class B type I (SR-BI), alpha-tocopherol transfer protein (TTP), and ATP binding cassette transporter A1 (ABCA1), were different in various PCa cells, which may contribute to cellular Vit E bioavailability. This notion is further supported by the findings that overexpression or knockdown of TTP could coordinately alter cellular alpha-Vit E levels in PCa cells. Antiproliferative efficacy of alpha-Vit E is correlated with its cellular bioavailability in PCa cells. Modulating the expression of the efflux or influx transporters could sensitize the growth inhibition efficacy of Vit E in prostate cancer cells.

Tissue factor expression as a possible determinant of thromboembolism in ovarian cancer

PubMed Central

Uno, K; Homma, S; Satoh, T; Nakanishi, K; Abe, D; Matsumoto, K; Oki, A; Tsunoda, H; Yamaguchi, I; Nagasawa, T; Yoshikawa, H; Aonuma, K

2007-01-01

Ovarian cancer, and clear cell carcinoma in particular, reportedly increases the risk of venous thromboembolism (VTE). However, the mechanisms remain unclear. Tissue factor (TF) supposedly represents a major factor in the procoagulant activities of cancer cells. The present study examined the involvement of TF expression in VTE for patients with ovarian cancer. Subjects comprised 32 consecutive patients (mean age 49.8 years) with histologically confirmed ovarian cancer. Presence of VTE was examined using a combination of clinical features, D-dimer levels and venous ultrasonography. Immunohistochemical analysis was used to evaluate TF expression into 4 degrees. Venous thromboembolism was identified in 10 of the 32 patients (31%), including five of the 11 patients with clear cell carcinoma. Tissue factor expression was detected in cancer tissues from 24 patients and displayed significant correlations with VTE development (P=0.0003), D-dimer concentration (P=0.003) and clear cell carcinoma (P<0.05). Multivariate analysis identified TF expression as an independent predictive factor of VTE development (P<0.05). Tissue factor (TF) expression is a possible determinant of VTE development in ovarian cancer. In particular, clear cell carcinoma may produce excessive levels of TF and is more likely to develop VTE. PMID:17211468

A novel eQTL-based analysis reveals the biology of breast cancer risk loci

PubMed Central

Li, Qiyuan; Seo, Ji-Heui; Stranger, Barbara; McKenna, Aaron; Pe'er, Itsik; LaFramboise, Thomas; Brown, Myles; Tyekucheva, Svitlana; Freedman, Matthew L.

2014-01-01

Summary Germline determinants of gene expression in tumors are less studied due to the complexity of transcript regulation caused by somatically acquired alterations. We performed expression quantitative trait locus (eQTL) based analyses using the multi-level information provided in The Cancer Genome Atlas (TCGA). Of the factors we measured, cis-acting eQTL saccounted for 1.2% of the total variation of tumor gene expression, while somatic copy number alteration and CpG methylation accounted for 7.3% and 3.3%, respectively. eQTL analyses of 15 previously reported breast cancer risk loci resulted in discovery of three variants that are significantly associated with transcript levels (FDR<0.1). In a novel trans- based analysis, an additional three risk loci were identified to act through ESR1, MYC, and KLF4. These findings provide a more comprehensive picture of gene expression determinants in breast cancer as well as insights into the underlying biology of breast cancer risk loci. PMID:23374354

Adrenal-kidney-gonad complex measurements may not predict gonad-specific changes in gene expression patterns during temperature-dependent sex determination in the red-eared slider turtle (Trachemys scripta elegans).

PubMed

Ramsey, Mary; Crews, David

2007-08-01

Many turtles, including the red-eared slider turtle (Trachemys scripta elegans) have temperature-dependent sex determination in which gonadal sex is determined by temperature during the middle third of incubation. The gonad develops as part of a heterogenous tissue complex that comprises the developing adrenal, kidney, and gonad (AKG complex). Owing to the difficulty in excising the gonad from the adjacent tissues, the AKG complex is often used as tissue source in assays examining gene expression in the developing gonad. However, the gonad is a relatively small component of the AKG, and gene expression in the adrenal-kidney (AK) compartment may interfere with the detection of gonad-specific changes in gene expression, particularly during early key phases of gonadal development and sex determination. In this study, we examine transcript levels as measured by quantitative real-time polymerase chain reaction for five genes important in slider turtle sex determination and differentiation (AR, ERalpha, ERbeta, aromatase, and Sf1) in AKG, AK, and isolated gonad tissues. In all cases, gonad-specific gene expression patterns were attenuated in AKG versus gonad tissue. All five genes were expressed in the AK in addition to the gonad at all stages/temperatures. Inclusion of the AK compartment masked important changes in gonadal gene expression. In addition, AK and gonad expression patterns are not additive, and gonadal gene expression cannot be predicted from intact AKG measurements. (c) 2007 Wiley-Liss, Inc.

Expression of vitamin D receptor (VDR), cyclooxygenase-2 (COX-2) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in benign and malignant ovarian tissue and 25-hydroxycholecalciferol (25(OH2)D3) and prostaglandin E2 (PGE2) serum level in ovarian cancer patients.

PubMed

Thill, Marc; Fischer, Dorothea; Kelling, Katharina; Hoellen, Friederike; Dittmer, Christine; Hornemann, Amadeus; Salehin, Darius; Diedrich, Klaus; Friedrich, Michael; Becker, Steffi

2010-07-01

Ovarian carcinomas are associated with increased inflammation which is based upon an up-regulation of inducible cyclooxygenase-2 (COX-2). Moreover, based on our previous published data, the extra-renal vitamin D metabolism seems to be dysregulated in comparison to healthy tissue. In order to gain further insight into the prostaglandin (PG)- and vitamin D-metabolism in ovarian carcinomas, the study aimed to evaluate the expression of the PG metabolising enzymes COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) compared to the vitamin D receptor (VDR) in benign and malignant ovarian tissues. Additionally, we determined the 25-hydroxycholecalciferol (25(OH2)D3) serum levels. Expression of VDR, COX-2 and 15-PGDH was determined by Western blot analysis. Serum levels of 25(OH2)D3 and PGE2 were measured by chemiluminescence-based and colorimetric immunoassay. We detected significantly higher expressions of the PG metabolising enzymes 15-PGDH and COX-2 in malignant tissue and PGE2 serum levels were 2-fold higher in tumour patients. Furthermore, we found an inverse correlation to the VDR-expression which was 62.1% lower in malignant tissues compared to that in benign tissues. Surprisingly, we could not detect any differences between the 25(OH2)D3 serum levels in either group (n=20). These data suggest a correlation between PG- and vitamin D-metabolism in ovarian carcinomas. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

In vitro responsiveness of human muscle cell peroxisome proliferator-activated receptor δ reflects donors' insulin sensitivity in vivo.

PubMed

Ordelheide, Anna-Maria; Heni, Martin; Thamer, Claus; Machicao, Fausto; Fritsche, Andreas; Stefan, Norbert; Häring, Hans-Ulrich; Staiger, Harald

2011-12-01

Peroxisome proliferator-activated receptor δ (PPARδ) activation enhances muscular fatty acid oxidation and oxidative phosphorylation, and muscle's oxidative capacity positively associates with whole-body insulin sensitivity. Therefore, we asked here whether human muscle cell PPARD expression is a determinant of donors' insulin sensitivity. Skeletal muscle cells derived from 38 nondiabetic donors were differentiated in vitro to myotubes, and gene (mRNA) expression was quantified by real-time RT-PCR. Donors' insulin sensitivity was calculated from plasma insulin and glucose levels during oral glucose tolerance test (OGTT) and hyperinsulinemic-euglycemic clamp. Basal myotube PPARD expression was closely related to the expression of its target genes PDK4 and ANGPTL4 (P = 0·0312 and P = 0·0003, respectively). Basal PPARD, PDK4 and ANGPTL4 expression levels were not associated with donors' insulin sensitivity (P > 0·2, all). Treatment of myotubes with a selective high-affinity PPARδ agonist (GW501516) did not change mean PPARD, but enhanced mean PDK4 and ANGPTL4 expression 13- and 16-fold, respectively (P < 0·0001, both). The individual PDK4 and ANGPTL4 expression levels reached upon GW501516 treatment were associated with donors' insulin sensitivity neither (P > 0·2, both). However, GW501516-mediated fold increments in PDK4 and ANGPTL4 expression, reflecting PPARδ responsiveness, were positively associated with donors' insulin sensitivity derived from OGTT (P = 0·0182 and P = 0·0231, respectively) and hyperinsulinemic-euglycemic clamp (P = 0·0046 and P = 0·0258, respectively). Using a highly selective pharmacological tool, we show here that the individual responsiveness of human muscle cell PPARδ, rather than the absolute PPARD expression level, represents a major determinant of insulin sensitivity. © 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.

The expression of 11β-hydroxysteroid dehydrogenase type 1 and 2 in nasal polyp-derived epithelial cells and its possible contribution to glucocorticoid activation in nasal polyp.

PubMed

Kook, Jin Ho; Kim, Hyun Jin; Kim, Kyung Won; Park, Se Jin; Kim, Tae Hoon; Lim, Sae Hee; Kang, Sung Hoon; Lee, Sang Hag

2015-01-01

The actions of glucocorticoids in target tissues depend on the local metabolism of glucocorticoids catalyzed by 11β hydroxysteroid dehydrogenase (HSD) 1 and 2. Glucocorticoids are the most effective anti-inflammatory drugs in the treatment of nasal polyps. However, the mechanisms that underlie the anti-inflammatory effects are unclear. The present study analyzed the expression of 11β-HSD1, 11β-HSD2, and steroidogenic enzymes (cytochrome P450, family 11, subfamily B, polypeptide 1 [CYP11B1]; cytochrome P450, family 11, subfamily A, polypeptide 1 [CYP11A1]) in nasal polyp tissues, and endogenous cortisol levels in nasal polyp-derived epithelial cells. The expression levels and distribution pattern of 11β-HSD1, 11β-HSD2, CYP11B1, and CYP11A1 were determined in nasal polyp tissues or nasal polyp-derived epithelial cells by using real-time polymerase chain reaction, Western blot, and immunohistochemistry testing. The expression levels of cortisol by using enzyme-linked immunosorbent assay were determined in cultured polyp-derived epithelial cells treated with adrenocorticotrophic hormone (ACTH), 11β-HSD1 inhibitor, or small interfering ribonucleic acid technique. The effect of glucocorticoids on the expression levels of these enzymes was investigated in cultured cells. Expressed in nasal polyp tissues and nasal polyp-derived epithelial cells were 11β-HSD1, 11β-HSD2, CYP11B1, and CYP11A1. Cortisol production in cultured epithelial cells was decreased in cells treated with 11β-HSD1 small interfering ribonucleic acid or inhibitor, compared with nontreated cells. Cultured cells treated with adrenocorticotropic hormone induced increased cortisol production. 11β-HSD1 expression levels were upregulated in cells treated with glucocorticoid. Analysis of these results indicated that 11β-HSD1 expressed in polyp-derived epithelial cells may be involved in the anti-inflammatory function of glucocorticoid in the treatment of nasal polyps, which contributes to increased levels of endogenous cortisol.

Enhancement of ICAM-1 via the JAK2/STAT3 signaling pathway in a rat model of severe acute pancreatitis-associated lung injury

PubMed Central

HAN, XIAO; WANG, YUXI; CHEN, HAILONG; ZHANG, JINGWEN; XU, CAIMING; LI, JIAN; LI, MINGYUE

2016-01-01

Acute lung injury (ALI), which is associated with severe acute pancreatitis (SAP), results from damage to the pulmonary microvascular endothelial cells (PMVECs), which in turn leads to high levels of inflammatory cytokines that destroy PMVECs. However, the molecular mechanisms underlying SAP-associated ALI (SAP-ALI) are currently not well understood. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the persistent migration and accumulation of neutrophils and macrophages, which in turn has been associated with the increased permeability of microvascular endothelial cells. Signal transduction via the Janus kinase-2 (JAK2)/signal transducer and activator of transcription-3 (STAT3) transcription factors has been shown to be involved in inflammation. The present study aimed to investigate the expression levels of ICAM-1 and JAK2/STAT3 signaling components in a rat model of SAP-ALI. SAP was induced in the rat model, and dexamethasone (DEX) was administered to the treatment group. Subsequently, ICAM-1, interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, JAK2, STAT3 and nuclear factor (NF)-κB mRNA expression levels were determined using reverse transcription-polymerase chain reaction; ICAM-1 protein expression levels were determined using western blotting; and IL-6, IL-8 and TNF-α levels were measured via an enzyme-linked immunosorbent assay. In addition, an immunohistochemical analysis of ICAM-1, NF-κB, JAK2 and STAT3 was conducted, and the protein expression and cell morphology of the lungs in all rats was analyzed. ICAM-1 mRNA and protein expression levels were significantly increased following induction of SAP, and were significantly decreased in the DEX-treated group. Furthermore, treatment with DEX significantly reduced serum expression levels of IL-6, IL-8 and TNF-α and decreased expression levels of NF-κB, JAK2 and STAT3 in the lung tissue, as compared with the untreated SAP group. The present study demonstrated that DEX treatment was able to suppress ICAM-1 mRNA and protein expression in a rat model of SAP-ALI via the inhibition of IL-6 and TNF-α-induced JAK2/STAT3 activation; thus suggesting that DEX treatment may be considered a potential strategy in the treatment of patients with SAP-ALI. PMID:26997994

Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release.

PubMed

Matsunaga, James; Medeiros, Marco A; Sanchez, Yolanda; Werneid, Kristian F; Ko, Albert I

2007-10-01

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

PubMed

Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

2015-01-01

The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

Cellular Fibronectin Expression in Human Trabecular Meshwork and Induction by Transforming Growth Factor-β2

PubMed Central

Medina-Ortiz, Wanda E.; Belmares, Ricardo; Neubauer, Sandra; Wordinger, Robert J.; Clark, Abbot F.

2013-01-01

Purpose. Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells. Methods. Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. Results. Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. Conclusions. This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma. PMID:24030464

Ursolic acid improves podocyte injury caused by high glucose.

PubMed

Xu, Li; Fan, Qiuling; Wang, Xu; Li, Lin; Lu, Xinxing; Yue, Yuan; Cao, Xu; Liu, Jia; Zhao, Xue; Wang, Lining

2017-08-01

Autophagy plays an important role in the maintenance of podocyte homeostasis. Reduced autophagy may result in limited renal cell function during exposure to high glucose conditions. In this study we investigated the effects of ursolic acid (UA) on autophagy and podocyte injury, which were induced by high glucose. Conditionally immortalized murine podocytes were cultured in media supplemented with high glucose and the effects of the PI3K inhibitor LY294002 and UA on protein expression were determined. miR-21 expression was detected by real-time RT-PCR. Activation of the PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related proteins and expression of podocyte marker proteins were determined by western blot. Immunofluorescence was used to monitor the accumulation of LC3 puncta. Autophagosomes were also observed by transmission electron microscopy. During exposure to high glucose conditions, the normal level of autophagy was reduced in podocytes, and this defective autophagy induced podocyte injury. Increased miR-21 expression, decreased PTEN expression and abnormal activation of the PI3K/Akt/mTOR pathway were observed in cells that were cultured in high glucose conditions. UA and LY294002 reduced podocyte injury through the restoration of defective autophagy. Our data suggest that UA inhibits miR-21 expression and increases PTEN expression, which in turn inhibits Akt and mTOR and restores normal levels of autophagy. Our data suggest that podocyte injury is associated with reduced levels of autophagy during exposure to high glucose conditions, UA attenuated podocyte injury via an increase in autophagy through miR-21 inhibition and PTEN expression, which inhibit the abnormal activation of the PI3K/Akt/mTOR pathway. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

miR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice

PubMed Central

Goedeke, Leigh; Rotllan, Noemi; Ramírez, Cristina M.; Aranda, Juan F.; Canfrán-Duque, Alberto; Araldi, Elisa; Fernández-Hernando, Ana; Langhi, Cedric; de Cabo, Rafael; Baldán, Ángel; Suárez, Yajaira; Fernández-Hernando, Carlos

2015-01-01

Rationale Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. Methods and Results Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50–fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10–20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. Conclusions The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels. PMID:26520906

Effects of dexmedetomidine postconditioning on myocardial ischemia and the role of the PI3K/Akt-dependent signaling pathway in reperfusion injury.

PubMed

Cheng, Xiang Yang; Gu, Xiao Yu; Gao, Qin; Zong, Qiao Feng; Li, Xiao Hong; Zhang, Ye

2016-07-01

The present study aimed to determine whether post-ischemic treatment with dexmedetomidine (DEX) protected the heart against acute myocardial ischemia/reperfusion (I/R)‑induced injury in rats. The phosphatidylinositol‑3 kinase/protein kinase B(PI3K/Akt)‑dependent signaling pathway was also investigated. Male Sprague Dawley rats (n=64) were subjected to ligation of the left anterior descending artery (LAD), which produced ischemia for 25 min, followed by reperfusion. Following LAD ligation, rats were treated with DEX (5, 10 and 20 µg/kg) or underwent post‑ischemic conditioning, which included three cycles of ischemic insult. In order to determine the role of the PI3K/Akt signaling pathway, wortmannin (Wort), a PI3K inhibitor, was used to treat a group of rats that had also been treated with DEX (20 µg/kg). Post‑reperfusion, lactate dehydrogenase (LDH), cardiac troponin I (cTnI), creatine kinase isoenzymes (CK‑MB), superoxide dismutase (SOD) and malondialdehyde (MDA) serum levels were measured using an ultraviolet spectrophotometer. The protein expression levels of phosphorylated (p)‑Akt, Ser9‑p‑glycogen synthase kinase‑3β (p‑GSK‑3β) and cleaved caspase‑3 were detected in heart tissue by western blotting. The mRNA expression levels of B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein (Bax) were detected using reverse transcription‑polymerase chain reaction. At the end of the experiment, the hearts were removed and perfused in an isolated perfusion heart apparatus with Evans blue (1%) in order to determine the non‑ischemic areas. The risk and infarct areas of the heart were not dyed. As expected, I/R induced myocardial infarction, as determined by the increased serum levels of cTnI, CK‑MB and MDA, and the decreased levels of SOD. Post‑ischemic treatment with DEX increased the expression levels of p‑Akt and p‑GSK‑3β, whereas caspase‑3 expression was reduced following DEX treatment compared with in the I/R group. Compared with the I/R group, the ratio of Bcl‑2/Bax at the mRNA level was elevated in the DEX and ischemic post‑conditioning groups, whereas the expression levels of Bax were decreased. Conversely, the effects of DEX were attenuated by Wort. These results indicated that, similar to post‑ischemic conditioning, post‑ischemic treatment with DEX protects the heart against I/R via the PI3K/Akt‑dependent signaling pathway, possibly by activating GSK‑3β.

Effects of dexmedetomidine postconditioning on myocardial ischemia and the role of the PI3K/Akt-dependent signaling pathway in reperfusion injury

PubMed Central

CHENG, XIANG YANG; GU, XIAO YU; GAO, QIN; ZONG, QIAO FENG; LI, XIAO HONG; ZHANG, YE

2016-01-01

The present study aimed to determine whether post-ischemic treatment with dexmedetomidine (DEX) protected the heart against acute myocardial ischemia/reperfusion (I/R)-induced injury in rats. The phosphatidylinositol-3 kinase/protein kinase B(PI3K/Akt)-dependent signaling pathway was also investigated. Male Sprague Dawley rats (n=64) were subjected to ligation of the left anterior descending artery (LAD), which produced ischemia for 25 min, followed by reperfusion. Following LAD ligation, rats were treated with DEX (5, 10 and 20 µg/kg) or underwent post-ischemic conditioning, which included three cycles of ischemic insult. In order to determine the role of the PI3K/Akt signaling pathway, wortmannin (Wort), a PI3K inhibitor, was used to treat a group of rats that had also been treated with DEX (20 µg/kg). Post-reperfusion, lactate dehydrogenase (LDH), cardiac troponin I (cTnI), creatine kinase isoenzymes (CK-MB), superoxide dismutase (SOD) and malondialdehyde (MDA) serum levels were measured using an ultraviolet spectrophotometer. The protein expression levels of phosphorylated (p)-Akt, Ser9-p-glycogen synthase kinase-3β (p-GSK-3β) and cleaved caspase-3 were detected in heart tissue by western blotting. The mRNA expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were detected using reverse transcription-polymerase chain reaction. At the end of the experiment, the hearts were removed and perfused in an isolated perfusion heart apparatus with Evans blue (1%) in order to determine the non-ischemic areas. The risk and infarct areas of the heart were not dyed. As expected, I/R induced myocardial infarction, as determined by the increased serum levels of cTnI, CK-MB and MDA, and the decreased levels of SOD. Post-ischemic treatment with DEX increased the expression levels of p-Akt and p-GSK-3β, whereas caspase-3 expression was reduced following DEX treatment compared with in the I/R group. Compared with the I/R group, the ratio of Bcl-2/Bax at the mRNA level was elevated in the DEX and ischemic post-conditioning groups, whereas the expression levels of Bax were decreased. Conversely, the effects of DEX were attenuated by Wort. These results indicated that, similar to post-ischemic conditioning, post-ischemic treatment with DEX protects the heart against I/R via the PI3K/Akt-dependent signaling pathway, possibly by activating GSK-3β. PMID:27221008

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

PubMed Central

Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

2015-01-01

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

[Expression of FAP and alpha-SMA during the incised wound healing in mice skin].

PubMed

Gao, Yang; Peng, Xue; Jin, Zhan-Fen; Fu, Zhi-Jun

2009-12-01

OBJECTIVE To investigate the time-dependent expression of fibroblast activation protein (FAP) and alpha-smooth muscle actin(alpha-SMA) during the incised wound healing of the skin in mice. The expression of FAP and alpha-SMA in incised wound of mice skin was detected by immunohistochemistry and Western blot. By immunohistochemistry, the expression of FAP and alpha-SMA in the normal skin and the skin 1 h after injury maintained at a very low level, but the positive cells expressing FAP and alpha-SMA started to elevate 6 h after injury and reached its peak on 5 d for FAP and on 3 d for alpha-SMA, then gradually decreased to the normal level on 14 d. The expression of FAP and alpha-SMA was observed throughout the wound healing stages 1 d after injuries by Western blot as well with a peak expression occurring on 5 d for FAP and on 3 d for alpha-SMA after injury. FAP may be a potentially useful marker for wound age determination and alpha-SMA may be used as an effective indicator for the mid- and late stage incised wound of mice skin. The combination use of FAP and alpha-SMA may be potentially effective indicators for wound age determination.

Neurokinin-2 receptor levels correlate with intensity, frequency, and duration of pain in chronic pancreatitis.

PubMed

Michalski, Christoph W; Shi, Xin; Reiser, Carolin; Fachinger, Patrick; Zimmermann, Arthur; Büchler, Markus W; Di Sebastiano, Pierluigi; Friess, Helmut

2007-11-01

Generation and maintenance of pain in chronic pancreatitis (CP) have been shown to be partially attributable to neuroimmune interactions, which involve neuropeptides such as substance P (SP). So far, expression of SP receptors NK-2R, NK-3R, the SP-encoding gene preprotachykinin A (PPT-A), and the SP degradation enzyme neutral endopeptidase (NEP) and their relation to pain in CP have not been determined. Tissue samples from patients with CP (n = 25) and from healthy donors (n = 20) were analyzed for PPT-A, NK-2R, NK-3R, and NEP expression using quantitative RT-PCR. NEP protein levels were examined by immunoblot analysis and its localization was determined using immunohistochemistry. A scoring system was used to grade the extent of fibrosis on hematoxylin and eosin- and Masson-Trichrome-stained sections. Messenger RNA levels and the extent of pain were analyzed for correlations. In CP tissues, NK-2R and PPT-A expression was increased, whereas NK-3R and NEP mRNA levels were comparable with normal pancreas. Overexpression of NK-2R was related to the intensity, frequency, and duration of pain in CP patients. NK-1R and NEP expression was significantly related to the extent of fibrosis. Expression of NK-2R and PPT-A is increased in CP and is associated with pain. Failure to up-regulate NEP may contribute to the disruption of the neuropeptides loop balance in CP and thus may exacerbate the severe pain syndrome.

AhR and SHP regulate phosphatidylcholine and S-adenosylmethionine levels in the one-carbon cycle.

PubMed

Kim, Young-Chae; Seok, Sunmi; Byun, Sangwon; Kong, Bo; Zhang, Yang; Guo, Grace; Xie, Wen; Ma, Jian; Kemper, Byron; Kemper, Jongsook Kim

2018-02-07

Phosphatidylcholines (PC) and S-adenosylmethionine (SAM) are critical determinants of hepatic lipid levels, but how their levels are regulated is unclear. Here, we show that Pemt and Gnmt, key one-carbon cycle genes regulating PC/SAM levels, are downregulated after feeding, leading to decreased PC and increased SAM levels, but these effects are blunted in small heterodimer partner (SHP)-null or FGF15-null mice. Further, aryl hydrocarbon receptor (AhR) is translocated into the nucleus by insulin/PKB signaling in the early fed state and induces Pemt and Gnmt expression. This induction is blocked by FGF15 signaling-activated SHP in the late fed state. Adenoviral-mediated expression of AhR in obese mice increases PC levels and exacerbates steatosis, effects that are blunted by SHP co-expression or Pemt downregulation. PEMT, AHR, and PC levels are elevated in simple steatosis patients, but PC levels are robustly reduced in steatohepatitis-fibrosis patients. This study identifies AhR and SHP as new physiological regulators of PC/SAM levels.

[The influence of HOXB2 anti-sense oligodeoxynucleotides on the proliferation and expression of human umbilical vein endothelial cells].

PubMed

Zhang, X; Liu, X; Liu, L

2001-12-01

To explore the effects of HOXB2 anti-sense oligodeoxynucleotides (asodn) on the proliferation and the expression of human umbilical vein endothelial cells (HUVECs). Various concentrations of HOXB2 ASODN modified by thiophosphate were transfected into HUVECs by liposome mediation. MTT and RT-PCR methods were employed to determine the influence of different concentrations of ASODN on endothelial proliferation and the expression level of HOXB2 mRNA. After the transfection of HOXB2 ASODN, the endothelial proliferation was inhibited in dose-dependent manner. Simultaneously, the expression level of HOXB2 mRNA decreased significantly. HOXB2 might play important roles in the proliferation of endothelial cells.

The effect of FcγRIIA and FcγRIIB on coronary artery lesion formation and intravenous immunoglobulin treatment responses in children with Kawasaki disease

PubMed Central

Chang, Ling-Sai; Lo, Mao-Hung; Li, Sung-Chou; Yang, Ming-Yu; Hsieh, Kai-Sheng; Kuo, Ho-Chang

2017-01-01

Previous research has found patients with the FcγRIIIB NA1 variant having increased risk of intravenous immunoglobulin (IVIG) resistance in Kawasaki disease (KD). Our previous studies revealed that elevated FcγRIIA expression correlated with the susceptibility of KD patients. We conducted this research to determine whether and how Fcγ receptors affect the susceptibility, IVIG treatment response, and coronary artery lesions (CAL) of KD patients. The activating FcγRIIA and inhibitory FcγRIIB methylation levels of seven patients with KD and four control subjects were examined using HumanMethylation27 BeadChip. We enrolled a total of 44 KD patients and 10 control subjects with fevers. We performed real-time RT-PCR to determine the FcγRIIA and FcγRIIB expression levels, as well as a luciferase assay of FcγRIIA. We found a considerable increase in methylation of both FcγRIIA and FcγRIIB in KD patients undergoing IVIG treatment. Promoter methylation of FcγRIIA inhibited reporter activity in K562 cells using luciferase assay. The FcγRIIB mRNA expression levels were not found to increase susceptibility, CAL formation, or IVIG resistance. FcγRIIA mRNA expression levels were significantly higher in IVIG-resistant patients than in those that responded to IVIG during the pre-treatment period. Furthermore, the FcγRIIA/IIB mRNA expression ratio was considerably higher in KD patients with CAL than in those without CAL. FcγRIIA and FcγRIIB both demonstrated increased methylation levels in KD patients that underwent IVIG treatment. FcγRIIA expression influenced the IVIG treatment response of KD patients. The FcγRIIA/IIB mRNA expression ratio was greater in KD patients with CAL formation. PMID:27893416

Differential expression of 11β-hydroxysteroid dehydrogenase type 1 and 2 in mild and moderate/severe persistent allergic nasal mucosa and regulation of their expression by Th2 cytokines: asthma and rhinitis.

PubMed

Jun, Y J; Park, S J; Hwang, J W; Kim, T H; Jung, K J; Jung, J Y; Hwang, G H; Lee, S H; Lee, S H

2014-02-01

Glucocorticoids are used to treat allergic rhinitis, but the mechanisms by which they induce disease remission are unclear. 11β-hydroxysteroid dehydrogenase (11β-HSD) is a tissue-specific regulator of glucocorticoid responses, inducing the interconversion of inactive and active glucocorticoids. We analysed the expression and distribution patterns of 11β-HSD1, 11β-HSD2, and steroidogenic enzymes in normal and allergic nasal mucosa, and cytokine-driven regulation of their expression. The production levels of cortisol in normal, allergic nasal mucosa and in cultured epithelial cells stimulated with cytokines were also determined. The expression levels of 11β-HSD1, 11β-HSD2, steroidogenic enzymes (CYP11B1, CYP11A1), and cortisol in normal, mild, and moderate/severe persistent allergic nasal mucosa were assessed by real-time PCR, Western blot, immunohistochemistry, and ELISA. The expression levels of 11β-HSD1, 11β-HSD2, CYP11B1, CYP11A1, and cortisol were also determined in cultured nasal epithelial cell treated with IL-4, IL-5, IL-13, IL-17A, and IFN-γ. Conversion ratio of cortisone to cortisol was evaluated using siRNA technique, 11β-HSD1 inhibitor, and the measurement of 11β-HSD1 activity. The expression levels of 11β-HSD1, CYP11B1, and cortisol were up-regulated in mild and moderate/severe persistent allergic nasal mucosa. By contrast, 11β-HSD2 expression was decreased in allergic nasal mucosa. In cultured epithelial cells treated with IL-4, IL-5, IL-13, and IL-17A, 11β-HSD1 expression and activity increased in parallel with the expression levels of CYP11B1 and cortisol, but the production of 11β-HSD2 decreased. CYP11A1 expression level was not changed in allergic nasal mucosa or in response to stimulation with cytokines. SiRNA technique or the measurement of 11β-HSD1 activity showed that nasal epithelium activates cortisone to cortisol in a 11β-HSD-dependent manner. These results indicate that the localized anti-inflammatory effects of glucocorticoids are regulated by inflammatory cytokines, which can modulate the expression of 11β-HSD1, 11β-HSD2, and CYP11B1, and by the intracellular concentrations of bioactive glucocorticoids. © 2013 John Wiley & Sons Ltd.

Decreased miR-17-92 cluster expression level in serum and granulocytes preceding onset of antithyroid drug-induced agranulocytosis.

PubMed

Yang, Jing; Lv, Yuncheng; Zhang, Yi; Li, Jiaoyang; Chen, Yajun; Liu, Chang; Zhong, Jing; Xiao, Xinhua; Liu, Jianghua; Wen, Gebo

2018-01-01

We aimed to determine changes in miR-17-92 cluster expression in serum and granulocytes from patients with antithyroid drug (ATD)-induced agranulocytosis. In this study, real-time polymerase chain reaction (PCR) was used to detect serum miR-17-92 expression levels in 20 ATD-induced agranulocytosis and 16 control patients. Importantly, dynamic changes in neutrophil counts from granulocytopenia to agranulocytosis were observed in 6 of the 20 patients. miR-17-92 expression levels in granulocytes of those six patients under the granulocytopenia condition were measured and compared with corresponding granulocyte samples after recovery. Additionally, the expression levels of these miRNAs in patients with type I or type II bone marrow characteristics were analyzed, and the correlation between miR-17-92 and serum free thyroxine level was analyzed. We found that levels of miR-17-92 expression decreased in both serum and pre-agranulocytosis granulocytes from patients with ATD-induced agranulocytosis compared with those in serum and granulocytes from both recovered patients and control patients. However, no difference among patients with either type of bone marrow characteristics was observed, and no correlation between serum miR-17-92 and free thyroxine levels was found. In ATD-induced agranulocytosis, expression of the miR-17-92 cluster is reduced in both serum and granulocytes, though this alteration does not correlate with bone marrow characteristics or thyroid function.

Neural restrictive silencer factor and choline acetyltransferase expression in cerebral tissue of Alzheimer’s Disease patients: A pilot study

PubMed Central

González-Castañeda, Rocío E.; Sánchez-González, Víctor J.; Flores-Soto, Mario; Vázquez-Camacho, Gonzalo; Macías-Islas, Miguel A.; Ortiz, Genaro G.

2013-01-01

Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer’s Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p < 0.05, U test). ChAT protein levels were reduced by 17% (p = 0.02, U test). NRSF gene expression levels were 86% higher in the AD group (p = 0.001, U test) as compared to the control group. In the AD subjects, the NRSF protein levels were 57% higher (p > 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels. PMID:23569405

Research in Undergraduate Instruction: A Biotech Lab Project for Recombinant DNA Protein Expression in Bacteria

NASA Astrophysics Data System (ADS)

Brockman, Mark; Ordman, Alfred B.; Campbell, A. Malcolm

1996-06-01

In the sophomore-level Molecular Biology and Biotechnology course at Beloit College, students learn basic methods in molecular biology in the context of pursuing a semester-long original research project. We are exploring how DNA sequence affects expression levels of proteins. A DNA fragment encoding all or part of the guanylate monokinase (gmk) sequence is cloned into pSP73 and expressed in E. coli. A monoclonal antibody is made to gmk. The expression level of gmk is determined by SDS gel elctrophoresis, a Western blot, and an ELISA assay. Over four years, an increase in enrollment in the course from 9 to 34 students, the 85% of majors pursuing advanced degrees, and course evaluations all support the conclusion that involving students in research during undergraduate courses encourages them to pursue careers in science.

Environmental effects on resistance gene expression in milk stage popcorn kernels and associations with mycotoxin production.

PubMed

Dowd, Patrick F; Johnson, Eric T

2015-05-01

Like other forms of maize, popcorn is subject to increased levels of contamination by a variety of different mycotoxins under stress conditions, although levels generally are less than dent maize under comparable stress. Gene array analysis was used to determine expression differences of disease resistance-associated genes in milk stage kernels from commercial popcorn fields over 3 years. Relatively lower expression of resistance gene types was noted in years with higher temperatures and lower rainfall, which was consistent with prior results for many previously identified resistance response-associated genes. The lower rates of expression occurred for genes such as chitinases, protease inhibitors, and peroxidases; enzymes involved in the synthesis of cell wall barriers and secondary metabolites; and regulatory proteins. However, expression of several specific resistance genes previously associated with mycotoxins, such as aflatoxin in dent maize, was not affected. Insect damage altered the spectrum of resistance gene expression differences compared to undamaged ears. Correlation analyses showed expression differences of some previously reported resistance genes that were highly associated with mycotoxin levels and included glucanases, protease inhibitors, peroxidases, and thionins.

Expression profiling and functional analysis of Toll-like receptors in primary healthy human nasal epithelial cells shows no correlation and a refractory LPS response.

PubMed

van Tongeren, J; Röschmann, K I L; Reinartz, S M; Luiten, S; Fokkens, W J; de Jong, E C; van Drunen, C M

2015-01-01

Innate immune recognition via Toll-like receptors (TLRs) on barrier cells like epithelial cells has been shown to influence the regulation of local immune responses. Here we determine expression level variations and functionality of TLRs in nasal epithelial cells from healthy donors. Expression levels of the different TLRs on primary nasal epithelial cells from healthy donors derived from inferior turbinates was determined by RT-PCR. Functionality of the TLRs was determined by stimulation with the respective ligand and evaluation of released mediators by Luminex ELISA. Primary nasal epithelial cells express different levels of TLR1-6 and TLR9. We were unable to detect mRNA of TLR7, TLR8 and TLR10. Stimulation with Poly(I:C) resulted in a significant increased secretion of IL-4, IL-6, RANTES, IP-10, MIP-1β, VEGF, FGF, IL-1RA, IL-2R and G-CSF. Stimulation with PGN only resulted in significant increased production of IL-6, VEGF and IL-1RA. Although the expression of TLR4 and co-stimulatory molecules could be confirmed, primary nasal epithelial cells appeared to be unresponsive to stimulation with LPS. Furthermore, we observed huge individual differences in TLR agonist-induced mediator release, which did not correlate with the respective expression of TLRs. Our data suggest that nasal epithelium seems to have developed a delicate system of discrimination and recognition of microbial patterns. Hypo-responsiveness to LPS could provide a mechanism to dampen the inflammatory response in the nasal mucosa in order to avoid a chronic inflammatory response. Individual, differential expression of TLRs on epithelial cells and functionality in terms of released mediators might be a crucial factor in explaining why some people develop allergies to common inhaled antigens, and others do not.

Mangiferin induces cell cycle arrest at G2/M phase through ATR-Chk1 pathway in HL-60 leukemia cells.

PubMed

Peng, Z G; Yao, Y B; Yang, J; Tang, Y L; Huang, X

2015-05-12

This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.

Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

PubMed

Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

2011-01-25

Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity patterns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation. 2010 Elsevier B.V. All rights reserved.

Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis.

PubMed

Sagor, G H M; Berberich, Thomas; Kojima, Seiji; Niitsu, Masaru; Kusano, Tomonobu

2016-06-01

Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine. Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

Shikonin suppresses the migratory ability of hepatocellular carcinoma cells.

PubMed

Wei, Po-Li; Tu, Chao-Chiang; Chen, Ching-Hsein; Ho, Yuan-Soon; Wu, Chun-Te; Su, Hou-Yu; Chen, Wei-Yu; Liu, Jun-Jen; Chang, Yu-Jia

2013-08-28

Shikonin is a traditional Oriental medical herb extracted from Lithospermum erythrorhizon. Many studies have shown that shikonin possesses anticancer ability against many different cancers, including hepatocellular carcinoma (HCC). Recently, tumor metastasis has been become an important clinical obstacle. However, the effect of shikonin on metastasis by HCC is unknown. The 50% inhibitory concentration (IC50) of shikonin on HCC cells was determined by an MTT assay and the xCELLigence biosensor system. The migratory ability of HCC cells was detected by a transwell migration assay and the xCELLigence biosensor system. Matrix metalloproteinase-2 and -9 (MMP-2 and -9) expression levels were determined by Western blotting, and the activities of MMP-2 and -9 were determined by gelatin zymography. We found that IC50 values of HepJ5 and Mahlavu cells to shikonin treatment were around 2 μM. Exposure to a low dose of shikonin (0-0.4 μM) did not influence the survival of HCC cells. Interestingly, exposure to a low dose of shikonin inhibited the migratory ability on HepJ5 and Mahlavu cells. To further dissect the mechanism, we found that treatment with a low dose of shikonin reduced the activities and expression levels of MMP-2 and -9, which were correlated with the decreased cell migratory ability of HCC cells. In addition, we found a decrease of vimnetin expression, but no influence on the expression levels of N-cadherin, TWIST, or GRP78. In mechanism dissecting, we found that shikonin treatment may suppress the phosphorylation of AKT and then reduce the NF-κB (NF = nuclear factor) levels, but has no influence on the levels of c-Fos and c-Jun. Furthermore, we also found that shikonin may also reduce the phosphorylation of IκB. We concluded that a low dose of shikonin can suppress the migratory ability of HCC cells through downregulation of expression levels of vimentin and MMP-2 and -9. Our findings suggest that shikonin may be a new compound to prevent the migration of HCC cells.

Expression of the FAP gene in non-fibroblast human cell lines. Development of cancer-associated fibroblast models.

PubMed

Tyulkina, D V; Pleshkan, V V; Alekseenko, I V; Kopantseva, M R; Sverdlov, E D

2016-09-01

The fibroblast activation protein (FAP) is selectively expressed in cancer-associated fibroblasts (CAFs) and facilitates tumor progression, which makes this protein an attractive therapeutic target. There are difficulties in obtaining CAFs for studying the function and suppression of FAP. In this work, the expression level of FAP was determined by PCR assay in 25 human cell lines and 8 surgical samples of tumor stroma. The expression of FAP was observed in all tumor stroma samples and in four cell lines: NGP-127, SJCRH30, SJSA-1, and A375. The level of FAP expression in NGP-127, SJCRH30, and SJSA-1 lines as well as in CAFs of patients was comparable, which makes these cell lines a possible model for studying FAP.

Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability

PubMed Central

Tani, Hidenori; Imamachi, Naoto; Salam, Kazi Abdus; Mizutani, Rena; Ijiri, Kenichi; Irie, Takuma; Yada, Tetsushi; Suzuki, Yutaka; Akimitsu, Nobuyoshi

2012-01-01

UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors. PMID:23064114

Relationships between oral MUC1 expression and salivary hormones in burning mouth syndrome.

PubMed

Kang, Jeong-Hyun; Kim, Yoon-Young; Chang, Ji-Youn; Kho, Hong-Seop

2017-06-01

To investigate possible relationships among oral mucosal epithelial MUC1 expression, salivary female gonadal hormones and stress markers, and clinical characteristics in patients with burning mouth syndrome (BMS). Thirty post-menopausal female patients with BMS (60.0±5.0 years) were included. Clinical and psychological evaluations were performed and the expression level of oral mucosal epithelial MUC1 was analyzed. The levels of cortisol, dehydroepiandrosterone (DHEA), 17β-estradiol, progesterone, chromogranin A, and blood contamination were determined from unstimulated whole saliva (UWS) and stimulated whole saliva (SWS) samples. Salivary progesterone level had significant positive correlations with oral mucosal epithelial MUC1 expression level and with salivary cortisol and DHEA levels. The salivary level of 17β-estradiol showed significant positive correlations with period of symptom duration, severity of effects of oral complaints on daily life, and results from psychological evaluations. Cortisol level in UWS and cortisol/DHEA ratio in UWS and SWS had negative correlations with severity of oral burning sensation significantly. The severity of taste disturbance had positive correlations with results from psychometry significantly. Dysregulated psychoendocrinological interactions might affect oral mucosal MUC1 expression and severity of oral burning sensation in post-menopausal BMS patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Hypercholesterolemia Up-Regulates the Expression of Intermedin and Its Receptor Components in the Aorta of Rats via Inducing the Oxidative Stress.

PubMed

Meng, Qingtao; Shi, Di; Feng, Jiayue; Su, Yanling; Long, Yang; He, Sen; Wang, Si; Wang, Yong; Zhang, Xiangxun; Chen, Xiaoping

2016-01-01

Hypercholesterolemia can cause damage to the artery. Intermedin (IMD) is a novel member of the calcitonin gene-related peptide family. This study aims to investigate the aortic expression of IMD and its receptors in hypercholesterolemia without atherosclerosis. Male Wistar rats were fed with high cholesterol diet, with or without simvastatin and vitamin C. Both the malondialdehyde (MDA) and superoxide dismutase (SOD) in plasma and aorta were determined as the oxidative stress biomarkers. The plasma IMD was assessed by radioimmunoassay. Within the aorta, the mRNA expression of IMD along with its receptor components was determined, and the corresponding protein level of the CRLR/RAMPs was also assessed. The hypercholesterolemia rats without atherosclerotic lesion manifested a higher level of MDA and SOD and the plasma IMD elevated. Increased expression of IMD and all its receptor components (CRLR, RAMP1, RAMP2, and RAMP3) were displayed within the aorta. The simvastatin indirectly attenuated oxidative stress by improving lipid profiles, while the vitamin C directly reduced oxidative stress without interfering with the serum lipids. Both simvastatin and vitamin C ameliorated the aortic injury, decreased the plasma IMD level, and recovered the expression of IMD and its receptors within the aorta. The up-regulated expression of IMD is observed within the aorta of the hypercholesterolemia rats. In addition, the oxidative stress participates in the up-regulation. © 2016 by the Association of Clinical Scientists, Inc.

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture*

PubMed Central

Mauceri, Daniela; Hagenston, Anna M.; Schramm, Kathrin; Weiss, Ursula; Bading, Hilmar

2015-01-01

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines. PMID:26231212

Histone posttranslational modifications and cell fate determination: lens induction requires the lysine acetyltransferases CBP and p300

PubMed Central

Wolf, Louise; Harrison, Wilbur; Huang, Jie; Xie, Qing; Xiao, Ningna; Sun, Jian; Kong, Lingkun; Lachke, Salil A.; Kuracha, Murali R.; Govindarajan, Venkatesh; Brindle, Paul K.; Ashery-Padan, Ruth; Beebe, David C.; Overbeek, Paul A.; Cvekl, Ales

2013-01-01

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300−/− ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens. PMID:24038357

Effect of stachydrine on endoplasmic reticulum stress-induced apoptosis in rat kidney after unilateral ureteral obstruction.

PubMed

Zhang, Cui; Lu, Ying; Tong, Qian-Qian; Zhang, Lan; Guan, Yu-Fei; Wang, Shu-Jing; Xing, Zhi-Hua

2013-01-01

Our study aimed at determining the effect of stachydrine on the PERK, CHOP, and caspase-3 in rat kidney with RIF. Rats were randomly divided into control group, model group, enalapril group, high stachydrine group, medium stachydrine group, and low stachydrine group. RIF models of five groups were developed by unilateral ureteral obstruction except the control group. The rats were sacrificed 12 days after surgery and blood samples were collected. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were detected. Renal tubular damage index was determined by HE staining. The area percentage of RIF was determined by the Masson method. Expressions of PERK, CHOP, and caspase-3 in kidney were determined by immunohistochemistry. Tubulointerstitial injury index, RIF, serum Scr, BUN level, and expressions of PERK, CHOP, and caspase-3 were different between the model and treatment groups (P < 0.05; P < 0.01). The expressions of PERK, CHOP, and caspase-3 in nephridial tissue were reduced (P < 0.05), tubulointerstitial injury and RIF were reduced (P < 0.05), and Scr and BUN were lower (P < 0.05) in the high stachydrine group than those in the enalapril group. The expressions of PERK, CHOP, and caspase-3 were reduced in the endoplasmic reticulum stress-related apoptosis pathway after stachydrine treatment. Consequently, apoptosis was prevented, and RIF was inhibited.

PDGF-α stimulates intestinal epithelial cell turnover after massive small bowel resection in a rat.

PubMed

Sukhotnik, Igor; Mogilner, Jorge G; Pollak, Yulia; Blumenfeld, Shiri; Bejar, Jacob; Coran, Arnold G

2012-06-01

Numerous cytokines have been shown to affect epithelial cell differentiation and proliferation through epithelial-mesenchymal interaction. Growing evidence suggests that platelet-derived growth factor (PDGF) signaling is an important mediator of these interactions. The purpose of this study was to evaluate the effect of PDGF-α on enterocyte turnover in a rat model of short bowel syndrome (SBS). Male rats were divided into four groups: Sham rats underwent bowel transection, Sham-PDGF-α rats underwent bowel transection and were treated with PDGF-α, SBS rats underwent a 75% bowel resection, and SBS-PDGF-α rats underwent bowel resection and were treated with PDGF-α. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined at euthanasia. Illumina's Digital Gene Expression analysis was used to determine PDGF-related gene expression profiling. PDGF-α and PDGF-α receptor (PDGFR-α) expression was determined by real-time PCR. Western blotting was used to determine p-ERK, Akt1/2/3, bax, and bcl-2 protein levels. SBS rats demonstrated a significant increase in PDGF-α and PDGFR-α expression in jejunum and ileum compared with sham animals. SBS-PDGF-α rats demonstrated a significant increase in bowel and mucosal weight, villus height, and crypt depth in jejunum and ileum compared with SBS animals. PDGF-α receptor expression in crypts increased in SBS rats (vs. sham) and was accompanied by an increased cell proliferation following PDGF-α administration. A significant decrease in cell apoptosis in this group was correlated with lower bax protein levels. In conclusion, in a rat model of SBS, PDGF-α stimulates enterocyte turnover, which is correlated with upregulated PDGF-α receptor expression in the remaining small intestine.

Arginase: A Novel Proliferative Determinant in Prostate Cancer

DTIC Science & Technology

2005-04-01

neoplastic prostate samples. The purpose of the present research funded by USAMRMC is to examine the expression of All in a wider range of benign and - malignant prostate...of polyamine synthesis levels in these lines, and our measurement and localization of arginase expression in benign and malignant prostate tissue samples.

Cytokine mRNA expression in normal skin of various age populations before and after engraftment onto nude mice.

PubMed

Gilhar, A; Ullmann, Y; Shalagino, R; Weisinger, G

1998-01-01

Whether the impact of skin biological age on cytokine expression is a result of this tissue's proliferation potential or not is an important issue in dermatology. We investigated these questions by monitoring cytokine marker mRNA expression from human skin samples from healthy groups of individuals. The skin samples studied represented three age groups: fetal (17-21 weeks), young (18-35 years) and aged (76-88 years). Furthermore, upon skin transplantation of tissue from different age groups onto nude mice, we investigated whether cytokine marker RNA levels would change or normalize. Interestingly, both TNF-alpha and P53 mRNA showed a similar pattern of expression. Both were significantly higher in fetal skin (p < 0.0001 and p < 0.05, respectively), and no difference was noted between aged versus young skin. In contrast to this, IL1-alpha mRNA was expressed at its lowest and highest levels in fetal and young skin, respectively. Following skin transplantation, cytokines and P53 mRNA expression were normalized to similar levels in all age groups. This study implies that when cytokine expression was determined directly at the mRNA level, post-natal expression was not significantly different at either age group. Furthermore, it seems that the environmental conditions surrounding the grafted human skin found on nude mice encouraged normalization of donor cytokine expression.

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

PubMed

Karbowska, Joanna; Kochan, Zdzislaw

2012-03-01

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

Annona muricata modulate brain-CXCL10 expression during cerebral malaria phase

NASA Astrophysics Data System (ADS)

Djamiatun, Kis; Matug, Sumia M. A.; Prasetyo, Awal; Wijayahadi, Noor; Nugroho, Djoko

2017-02-01

Cerebral malaria (CM) contributes in malaria mortality. People in endemic region get benefices by using A. muricata-leaf extract (AME) before qualified for receiving standard anti-malaria, because AME restrains malaria infection and modulate immune responses. CXCL10 expressed by astrocytes limit brain inflammation. Vascular leakage was found in the brain of experimental CM. Additionally, biomarker related with vascular leakage, angiopoietin-2 (Ang-2) levels increase in CM-patients. Objectives of this study were to determine the efficacy of ethanolic-AME in regulating brain-CXCL10-expression and Ang-2 levels during CM-phase. The study was post-test-only-control-group design. Thirty Swiss-mice were randomly divided in 6 groups. C+ and C- groups were PbA-inoculated and healthy-mice, respectively. X1 and X2 groups were healthy-mice treated with AME 100 and 150 mg/Kg BW/day, respectively. X3 and X4 groups were PbA-inoculated and received either dose mentioned above. CXCL10 was stained by IHC, and determined by Allred score. Plasma-Ang-2 was measured by elisa-method. Kruskal-Wallis-test showed the difference of CXCL10-expression among the studied groups (p=0.003). CXCL10-expression of C+ group was lower than healthy-mice which were C-, X1 and X2 groups (p=0.008, p=0.045, and p=0.012). CXCL10-expression of X3 was comparable to healthy mice (C-, X1 and X2), and was higher than C+ and X4 groups (p=0.012 and p=0.028). CXCL10-expression of X4 group was lower than C- and X2 groups (p=0.011 and p=0.016). Kruskal-Wallis-test showed no difference of Ang-2-levels among 6 groups (p = 0.175). The conclusion is A. muricata influences brain-CXCL10 expression during CM phase, but has no association with Ang-2 levels during CM phase.

Pathogenicity of Different Rabies Virus Variants Inversely Correlates with Apoptosis and Rabies Virus Glycoprotein Expression in Infected Primary Neuron Cultures

PubMed Central

Morimoto, Kinjiro; Hooper, D. Craig; Spitsin, Sergei; Koprowski, Hilary; Dietzschold, Bernhard

1999-01-01

The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein. PMID:9847357

Nandrolone, an anabolic steroid, stabilizes Numb protein through inhibition of mdm2 in C2C12 myoblasts.

PubMed

Liu, Xin-Hua; Yao, Shen; Levine, Alice C; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Collier, Lauren; Bauman, William A; Cardozo, Christopher P

2012-01-01

Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.

ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

PubMed Central

Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

2015-01-01

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

Expression of TRPC5 is decreased in the sperm of patients with varicocele-associated asthenozoospermia

PubMed Central

Zhu, Guangbin; Xie, Changying; Yang, Zhonghua; Wang, Yongzhi; Chen, Dong; Wang, Xinghuan

2018-01-01

The present study aimed to determine whether the expression of transient receptor potential channel 5 (TRPC5) protein is altered in spermatozoa of patients with varicocele-associated asthenozoospermia. TRPC5 expression in spermatozoa was determined by polymerase chain reaction and western blotting analyses, and indirect immunofluorescence was used for identification and immunolocalization of the TRPC5 channel in human sperm. Sperm motility and superoxide dismutase (SOD) activity were also determined with a computer-assisted semen analysis system and assay kit, respectively. Compared with levels in control subjects, it was identified that TRPC5 protein expression, SOD activity and cellular motility in the sperm of patients with varicocele-associated asthenozoospermia were reduced (P<0.001). Furthermore, the expression of TRPC5 was positively correlated with sperm motility (r=0.781, P<0.001) and SOD activity (r=0.933, P<0.001), indicated by partial correlation analysis. The present study may provide a novel target for the study and treatment of varicocele-associated asthenozoospermia.

Decreased expression of dual specificity phosphatase 22 in colorectal cancer and its potential prognostic relevance for stage IV CRC patients.

PubMed

Yu, Dan; Li, Zhenli; Gan, Meifu; Zhang, Hanyun; Yin, Xiaoyang; Tang, Shunli; Wan, Ledong; Tian, Yiping; Zhang, Shuai; Zhu, Yimin; Lai, Maode; Zhang, Dandan

2015-11-01

Dual specificity phosphatase 22 (DUSP22) is a novel dual specificity phosphatase that has been demonstrated to be a cancer suppressor gene associated with numerous biological and pathological processes. However, little is known of DUSP22 expression profiling in colorectal cancer and its prognostic value. Our study aims to investigate the role of DUSP22 expression in the prognosis of colorectal cancer. We detected the mRNA expression in 92 paired primary colorectal cancer tissues and the corresponding adjacent normal tissues by using QuantiGenePlex assay. The Friedman test was used to determine the statistical difference of gene expression. Kaplan-Meier survival analysis was performed. Mann-Whitney test and Kruskal-Wallis test were used to conduct data analyses to determine the prognostic value. Statistical significance was set at P < 0.05. In 74 of 92 cases, DUSP22 mRNA was reduced in primary colorectal cancer tissues, compared to the adjacent normal tissues. The mRNA levels of DUSP22 were significantly lower in colorectal cancer tissues than in adjacent normal tissues (0.0290 vs. 0.0658; P < 0.001). Low expression of DUSP22 correlated significantly with large tumor size (P = 0.013). No association was observed between DUSP22 mRNA expression and differentiation, histopathological type, tumor invasion, lymph node metastases, metastases, TNM stage, and Duke's phase (all P > 0.05). Kaplan-Meier analysis indicated that DUSP22 expression had no significant relationship with overall survival in all patients (P > 0.05). Interestingly, low expression level of DUSP22 in stage IV patients had a poor survival measures with a marginal P value (P = 0.07). Reduced DUSP22 expression was found in colorectal cancer specimens. Low expression level of DUSP22 in stage IV patients had a poor survival outcome. Further study is required for the investigation of the role of DUSP22 in colorectal cancer.

Expression of β-catenin protein in hepatocellular carcinoma and its relationship with alpha-fetoprotein.

PubMed

Ren, Ya-Jun; Huang, Tao; Yu, Hong-Lu; Zhang, Li; He, Qian-Jin; Xiong, Zhi-Fan; Peng, Hua

2016-12-01

This study aimed to investigate the expression of β-catenin in hepatocellular carcinoma (HCC) tissues and its relationship with α-fetoprotein (AFP) in HCC. Immunohistochemistry was used to determine the expression of β-catenin in normal liver tissues (n=10), liver cirrhosis tissues (n=20), and primary HCC tissues (n=60). The relationship between β-catenin expression and clinical parameters of HCC was investigated. Real-time PCR and Western blotting were used to detect the mRNA and protein expression levels of β-catenin in the liver cancer cell line SMMC-7721 transfected with a plasmid encoding AFP, and also the mRNA and protein expression levels of β-catenin were measured in the liver cancer cell line Huh7 before and after the transfection with AFP shRNA plasmids. The results showed that β-catenin was only expressed on the cell membrane in normal liver tissues. Its localization to the cytoplasm and nucleus of cells was observed in a small proportion of cirrhotic tissues or adjacent HCC tissues, and such ectopic expression of β-catenin was predominant in HCC tissues. The abnormal expression of β-catenin was correlated with serum AFP levels, cancer cell differentiation and vascular invasion (P<0.05). Additionally, the increased expression of AFP resulted in the upregulation of β-catenin mRNA and protein levels, while knockdown of AFP with AFP shRNA led to significantly decreased β-catenin mRNA and protein levels (P<0.05). It was suggested that the abnormal expression of β-catenin is implicated in hepatic carcinogenesis and development. AFP can lead to increased expression of β-catenin, which may account for the poor prognosis of AFP-associated HCC patients.

Active RNA replication of hepatitis C virus downregulates CD81 expression.

PubMed

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

PubMed Central

Ke, Po-Yuan; Chen, Steve S.-L.

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81. PMID:23349980

Association between MDR1 gene of gastrointestinal tumors, the expression of P-glycoprotein and resistance to chemotherapeutic drugs.

PubMed

Su, Jian-Li; Wang, Cheng-Hong; Kang, Hong-Gang; Zhang, Jing; Wang, Bao-Zhong; Liu, Mei-Rong; Zhao, Jun; Liu, Lin

2017-09-01

The aim of the present study was to examine and discuss the association between multidrug resistance 1 gene ( MDR1 ) of gastrointestinal tumors, the expression of P-glycoprotein and resistance to chemotherapeutic drugs. In this study, 126 cases of patients with gastrointestinal tumors admitted to hospital from February 2013 to February 2015 were selected. The expression levels of MDR1 gene were obsreved in the control population and patients before and after treatment by fluoresecent quantitative PCR. The protein expression level of P-glycoprotein was determined using western blotting and enzyme-linked immunosorbent assay. In addition, drug resistance was assessed by ATP-TCA chemosensitivity experiments. The results showed that before treatment, the expression of mRNA in MDR1 of tissues of gastrointestinal tract of the 126 cases was 108-fold larger than that of the gastrointestinal tract of the controls (p<0.05), P-glycoprotein was 87-fold larger than the expression level of the controls (p<0.05). The sensitivity of 126 tumor tissues to different chemotherapeutic drugs was determined, and the results showed that most of the tumor tissues were sensitive to chemotherapeutic drugs, and the sensitivity rate reached 96.4%. Following chemotherapy, the expression of mRNA in MDR1 of tumor tissues and the expression of P-glycoprotein decreased (p<0.05). In conclusion, the MDR1 gene and P-glycoprotein have a positive correlation with the occurrence of gastrointestinal tumors, and a negative correlation between the MDR1 gene and P-glycoprotein with resistance of chemotherapeutic drugs. Therefore, the MDR1 gene and P-glycoprotein can be used as references in the identification and diagnosis of gastrointestinal tumors.

The orphan nuclear receptor NR4A1 attenuates oxidative stress-induced β cells apoptosis via up-regulation of glutathione peroxidase 1.

PubMed

Yang, Yingfeng; Xie, Fangyu; Qin, Dandan; Zong, Chen; Han, Feng; Pu, Zeqing; Liu, Dong; Li, Xia; Zhang, Yuchao; Liu, Yuantao; Wang, Xiangdong

2018-06-15

Our previous study showed that NR4A1 protects against oxidative stress-induced cell apoptosis. However, the targets downstream of NR4A1 are incompletely known. Glutathione peroxidase 1 (GPX1) is the most common antioxidant enzyme in the glutathione peroxidase class. In this study, we aimed to investigate whether GPX1 is a mediator of the protective effects of NR4A1 in pancreatic β cells. A pancreatic β cell line, MIN6, was used to generate NR4A1 over-expression cell line. GPX1 expression and GPX1 promoter trans-activation in these cells was determined. These cells were then treated with H 2 O 2 , and the active caspase3 level was determined. NR4A1 over-expression in MIN6 cells resulted in increased GPX1 expression at both mRNA and protein levels. Dual luciferase assay showed that NR4A1 over-expression was able to enhance the trans-activation of GPX1 promoter, and the critical regulatory elements were narrowed down between 0 to -2000 bp in GPX1 promoter with a putative NR4A1 binding site (-273 to -268). ChIP assays demonstrated that NR4A1 physically associates with the GPX1 promoter. Over-expression of GPX1 reduced the active level of Caspase3 after H 2 O 2 treatment. NR4A1 increases the expression of GPX1 by enhancing the trans-activation of GPX1 promoter through binding to the putative binding site on GPX1 promoter. NR4A1 potentially protects pancreatic β cells against oxidative stress-induced apoptosis by increasing GPX1 expression. Copyright © 2018 Elsevier Inc. All rights reserved.

CD133 expression in osteosarcoma and derivation of CD133⁺ cells.

PubMed

Li, Ji; Zhong, Xiao-Yan; Li, Zong-Yu; Cai, Jin-Fang; Zou, Lin; Li, Jian-Min; Yang, Tao; Liu, Wei

2013-02-01

Cluster of differentiation 133 (CD133) is recognized as a stem cell marker for normal and cancerous tissues. Using cell culture and real‑time fluorescent polymerase chain reaction, CD133 expression was analyzed in osteosarcoma tissue and Saos‑2 cell lines. In addition, cancer stem cell‑related gene expression in the Saos‑2 cell line was determined to explore the mechanisms underlying tumorigenesis and high drug resistance in osteosarcoma. CD133+ cells were found to be widely distributed in various types of osteosarcoma tissue. Following cell culture, cells entered the G2/M and S cell cycle stages from G0/G1. Levels of CD133+ cells decreased to normal levels rapidly over the course of cell culture. Colony forming efficiency was higher in the CD133+ compared with the CD133‑ subpopulation of Saos‑2 cells. Expression levels of stem cell‑related genes, including multidrug resistance protein 1 (MDR1) and sex determining region Y‑box 2 (Sox2) in the CD133+ subpopulation of cells were found to be significantly higher compared with the CD133‑ subpopulation. These observations indicate that CD133+ Saos‑2 cells exhibit stem cell characteristics, including low abundance, quiescence and a high potential to undergo differentiation, as well as expression of key stem cell regulatory and drug resistance genes, which may cause osteosarcoma and high drug resistance.

Functional characterization of the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants.

PubMed

Yang, Qingjie; Yuan, Dawei; Shi, Lianxuan; Capell, Teresa; Bai, Chao; Wen, Nuan; Lu, Xiaodan; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

2012-10-01

The accumulation of carotenoids in plants depends critically on the spatiotemporal expression profiles of the genes encoding enzymes in the carotenogenic pathway. We cloned and characterized the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter to determine its role in the regulation of carotenogenesis, because the native gene is expressed at high levels in petals, which contain abundant chromoplasts. We transformed tomato (Solanum lycopersicum cv. Micro-Tom) plants with the gusA gene encoding the reporter enzyme β-glucuronidase (GUS) under the control of the GlZEP promoter, and investigated the reporter expression profile at the mRNA and protein levels. We detected high levels of gusA expression and GUS activity in chromoplast-containing flowers and fruits, but minimal levels in immature fruits containing green chloroplasts, in sepals, leaves, stems and roots. GlZEP-gusA expression was strictly associated with fruit development and chromoplast differentiation, suggesting an evolutionarily-conserved link between ZEP and the differentiation of organelles that store carotenoid pigments. The impact of our results on current models for the regulation of carotenogenesis in plants is discussed.

Expression of Clock genes in the pineal glands of newborn rats with hypoxic-ischemic encephalopathy☆

PubMed Central

Sun, Bin; Feng, Xing; Ding, Xin; Bao, Li; Li, Yongfu; He, Jun; Jin, Meifang

2012-01-01

Clock genes are involved in circadian rhythm regulation, and surviving newborns with hypoxic-ischemic encephalopathy may present with sleep-wake cycle reversal. This study aimed to determine the expression of the clock genes Clock and Bmal1, in the pineal gland of rats with hypoxic-ischemic brain damage. Results showed that levels of Clock mRNA were not significantly changed within 48 hours after cerebral hypoxia and ischemia. Expression levels of CLOCK and BMAL1 protein were significantly higher after 48 hours. The levels of Bmal1 mRNA reached a peak at 36 hours, but were significantly reduced at 48 hours. Experimental findings indicate that Clock and Bmal1 genes were indeed expressed in the pineal glands of neonatal rats. At the initial stage (within 36 hours) of hypoxic-ischemic brain damage, only slight changes in the expression levels of these two genes were detected, followed by significant changes at 36–48 hours. These changes may be associated with circadian rhythm disorder induced by hypoxic-ischemic brain damage. PMID:25538743

Multidisciplinary Biomarkers of Early Mammary Carcinogenesis

DTIC Science & Technology

2011-04-01

cDNA prepared. Quantitative real-time PCR (qRT-PCR) was then performed on the cDNA. All qRT-PCR reactions were performed in triplicate. ESR1 ...in Figure 4, all ER(+) cells express ESR1 at high levels (at least 4 fold higher than ER(-) cell lines). A Pearson correlation coefficient was...calculated to determine the linear relationship between the optical redox ratio and ESR1 expression levels and found to be significant (p = 0.0024, r

The Influence of Glycated Hemoglobin on the Cross Susceptibility Between Type 1 Diabetes Mellitus and Periodontal Disease.

PubMed

Lappin, David F; Robertson, Douglas; Hodge, Penny; Treagus, David; Awang, Raja A; Ramage, Gordon; Nile, Christopher J

2015-11-01

Periodontal disease is a major complication of type 1 diabetes mellitus (T1DM). The aim of the present study is to investigate the relationship between glycated hemoglobin and circulating levels of interleukin (IL)-6, IL-8, and C-X-C motif chemokine ligand 5 (CXCL5) in non-smoking patients suffering from T1DM, with and without periodontitis. In addition, to determine the effect of advanced glycation end products (AGE) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) on IL-6, IL-8, and CXCL5 expression by THP-1 monocytes and OKF6/TERT-2 cells. There were 104 participants in the study: 19 healthy volunteers, 23 patients with periodontitis, 28 patients with T1DM, and 34 patients with T1DM and periodontitis. Levels of blood glucose/glycated hemoglobin (International Federation of Clinical Chemistry [IFCC]) were determined by high-performance liquid chromatography. Levels of IL-6, IL-8, and CXCL5 in plasma were determined by enzyme-linked immunosorbent assay (ELISA). In vitro stimulation of OKF6/TERT-2 cells and THP-1 monocytes was performed with combinations of AGE and P. gingivalis LPS. Changes in expression of IL-6, IL-8, and CXCL5 were monitored by ELISA and real-time polymerase chain reaction. Patients with diabetes and periodontitis had higher plasma levels of IL-8 than patients with periodontitis alone. Plasma levels of IL-8 correlated significantly with IFCC units, clinical probing depth, and attachment loss. AGE and LPS, alone or in combination, stimulated IL-6, IL-8, and CXCL5 expression in both OKF6/TERT-2 cells and THP-1 monocytes. Elevated plasma levels of IL-8 potentially contribute to the cross-susceptibility between periodontitis and T1DM. P. gingivalis LPS and AGE in combination caused significantly greater expression of IL-6, IL-8, and CXCL5 from THP-1 monocytes and OKF6/TERT-2 cells than LPS alone.

Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

NASA Astrophysics Data System (ADS)

Canuto, K. S.; Sergio, L. P. S.; Paoli, F.; Mencalha, A. L.; Fonseca, A. S.

2016-03-01

Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

[Expression of MiR-130a in Serum Samples of Patients with Epithelial Ovarian Cancer and Its Association with Platinum Resistance].

PubMed

Chen, Cen; Wang, Hong-jing; Yang, Ling-Yun; Jia, Xi-biao; Xu, Pan; Chen, Jing; Liu, Ya

2016-01-01

To determine the expression of miR-130a in patients with epithelial ovarian cancer and its association with platinum resistance. 32 patients with platinum resistance and 30 patients without platinum resistance were recruited in this study. Real-time PCR was performed to detect the expression of miR-130a in the serum samples of the patients. ELISA was used to measure the expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and B-cell lymphoma-2 (BCL-2). Platinum-resistant patients had significantly higher levels of expression of miR-130a and BCL-2, and lower level of PTEN than platinum-sensitive patients (P < 0.05). The expression level of miR-130a increased with increased severity in histological classification and appearance of lymph node metastasis in the platinum-resistant patients (P < 0.05). MiR-130a may mediate the generation of platinum resistance in epithelial ovarian cancer through inhibiting PTEN to activate PI3K/AKT signaling pathway and increasing BCL-2 to inhibit tumor cell apoptosis. MiR-130a may be a new potential target of gene therapy in platinum-resistant ovarian cancers.

Increased OGA Expression and Activity in Leukocytes from Patients with Diabetes: Correlation with Inflammation Markers.

PubMed

Pagesy, Patrick; Tachet, Caroline; Mostefa-Kara, Ali; Larger, Etienne; Issad, Tarik

2018-06-11

O-linked-β-N-Acetylglucosaminylation (O-GlcNAcylation), a reversible post-translational modification involved in diabetic complications, is regulated by only two enzymes, O-linked N-acetylglucosamine transferase (OGT) and β-N-Acetylglucosaminidase (OGA). Increased OGA expression has been described previously in blood cells from patients with diabetes and was interpreted as an adaptative response to hyperglycemia-induced O-GlcNAcylation. OGA expression was thus proposed to have potential utility as a diagnostic marker. The present work was undertaken to determine whether determination of OGA enzymatic activity in blood cells could constitute a more rapidly accessible marker than OGA expression level measurements.Blood samples were obtained from patients with type 2 diabetes from the Department of Diabetology of the Cochin Hospital and healthy volunteers from the French blood Agency. OGA enzymatic activity and OGA mRNA expression levels were evaluated in leucocytes from patients with type 2 diabetes and from healthy donors.OGA activity was higher in leucocytes from patients with diabetes compared to control individuals. Surprisingly, OGA activity was not correlated hyperglycaemia markers (blood glucose, fructosamine, HbA 1c ) but was positively correlated with the inflammatory marker C-reactive protein. OGA mRNA levels were also increased in leucocytes from patients with diabetes and were correlated with mRNA coding for two pro-inflammatory proteins, TNFα and TxNIP.Therefore, OGA activity in leucocytes might be a more easily accessible biomarker than OGA expression levels. However, changes in OGA activity observed in patients with type 2 diabetes may reflect the inflammatory rather than the glycaemic status of these patients. © Georg Thieme Verlag KG Stuttgart · New York.

Downregulation of the glucocorticoid-induced leucine zipper (GILZ) promotes vascular inflammation.

PubMed

Hahn, Rebecca T; Hoppstädter, Jessica; Hirschfelder, Kerstin; Hachenthal, Nina; Diesel, Britta; Kessler, Sonja M; Huwer, Hanno; Kiemer, Alexandra K

2014-06-01

Glucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. Degenerated aortocoronary saphenous vein bypass grafts (n = 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n = 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-α in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-κB activation after GILZ knockdown by siRNA and found that NF-κB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-α-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-α-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. Our data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Gene expression analyses determine two different subpopulations in KIT-negative GIST-like (KNGL) patients.

PubMed

Moura, David S; Ramos, Rafael; Fernandez-Serra, Antonio; Serrano, Teresa; Cruz, Julia; Alvarez-Alegret, Ramiro; Ortiz-Duran, Rosa; Vicioso, Luis; Gomez-Dorronsoro, Maria Luisa; Garcia Del Muro, Xavier; Martinez-Trufero, Javier; Rubio-Casadevall, Jordi; Sevilla, Isabel; Lainez, Nuria; Gutierrez, Antonio; Serrano, Cesar; Lopez-Alvarez, Maria; Hindi, Nadia; Taron, Miguel; López-Guerrero, José Antonio; Martin-Broto, Javier

2018-04-03

There are limited findings available on KIT-negative GIST-like (KNGL) population. Also, KIT expression may be post-transcriptionally regulated by miRNA221 and miRNA222. Hence, the aim of this study is to characterize KNGL population, by differential gene expression, and to analyze miRNA221/222 expression and their prognostic value in KNGL patients. KIT , PDGFRA , DOG1 , IGF1R , MIR221 and MIR222 expression levels were determined by qRT-PCR. We also analyzed KIT and PDGFRA mutations, DOG1 expression, by immunohistochemistry, along with clinical and pathological data. Disease-free survival (DFS) and overall survival (OS) differences were calculated using Log-rank test. Hierarchical cluster analyses from gene expression data identified two groups: group I had KIT , DOG1 and PDGFRA overexpression and IGF1R underexpression and group II had overexpression of IGF1R and low expression of KIT , DOG1 and PDGFRA . Group II had a significant worse OS ( p = 0.013) in all the series, and showed a tendency for worse OS ( p = 0.11), when analyzed only the localized cases. MiRNA222 expression was significantly lower in a control subset of KIT-positive GIST ( p < 0.001). OS was significantly worse in KNGL cases with higher expression of MIR221 ( p = 0.028) or MIR222 ( p = 0.014). We identified two distinct KNGL subsets, with a different prognostic value. Increased levels of miRNA221/222, which are associated with worse OS, could explain the absence of KIT protein expression of most KNGL tumors.

Regulation of Hippocampal α1d Adrenergic Receptor mRNA by Corticosterone in Adrenalectomized Rats

PubMed Central

Day, Heidi E.W.; Kryskow, Elisa M.; Watson, Stanley J.; Akil, Huda; Campeau, Serge

2008-01-01

The hippocampal formation receives extensive noradrenergic projections and expresses high levels of mineralocorticoid (MR) and glucocorticoid (GR) receptors. Considerable evidence suggests that the noradrenergic system influences hippocampal corticosteroid receptors. However, there is relatively little data describing the influence of glucocorticoids on noradrenergic receptors in the hippocampal formation. α1d adrenergic receptor (ADR) mRNA is expressed at high levels in the hippocampal formation, within cells that express MR or GR. In order to determine whether expression of α1d ADR mRNA is influenced by circulating glucocorticoids, male rats underwent bilateral adrenalectomy (ADX) or sham surgery, and were killed after 1, 3, 7 or 14 days. Levels of α1d ADR mRNA were profoundly decreased in hippocampal subfields CA1, CA2 and CA3 and the medial and lateral blades of the dentate gyrus, as early as 1 day after ADX, as determined by in situ hybridization. The effect was specific for the hippocampal formation, with levels of α1d mRNA unaltered by ADX in the lateral amygdala, reticular thalamic nucleus, retrosplenial cortex or primary somatosensory cortex. Additional rats underwent ADX or sham surgery and received a corticosterone pellet (10 or 50 mg) or placebo for 7 days. Corticosterone replacement prevented the ADX-induced decrease in hippocampal α1d ADR mRNA, with the magnitude of effect depending on corticosterone dose and hippocampal subregion. These data indicate that α1d ADR mRNA expression in the hippocampal formation is highly sensitive to circulating levels of corticosterone, and provides further evidence for a close interaction between glucocorticoids and the noradrenergic system in the hippocampus. PMID:18534559

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis

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

Xu, Nanwei; Wang, Yuji, E-mail: yujiwang@sohu.com; State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433

Research highlights: {yields} Elevated MDM4 mRNA and protein levels in FLS from patients with RA and OA. {yields} Strong MDM4 staining in synovial cells of inflammatory synovium. {yields} MDM4 knockdown increased p53 and p21 levels, and inhibited the proliferation of RA FLS. {yields} MDM4 overexpression increased p53 while decreased p21 levels, and promoted the growth of RA FLS. -- Abstract: Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a majormore » negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.« less

Abnormal mRNA Expression Levels of Telomere-Binding Proteins Represent Biomarkers in Myelodysplastic Syndromes: A Case-Control Study.

PubMed

Liu, Baoshan; Yan, Rongdi; Zhang, Jie; Wang, Bin; Sun, Hu; Cui, Xing

2017-08-02

As evidence was shown that abnormal shortening of telomeres begins to accumulate in myelodysplastic syndrome (MDS) patients, this study was conducted to determine the relationship between the mRNA expression levels of telomere-binding proteins (TRF1/TRF2/TIN2/TPP1/POT1/RAP1) and the risk level in MDS. There were 40 patients with MDS and 40 normal controls in this study. Methods including telomere content assays and quantitative reverse transcription-polymerase chain reaction were used to examine the mRNA levels of TRF1/TRF2/TIN2/TPP1/POT1/RAP1 in patients with MDS. Compared to the normal group used as a control, the mRNA expression levels of RAP1/POT1/TPP1 of the patients with MDS were decreased, whereas their levels of TRF1/TRF2 and TIN2 were increased. A positive correlation was found between the TRF1, TRF2, and TIN2 mRNA expression levels and the risk level of the International Prognostic Scoring System (IPSS) and the World Health Organization Prognostic Scoring System (WPSS) criteria; however, a negative correlation was found between RAP1/POT1/TPP1 mRNA expression levels and the risk levels of IPSS and WPSS criteria. Because the reduction of TRF1/TRF2/TIN2 mRNA expression and the increase of RAP1/POT1/TPP1 mRNA expression are closely related to the risk levels of the IPSS and WPSS criteria in MDS, it is thought that these telomere-binding proteins could lead to abnormal telomere length and function, which cause chromosomal abnormalities in MDS. With this evidence, we suggest that those proteins' mRNA expressions could be used as biomarkers for the assessment of the risk degree of MDS patients.

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

PubMed

Amendola, R

1994-11-01

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

Expression of fas protein on CD4+T cells irradiated by low level He-Ne

NASA Astrophysics Data System (ADS)

Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

2005-07-01

Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min、60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2（60 minutes）and 45.84J/cm2（120minutes）, the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

Salivary FOXP2 expression and oral feeding success in premature infants.

PubMed

Zimmerman, Emily; Maki, Monika; Maron, Jill

2016-01-01

The objective of the study is to determine whether salivary FOXP2 gene expression levels at the initiation of oral feeding attempts are predictive of oral feeding success in the premature newborn. In this prospective study, saliva samples from 21 premature infants (13 males; birth gestational age [GA]: 30-34 wk) were collected around the initiation of oral feeding trials. Total RNA was extracted and underwent reverse transcription-quantitative polymerase chain reaction amplification for FOXP2. Oral feeding success was denoted by the days required to attain full oral feeds. A linear regression model, controlling for sex, birth GA, and weight at salivary collection, revealed that FOXP2 expression was significantly associated with oral feeding success (P = 0.002). The higher the expression level of FOXP2, the shorter the duration to feed. Salivary FOXP2 expression levels are significantly associated with oral feeding success in the preterm infant. FOXP2 may serve as a novel and informative biomarker to noninvasively assess infant feeding skills to reduce morbidities and length of stay.

Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants.

PubMed Central

Dean, C; Jones, J; Favreau, M; Dunsmuir, P; Bedbrook, J

1988-01-01

The petunia rbcS gene SSU301 was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. The time at which rbcS expression was maximal after transfer of the tobacco plants to the greenhouse was determined. The expression level of the SSU301 gene varied up to 9 fold between individual tobacco plants which had been standardized physiologically as much as possible. The presence of adjacent pUC plasmid sequences did not affect the expression of the SSU301 gene. In an attempt to reduce the between-transformant variability in expression, the SSU301 gene was introduced into tobacco surrounded by 10kb of 5' and 13 kb of 3' DNA sequences which normally flank SSU301 in petunia. The longer flanking regions did not reduce the between-transformant variability of SSU301 gene expression. Images PMID:3174450

Cloning and characterization of acid invertase genes in the roots of the metallophyte Kummerowia stipulacea (Maxim.) Makino from two populations: Differential expression under copper stress.

PubMed

Zhang, Luan; Xiong, Zhi-ting; Xu, Zhong-rui; Liu, Chen; Cai, Shen-wen

2014-06-01

The roots of metallophytes serve as the key interface between plants and heavy metal-contaminated underground environments. It is known that the roots of metallicolous plants show a higher activity of acid invertase enzymes than those of non-metallicolous plants when under copper stress. To test whether the higher activity of acid invertases is the result of increased expression of acid invertase genes or variations in the amino acid sequences between the two population types, we isolated full cDNAs for acid invertases from two populations of Kummerowia stipulacea (from metalliferous and non-metalliferous soils), determined their nucleotide sequences, expressed them in Pichia pastoris, and conducted real-time PCR to determine differences in transcript levels during Cu stress. Heterologous expression of acid invertase cDNAs in P. pastoris indicated that variations in the amino acid sequences of acid invertases between the two populations played no significant role in determining enzyme characteristics. Seedlings of K. stipulacea were exposed to 0.3µM Cu(2+) (control) and 10µM Cu(2+) for 7 days under hydroponics׳ conditions. The transcript levels of acid invertases in metallicolous plants were significantly higher than in non-metallicolous plants when under copper stress. The results suggest that the expression of acid invertase genes in metallicolous plants of K. stipulacea differed from those in non-metallicolous plants under such conditions. In addition, the sugars may play an important role in regulating the transcript level of acid invertase genes and acid invertase genes may also be involved in root/shoot biomass allocation. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression of putative sex-determining genes during the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina.

PubMed

Rhen, T; Metzger, K; Schroeder, A; Woodward, R

2007-01-01

Modes of sex determination are quite variable in vertebrates. The developmental decision to form a testis or an ovary can be influenced by one gene, several genes, environmental variables, or a combination of these factors. Nevertheless, certain morphogenetic aspects of sex determination appear to be conserved in amniotes. Here we clone fragments of nine candidate sex-determining genes from the snapping turtle Chelydra serpentina, a species with temperature-dependent sex determination (TSD). We then analyze expression of these genes during the thermosensitive period of gonad development. In particular, we compare gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature. Expression of Dmrt1 and Sox9 mRNA increased gradually at the male-producing temperature, but was suppressed at the female-producing temperature. This finding suggests that Dmrt1 and Sox9 play a role in testis development. In contrast, expression of aromatase, androgen receptor (Ar), and Foxl2 mRNA was constant at the male-producing temperature, but increased several-fold in embryos at the female-producing temperature. Aromatase, Ar, and Foxl2 may therefore play a role in ovary development. In addition, there was a small temperature effect on ER alpha expression with lower mRNA levels found in embryos at the female-producing temperature. Finally, Dax1, Fgf9, and SF-1 were not differentially expressed during the sex-determining period, suggesting these genes are not involved in sex determination in the snapping turtle. Comparison of gene expression profiles among amniotes indicates that Dmrt1 and Sox9 are part of a core testis-determining pathway and that Ar, aromatase, ER alpha, and Foxl2 are part of a core ovary-determining pathway. 2007 S. Karger AG, Basel

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

PubMed

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Increased expression of sex determining region Y-box 11 (SOX11) in cutaneous malignant melanoma.

PubMed

Jian, Jiao; Guoying, Wang; Jing, Zhao

2013-08-01

To observe sex determining region Y-box 11 (SOX11) gene expression in cutaneous malignant melanoma and its effect on tumour cell proliferation. Clinicopathological data and tissue samples from patients with cutaneous malignant melanoma, together with tissue samples from healthy volunteers (controls), were retrospectively reviewed. Protein levels of SOX11 and the antigen identified by monoclonal antibody Ki-67 (Ki-67) in skin lesions were analysed using immunohistochemistry. The correlation between protein levels and clinipathological parameters was investigated. Out of 40 patient samples, 25 (62.5%) were positive for SOX11 protein in malignant melanoma tissue. This was significantly higher than in 40 control tissue samples, in which no SOX11 protein was detected. Presence of SOX11 protein was positively related to the proliferation index of cutaneous malignant melanoma tumour cells. Presence of SOX11 protein in cutaneous malignant melanoma was related to tumour type, tumour location, lymph node metastasis and 5-year survival rate. Human cutaneous malignant melanoma tissues expressed high levels of SOX11 compared with healthy controls, suggesting that SOX11 may be a new prognostic marker for malignant melanoma.

Decreased concentration of serum melatonin in nighttime compared with daytime female medical technologists in South Korea.

PubMed

Song, GiSeon; Yoon, Kyong-Ah; Chi, HyunYoung; Roh, Jaehoon; Kim, Jin-Hee

2016-01-01

Working during the night can disrupt the normal circadian rhythm by altering the melatonin level. A low level of melatonin is associated with an increased risk of cancer, possibly by decreasing the expression of tumor-suppressor genes, such as p53. To determine whether nighttime work is associated with melatonin level in serum as well as the expression of related genetic markers, we enrolled 100 female nighttime medical technologists employed at a hospital in South Korea. Melatonin concentration and melatonin receptor 1 (MT1) expression were significantly lower in nighttime than in daytime workers (1.84 pg/mL versus 4.04 pg/mL; 1.16 versus 1.61, respectively). However, p53 expression showed no difference between the groups. In summary, nighttime work could be an important risk factor for circadian disruption, but not a direct risk factor for cancer in medical technologists in South Korea.

Total bile acids in the maternal and fetal compartment in relation to placental ABCG2 expression in preeclamptic pregnancies complicated by HELLP syndrome.

PubMed

Jebbink, Jiska; Veenboer, Geertruda; Boussata, Souad; Keijser, Remco; Kremer, Andreas E; Elferink, Ronald Oude; van der Post, Joris; Afink, Gijs; Ris-Stalpers, Carrie

2015-01-01

To investigate total bile acid (TBA) levels in maternal (MB) and umbilical cord blood (UCB) in normotensive, preeclamptic (PE), and PE pregnancies complicated by hemolysis elevated liver enzymes and low platelets (HELLP) syndrome in the context of ABCG2 placental gene expression levels, a recently reported placental bile acid transporter. TBA levels were determined in 83 paired MB and UCB samples of normotensive, PE and PE/HELLP pregnancies and in 22 paired arterial and venous UCB samples from uncomplicated term pregnancies. ABCG2 gene expression was measured in 104 human placentas by reverse transcriptase quantitative polymerase chain reaction. Overall, TBA levels in MB are higher compared to levels in UCB (p<0.0001), but this comparison looses statistical significance for the 11 PE/HELLP cases. TBA levels in maternal blood are increased in PE/HELLP compared to PE pregnancies (p=0.016). TBA levels in arterial and venous UCB from 22 normotensive pregnancies are not statistically different. ABCG2 expression is reduced in pregnancies where preeclampsia is further complicated by HELLP syndrome. ABCG2 expression in human placenta is not correlated with TBA levels in either the maternal or fetal compartment. Increased maternal TBA levels in PE/HELLP pregnancies indicate a relation between bile acids in the maternal circulation and HELLP syndrome. As overall TBA levels in maternal blood are increased compared to UCB, we conclude that the placenta partly protects the fetus from increased maternal TBA levels. This consistent difference in TBA levels between the maternal and fetal compartment is unrelated to the placental expression of ABCG2. Copyright © 2014 Elsevier B.V. All rights reserved.

Differences in otosclerotic and normal human stapedial osteoblast properties are normalized by alendronate in vitro.

PubMed

Gronowicz, Gloria; Richardson, Yvonne L; Flynn, John; Kveton, John; Eisen, Marc; Leonard, Gerald; Aronow, Michael; Rodner, Craig; Parham, Kourosh

2014-10-01

Identify and compare phenotypic properties of osteoblasts from patients with otosclerosis (OSO), normal bones (HOB), and normal stapes (NSO) to determine a possible cause for OSO hypermineralization and assess any effects of the bisphosphonate, alendronate. OSO (n = 11), NSO (n = 4), and HOB (n = 13) cultures were assayed for proliferation, adhesion, mineralization, and gene expression with and without 10(-10)M-10(-8)M alendronate. Academic hospital. Cultures were matched for age, sex, and passage number. Cell attachment and proliferation + alendronate were determined by Coulter counting cells and assaying tritiated thymidine uptake, respectively. At 7, 14, and 21 days of culture + alendronate, calcium content and gene expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were determined. OSO had significantly more cells adhere but less proliferation than NSO or HOB. Calcification was significantly increased in OSO compared to HOB and NSO. NSO and HOB had similar cell adhesion and proliferation rates. A dose-dependent effect of alendronate on OSO adhesion, proliferation, and mineralization was found, resulting in levels equal to NSO and HOB. All cultures expressed osteoblast-specific genes such as RUNX2, alkaline phosphatase, type I collagen, and osteocalcin. However, osteopontin was dramatically reduced, 9.4-fold at 14 days, in OSO compared to NSO. Receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG), important in bone resorption, was elevated in OSO with decreased levels of OPG levels. Alendronate had little effect on gene expression in HOB but in OSO increased osteopontin levels and decreased RANKL/OPG. OSO cultures displayed properties of hypermineralization due to decreased osteopontin (OPN) and also had increased RANKL/OPG, which were normalized by alendronate. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014.

Distinctive genes determine different intramuscular fat and muscle fiber ratios of the longissimus dorsi muscles in Jinhua and landrace pigs.

PubMed

Wu, Ting; Zhang, Zhenhai; Yuan, Zhangqin; Lo, Li Jan; Chen, Jun; Wang, Yizhen; Peng, Jinrong

2013-01-01

Meat quality is determined by properties such as carcass color, tenderness and drip loss. These properties are closely associated with meat composition, which includes the types of muscle fiber and content of intramuscular fat (IMF). Muscle fibers are the main contributors to meat mass, while IMF not only contributes to the sensory properties but also to the plethora of physical, chemical and technological properties of meat. However, little is known about the molecular mechanisms that determine meat composition in different pig breeds. In this report we show that Jinhua pigs, a Chinese breed, contains much higher levels of IMF than do Landrace pigs, a Danish breed. We analyzed global gene expression profiles in the longissimus dorsi muscles in Jinhua and Landrace breeds at the ages of 30, 90 and 150 days. Cross-comparison analysis revealed that genes that regulate fatty acid biosynthesis (e.g., fatty acid synthase and stearoyl-CoA desaturase) are expressed at higher levels in Jinhua pigs whereas those that regulate myogenesis (e.g., myogenic factor 6 and forkhead box O1) are expressed at higher levels in Landrace pigs. Among those genes which are highly expressed in Jinhua pigs at 90 days (d90), we identified a novel gene porcine FLJ36031 (pFLJ), which functions as a positive regulator of fat deposition in cultured intramuscular adipocytes. In summary, our data showed that the up-regulation of fatty acid biosynthesis regulatory genes such as pFLJ and myogenesis inhibitory genes such as myostatin in the longissimus dorsi muscles of Jinhua pigs could explain why this local breed produces meat with high levels of IMF.

Distinctive Genes Determine Different Intramuscular Fat and Muscle Fiber Ratios of the longissimus dorsi Muscles in Jinhua and Landrace Pigs

PubMed Central

Yuan, Zhangqin; Lo, Li Jan; Chen, Jun; Wang, Yizhen; Peng, Jinrong

2013-01-01

Meat quality is determined by properties such as carcass color, tenderness and drip loss. These properties are closely associated with meat composition, which includes the types of muscle fiber and content of intramuscular fat (IMF). Muscle fibers are the main contributors to meat mass, while IMF not only contributes to the sensory properties but also to the plethora of physical, chemical and technological properties of meat. However, little is known about the molecular mechanisms that determine meat composition in different pig breeds. In this report we show that Jinhua pigs, a Chinese breed, contains much higher levels of IMF than do Landrace pigs, a Danish breed. We analyzed global gene expression profiles in the longissimus dorsi muscles in Jinhua and Landrace breeds at the ages of 30, 90 and 150 days. Cross-comparison analysis revealed that genes that regulate fatty acid biosynthesis (e.g., fatty acid synthase and stearoyl-CoA desaturase) are expressed at higher levels in Jinhua pigs whereas those that regulate myogenesis (e.g., myogenic factor 6 and forkhead box O1) are expressed at higher levels in Landrace pigs. Among those genes which are highly expressed in Jinhua pigs at 90 days (d90), we identified a novel gene porcine FLJ36031 (pFLJ), which functions as a positive regulator of fat deposition in cultured intramuscular adipocytes. In summary, our data showed that the up-regulation of fatty acid biosynthesis regulatory genes such as pFLJ and myogenesis inhibitory genes such as myostatin in the longissimus dorsi muscles of Jinhua pigs could explain why this local breed produces meat with high levels of IMF. PMID:23301040

Effects of celecoxib on proliferation and tenocytic differentiation of tendon-derived stem cells

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

Zhang, Kairui; Zhang, Sheng; Li, Qianqian

Highlights: • Celecoxib has no effects on TDSCs cell proliferation in various concentrations. • Celecoxib reduced mRNAs levels of tendon associated transcription factor. • Celecoxib reduced mRNAs levels of main tendon associated collagen. • Celecoxib reduced mRNAs levels of tendon associated molecules. - Abstract: NSAIDs are often ingested to reduce the pain and improve regeneration of tendon after tendon injury. Although the effects of NSAIDs in tendon healing have been reported, the data and conclusions are not consistent. Recently, tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and has been suggested involved in tendon repair. Our study aimsmore » to determine the effects of COX-2 inhibitor (celecoxib) on the proliferation and tenocytic differentiation of TDSCs. TDSCs were isolated from mice Achilles tendon and exposed to celecoxib. Cell proliferation rate was investigated at various concentrations (0.1, 1, 10 and 100 μg/ml) of celecoxib by using hemocytometer. The mRNA expression of tendon associated transcription factors, tendon associated collagens and tendon associated molecules were determined by reverse transcription-polymerase chain reaction. The protein expression of Collagen I, Collagen III, Scleraxis and Tenomodulin were determined by Western blotting. The results showed that celecoxib has no effects on TDSCs cell proliferation in various concentrations (p > 0.05). The levels of most tendon associated transcription factors, tendon associated collagens and tendon associated molecules genes expression were significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). Collagen I, Collagen III, Scleraxis and Tenomodulin protein expression were also significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). In conclusion, celecoxib inhibits tenocytic differentiation of tendon-derived stem cells but has no effects on cell proliferation.« less

Effects of mutant human Ki-ras{sup G12C} gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

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

Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.

2008-08-15

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras{sup G12C} allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 {mu}g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras{sup G12C} allele in the lung, and resulted in themore » development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 {mu}g/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 {mu}g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 {mu}g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras{sup G12C} expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.« less

Independent effects of apolipoprotein AV and apolipoprotein CIII on plasma triglyceride concentrations

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

Baroukh, Nadine N.; Bauge, Eric; Akiyama, Jennifer

2003-08-15

Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered Both the apolipoprotein A5 and C3 genes have repeatedly been shownmore » to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered triglycerides. To overcome these confounding factors and address their relationship, we generated independent lines of mice that either over-expressed (''double transgenic'') or completely lacked (''double knockout'') both apolipoprotein genes. We report that both ''double transgenic'' and ''double knockout'' mice display intermedia tetriglyceride concentrations compared to over-expression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the ''double transgenic'' mice are approximately 500-fold lower than human ApoCIII levels, supporting ApoAV is a potent triglyceride modulator despite its low concentration. Together, these data indicate that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.« less

Differential regulation of cyclo-oxygenase-2 and 5-lipoxygenase-activating protein (FLAP) expression by glucocorticoids in monocytic cells.

PubMed

Goppelt-Struebe, M; Schaefer, D; Habenicht, A J

1997-10-01

1. The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP). The human monocytic cell line THP-1 was used as a model system. mRNA and protein levels of COX-2 and FLAP were determined by Northern and Western blot analyses, respectively. 2. Low levels of COX-2 and FLAP mRNA were expressed in undifferentiated THP-1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days. 3. Coincubation of the undifferentiated cells with dexamethasone (10(-9) - 10(-6) M) and phorbol ester prevented induction of COX-2 mRNA, but did not affect the induction of FLAP mRNA. 4. Dexamethasone downregulated COX-2 mRNA and protein in differentiated, monocyte-like THP-1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP-1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5-lipoxygenase mRNA expression. 5. Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP-1 cells by ELISA. Calcium ionophore-dependent PGE2 synthesis was associated with COX-2 expression, whereas COX-1 and COX-2 seemed to participate in arachidonic acid-dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP-1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release. 6. These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid-mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5-lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.

Abnormal expression and functional characteristics of cyclic adenosine monophosphate response element binding protein in postmortem brain of suicide subjects.

PubMed

Dwivedi, Yogesh; Rao, Jagadeesh Sridhara; Rizavi, Hooriyah S; Kotowski, Jacek; Conley, Robert R; Roberts, Rosalinda C; Tamminga, Carol A; Pandey, Ghanshyam N

2003-03-01

Cyclic adenosine monophosphate response element binding protein (CREB) is a transcription factor that, on phosphorylation by protein kinases, is activated, and in response, regulates the transcription of many neuronally expressed genes. In view of the recent observations that catalytic properties and/or expression of many kinases that mediate their physiological responses through the activation of CREB are altered in the postmortem brain of subjects who commit suicide (hereafter referred to as suicide subjects), we examined the status of CREB in suicidal behavior. These studies were performed in Brodmann area (BA) 9 and hippocampus obtained from 26 suicide subjects and 20 nonpsychiatric healthy control subjects. Messenger RNA levels of CREB and neuron-specific enolase were determined in total RNA by means of quantitative reverse transcriptase-polymerase chain reaction. Protein levels and the functional characteristics of CREB were determined in nuclear fractions by means of Western blot and cyclic adenosine monophosphate response element (CRE)-DNA binding activity, respectively. In the same nuclear fraction, we determined the catalytic activity of cyclic adenosine monophosphate-stimulated protein kinase A by means of enzymatic assay. We observed a significant reduction in messenger RNA and protein levels of CREB, CRE-DNA binding activity, and basal and cyclic adenosine monophosphate-stimulated protein kinase A activity in BA 9 and hippocampus of suicide subjects, without any change in messenger RNA levels of neuron-specific enolase in BA 9. Except for protein kinase A activity, changes in CREB expression and CRE-DNA binding activity were present in all suicide subjects, irrespective of diagnosis. These changes were unrelated to postmortem intervals, age, sex, or antidepressant treatment. Given the significance of CREB in mediating various physiological functions through gene transcription, our results of decreased expression and functional characteristics of CREB in postmortem brain of suicide subjects suggest that CREB may play an important role in suicidal behavior.

Adenovirus36 infection expresses cellular APMI and Visfatin genes in overweight Uygur individuals

PubMed Central

2014-01-01

Objective This study is to determine if Adenovirus type 36 (Ad36) infection is related to macrophage infiltration in the obese group and non-obese group and the related molecular mechanisms. Methods Ninety obesity patients and 95 non-obesity Uygur individuals were enrolled in this study. CD68 levels in abdominal subcutaneous and omental adipose tissues were detected by immunohistochemistry. The cytokine expression levels of adiponectin (APMI) and visfatin in serum were measured by enzyme-linked immunosorbent assay. Infection of 3T3-L1 cells with Ad36 was performed. Real-time PCR was performed to determine expression levels of APMI and Visfatin genes in the 3T3-L1 preadipocytes infected with Ad36. Results In the obese individuals infected with Ad36, the expression levels of adiponectin and visfatin in serum was elevated. For the individuals infected with Ad36, the macrophage infiltration (as indicated by CD68 level) in the obese group was also significantly higher than that in the non-obese group (P < 0.05) in both abdominal subcutaneous and omental adipose tissues. The real-time PCR results indicated that APMI mRNA levels and Visfatin mRNA levels in Ad36 infected cells were significantly increased. Conclusions Ad36 infection may be a factor related with macrophage infiltration in adipose tissues of the obese patients. The APMI and Visfatin genes may be involved in the mechanism underlying the effect of Ad36 infection on the obese patients. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1849614638119816 PMID:24739504

Career satisfaction of Jordanian dental hygienists.

PubMed

Malkawi, Z A

2016-11-01

The aim of this study was to determine the factors that affect Jordanian dental hygienists with their career satisfaction including financial issues, employment settings and policies. Randomized sample of 102 dental hygienists with a bachelor's degree were selected from the entire population of Jordanian dental hygienists. Participants received a cover letter with a questionnaire. Findings were analysed using descriptive data techniques. Chi-square test was used to determine the statistically significant differences across demographic variables and career satisfaction's factors. About 22.5% of the participants are not working as dental hygienist. Dental hygiene profession in Jordan includes predominantly (74.0%) females. Majority of them (51.9%) were employed in JUST, and minority (6.3%) in MOH. Most of them (56.4%) were aged 24-29 years old, and mostly 62.2% with ≤1 child. About 53.1% employed by general dentist. Almost 35.3% had ≥4 years' job experience. Majority (47.6%) expressed high level of satisfaction with dental materials and equipment to practice work; however, only 2.0% expressed very high level of satisfaction with employment policies. Almost 32.4% expressed low level of satisfaction with salary level. Minority (2.0%) expressed dissatisfaction with quality of dentist's work. Statistically significant association was found between workplace, and dental materials and equipment to practice work, salary level, employment policies (P = 0.003, P = 0.003, P = 0.026), and number of children with flexibility in work hours (P = 0.001). Jordanian dental hygienists' workplacesatisfaction w as significantly associated with dental materials and equipment to practice work, salary level, and employment policies. Understanding the working patterns of dental hygienists in Jordan is important to increase their career satisfaction levels. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The level of HER2 expression is a predictor of antibody-HER2 trafficking behavior in cancer cells

PubMed Central

Ram, Sripad; Kim, Dongyoung; Ober, Raimund J; Ward, E Sally

2014-01-01

The receptor tyrosine kinase HER2 is known to play a central role in mitogenic signaling, motivating the development of targeted, HER2-specific therapies. However, despite the longstanding use of antibodies to target HER2, controversies remain concerning antibody/HER2 trafficking behavior in cancer cells. Understanding this behavior has direct relevance to the mechanism of action and effective design of such antibodies. In the current study, we analyzed the intracellular dynamics of trastuzumab, a marketed HER2-targeting antibody, in a panel of breast and prostate cancer cell lines that have a wide range of HER2 expression levels. Our results reveal distinct post-endocytic trafficking behavior of antibody-HER2 complexes in cells with different HER2 expression levels. In particular, HER2-overexpressing cells exhibit efficient HER2 recycling and limited reductions in HER2 levels upon antibody treatment, and consequently display a high level of antibody persistence on their plasma membrane. By contrast, in cells with low HER2 expression, trastuzumab treatment results in rapid antibody clearance from the plasma membrane combined with substantial decreases in HER2 levels and undetectable levels of recycling. A cell line with intermediate levels of HER2 expression exhibits both antibody recycling and clearance from the cell surface. Significantly, these analyses demonstrate that HER2 expression levels, rather than cell origin (breast or prostate), is a determinant of subcellular trafficking properties. Such studies have relevance to optimizing the design of antibodies to target HER2. PMID:25517306

Quantitative comparison of the expression of myogenic regulatory factors in flounder ( Paralichthys olivaceus) embryos and adult tissues

NASA Astrophysics Data System (ADS)

Zhang, Yuqing; Tan, Xungang; Xu, Peng; Sun, Wei; Xu, Yongli; Zhang, Peijun

2010-03-01

MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal differentiation and lineage maintenance. To better understand the function of myogenic regulatory factors in muscle development of flounder, an important economic fish in Asia, real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD, Myf5, and myogenin at early stages of embryo development, and in different tissues of the adult flounder. The results show that, Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions of Myf5, yoD, and myogenin at the early stages have a common characteristic: expression gradually increased to a peak level, and then gradually decreased to an extremely low level. In the adult flounder, the expression of the three genes in muscle is much higher than that in other tissues, indicating that they are important for muscle growth and maintenance of grown fish. During embryonic stages, the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation. When the MyoD expression is over 30% of its highest level, the muscle cells enter the differentiation stage.

Expression of lnterleukin-8 Correlates with Angiogenesis, Tumorigenicity, and Metastasis of Human Prostate Cancer Cells Implanted Orthotopically in Nude Mice1

PubMed Central

Sun, Jin Kim; Uehara, Hisanori; Karashima, Takashi; Mccarty, Marya; Shih, Nancy; Fidler, Isaiah J

2001-01-01

Abstract We determined whether the expression of interleukin-8 (IL-8) by human prostate cancer cells correlates with induction of angiogenesis, tumorigenicity, and production of metastasis. Low and high IL-8-producing clones were isolated from the heterogeneous PC-3 human prostate cancer cell line. The secretion of IL-8 protein correlated with transcriptional activity and levels of IL-8 mRNA. All PC-3 cells expressed both IL-8 receptors, CXCR1 and CXCR2. The low and high IL-8-producing clones were injected into the prostate of nude mice. Titration studies indicated that PC-3 cells expressing high levels of IL-8 were highly tumorigenic, producing rapidly growing, highly vascularized prostate tumors with and a 100% incidence of lymph node metastasis. Low IL-8-expressing PC-3 cells were less tumorigenic, producing slower growing and less vascularized primary tumors and a significantly lower incidence of metastasis. In situ hybridization (ISH) analysis of the tumors for expression of genes that regulate angiogenesis and metastasis showed that the expression level of IL-8, matrix metalloproteinases, vascular endothelial growth factor (VEGF), and E-cadherin corresponded with microvascular density and biological behavior of the prostate cancers in nude mice. Collectively, the data show that the expression level of IL-8 in human prostate cancer cells is associated with angiogenesis, tumorigenicity, and metastasis. PMID:11326314

Determination of internal controls for quantitative gene expression of Isochrysis zhangjiangensis at nitrogen stress condition

NASA Astrophysics Data System (ADS)

Wu, Shuang; Zhou, Jiannan; Cao, Xupeng; Xue, Song

2016-02-01

Isochrysis zhangjiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under nitrogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction (qPCR) provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin (ACT), α-tubulin (TUA), ß-tubulin (TUB), ubiquitin (UBI), 18S rRNA (18S) and one target gene, diacylglycerol acyltransferase (DGAT), were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed I. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experimental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expression under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of I. zhangjiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.

Expressed Emotion-Criticism and Risk of Depression Onset in Children

ERIC Educational Resources Information Center

Burkhouse, Katie L.; Uhrlass, Dorothy J.; Stone, Lindsey B.; Knopik, Valerie S.; Gibb, Brandon E.

2012-01-01

The primary goal of the current study was to examine the impact of maternal criticism (expressed emotion-criticism; EE-Crit) on the prospective development of depressive episodes in children. In addition to examining baseline levels of EE-Crit, we also sought to determine whether distinct subgroups (latent classes) of mothers could be identified…

Optimal Levels of Emotional Arousal in Experiential Therapy of Depression

ERIC Educational Resources Information Center

Carryer, Jonathan R.; Greenberg, Leslie S.

2010-01-01

Objective: To determine the relationship between length of time spent expressing highly aroused emotion and therapeutic outcome. Method: Thirty-eight clients (14 male, 24 female) between the ages of 22 and 60 years (M = 39.5, SD = 9.71), treated for depression with experiential therapy, were rated on working alliance and expressed emotional…

PAI-1 mRNA expression and plasma level in rheumatoid arthritis: relationship with 4G/5G PAI-1 polymorphism.

PubMed

Muñoz-Valle, José Francisco; Ruiz-Quezada, Sandra Luz; Oregón-Romero, Edith; Navarro-Hernández, Rosa Elena; Castañeda-Saucedo, Eduardo; De la Cruz-Mosso, Ulises; Illades-Aguiar, Berenice; Leyva-Vázquez, Marco Antonio; Castro-Alarcón, Natividad; Parra-Rojas, Isela

2012-12-01

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the synovial membrane, cartilage and bone. PAI-1 is a key regulator of the fibrinolytic system through which plasminogen is converted to plasmin. The plasmin activates the matrix metalloproteinase system, which is closely related with the joint damage and bone destruction in RA. The aim of this study was to investigate the relationship between 4G/5G PAI-1 polymorphism with mRNA expression and PAI-1 plasma protein levels in RA patients. 113 RA patients and 123 healthy subjects (HS) were included in the study. The 4G/5G PAI-1 polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism method; the PAI-1 mRNA expression was determined by real-time PCR; and the soluble PAI-1 (sPAI-1) levels were quantified using an ELISA kit. No significant differences in the genotype and allele frequencies of 4G/5G PAI-1 polymorphism were found between RA patients and HS. However, the 5G/5G genotype was the most frequent in both studied groups: RA (42%) and HS (44%). PAI-1 mRNA expression was slightly increased (0.67 fold) in RA patients with respect to HS (P = 0.0001). In addition, in RA patients, the 4G/4G genotype carriers showed increased PAI-1 mRNA expression (3.82 fold) versus 4G/5G and 5G/5G genotypes (P = 0.0001), whereas the sPAI-1 plasma levels did not show significant differences. Our results indicate that the 4G/5G PAI-1 polymorphism is not a marker of susceptibility in the Western Mexico. However, the 4G/4G genotype is associated with high PAI-1 mRNA expression but not with the sPAI-1 levels in RA patients.

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.

Circadian gene expression in peripheral blood leukocytes of rotating night shift nurses.

PubMed

Reszka, Edyta; Peplonska, Beata; Wieczorek, Edyta; Sobala, Wojciech; Bukowska, Agnieszka; Gromadzinska, Jolanta; Lie, Jenny-Anne; Kjuus, Helge; Wasowicz, Wojciech

2013-03-01

It has been hypothesized that the underlying mechanism of elevated breast cancer risk among long-term, night-working women involves circadian genes expression alteration caused by exposure to light at night and/or irregular work hours. The aim of the present study was to determine the effect of rotating night shift work on expression of selected core circadian genes. The cross-sectional study was conducted on 184 matched nurses and midwives, who currently work either day or rotating night shifts, to determine the effect of irregular work at night on circadian gene expression in peripheral blood leukocytes. Transcript levels of BMAL1, CLOCK, CRY1, CRY2, PER1, PER2, and PER3 were determined by means of quantitative real-time polymerase chain reaction (PCR). After adjusting for hour of blood collection, there were no statistically significant changes of investigated circadian genes among nurses and midwives currently working rotating night shifts compared to nurses working day shifts. The highest expression of PER1 messenger ribonucleic acid (mRNA) was observed for women currently working shifts who had worked >15 years in rotating night shift work. PER1 gene expression was associated with the lifetime duration of rotating night shift work among women currently working night shifts (P=0.04). PER1 and PER3 transcript levels in blood leukocytes were significantly down-regulated in the later versus early hours of the morning between 06.00-10.00 hours (β-coefficient -0.226, P=0.001 and β-coefficient -0.181, P<0.0001, respectively). These results suggest that current rotating night shift work does not affect circadian gene expression in human circulating leukocytes. In analysis of the peripheral clock in human studies, the hour of blood collection should be precisely specified.

MicroRNA Expression in Alpha and Beta Cells of Human Pancreatic Islets

PubMed Central

Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L.

2013-01-01

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels. In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology. PMID:23383059

MicroRNA expression in alpha and beta cells of human pancreatic islets.

PubMed

Klein, Dagmar; Misawa, Ryosuke; Bravo-Egana, Valia; Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L

2013-01-01

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

The caspase-1 inhibitor CARD18 is specifically expressed during late differentiation of keratinocytes and its expression is lost in lichen planus.

PubMed

Qin, Haihong; Jin, Jiang; Fischer, Heinz; Mildner, Michael; Gschwandtner, Maria; Mlitz, Veronika; Eckhart, Leopold; Tschachler, Erwin

2017-08-01

CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis.

PubMed

Reséndiz-Martínez, Judith; Asbun-Bojalil, Juan; Huerta-Yepez, Sara; Vega, Mario

2017-05-01

Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick‑end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1 expression. Furthermore, YY1 and Fas expression in PBMCs may be used to as prognostic markers.

Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis

PubMed Central

Reséndiz-Martínez, Judith; Asbun-Bojalil, Juan; Huerta-Yepez, Sara; Vega, Mario

2017-01-01

Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1 expression. Furthermore, YY1 and Fas expression in PBMCs may be used to as prognostic markers. PMID:28447715

Heterochronic shift in Hox-mediated activation of sonic hedgehog leads to morphological changes during fin development.

PubMed

Sakamoto, Koji; Onimaru, Koh; Munakata, Keijiro; Suda, Natsuno; Tamura, Mika; Ochi, Haruki; Tanaka, Mikiko

2009-01-01

We explored the molecular mechanisms of morphological transformations of vertebrate paired fin/limb evolution by comparative gene expression profiling and functional analyses. In this study, we focused on the temporal differences of the onset of Sonic hedgehog (Shh) expression in paired appendages among different vertebrates. In limb buds of chick and mouse, Shh expression is activated as soon as there is a morphological bud, concomitant with Hoxd10 expression. In dogfish (Scyliorhinus canicula), however, we found that Shh was transcribed late in fin development, concomitant with Hoxd13 expression. We utilized zebrafish as a model to determine whether quantitative changes in hox expression alter the timing of shh expression in pectoral fins of zebrafish embryos. We found that the temporal shift of Shh activity altered the size of endoskeletal elements in paired fins of zebrafish and dogfish. Thus, a threshold level of hox expression determines the onset of shh expression, and the subsequent heterochronic shift of Shh activity can affect the size of the fin endoskeleton. This process may have facilitated major morphological changes in paired appendages during vertebrate limb evolution.

NADPH Oxidase-Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature.

PubMed

Kida, Teruyo; Oku, Hidehiro; Horie, Taeko; Matsuo, Junko; Kobayashi, Takatoshi; Fukumoto, Masanori; Ikeda, Tsunehiko

2015-10-01

To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO. Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using real-time PCR. Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions. The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

PubMed

Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

2015-11-01

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

PD-L1 expression is associated with p16INK4A expression in non-oropharyngeal head and neck squamous cell carcinoma

PubMed Central

Chen, San-Chi; Chang, Peter Mu-Hsin; Wang, Hsiao-Jung; Tai, Shyh-Kuan; Chu, Pen-Yuan; Yang, Muh-Hwa

2018-01-01

PD-L1 expression is critical in helping tumor cells evade the immune system. However, the level of PD-L1 expression in non-oropharyngeal head and neck squamous cell carcinoma (non-OPHNSCC) and its association with patient prognosis remains unclear. A retrospective clinicopathological analysis was performed on 106 patients with non-OPHNSCC diagnosed between 2007 and 2014. In the current study, tissue arrays from paraffin-embedded non-OPHNSCC samples obtained from patients were constructed, and PD-L1 and p16INK4A expression were determined using immunohistochemistry. Systemic inflammatory factors, including C-reactive protein, serum white blood cell, neutrophil, monocyte and lymphocyte counts were also analyzed. The current study demonstrated that PD-L1 was overexpressed in 32.1% (34/106) and p16INK4A in 20.8% (22/106) of patients. The expression of PD-L1 was associated with p16INK4A expression (P<0.01) but was not associated with levels of systemic inflammatory factors. Tumor stage was determined to be a significant prognostic value (stage I/II vs. III/IV, P=0.03), however, PD-L1, p16INK4A or other clinicopathological factors were not. The current study identified an association between PD-L1 and p16INK4A expression in non-OPHNSCC. This may facilitate the development of anti-PD1/PDL1 therapies to treat patients with head and neck cancer. PMID:29434933

Hepatitis B virus inhibits the in vivo and in vitro synthesis and secretion of apolipoprotein C3.

PubMed

Zhu, Chengliang; Zhu, Hengcheng; Song, Hui; Xu, Limin; Li, Longxuan; Liu, Fang; Liu, Xinghui

2017-11-13

Hepatitis B virus (HBV) infection in the body can damage liver cells and cause disorders in blood lipid metabolism. Apolipoprotein C3 (ApoC3) plays an important role in the regulation of lipid metabolism, but no study on the HBV regulation of ApoC3 has been reported. This purpose of this study was to investigate the effect of HBV on ApoC3 expression and its regulatory mechanism. The expression levels of ApoC3 mRNA and protein in the human hepatoma cell lines HepG2 and HepG2.2.15 were determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The HepG2 cells were co-transfected with the ApoC3 gene promoter and either HBV-infected clone pHBV1.3 or its individual genes. The changes in luciferase activity were assayed. The expression levels of ApoC3 mRNA and protein were determined using RT-qPCR and Western blot. The content of ApoC3 in the supernatant of the cultured cells was determined using an enzyme-linked immunosorbent assay (ELISA). The sera were collected from 149 patients with HBV infection and 102 healthy subjects at physical examination as the normal controls. The serological levels of ApoC3 in the HBV group and the normal control group were determined using ELISA. The contents of serum triglyceride (TG) and very-low-density lipoprotein (VLDL) in the HBV patients and the normal control were determined using an automatic biochemical analyser. The expression levels of ApoC3 mRNA and protein were lower in the HepG2.2.15 cells than in the HepG2 cells. pHBV1.3 and its X gene could inhibit the activity of the ApoC3 promoter and its mRNA and protein expression. The serum levels of ApoC3, VLDL and TG were 65.39 ± 7.48 μg/ml, 1.24 ± 0.49 mmol/L, and 0.46 ± 0.10 mmol/L in the HBV patients and 41.02 ± 6.88 μg/ml, 0.76 ± 0.21 mmol/L, 0.29 ± 0.05 mmol/L in the normal controls, respectively, statistical analysis revealed significantly lower serum levels of ApoC3, VLDL and TG in HBV patients than in the normal controls (P < 0.05). HBV can inhibit the in vivo and in vitro synthesis and secretion of ApoC3.

Dysregulation of Lysyl Oxidase Expression in Lesions and Endometrium of Women With Endometriosis

PubMed Central

Ruiz, Lynnette A.; Báez-Vega, Perla M.; Ruiz, Abigail; Peterse, Daniëlle P.; Monteiro, Janice B.; Bracero, Nabal; Beauchamp, Pedro; Fazleabas, Asgerally T.; Flores, Idhaliz

2015-01-01

Lysyl oxidases (LOXs) are enzymes involved in collagen deposition, extracellular membrane remodeling, and invasive/metastatic potential. Previous studies reveal an association of LOXs and endometriosis. We aimed to identify the mechanisms activated by upregulation of lysyl oxidases (LOX) in endometriotic cells and tissues. We hypothesized that LOX plays a role in endometriosis by promoting invasiveness and epithelial to mesenchymal transition (EMT). Methods: The LOX protein expression levels were measured by immunohistochemistry in lesions and endometrium on a tissue microarray (TMA) and in endometrial biopsies from patients and controls during the window of implantation (WOI). Estradiol regulation of LOX expression was determined by quantitative polymerase chain reaction (qPCR). Proliferation, invasion, and migration assays were performed in epithelial (endometrial epithelial cell), endometrial (human endometrial stromal cell), and endometriotic cell lines (ECL and 12Z). Pathway-focused multiplex qPCR was used to determine transcriptome changes due to LOX overexpression. Results: LOX protein was differentially expressed in ovarian versus peritoneal lesions. During WOI, LOX levels were higher in luminal epithelium of patients with endometriosis-associated infertility compared to controls. Invasive epithelial cell lines expressed higher levels of LOX than noninvasive ones. Transfection of LOX into noninvasive epithelial cells increased their migration in an LOX inhibitor-sensitive manner. Overexpression of LOX did not fully induce EMT but the expression of genes related to fibrosis and extracellular matrix remodeling were dysregulated. Conclusions: This study documents that expression of LOX is differentially regulated in endometriotic lesions and endometrium. A role for LOX in mediating proliferation, migration, and invasion of endometrial and endometriotic cells was observed, which may be implicated in the establishment and progression of endometriotic lesions. PMID:25963914

Relationship between aquaporin-5 expression and saliva flow in streptozotocin-induced diabetic mice?

PubMed

Soyfoo, M S; Bolaky, N; Depoortere, I; Delporte, C

2012-07-01

To investigate the expression and distribution of AQP5 in submandibular acinar cells from sham- and streptozotocin (STZ)-treated mice in relation to the salivary flow. Mice were sham or STZ injected. Distribution of AQP5 subcellular expression in submandibular glands was determined by immunohistochemistry. AQP5 labelling indices (LI), reflecting AQP5 subcellular distribution, were determined in acinar cells. Western blotting was performed to determine the expression of AQP5 in submandibular glands. Blood glycaemia and osmolality and saliva flow rates were also determined. AQP5 immunoreactivity was primarily located at the apical and apical-basolateral membranes of submandibular gland acinar cells from sham- and STZ-treated mice. No significant differences in AQP5 protein levels were observed between sham- and STZ-treated mice. Compared to sham-treated mice, STZ-treated mice had significant increased glycaemia, while no significant differences in blood osmolality were observed. Saliva flow rate was significantly decreased in STZ-treated mice as compared to sham-treated mice. In STZ-treated mice, significant reduction in salivary flow rate was observed without any concomitant modification in AQP5 expression and localization. © 2011 John Wiley & Sons A/S.

Ocular and extraocular expression of opsins in the rhopalium of Tripedalia cystophora (Cnidaria: Cubozoa).

PubMed

Bielecki, Jan; Zaharoff, Alexander K; Leung, Nicole Y; Garm, Anders; Oakley, Todd H

2014-01-01

A growing body of work on the neuroethology of cubozoans is based largely on the capabilities of the photoreceptive tissues, and it is important to determine the molecular basis of their light sensitivity. The cubozoans rely on 24 special purpose eyes to extract specific information from a complex visual scene to guide their behavior in the habitat. The lens eyes are the most studied photoreceptive structures, and the phototransduction in the photoreceptor cells is based on light sensitive opsin molecules. Opsins are photosensitive transmembrane proteins associated with photoreceptors in eyes, and the amino acid sequence of the opsins determines the spectral properties of the photoreceptors. Here we show that two distinct opsins (Tripedalia cystophora-lens eye expressed opsin and Tripedalia cystophora-neuropil expressed opsin, or Tc-leo and Tc-neo) are expressed in the Tripedalia cystophora rhopalium. Quantitative PCR determined the level of expression of the two opsins, and we found Tc-leo to have a higher amount of expression than Tc-neo. In situ hybridization located Tc-leo expression in the retinal photoreceptors of the lens eyes where the opsin is involved in image formation. Tc-neo is expressed in a confined part of the neuropil and is probably involved in extraocular light sensation, presumably in relation to diurnal activity.

Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Single-Nucleotide Polymorphisms Identified in Clinical Samples Can Affect MicroRNA Processing, Level of Expression, and Silencing Activity

PubMed Central

Han, Soo-Jin; Marshall, Vickie; Barsov, Eugene; Quiñones, Octavio; Ray, Alex; Labo, Nazzarena; Trivett, Matthew; Ott, David; Renne, Rolf

2013-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645–659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies. PMID:24006441

Activation of hepatic Nogo-B receptor expression—A new anti-liver steatosis mechanism of statins

PubMed Central

Zhang, Wenwen; Yang, Xiaoxiao; Chen, Yuanli; Hu, Wenquan; Liu, Lipei; Zhang, Xiaomeng; Liu, Mengyang; Sun, Lei; Liu, Ying; Yu, Miao; Li, Xiaoju; Li, Luyuan; Zhu, Yan; Miao, Qing Robert; Han, Jihong; Duan, Yajun

2017-01-01

Deficiency of hepatic Nogo-B receptor (NgBR) expression activates liver X receptor α (LXRα) in an adenosine monophosphate-activated protein kinase α (AMPKα)-dependent manner, thereby inducing severe hepatic lipid accumulation and hypertriglyceridemia. Statins have been demonstrated non-cholesterol lowering effects including anti-nonalcoholic fatty liver disease (NAFLD). Herein, we investigated if the anti-NAFLD function of statins depends on activation of NgBR expression. In vivo, atorvastatin protected apoE deficient or NgBR floxed, but not hepatic NgBR deficient mice, against Western diet (WD)-increased triglyceride levels in liver and serum. In vitro, statins reduced lipid accumulation in nonsilencing small hairpin RNA-transfected (shNSi), but not in NgBR small hairpin RNA-transfected (shNgBRi) HepG2 cells. Inhibition of cellular lipid accumulation by atorvastatin is related to activation of AMPKα, and inactivation of LXRα and lipogenic genes. Statin also inhibited expression of oxysterol producing enzymes. Associated with changes of hepatic lipid levels by WD or atorvastatin, NgBR expression was inversely regulated. At cellular levels, statins increased NgBR mRNA and protein expression, and NgBR protein stability. In contrast to reduced cellular cholesterol levels by statin or β-cyclodextrin, increased cellular cholesterol levels decreased NgBR expression suggesting cholesterol or its synthesis intermediates inhibit NgBR expression. Indeed, mevalonate, geranylgeraniol or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or farnesol, blocked atorvastatin-induced NgBR expression. Furthermore, we determined that induction of hepatic NgBR expression by atorvastatin mainly depended on inactivation of extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (Akt). Taken together, our study demonstrates that statins inhibit NAFLD mainly through activation of NgBR expression. PMID:29217477

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture.

PubMed

Mauceri, Daniela; Hagenston, Anna M; Schramm, Kathrin; Weiss, Ursula; Bading, Hilmar

2015-09-18

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Influence of Tumor-Host Interactions in the Stromal Cell-Derived Factor-1/CXCR4 Ligand/Receptor Axis in Determining Metastatic Risk in Breast Cancer

PubMed Central

Hassan, Saima; Ferrario, Cristiano; Saragovi, Uri; Quenneville, Louise; Gaboury, Louis; Baccarelli, Andrea; Salvucci, Ombretta; Basik, Mark

2009-01-01

The chemokine stromal cell-derived factor-1 (SDF-1) may function to attract CXCR4-expressing cancer cells to metastatic organs. We have previously demonstrated that low plasma SDF-1, a host-derived marker, increases distant metastatic risk in breast cancer. We therefore hypothesized that tumors overexpressing the SDF-1 receptor CXCR4 have an enhanced ability to metastasize in patients with low plasma SDF-1 levels. In this study, we determined the prognostic significance of activated CXCR4, or phosphorylated CXCR4 (p-CXCR4), and CXCR7, another receptor for SDF-1. Immunohistochemistry was performed on a tissue microarray built using 237 samples from the same cohort of patients for which we measured plasma SDF-1 levels. We found that the prognostic value of p-CXCR4 expression (hazard ratio or HR, 3.95; P = 0.004) was superior to total CXCR4 expression (HR, 3.20; P = 0.03). The rate of breast cancer-specific mortality was much higher in patients with both high p-CXCR4 expression and low plasma SDF-1 levels (HR, 5.96; P < 0.001) than either low plasma SDF-1 (HR, 3.59; P = 0.01) or high p-CXCR4 expression (HR, 3.83; P = 0.005) alone. The added prognostic value of low plasma SDF-1 was only effective in patients with high p-CXCR4 expression, and as such, provides clinical validation for modulation of the metastatic potential of tumor cells by an inherent host-derived metastatic risk factor. PMID:19497995

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton's jelly stem cells (hWJSCs).

PubMed

Mostafavi-Pour, Zohreh; Ashrafi, Mohammad Reza; Talaei-Khozani, Tahereh

2018-06-01

Human Wharton's jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P < 0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.

Evaluation of the Role of -137G/C Single Nucleotide Polymorphism (rs187238) and Gene Expression Levels of the IL-18 in Patients with Coronary Artery Disease.

PubMed

Hoseini, Fatemeh; Mahmazi, Sanaz; Mahmoodi, Khalil; Jafari, Gholam Ali; Soltanpour, Mohammad Soleiman

2018-03-01

Interleukin-18 (IL-18) is a proinflammatory and proatherogenic cytokine, and its genetic variations may contribute to the development of coronary artery disease (CAD). We sought to investigate the role of -137G/C polymorphism and gene expression levels of IL-18 in patients with CAD. The study population included 100 patients with angiographically proven CAD and 100 matched controls. Total RNA and DNA were extracted from leukocytes using appropriate kits. The genotype of -137G/C polymorphism and gene expression level of IL-18 was determined using allele-specific polymerase chain reaction (PCR) and real-time (RT)-PCR assay, respectively. The genotypic and allelic distribution of IL-18 -137G/C polymorphism was not significantly different between the two groups ( p > 0.050). Moreover, the -137G/C polymorphism did not increase the risk of CAD in dominant and recessive genetic models ( p > 0.050). However, subgroup analysis of CAD patients revealed that the IL-18 -137G/C polymorphism was significantly associated with increased risk of CAD in hypertensive patients (odds ratio (OR) = 7.51; 95% confidence interval (CI): 1.24-25.17; p = 0.019) and smokers (OR = 4.90; 95% CI: 1.21-19.70; p = 0.031) but not in the diabetic subpopulation ( p = 0.261). The genotype distribution of IL-18 -137G/C genetic polymorphism was significantly different among patients with one, two, and three stenotic vessels ( p < 0.050). The gene expression level of IL-18 was significantly higher in the CAD group than the control group ( p < 0.001). Moreover, the carriers of CC genotype had significantly lower gene expression levels of IL-18 than carriers of GG genotype ( p < 0.050). The -137G/C polymorphism of IL-18 may be associated with the CAD risk in hypertensive and smoker subgroup of CAD patients. The -137G/C polymorphism seems to play an important role in determining the severity of CAD. Increased IL-18 gene expression level is a significant risk factor for the development of CAD. The CC genotype of -137G/C polymorphism is associated with lower IL-18 gene expression levels.

A Novel Role for SIRT3 in Regulating Mediators Involved in the Terminal Pathways of Human Labor and Delivery.

PubMed

Lim, Ratana; Barker, Gillian; Menon, Ramkumar; Lappas, Martha

2016-11-01

Preterm birth remains the major cause of neonatal mortality and morbidity, mediated largely by an inflammatory process. The sirtuin (SIRT) family of cellular regulators has been implicated as key inhibitors of inflammation. We have previously reported a role for SIRT1, SIRT2, and SIRT6 in regulating inflammation-induced prolabor mediators. In this study, we determined the effect of term labor and pro-inflammatory cytokines on SIRT3, SIRT4, SIRT5, and SIRT7 expression in human myometrium. Functional studies were also used to investigate the effect of small interfering RNA (siRNA) knockdown of SIRTs in regulating inflammation-induced prolabor mediators. Western blot analysis and qRT-PCR were used to determine SIRT3, SIRT4, SIRT5, and SIRT7 mRNA and protein expression in human myometrium. Small interfering RNA knockdown of SIRT3 in myometrial primary cells determined its role in response to inflammatory stimuli IL1B and TNF. SIRT3 mRNA and protein expression levels were significantly lower in term laboring myometrium compared with term nonlaboring myometrium. There was no effect of labor on SIRT4, SIRT5 or SIRT7 protein expression. The pro-inflammatory cytokines IL1B and TNF significantly decreased levels of SIRT3 mRNA and protein expression. SIRT3 knockdown by siRNA significantly augmented IL1B- and TNF-stimulated IL6, CXCL8, and CCL2 mRNA expression and release; PTGS2 mRNA expression and subsequent PGF 2alpha release; the mRNA expression and secretion of the adhesion molecule ICAM1 and the extracellular matrix remodeling enzyme MMP9; and nuclear factor kappa B1 (NFkappaB1) transcriptional activity. In human myometrium, SIRT3 expression decreases with term labor and regulates the mediators involved in the terminal effector pathways of human labor and delivery through the NFkappaB1 pathway. © 2016 by the Society for the Study of Reproduction, Inc.

Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line.

PubMed

Schelman, William R; Andres, Robert D; Sipe, Kimberly J; Kang, Evan; Weyhenmeyer, James A

2004-09-28

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of control. Taken together, these findings provide additional evidence for the reciprocal regulation of Bcl-2 and Bax expression by glutamate and suggest that neuronal excitotoxicity may, in part, result from the inverse regulation of these proteins.

The rise of soluble TWEAK levels in severely obese subjects after bariatric surgery may affect adipocyte-cytokine production induced by TNFα.

PubMed

Maymó-Masip, Elsa; Fernández-Veledo, Sonia; Garcia España, Antonio; Vázquez-Carballo, Ana; Tinahones, Francisco Jóse; García-Fuentes, Eduardo; Garrifo-Sanchez, Lourdes; Rodriguez, Maria del Mar; Vendrell, Joan; Chacón, Matilde R

2013-08-01

Soluble TNF-like weak inducer of apoptosis (sTWEAK) is generated by the intracellular proteolytic cleavage of full-length membrane-bound TNF-like weak inducer of apoptosis (mTWEAK). sTWEAK levels are reduced in diseases with an inflammatory component. Additionally, sTWEAK hampers TNFα activity in human cells. The objectives of the study were as follows: 1) to determine circulating sTWEAK in severe obesity and after bariatric surgery; 2) to study m/sTWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) protein expression in sc adipose tissue (SAT) of severely obese subjects, in SAT stromal vascular fraction (SVF), and isolated adipocytes and in human monocyte-derived macrophages; and 3) to explore, on human adipocytes, the sTWEAK effect on TNFα proinflammatory activity. sTWEAK levels were measured in cohort 1: severely obese subjects (n = 23) and a control group (n = 35); and in cohort 2: (n = 23) severely obese subjects before and after surgery. The m/sTWEAK and Fn14 expressions were determined in SAT biopsies, SVF, and isolated adipocytes from severely obese and control subjects and in human monocyte-derived macrophages. In human primary cultured adipocytes, sTWEAK pretreated and TNFα challenged, IL-6, IL-8, and adiponectin protein and gene expressions were determined and nuclear factor-κ B and MAPK signaling analyzed. sTWEAK levels were reduced in severely obese subjects. After surgery, sTWEAK levels rose in 69% of patients. mTWEAK protein expression was increased in SAT and SVF of severely obese subjects, whereas Fn14 was up-regulated in isolated adipocytes. M2 human monocyte-derived macrophages overexpress mTWEAK. In human adipocytes, sTWEAK down-regulates TNFα cytokine production by hampering TNFα intracellular signaling events. The decrease of sTWEAK in severely obese patients may favor the proinflammatory activity elicited by TNFα.

Alleviation of Acute Lung Injury in Rats with Sepsis by Resveratrol via the Phosphatidylinositol 3-Kinase/Nuclear Factor-Erythroid 2 Related Factor 2/Heme Oxygenase-1 (PI3K/Nrf2/HO-1) Pathway.

PubMed

Wang, Yu; Wang, Xiaofeng; Zhang, Lichun; Zhang, Rong

2018-05-30

BACKGROUND Resveratrol (Res) is a type of polyphenol found in many plants, which can protect important organs from the damage induced by sepsis. However, the exact mechanism of its protective effect has not been established. This study investigated the effect of Res on the PI3K/Nrf2/HO-1 signaling pathway in rats with sepsis-induced acute lung injury (ALI). MATERIAL AND METHODS Male Wistar rats were treated with 30 mg/kg Res by intraperitoneal administration for 1 hour immediately after cecal ligation and puncture. Levels of MIP-2, IL-18, and IL-10 in bronchoalveolar lavage fluid (BALF) were determined. Lung tissues were collected to measure the wet-to-dry (W/D) ratios, oxidative stress index, and lung injury scores. Expression levels of Akt, p-Akt, HO-1, Nrf-2, and active caspase-3 proteins were determined by western blotting; expression of HO-1 mRNA was determined by RT-PCR. RESULTS Treatment with Res significantly decreased the levels of MIP-2 and IL-18 and increased IL-10 in the BALF of rats with sepsis-induced ALI. In addition, Res also effectively reduced the W/D lung weight ratio, lung injury score, and the levels of MDA (malondialdehyde) and 8-OHdG. Conversely, Res increased SOD (superoxide dismutase) activity in the lung tissue. Moreover, Res significantly induced higher HO-1 mRNA expression, upregulated HO-1 and Nrf-2 protein expression, and the phosphorylation of Akt in the lung tissue. In contrast, the levels of activated caspase-3 protein were decreased in Res-treated rats (P<0.05). CONCLUSIONS Res could inhibit inflammation, oxidative stress, and cell apoptosis to alleviate ALI in septic rats through the inhibition of the PI3K/Nrf2/HO-1 signaling pathway.

[Effect of NOR1 gene knockdown on the biological behavior of HeLa cells].

PubMed

Tan, Yixin; Li, Wenjuan; Yi, Mei; Wang, Wei; Zheng, Pan; Zhang, Haijing; Xiang, Bo; Li, Guiyuan

2014-08-01

To explore the effect of the oxidored nitro domain containing protein 1 (NOR1) gene knockdown on the biological behavior of HeLa cells in cervical carcinoma. The recombinant plasmids pSUPER-shNOR1-1, pSUPER-shNOR1-2 and pSUPERscramble, which targeted to NOR1 gene, were constructed by pSUPER.neo+GFP vector, transfected into HeLa cells respectively using Lipofectamine 2000 reagent, and followed by G418 selection. The expression level of NOR1 mRNA and protein were determined by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the growth curve of cell viability. The stable transfectants were treated with H₂O₂ and cell apoptosis was determined by Hoechst 33258 staining and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. The expression levels of Bcl-2, cleaved caspase 9 and poly ADP-ribose polymerase (PARP) were measured by Western blot. NOR1- knockdown HeLa cells were successfully constructed by transfection of pSUPER-shNOR1-1 or pSUPER-shNOR1-2 plasmids into HeLa cells. MTT assay showed that the silence of endogenous NOR1 in HeLa cells could lead to the increase in cell viability and proliferation, and the inhibition of H₂O₂-induced apoptosis compared with the negative control. Western blot showed that the expression level of active caspase 9 and cleaved PARP was inhibited in NOR1-knockdown cells when they were treated with H₂O₂ while the expression level of Bcl-2 protein increased. Silence of endogenous NOR1 facilitates the cell viability and growth of HeLa cells, and attenuates HeLa cells apoptosis induced by H₂O₂, which might be mediated by up-regulation of Bcl-2 level and down-regulation of the cleaved caspase 9 cascade.

An approach to the determination of aircraft handling qualities using pilot transfer functions

NASA Technical Reports Server (NTRS)

Adams, J. J.; Hatch, H. G., Jr.

1978-01-01

It was shown that a correlation exists between pilot-aircraft system closed-loop characteristics, determined by using analytical expressions for pilot response along with the analytical expression for the aircraft response, and pilot ratings obtained in many previous flight and simulation studies. Two different levels of preferred pilot response were used. These levels were: (1) a static gain and a second-order lag function with a lag time constant of 0.2 second; and (2) a static gain, a lead time constant of 1 second, and a 0.2-second lag time constant. If a system response with a pitch-angle time constant of 2.6 seconds and a stable oscillatory mode of motion with a period of 2.5 seconds could be achieved with the first-level pilot model, it was shown that the pilot rating will be satisfactory for that vehicle.

Development-related expression patterns of protein-coding and miRNA genes involved in porcine muscle growth.

PubMed

Wang, F J; Jin, L; Guo, Y Q; Liu, R; He, M N; Li, M Z; Li, X W

2014-11-27

Muscle growth and development is associated with remarkable changes in protein-coding and microRNA (miRNA) gene expression. To determine the expression patterns of genes and miRNAs related to muscle growth and development, we measured the expression levels of 25 protein-coding and 16 miRNA genes in skeletal and cardiac muscles throughout 5 developmental stages by quantitative reverse transcription-polymerase chain reaction. The Short Time-Series Expression Miner (STEM) software clustering results showed that growth-related genes were downregulated at all developmental stages in both the psoas major and longissimus dorsi muscles, indicating their involvement in early developmental stages. Furthermore, genes related to muscle atrophy, such as forkhead box 1 and muscle ring finger, showed unregulated expression with increasing age, suggesting a decrease in protein synthesis during the later stages of skeletal muscle development. We found that development of the cardiac muscle was a complex process in which growth-related genes were highly expressed during embryonic development, but they did not show uniform postnatal expression patterns. Moreover, the expression level of miR-499, which enhances the expression of the β-myosin heavy chain, was significantly different in the psoas major and longissimus dorsi muscles, suggesting the involvement of miR-499 in the determination of skeletal muscle fiber types. We also performed correlation analyses of messenger RNA and miRNA expression. We found negative relationships between miR-486 and forkhead box 1, and miR-133a and serum response factor at all developmental stages, suggesting that forkhead box 1 and serum response factor are potential targets of miR-486 and miR-133a, respectively.

Palm is expressed in both developing and adult mouse lens and retina

PubMed Central

Castellini, Meryl; Wolf, Louise V; Chauhan, Bharesh K; Galileo, Deni S; Kilimann, Manfred W; Cvekl, Ales; Duncan, Melinda K

2005-01-01

Background Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6. Methods The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR. Results In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens. Conclusion Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation. PMID:15969763

Chitinase mRNA Levels Determined by QPCR in Crab-Eating Monkey (Macaca fascicularis) Tissues: Species-Specific Expression of Acidic Mammalian Chitinase and Chitotriosidase.

PubMed

Uehara, Maiko; Tabata, Eri; Ishii, Kazuhiro; Sawa, Akira; Ohno, Misa; Sakaguchi, Masayoshi; Matoska, Vaclav; Bauer, Peter O; Oyama, Fumitaka

2018-05-09

Mice and humans express two active chitinases: acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Both chitinases are thought to play important roles in specific pathophysiological conditions. The crab-eating monkey ( Macaca fascicularis ) is one of the most frequently used nonhuman primate models in basic and applied biomedical research. Here, we performed gene expression analysis of two chitinases in normal crab-eating monkey tissues by way of quantitative real-time polymerase chain reaction (qPCR) using a single standard DNA molecule. Levels of AMCase and CHIT1 messenger RNAs (mRNAs) were highest in the stomach and the lung, respectively, when compared to other tissues. Comparative gene expression analysis of mouse, monkey, and human using monkey⁻mouse⁻human hybrid standard DNA showed that the AMCase mRNA levels were exceptionally high in mouse and monkey stomachs while very low in the human stomach. As for the CHIT1 mRNA, we detected higher levels in the monkey lung when compared with those of mouse and human. The differences of mRNA expression between the species in the stomach tissues were basically reflecting the levels of the chitinolytic activities. These results indicate that gene expression of AMCase and CHIT1 differs between mammalian species and requiring special attention in handling data in chitinase-related studies in particular organisms.

Effects of Pax3 and Pax7 expression on muscle mass in the Pekin duck (Anas platyrhynchos domestica).

PubMed

Wang, Y; Zhang, R P; Zhao, Y M; Li, Q Q; Yan, X P; Liu, J Y; Gou, H; Li, L

2015-09-28

This study aimed to investigate whether the differential expression of muscle development-related genes is one of the reasons why muscle development differs between Pekin, Jianchang, and Heiwu ducks, which are all domesticated duck breeds (Anas platyrhynchos domestica) breeds. At 2 weeks of age, the RNA expression of paired box 7 (Pax7), paired box 3 (Pax3), myogenic differentiation antigen (MYOD), and myogenin (MYOG) genes were measured by quantitative polymerase chain reaction, and Pax3 and Pax7 protein levels were detected by western blot assay. Myofiber morphology was investigated using paraffin-embedded muscle sections. At 8 weeks of age, 30 ducks of each breed were slaughtered for meat quality determination. The results revealed that Pax3 and Pax7 expression levels at both the RNA and protein levels were high in the Pekin duck. In addition, MYOG expression levels in the Jianchang duck were significantly higher than in the other two duck breeds (P < 0.05). There were no significant differences in MYOD expression levels between the breeds (P > 0.05). Myofiber diameter and cross-sectional area were the largest in the Pekin duck and the smallest in the Heiwu duck. There were significant differences in slaughter data between these breeds, and muscle content was greatest in the Pekin duck. The results indicate that the muscle content of three different duck breeds is associated with the expression of satellite-cell marker genes.

Differential Regulation of the Ascorbic Acid Transporter SVCT2 during Development and in Response to Ascorbic Acid Depletion

PubMed Central

Meredith, M. Elizabeth; Harrison, Fiona E.; May, James M.

2011-01-01

The sodium-dependent vitamin C transporter-2 (SVCT2) is the only ascorbic acid (ASC) transporter significantly expressed in brain. It is required for life and critical during brain development to supply adequate levels of ASC. To assess SVCT2 function in the developing brain, we studied time-dependent SVCT2 mRNA and protein expression in mouse brain, using liver as a comparison tissue because it is the site of ASC synthesis. We found that SVCT2 expression followed an inverse relationship with ASC levels in the developing brain. In cortex and cerebellum, ASC levels were high throughout late embryonic stages and early post-natal stages and decreased with age, whereas SVCT2 mRNA and protein levels were low in embryos and increased with age. A different response was observed for liver, in which ASC levels and SVCT2 expression were both low throughout embryogenesis and increased post-natally. To determine whether low intracellular ASC might be capable of driving SVCT2 expression, we depleted ASC by diet in adult mice unable to synthesize ASC. We observed that SVCT2 mRNA and protein were not affected by ASC depletion in brain cortex, but SVCT2 protein expression was increased by ASC depletion in the cerebellum and liver. The results suggest that expression of the SVCT2 is differentially regulated during embryonic development and in adulthood. PMID:22001929

Determination of dietary iron requirements by full expression of iron-containing cytochrome c oxidase in the heart of broilers from 22 to 42 d of age.

PubMed

Liao, Xiudong; Ma, Chunyan; Lu, Lin; Zhang, Liyang; Luo, Xugang

2017-10-01

The present study was carried out to determine dietary Fe requirements for the full expression of Fe-containing enzyme in broilers chicks from 22 to 42 d of age. At 22 d of age, 288 Arbor Acres male chicks were randomly assigned to one of six treatments with six replicates and fed a basal maize-soyabean-meal diet (control, containing 47·0 mg Fe/kg) or the basal diet supplemented with 20, 40, 60, 80 or 100 mg Fe/kg from FeSO4.7H2O for 21 d. Regression analysis was performed to estimate the optimal dietary Fe level using quadratic models. Liver cytochrome c oxidase (Cox), heart Cox and kidney succinate dehydrogenase mRNA levels as well as heart COX activity were affected (P<0·08) by dietary Fe level, and COX mRNA level and activity in heart of broilers increased quadratically (P<0·03) as dietary Fe level increased. The estimates of dietary Fe requirements were 110 and 104 mg/kg for the full expression of Cox mRNA and for its activity in the heart of broilers, respectively. The results from this study indicate that COX mRNA level and activity in the heart are new and sensitive criteria to evaluate the dietary Fe requirements of broilers, and the dietary Fe requirements would be 104-110 mg/kg to support the full expression of COX in the heart of broiler chicks from 22 to 42 d of age, which are higher than the current National Research Council Fe requirement (80 mg/kg) of broiler chicks from 1 to 21 d or 22 to 42 d of age.

The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a.

PubMed

Carter, Javier A; Jiménez, Juan C; Zaldívar, Mercedes; Alvarez, Sergio A; Marolda, Cristina L; Valvano, Miguel A; Contreras, Inés

2009-10-01

The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded Wzz(pHS-2) proteins, respectively. In this study, genes wzzB, wzz(pHS-2) and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz(pHS-2) revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and Wzz(pHS-2) proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.

Expression and distribution of antimicrobial peptides in the skin of healthy beagles.

PubMed

Santoro, Domenico; Bunick, David; Graves, Thomas K; Campbell, Karen L

2011-02-01

Antimicrobial peptides (AMPs) are small proteins involved in defense against pathogenic organisms. Defensins and cathelicidin are the most frequently studied human AMPs. Our goals were to determine the distribution of AMPs and evaluate their mRNA expression in normal canine skin. Skin biopsies were taken from six healthy beagles. The relative transcript level of canine cathelicidin (cCath) and β-defensin (cBD)-1, cBD2 and cBD3 mRNA was quantified using quantitative real-time polymerase chain reaction. Indirect immunofluorescence (IIF), using polyclonal antibodies against cBD2, cBD3 and cCath, was used to evaluate protein localization in the skin of healthy dogs. The Pfaffl method, using experimentally determined primer efficiencies of amplification, was used to determine the expression level of cCath, cBD1 and cDB3 relative to cDB2. The levels of cCath, cBD3 and cBD1 mRNA were 358, 296 and 177 times higher than those of cBD2, respectively. Using IIF, cBD2 and cBD3 protein fluorescence was detected in all layers of the epidermis, whereas cCath was detected predominantly in the stratum granulosum and corneum. In addition, antimicrobial peptide detection was limited to the infundibular portion of the pilosebaceous units. We have validated useful methods to evaluate AMPs in canine skin. Further studies are needed to compare AMP expression in healthy dogs with that of dogs with inflammatory skin conditions. © 2010 The Authors. Journal compilation © 2010 ESVD and ACVD.

Methotrexate increases expression of cell cycle checkpoint genes via Jun-N-terminal kinase activation

PubMed Central

Spurlock, Charles F.; Tossberg, John T.; Fuchs, Howard A.; Olsen, Nancy J.; Aune, Thomas M.

2011-01-01

Objective To assess defects in expression of critical cell cycle checkpoint genes and proteins in subjects with rheumatoid arthritis relative to presence or absence of methotrexate medication and assess the role of Jun N-terminal kinase in methotrexate induction of these genes. Methods Flow cytometry analysis was used to quantify changes in intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) was employed to determine changes in cell cycle checkpoint target genes. Results RA subjects express lower baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and total JNK protein. MAPK9, TP53, CDKN1A, and CDKN1B mRNA expression, but not CHEK2, and RANGAP1, is higher in patients on low-dose MTX therapy. Further, JNK levels inversely correlate with CRP levels in RA patients. In tissue culture, MTX induces expression of both p53 and p21 by JNK2 and JNK1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 are not induced by MTX. MTX also induces ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocks these MTX-mediated effects. Conclusions Our findings support the notion that MTX restores some, but not all of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway. PMID:22183962

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

PubMed

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

2014-07-01

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

Correlation between p65 and TNF-α in patients with acute myelocytic leukemia.

PubMed

Dong, Qiao-Mei; Ling, Chun; Zhu, Jun-Fang; Chen, Xuan; Tang, Yan; Zhao, L I

2015-11-01

The correlation between the expression levels of p65 and TNF-α in patients with acute myelocytic leukemia (AML) and AML cell lines were investigated. The bone marrow samples of 30 AML patients and 10 non-leukemia controls were studied. The mRNA expression levels of p65 and TNF-α were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Pearson's Correlation test was used to demonstrate the correlation between TNF-α and p65 expression levels in AML specimens. Receiver operating characteristic (ROC) curves were plotted to determine whether TNF-α and p65 expression levels could be used to differentiate AML samples from non-leukemia samples. MG132 and anti-TNF-α antibody were used to inhibit the expression of p65 and TNF-α in the AML cell line, HL-60. The expression of p65 and TNF-α were detected by RT-qPCR and western blot analysis. The mRNA expression levels of p65 and TNF-α were significantly increased in AML patients compared with non-leukemia control bone marrow samples by RT-qPCR, and the two molecules expression pattern's exhibited sufficient predictive power to distinguish AML patients from non-leukemia control samples. Pearson's correlation analysis demonstrated that TNF-α expression was strongly correlated with p65 expression in AML bone marrow samples. In HL-60 cells, inhibition of TNF-α reduced the expression of p65; in addition, inhibition of p65 reduced the expression of TNF-α as assessed by RT-qPCR and western blot analysis. p65 and TNF-α were highly expressed in AML patients, and these 2 molecules were strongly correlated. The present study indicates that p65 and TNF-α have potential as molecular markers to distinguish AML patients from non-leukemia control samples, and that these 2 molecules may be useful prognostic factor for patients with AML.

B cell expression of the inhibitory Fc gamma receptor is unchanged in early MS.

PubMed

Comabella, Manuel; Montalban, Xavier; Kakalacheva, Kristina; Osman, Deeqa; Nimmerjahn, Falk; Tintoré, Mar; Lünemann, Jan D

2010-06-01

Expression of the inhibitory Fcgamma receptor IIB (FcgammaRIIB) has emerged as a late checkpoint during peripheral B cell development which prevents autoreactive memory B lymphocytes from becoming long-lived plasma cells. Decreased expression of FcgammaRIIB or non-functional FcgammaRIIB variants are associated with the development of autoimmune tissue inflammation. We determined the expression profile of FcgammaRIIB in peripheral blood cells in treatment-naïve patients with early MS. Twenty-five patients with clinically isolated syndrome (CIS) who converted to clinically definite MS (CDMS) and 25 demographically matched healthy donors were included in the study. Frequencies of peripheral blood monocytes and B cell subsets as well as FcgammaRIIB expression profile was determined by flow cytometry. FcgammaRIIB expression levels were higher in B cells compared to monocytes (p<0.0001) and higher in memory B cells compared to their naïve counterparts (p<0.0001). However, FcgammaRIIB expression in naïve and memory B cells as well as monocytes was unchanged in patients with early MS at onset of symptoms as well as after conversion to CDMS compared to controls. No significant correlations were found between FcgammaRIIB expression levels and brain MRI-derived metrics or EDSS progression during follow-up. These data indicate that FcgammaRIIB expression, a critical late B cell differentiation checkpoint preventing the occurrence of autoreactive long-lived plasma cells, is not impaired in treatment-naïve patients with MS, at least in the early phases of the disease. Copyright 2010 Elsevier B.V. All rights reserved.

Collagen Triple Helix Repeat Containing-1 (CTHRC1) Expression in Oral Squamous Cell Carcinoma (OSCC): Prognostic Value and Clinico-Pathological Implications

PubMed Central

Lee, Chia Ee; Vincent-Chong, Vui King; Ramanathan, Anand; Kallarakkal, Thomas George; Karen-Ng, Lee Peng; Ghani, Wan Maria Nabillah; Rahman, Zainal Ariff Abdul; Ismail, Siti Mazlipah; Abraham, Mannil Thomas; Tay, Keng Kiong; Mustafa, Wan Mahadzir Wan; Cheong, Sok Ching; Zain, Rosnah Binti

2015-01-01

BACKGROUND: Collagen Triple Helix Repeat Containing 1 (CTHRC1) is a protein often found to be over-expressed in various types of human cancers. However, correlation between CTHRC1 expression level with clinico-pathological characteristics and prognosis in oral cancer remains unclear. Therefore, this study aimed to determine mRNA and protein expression of CTHRC1 in oral squamous cell carcinoma (OSCC) and to evaluate the clinical and prognostic impact of CTHRC1 in OSCC. METHODS: In this study, mRNA and protein expression of CTHRC1 in OSCCs were determined by quantitative PCR and immunohistochemistry, respectively. The association between CTHRC1 and clinico-pathological parameters were evaluated by univariate and multivariate binary logistic regression analyses. Correlation between CTHRC1 protein expressions with survival were analysed using Kaplan-Meier and Cox regression models. RESULTS: Current study demonstrated CTHRC1 was significantly overexpressed at the mRNA level in OSCC. Univariate analyses indicated a high-expression of CTHRC1 that was significantly associated with advanced stage pTNM staging, tumour size ≥ 4 cm and positive lymph node metastasis (LNM). However, only positive LNM remained significant after adjusting with other confounder factors in multivariate logistic regression analyses. Kaplan-Meier survival analyses and Cox model demonstrated that patients with high-expression of CTHRC1 protein were associated with poor prognosis and is an independent prognostic factor in OSCC. CONCLUSION: This study indicated that over-expression of CTHRC1 potentially as an independent predictor for positive LNM and poor prognosis in OSCC. PMID:26664254

Abnormal histopathology, fat percent and hepatic apolipoprotein A I and apolipoprotein B100 mRNA expression in fatty liver hemorrhagic syndrome and their improvement by soybean lecithin.

PubMed

Song, Yalu; Ruan, Jiming; Luo, Junrong; Wang, Tiancheng; Yang, Fei; Cao, Huabin; Huang, Jianzhen; Hu, Guoliang

2017-10-01

To investigate the etiopathogenesis of fatty liver hemorrhagic syndrome (FLHS) and the protective effects of soybean lecithin against FLHS in laying hens, 135 healthy 300-day-old Hyline laying hens were randomly divided into groups: control (group 1), diseased (group 2), and protected (group 3). Each group contained 45 layers with 3 replicates. The birds in these 3 groups were fed a control diet, a high-energy/low-protein (HELP) diet or the HELP diet supplemented with 3% soybean lecithin instead of maize. The fat percent in the liver was calculated. Histopathological changes in the liver were determined by staining, and the mRNA expression levels of apolipoproteinA I (apoA I) and apolipoprotein B100 (apoB100) in the liver were determined by RT-PCR. The results showed that the fat percent in the liver of group 2 was much higher (P < 0.01) than that of group 1 and group 2 on d 30 and 60. The histology of the liver in group 2 on d 30 and 60 displayed various degrees of liver lesions, while the hepatocytes showed a normal structure in group 3 with mild microvesicular steatosis in the liver cell on d 30 and 60. The mRNA expression levels of apoA I and apoB100 in the livers were variable throughout the experiment. The expression level of apoA I in group 2 significantly decreased on d 60 (P < 0.05); the expression level of apoB100 slightly increased on d 30 in group 2, while it sharply decreased on d 60. Compared to group 1, the expression level of apoB100 showed no significant difference in group 3 (P < 0.05). This study indicated that FLHS induced pathological changes and abnormal expression of apoA I and apoB100 in the livers of laying hens and that soybean lecithin alleviated these abnormal changes. © 2017 Poultry Science Association Inc.

Characterization of the yeast copper-inducible promoter system in Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Granger, C. L.; Cyr, R. J.

2001-01-01

Inducible promoters or gene-switches are used to both spatially and temporally regulate gene expression. Such regulation can provide information concerning the function of a gene in a developmental context as well as avoid potential harmful effects due to overexpression. A gfp construct under the control of a copper-inducible promoter was introduced into Arabidopsis thaliana (L.) Heynh. and the regulatory parameters of this inducible promoter were determined. Here, we describe the time-course of up- and down-regulation of GFP expression in response to copper level, the optimal regulatory levels of copper, and the tissue specificity of expression in three transgenic lines. We conclude that the copper-inducible promoter system may be useful in regulating the time and location of gene expression in A. thaliana.

ATBF1-a messenger RNA expression is correlated with better prognosis in breast cancer.

PubMed

Zhang, Zhenhuan; Yamashita, Hiroko; Toyama, Tatsuya; Sugiura, Hiroshi; Ando, Yoshiaki; Mita, Keiko; Hamaguchi, Maho; Kawaguchi, Makoto; Miura, Yutaka; Iwase, Hirotaka

2005-01-01

The AT motif-binding factor 1 (ATBF1) gene was first identified as a suppressor of the alpha-fetoprotein (AFP) gene through its binding to an AT-rich enhancer element of this gene. The gene is located at chromosome 16q22.3-q23.1 where loss of heterozygosity has been observed in various malignant tumors, especially in breast cancer. It was also found that in highly malignant AFP-producing gastric cancer cells the expression of AFP is inhibited by ATBF1-A. This led us to hypothesize that there was a link between levels of ATBF1 expression and the metastatic potential of breast cancer and also, therefore, the prognosis of these patients. In the present study, the level of ATBF1-A mRNA expression was analyzed using quantitative real-time reverse transcriptase-PCR, in 153 female patients with invasive carcinoma of the breast. ATBF1-A protein expression was also determined by immunohistochemistry from available 90 cases of paired tissues. An association was sought between ATBF1-A expression and various clinicopathologic factors. ATBF1-A mRNA was expressed at significantly higher levels in breast cancer patients with no axillary lymph node involvement, with small tumors measuring <2 cm and in estrogen receptor-alpha-positive tumors. By contrast, no relationship was found between ATBF1-A mRNA expression and ATBF1-A protein expression, and also no relationship was found between ATBF1-A protein expression and any of the other clinicopathologic factors. Patients expressing high levels of ATBF1-A mRNA tended to have a better prognosis than those expressing low levels. Univariate and multivariate prognostic analyses showed that ATBF1-A mRNA expression is an independent prognostic factor for disease-free survival. In breast cancer, levels of ATBF1-A mRNA may serve as a predictive indicator of lymph node metastasis. The results of this study also imply that ATBF1-A gene expression may have potential both as a marker of endocrine responsiveness and also as a prognostic indicator for breast cancer progression.

Chronological gene expression of parathyroid hormone-related protein (PTHrP) in the stellate reticulum of the rat: implications for tooth eruption.

PubMed

Yao, Shaomian; Pan, Fenghui; Wise, Gary E

2007-03-01

Tooth eruption is a localized event that requires the expression of certain molecules at precise times to regulate bone resorption and bone formation. Parathyroid hormone-related protein (PTHrP) may be one of those molecules. Although PTHrP is produced in the stellate reticulum (SR) of the tooth and exerts its effect on the adjacent dental follicle, its expression pattern in the SR is unknown. Thus, it was the objectives of this study to determine the chronology of expression of PTHrP, and then to determine its effect on vascular endothelial growth factor (VEGF) expression for osteoclastogenesis and on bone morphogenetic protein-2 (BMP-2) for bone growth. Laser capture microdissection and RT-PCR were used to determine the chronological expression of PTHrP in vivo. In vitro, dental follicle cells were incubated with PTHrP and RT-PCR was conducted to determine its effect on VEGF and BMP-2 gene expression. PTHrP was maximally expressed at day 7 postnatally in the SR with the level of expression still high at day 9. In vitro, PTHrP upregulated VEGF120 and VEGF164 expression after 4h of incubation with a maximum effect at 6h. PTHrP upregulated BMP-2 gene expression with a maximal effect at 2h. Because the secondary burst of osteoclastogenesis needed for eruption occurs around day 10, it is possible that PTHrP is stimulating this osteoclastogenesis by upregulating VEGF. Concurrently, the upregulation of BMP-2 by PTHrP may stimulate bone growth at the base of the bony crypt to promote eruption.

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.

Expression and activity levels of chymase in mast cells of burn wound tissues increase during the healing process in a hamster model.

PubMed

Dong, Xianglin; Xu, Tao; Ma, Shaolin; Wen, Hao

2015-06-01

The present study aimed to investigate the changes in the expression levels and activity of mast cell chymase in the process of burn wound healing in a hamster model of deep second-degree burn. The hamster model was established by exposing a ~3 cm diameter area of bare skin to hot water (75°C) for 0, 6, 8, 10 or 12 sec. Tissue specimens were collected 24 h after burning and histological analysis revealed that hot water contact for 12 sec was required to produce a deep second-degree burn. Quantitative polymerase chain reaction and a radioimmunoassay were used to the determine changes in chymase mRNA expression levels and activity. The mRNA expression levels and activity of chymase were increased in the burn wound tissues when compared with the normal skin. However, no statistically significant differences were observed in mast cell chymase activity amongst the various post-burn stages. Chymase mRNA expression levels peaked at day 1 post-burn, subsequently decreasing at days 3 and 7 post-burn and finally increasing again at day 14 post-burn. In summary, a hamster model of deep second-degree burn can be created by bringing the skin into contact with water at 75°C for 12 sec. Furthermore, the mRNA expression levels and activity of chymase in the burn wound tissues increased when compared with those in normal skin tissues.

Expression and activity levels of chymase in mast cells of burn wound tissues increase during the healing process in a hamster model

PubMed Central

DONG, XIANGLIN; XU, TAO; MA, SHAOLIN; WEN, HAO

2015-01-01

The present study aimed to investigate the changes in the expression levels and activity of mast cell chymase in the process of burn wound healing in a hamster model of deep second-degree burn. The hamster model was established by exposing a ~3 cm diameter area of bare skin to hot water (75°C) for 0, 6, 8, 10 or 12 sec. Tissue specimens were collected 24 h after burning and histological analysis revealed that hot water contact for 12 sec was required to produce a deep second-degree burn. Quantitative polymerase chain reaction and a radioimmunoassay were used to the determine changes in chymase mRNA expression levels and activity. The mRNA expression levels and activity of chymase were increased in the burn wound tissues when compared with the normal skin. However, no statistically significant differences were observed in mast cell chymase activity amongst the various post-burn stages. Chymase mRNA expression levels peaked at day 1 post-burn, subsequently decreasing at days 3 and 7 post-burn and finally increasing again at day 14 post-burn. In summary, a hamster model of deep second-degree burn can be created by bringing the skin into contact with water at 75°C for 12 sec. Furthermore, the mRNA expression levels and activity of chymase in the burn wound tissues increased when compared with those in normal skin tissues. PMID:26136958

Elevated Expression of Immunoreceptor Tyrosine-Based Inhibitory Motif (TIGIT) on T Lymphocytes is Correlated with Disease Activity in Rheumatoid Arthritis.

PubMed

Luo, Qing; Deng, Zhen; Xu, Chuxin; Zeng, Lulu; Ye, Jianqing; Li, Xue; Guo, Yang; Huang, Zikun; Li, Junming

2017-03-10

BACKGROUND It is well known that lymphocytes play an important role in rheumatoid arthritis (RA). T cell immunoreceptors with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (TIGIT) have immunosuppressive co-stimulatory molecules that mediate inhibitory effects, but their roles in RA are poorly understood. MATERIAL AND METHODS Were recruited 76 patients with RA and 33 healthy controls (HC). Clinical manifestations, laboratory measurements, physical examination, and medical history of RA patients were recorded. The expression of TIGIT on CD3+ T lymphocytes, B lymphocytes, monocytes, neutrophils, CD3+CD4+ T lymphocytes, and CD3+CD8+ T lymphocytes was determined using flow cytometry. The expression of TIGIT on T lymphocytes in patients with RA was further analyzed to investigate its correlations with markers of autoimmune response, inflammation, and disease activity in RA. RESULTS Compared with HC, the expression levels of TIGIT on CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes were significantly increased in patients with RA (P < 0.01). The frequency of TIGIT-expressing CD3+CD4+ T lymphocytes was positively correlated with RF, increased ACPA, ESR, and CRP levels. The frequency of TIGIT-expressing CD3+CD8+ T lymphocytes was positively correlated with RF and ESR levels. Furthermore, the expression level of TIGIT on CD3+CD4+ T lymphocytes was positively correlated with the DAS28 score in RA. CONCLUSIONS The expression levels of TIGIT on T lymphocytes were elevated and correlated with disease activity in RA.

Molecular characterization of the gene feminizer in the stingless bee Melipona interrupta (Hymenoptera: Apidae) reveals association to sex and caste development.

PubMed

Brito, Diana V; Silva, Carlos Gustavo N; Hasselmann, Martin; Viana, Luciana S; Astolfi-Filho, Spartaco; Carvalho-Zilse, Gislene A

2015-11-01

In highly eusocial insects, development of reproductive traits are regulated not only by sex determination pathway, but it also depends on caste fate. The molecular basis of both mechanisms in stingless bees and possible interaction with each other is still obscure. Here, we investigate sex determination in Melipona interrupta, focusing on characterization and expression analysis of the feminizer gene (Mi-fem), and its association to a major component of caste determination, the juvenile hormone (JH). We present evidence that Mi-fem mRNA is sex-specifically spliced in which only the female splice variant encodes the full length protein, following the same principle known for other bee species. We quantified Mi-fem expression among developmental stages, sexes and castes. Mi-fem expression varies considerably throughout development, with higher expression levels in embryos. Also, fem levels in pupae and newly emerged adults were significantly higher in queens than workers and males. Finally, we ectopically applied JH in cocoon spinning larvae, which correspond to the time window where queen/worker phenotypes diverge. We observed a significantly increase in Mi-fem expression compared to control groups. Since up to 100% of females turn into queens when treated with JH (while control groups are composed mainly of workers), we propose that fem might act to regulate queens' development. Our findings provide support for the conserved regulatory function of fem in Melipona bees and demonstrate a significant correlation between key elements of sex and caste determination pathways, opening the avenue to further investigate the molecular basis of these complex traits. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dietary restriction decreases coenzyme Q and ubiquinol potentially via changes in gene expression in the model organism C. elegans.

PubMed

Fischer, Alexandra; Klapper, Maja; Onur, Simone; Menke, Thomas; Niklowitz, Petra; Döring, Frank

2015-05-06

Dietary restriction (DR) is a robust intervention that extends both health span and life span in many organisms. Ubiquinol and ubiquinone represent the reduced and oxidized forms of coenzyme Q (CoQ). CoQ plays a central role in energy metabolism and functions in several cellular processes including gene expression. Here we used the model organism Caenorhabditis elegans to determine level and redox state of CoQ and expression of genes in response to DR. We found that DR down-regulates the steady-state expression levels of several evolutionary conserved genes (i.e. coq-1) that encode key enzymes of the mevalonate and CoQ-synthesizing pathways. In line with this, DR decreases the levels of total CoQ and ubiquinol. This CoQ-reducing effect of DR is obvious in adult worms but not in L4 larvae and is also evident in the eat-2 mutant, a genetic model of DR. In conclusion, we propose that DR reduces the level of CoQ and ubiquinol via gene expression in the model organism C. elegans. © 2015 International Union of Biochemistry and Molecular Biology.

Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR

PubMed Central

Shi, Xu; Gao, Weimin; Chao, Shih-hui

2013-01-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition. PMID:23315741

Monitoring the single-cell stress response of the diatom Thalassiosira pseudonana by quantitative real-time reverse transcription-PCR.

PubMed

Shi, Xu; Gao, Weimin; Chao, Shih-hui; Zhang, Weiwen; Meldrum, Deirdre R

2013-03-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

Perivascular Delivery of Notch 1 siRNA Inhibits Injury-Induced Arterial Remodeling

PubMed Central

Redmond, Eileen M.; Liu, Weimin; Hamm, Katie; Hatch, Ekaterina; Cahill, Paul A.; Morrow, David

2014-01-01

Objectives To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling. Methods and Results Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-XL expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown. Conclusion These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA. PMID:24416200

Changes in Maternal Expressed Emotion toward Clinically Anxious Children following Cognitive Behavioral Therapy

ERIC Educational Resources Information Center

Gar, Natalie S.; Hudson, Jennifer L.

2009-01-01

The aim of this study was to determine whether maternal expressed emotion (criticism and emotional overinvolvement) decreased across treatment for childhood anxiety. Mothers of 48 clinically anxious children (aged 6-14 years) were rated on levels of criticism (CRIT) and emotional overinvolvement (EOI), as measured by a Five Minute Speech Sample…

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

PubMed

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

1996-10-01

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

Nicotine affects rat Leydig cell function in vivo and vitro via down-regulating some key steroidogenic enzyme expressions.

PubMed

Guo, Xiaoling; Wang, Huang; Wu, Xiaolong; Chen, Xianwu; Chen, Yong; Guo, Jingjing; Li, Xiaoheng; Lian, Qingquan; Ge, Ren-Shan

2017-12-01

Nicotine is consumed largely as a component of cigarettes and has a potential effect on pubertal development of Leydig cells in males. To investigate its effects, 49-day-old male Sprague Dawley rats received intraperitoneal injections of nicotine (0.5 or 1 mg/kg/day) for 2 weeks and immature Leydig cells were isolated from the testes of 35-day-old rats and treated with nicotine (0.05-50 μM). Serum hormones, Leydig cell number and related gene expression levels after in vivo treatment were determined and medium androgen levels were measured and cell cycle, apoptosis, mitochondrial membrane potential (△Ψm), and reactive oxygen species (ROS) of Leydig cells after in vitro treatment were measured. In vivo exposure to nicotine lowered serum luteinizing hormone, follicle stimulating hormone, and testosterone levels and reduced Leydig cell number and gene expression levels. Nicotine in vitro inhibited androgen production in Leydig cells by downregulating the expression levels of P450 cholesterol side cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1, and steroidogenic factor 1 at different concentration ranges. In conclusion, nicotine disrupts Leydig cell steroidogenesis during puberty possibly via down-regulating some key steroidogenic enzyme expressions. Copyright © 2017. Published by Elsevier Ltd.

Atorvastatin Protects Myocardium Against Ischemia-Reperfusion Injury Through Inhibiting miR-199a-5p.

PubMed

Zuo, YaBei; Wang, YuZhao; Hu, HaiJuan; Cui, Wei

2016-01-01

This study aimed to evaluate the protective effects of atorvastatin against myocardial ischemia/reperfusion (I/R) injury in cardiomyocytes and its possible underlying mechanism. Direct cytotoxic effect of OGD/R on cardiomyocytes with and without atorvastatin pretreatment was evaluated. Effects of atorvastatin on expression of GSK-3β and miR-199a-5p were determined using RT-PCR and Western blot. In addition, GSK-3β expression with miR-199a-5p upregulation and downregulation was detected using RT-PCR, Western blot, and immunohistochemistry. Pretreatment with atorvastatin significantly improved the recovery of cells viability from OGD/R (p<0.05). In addition, the atorvastatin pretreatment significantly increased GSK-3β expression both in mRNA level and protein level and decreased miR-199a-5p expression in mRNA level (p<0.05). Upregulation and downregulation of miR-199a-5p respectively decreased and increased GSK-3β expression both in mRNA level and protein level. These results suggested that atorvastatin provides the cardioprotective effects against I/R injury via increasing GSK-3β through inhibition of miR-199a-5p. © 2016 The Author(s) Published by S. Karger AG, Basel.

Effects of infrasound on the growth of bone marrow mesenchymal stem cells: a pilot study.

PubMed

He, Renhong; Fan, Jianzhong

2014-11-01

Poor viability of transplanted bone marrow mesenchymal stem cells (BMSCs) is well‑known, but developing methods for enhancing the viability of BMSCs requires further investigation. The aim of the present study was to elucidate the effects of infrasound on the proliferation and apoptosis of BMSCs, and to determine the association between survivin expression levels and infrasound on BMSCs. Primary BMSCs were derived from Sprague Dawley rats. The BMSCs, used at passage three, were divided into groups that received infrasound for 10, 30, 60, 90 or 120 min, and control groups, which were exposed to the air for the same durations. Infrasound was found to promote proliferation and inhibit apoptosis in BMSCs. The results indicated that 60 min was the most suitable duration for applied infrasound treatment to BMSCs. The protein and mRNA expression levels of survivin in BMSCs from the two treatment groups that received 60 min infrasound or air, were examined by immunofluorescence and quantitative polymerase chain reaction. Significant differences in survivin expression levels were identified between the two groups, as infrasound enhanced the expression levels of survivin. In conclusion, infrasound promoted proliferation and inhibited apoptosis in BMSCs, and one mechanisms responsible for the protective effects may be the increased expression levels of survivin.

Downregulation of BRAF-activated non-protein coding RNA in patients with hepatitis B virus-associated hepatocellular carcinoma.

PubMed

Zhao, Na-Na; Wang, Cheng; Lai, Cheng-Cai; Cheng, Si-Jie; Yan, Jin; Hong, Zhi-Xian; Yu, Lin-Xiang; Zhu, Zhen-Yu; Zhang, Pei-Rui; Wang, Zhao-Hai; Wang, Xi-Liang; Zhang, Shao-Geng; Yang, Peng-Hui

2018-05-01

Long non-coding RNAs (lncRNAs) have been investigated as a novel class of regulators of cellular processes, including cell growth, apoptosis and carcinogenesis. lncRNA BRAF-activated non-protein coding RNA (BANCR) has recently been revealed to be involved in tumorigenesis of numerous types of cancer, including papillary thyroid carcinoma, melanoma, non-small cell lung cancer and colorectal cancer. However, the expression profiles and biological relevance of lncRNA BANCR in hepatocellular carcinoma (HCC) has not yet been reported. In the present study, the expression level of BANCR in tumor tissues and para-cancerous tissues was determined by reverse transcription-quantitative polymerase chain reaction in patients with hepatitis B virus (HBV)-associated HCC, and its association with clinicopathological characteristics of patients was analyzed. The results demonstrated that the expression level of BANCR was significantly reduced in tumor tissues in comparison with in para-cancerous tissues (P<0.001). Furthermore, the present study demonstrated that BANCR expression level was closely associated with serum α-fetoprotein levels (P<0.01) and HCC tumor number (P<0.05). To the best of our knowledge, these results revealed for the first time that BANCR downregulated in patients with HBV-associated HCC and BANCR expression level may be a potential valuable diagnosis and therapeutic biomarker in HCC.

9 CFR 206.1 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... pricing determination included in a contract that establishes a minimum and/or maximum level of base price... premiums or discounts, expressed in dollars per unit. Contract. Any agreement, whether written or verbal... accrues a running positive or negative balance as a result of a pricing determination included in a...

Life-cycle and growth-phase-dependent regulation of the ubiquitin genes of Trypanosoma cruzi.

PubMed

Manning-Cela, Rebeca; Jaishankar, Sobha; Swindle, John

2006-07-01

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cycle that is accompanied by the stage-specific gene expression. At the molecular level, very little is known about gene regulation in trypanosomes. Complex gene organizations coupled with polycistronic transcription units make the analysis of regulated gene expression difficult in trypanosomes. The ubiquitin genes of T. cruzi are a good example of this complexity. They are organized as a single cluster containing five ubiquitin fusion (FUS) and five polyubiquitin (PUB) genes that are polycistronically transcribed but expressed differently in response to developmental and environmental changes. Gene replacements were used to study FUS and PUB gene expression at different stages of growth and at different points in the life cycle of T. cruzi. Based on the levels of reporter gene expression, it was determined that FUS1 expression was downregulated as the parasites approached stationary phase, whereas PUB12.5 polyubiquitin gene expression increased. Conversely, FUS1 expression increases when epimastigotes and amastigotes differentiate into trypomastigotes, whereas the expression of PUB12.5 decreases when epimastigotes differentiate into amastigotes and trypomastigotes. Although the level of CAT activity in logarithmic growing epimastigotes is six- to seven-fold higher when the gene was expressed from the FUS1 locus than when expressed from the PUB12.5 locus, the rate of transcription from the two loci was the same implying that post-transcriptional mechanisms play a dominant role in the regulation of gene expression.

Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro

PubMed Central

Yang, Dong-Ye; Lu, Fang-Gen; Tang, Xi-Xiang; Zhao, Shui-Ping; Ouyang, Chun-Hui; Wu, Xiao-Ping; Liu, Xiao-Wei; Wu, Xiao-Ying

2003-01-01

AIM: To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS: The full length cDNAs of both P40 and P35 subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1 (±) to construct plasmids of P (+)/IL-12, P (+)/P40 and P (-)/P35. These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P (+)/IL-12 and ASOR-PLL-P (+)/P40 + ASOR-PLL-P (-)/P35] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. RESULTS: Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P (+)/P40 and ASOR-PLL-P (-)/P35 was higher than that of ASOR-PLL-P (+)/IL-12 transfected group. CONCLUSION: The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level. PMID:12970883

aPKCλ/ι is a beneficial prognostic marker for pancreatic neoplasms.

PubMed

Kato, Shingo; Akimoto, Kazunori; Nagashima, Yoji; Ishiguro, Hitoshi; Kubota, Kensuke; Kobayashi, Noritoshi; Hosono, Kunihiro; Watanabe, Seitaro; Sekino, Yusuke; Sato, Takamitsu; Sasaki, Kazunori; Nakaigawa, Noboru; Kubota, Yoshinobu; Inayama, Yoshiaki; Endo, Itaru; Ohno, Shigeo; Maeda, Shin; Nakajima, Atsushi

2013-01-01

Pancreatic cancer is a lethal disease. Overall survival is typically 6 months from diagnosis. Determination of prognostic factors in pancreatic cancer that would allow identification of patients who could potentially benefit from aggressive treatment is important. However, until date, there are no established reliable prognostic factors for pancreatic cancer patients. Herein, we propose a beneficial biomarker which is significantly correlated with the prognosis in pancreatic cancer patients. Atypical protein kinase C λ/ι (aPKCλ/ι) is overexpressed and has been implicated in the progression of several cancers. We tested the expression levels of aPKCλ/ι in two types of pancreatic neoplasm, pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMNs), by immunohistochemistry. Examination of the aPKCλ/ι expression levels in surgically resected specimens of PDCA (n = 115) demonstrated that the expression levels of aPKCλ/ιin PDAC had prognostic implications, independent of the Tumor-Node-Metastasis classification and World Health Organization tumor grade. In the case of IPMNs (n = 46) also, the expression levels of aPKCλ/ιin IPMN were found to be of prognostic importance, independent of the World Health Organization histological grade or morphological type. Interestingly, high expression levels of aPKCλ/ι were significantly correlated with a worse histological grade (p = 0.010) and advanced stage of the tumor (p = 0.0050) in IPMN patients. These findings suggest that high expression levels of aPKCλ/ι could be involved in the malignant transformation of IPMNs. Based on these observations, we propose the expression level of aPKCλ/ι as a prognostic marker common to different types of pancreatic neoplasms. Copyright © 2013 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Piwil1 mediates meiosis during spermatogenesis in chicken.

PubMed

Xu, Lu; Chang, Guobin; Ma, Teng; Wang, Hongzhi; Chen, Jing; Li, Zhiteng; Guo, Xiaomin; Wan, Fang; Ren, Lichen; Lu, Wei; Chen, Guohong

2016-03-01

Piwil1 mediates spermatogenesis and ensures stable cell division rates in germline cells in mammals. However, the involvement of Piwil1 in poultry spermatogenesis and meiosis is poorly understood. In the present study, we used TaqMan RT-qPCR to characterize Piwil1 mRNA expression in different types of spermatogenic cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia cells (Sa), tetraploid cells (Tp), round sperm cells (Rs), mature sperm, and in PGCs treated with retinoic acid. Our results revealed that Piwil1 is differentially expressed during spermatogenesis in chicken. Compared to PGCs, SSCs, Tp, and Sa, Rs cells presented the highest Piwil1 mRNA expression levels. Retinoic acid significantly upregulated Piwil1 and Stra8 mRNA expression as well as Piwil1 levels in chicken PGCs. In addition, retinoic acid induced PGCs to progress through all the meiotic stages, eventually leading to haploid cell formation, which was determined using flow cytometry and western blot analysis. Taken together, our results showed that during spermatogenesis, Piwil1 was first expressed at low levels in germ stem cells, PGCs, and SSCs. Its expression levels increased during later meiosis stages. Finally, no expression was detected in mature sperm after meiosis. Treatment of PGCs with retinoic acid further demonstrated that Piwil1 plays a key role in meiosis during chicken spermatogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.

PubMed

Wert, S E; Glasser, S W; Korfhagen, T R; Whitsett, J A

1993-04-01

Transgenic animals bearing a chimeric gene containing 5'-flanking regions of the human surfactant protein C (SP-C) gene ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene were analyzed by in situ hybridization histochemistry to determine the temporal and spatial distribution of transgene expression during organogenesis of the murine lung. Ontogenic expression of the SP-C-CAT gene was compared to that of the endogenous SP-C gene and to the Clara cell CC10 gene. High levels of SP-C-CAT expression were observed as early as Day 10 of gestation in epithelial cells of the primordial lung buds. Low levels of endogenous SP-C mRNA were detected a day later, but only in the more distal epithelial cells of the newly formed, primitive, lobar bronchi. On Gestational Days 13 through 16, transcripts for both the endogenous and chimeric gene were restricted to distal epithelial elements of the branching bronchial tubules and were no longer detected in the more proximal regions of the bronchial tree. Although high levels of SP-C-CAT expression were maintained throughout organogenesis, endogenous SP-C expression increased dramatically on Gestational Day 15, coincident with acinar tubule differentiation at the lung periphery. Low levels of endogenous CC10 expression were detected by Gestational Day 16 in both lobar and segmental bronchi. By the time of birth, CC10 transcripts were expressed at high levels in the trachea and at all levels of the bronchial tree; endogenous SP-C mRNA was restricted to epithelial cells of the terminal alveolar saccules; and SP-C-CAT expression was now detected in both alveolar and bronchiolar epithelial cells. These results indicate that (1) cis-acting regulatory elements of the human SP-C gene can direct high levels of foreign gene expression to epithelial cells of the embryonic mouse lung; (2) expression of the human SP-C-CAT chimeric gene is developmentally regulated, exhibiting a morphogenic expression pattern similar, but not identical, to that of the endogenous murine SP-C gene; (3) the embryonic expression of endogenous SP-C and chimeric SP-C-CAT transcripts identifies progenitor cells of the distal respiratory epithelium; and (4) differentiation of bronchial epithelium is coincident with loss of SP-C expression and subsequent acquisition of CC10 expression in proximal regions of the developing bronchial tubules.

Application of anti-listerial bacteriocins: monitoring enterocin expression by multiplex relative reverse transcription-PCR.

PubMed

Williams, D Ross; Chanos, Panagiotis

2012-12-01

Listeriosis is a deadly food-borne disease, and its incidence may be limited through the biotechnological exploitation of a number of anti-listerial biocontrol agents. The most widely used of these agents are bacteriocins and the Class II enterocins are characterized by their activity against Listeria. Enterocins are primarily produced by enterococci, particularly Enterococcus faecium and many strains have been described, often encoding multiple bacteriocins. The use of these strains in food will require that they are free of virulence functions and that they exhibit a high level expression of anti-listerial enterocins in fermentation conditions. Multiplex relative RT (reverse transcription)-PCR is a technique that is useful in the discovery of advantageous expression characteristics among enterocin-producing strains. It allows the levels of individual enterocin gene expression to be monitored and determination of how expression is altered under different growth conditions.

Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells.

PubMed

Ugusman, Azizah; Zakaria, Zaiton; Hui, Chua Kien; Nordin, Nor Anita Megat Mohd

2010-07-01

Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). HUVECS WERE DIVIDED INTO FOUR GROUPS: control, treatment with 180 microM hydrogen peroxide (H(2)O(2)), treatment with 150 microg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H(2)O(2) for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.

Expression of hsp 27, hsp 60, hsc 70, and hsp 70 stress response genes in cultured human urothelial cells (UROtsa) exposed to lethal and sublethal concentrations of sodium arsenite.

PubMed

Rossi, Michael R; Somji, Seema; Garrett, Scott H; Sens, Mary Ann; Nath, Joginder; Sens, Donald A

2002-12-01

The stress response is one mechanism that the bladder urothelium could potentially employ to protect itself from cellular damage after exposure to arsenic and, in so doing, influence the shape of the dose-response curve at low concentrations of exposure to this environmental pollutant. In the present study, we used the cultured human urothelial cell line UROtsa, a model of human urothelium, to determine the expression of heat shock proteins hsp 27, hsp 60, hsc 70, and hsp 70 after acute and extended exposure of the cells to lethal and sublethal levels of sodium arsenite (NaAsO2). Acute exposure was modeled by exposing confluent cultures of UROtsa cells to 100 micro M NaAsO2 for 4 hr followed by a 48-hr recovery period. Extended exposure was modeled by exposing confluent UROtsa cells to 1, 4, and 8 micro M NaAsO2 for 16 days, with the highest concentration producing cell death by 4 days of exposure. The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined by reverse-transcription polymerase chain reaction and Western analysis. Cell viability was determined by the MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The results demonstrated that the expression of hsp 27, hsp 60, and hsc 70 mRNA and protein were not consistently increased by either acute or extended exposure to NaAsO2. In contrast, hsp 70 expression was induced by NaAsO2 after both acute and extended exposure. The degree and duration of the induction of the hsp 70 protein in the extended time course of exposure to NaAsO2 correlated directly with UROtsa cell cytotoxicity. The substantial level of basal expression of hsp 27, hsp 60, and hsc 70 shown previously in human bladder urothelium, coupled with the inducible expression of hsp 70, could provide the human urothelium with a mechanism to withstand and recover from a low level of arsenite exposure.

Sex difference in EGFR pathways in mouse kidney-potential impact on the immune system.

PubMed

Liu, Fengxia; Jiao, Yan; Jiao, Yun; Garcia-Godoy, Franklin; Gu, Weikuan; Liu, Qingyi

2016-11-24

Epidermal growth factor receptor (Egfr) has been the target of several drugs for cancers. The potential gender differences in genes in the Egfr axis have been suggested in humans and in animal models. Female and male mice from the same recombinant inbred (RI) strain have the same genomic components except the sex difference. A population of different RI mouse strains allows to conduct precise analysis of molecular pathways and regulation of Egfr between female and male mice. The whole genome expression profiles of 70 genetically diverse RI strains of mice were used to compare three major molecular aspects of Egfr gene: the relative expression levels, gene network and expression quantitative trait loci (eQTL) that regulate the expression of Egfr between female and male mice. Our data showed that there is a significant sex difference in the expression levels in kidney. A considerable number of genes in the gene network of Egfr are sex differentially expressed. The expression levels of Egfr in mice are statistical significant different between C57BL/6 J (B6) and DBA/2 J (D2) genotypes in male while no difference in female mice. The eQTLs that regulate the expression levels of Egfr between female and male mice are also different. Furthermore, the differential expression levels of Egfr showed significantly different correlations with two known biological traits between male and female mice. Overall there is a substantial sex difference in the Egfr pathways in mice. These data may have significant impact on drug target design, development, formulation, and dosage determinant for women and men in clinical trials.

TLR-4 polymorphisms and leukocyte TLR-4 expression in febrile UTI and renal scarring.

PubMed

Bayram, Meral Torun; Soylu, Alper; Ateş, Halil; Kızıldağ, Sefa; Kavukçu, Salih

2013-09-01

In this study, we aimed to determine the relation of TLR-4 Asp299Gly and Thr399Ile polymorphisms and monocyte/neutrophil TLR-4 expression to febrile urinary tract infection (UTI) and renal scar development in children. The study was performed in children with a history of febrile UTI. Patients with and without renal scarring were classified as group 1 and group 2, respectively, while the control cases in our previous study were used as the control group (group 3). All three groups were compared for the rate of TLR-4 Asp299Gly and Thr399Ile polymorphisms, and for basal and lipopolysaccharide-stimulated monocyte/neutrophil TLR-4 expression levels. There were 168 patients (86 in group 1, 82 in group 2) and 120 control cases. Monocyte/neutrophil TLR-4 expression levels were similar in groups 1 and 2. However, both groups had lower TLR-4 expression than group 3. The rate of TLR-4 Asp299Gly polymorphism was not different in all groups. TLR-4 Thr399Ile polymorphism was higher in groups 1 and 2 than in group 3 (14.0, 12.2, and 2.0 %, respectively), while group 1 and group 2 were not different. Furthermore, monocyte TLR-4 expression level was lower in those having TLR-4 Thr399Ile polymorphism than in those without this polymorphism. Patients with febrile UTI had more frequent TLR-4 Thr399Ile polymorphism and lower monocyte/neutrophil TLR-4 expression. These findings indicate that children carrying TLR-4 Thr399Ile polymorphism and/or having low level of monocyte/neutrophil TLR-4 expression have a tendency to develop febrile UTI. However, we could not show the association of TLR-4 polymorphisms and of TLR-4 expression level to renal scarring.

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

PubMed

Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

2017-01-01

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

FOXP3 expression is modulated by TGF-β1/NOTCH1 pathway in human melanoma

PubMed Central

Skarmoutsou, Eva; Bevelacqua, Valentina; D'Amico, Fabio; Russo, Angela; Spandidos, Demetrios A.; Scalisi, Aurora

2018-01-01

Forkhead box protein 3 (FOXP3) transcription factor is expressed by immune cells and several human cancers and is associated with tumor aggressiveness and unfavorable clinical outcomes. NOTCH and transforming growth factor-β (TGF-β) protumorigenic effects are mediated by FOXP3 expression in several cancer models; however, their interaction and role in melanoma is unknown. We investigated TGF-β-induced FOXP3 gene expression during NOTCH1 signaling inactivation. Primary (WM35) and metastatic melanoma (A375 and A2058) cell lines and normal melanocytes (NHEM) were used. FOXP3 subcellular distribution was evaluated by immuno cytochemical analysis. Gene expression levels were assessed by reverse transcription-quantitative polymerase chain reaction. Protein levels were assessed by western blot analysis. The γ-secretase inhibitor (GSI) was used for NOTCH1 inhibition and recombinant human (rh)TGF-β was used for melanoma cell stimulation. Cell proliferation and viability were respectively assessed by MTT and Trypan blue dye assays. FOXP3 mRNA and protein levels were progressively higher in WM35, A375 and A2058 cell lines compared to NHEM and their levels were further increased after stimulation with rh-TGF-β. TGF-β-mediated FOXP3 expression was mediated by NOTCH1 signaling. Inhibition of NOTCH1 with concomitant rh-TGF-β stimulation determined the reduction in gene expression and protein level of FOXP3. Finally, melanoma cell line proliferation and viability were reduced by NOTCH1 inhibition. The results show that nn increase in FOXP3 expression in metastatic melanoma cell lines is a potential marker of tumor aggressiveness and metastasis. NOTCH1 is a central mediator of TGF-β-mediated FOXP3 expression and NOTCH1 inhibition produces a significant reduction of melanoma cell proliferation and viability. PMID:29620159

Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds.

PubMed

Murfett, J; McClure, B A

1998-06-01

Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.

Expression level of miRNAs on chromosome 14q32.31 region correlates with tumor aggressiveness and survival of glioblastoma patients.

PubMed

Shahar, Tal; Granit, Avital; Zrihan, Daniel; Canello, Tamar; Charbit, Hanna; Einstein, Ofira; Rozovski, Uri; Elgavish, Sharona; Ram, Zvi; Siegal, Tali; Lavon, Iris

2016-12-01

The 54 microRNAs (miRNAs) within the DLK-DIO3 genomic region on chromosome 14q32.31 (cluster-14-miRNAs) are organized into sub-clusters 14A and 14B. These miRNAs are downregulated in glioblastomas and might have a tumor suppressive role. Any association between the expression levels of cluster-14-miRNAs with overall survival (OS) is undetermined. We randomly selected miR-433, belonging to sub-cluster 14A and miR-323a-3p and miR-369-3p, belonging to sub-cluster 14B, and assessed their role in glioblastomas in vitro and in vivo. We also determined the expression level of cluster-14-miRNAs in 27 patients with newly diagnosed glioblastoma, and analyzed the association between their level of expression and OS. Overexpression of miR-323a-3p and miR-369-3p, but not miR-433, in glioblastoma cells inhibited their proliferation and migration in vitro. Mice implanted with glioblastoma cells overexpressing miR323a-3p and miR369-3p, but not miR433, exhibited prolonged survival compared to controls (P = .003). Bioinformatics analysis identified 13 putative target genes of cluster-14-miRNAs, and real-time RT-PCR validated these findings. Pathway analysis of the putative target genes identified neuregulin as the most enriched pathway. The expression level of cluster-14-miRNAs correlated with patients' OS. The median OS was 8.5 months for patients with low expression levels and 52.7 months for patients with high expression levels (HR 0.34; 95 % CI 0.12-0.59, P = .003). The expression level of cluster-14-miRNAs correlates directly with OS, suggesting a role for this cluster in promoting aggressive behavior of glioblastoma, possibly through ErBb/neuregulin signaling.

Effects of massage on the expression of proangiogenic markers in rat skin.

PubMed

Ratajczak-Wielgomas, Katarzyna; Kassolik, Krzysztof; Grzegrzolka, Jedrzej; Halski, Tomasz; Piotrowska, Aleksandra; Mieszala, Katarzyna; Wilk, Iwona; Podhorska-Okolow, Marzenna; Dziegiel, Piotr; Andrzejewski, Waldemar

2018-05-17

Massage is a physiotherapeutic treatment, commonly used in both therapy and restoration of normal body functions. The aim of this work was to determine the effects of skin massage on stimulating the expression of angiogenesis-initiating factors, i.e. VEGF-A, FGF-2 (bFGF) and CD34 and on skin regeneration processes. The study was conducted on 48 Buffalo strain rats, randomly divided into two groups. In the first group (M, the massaged group), massage was applied five times a week for 7 weeks. In the second study group (C, the control group), the massage was omitted. Massage consisted of spiral movements at the plantar surface of skin for 5 min on each rear extremity. The gene expression of proangiogenic factors, including VEGF-A, FGF-2, CD34 at the mRNA level was determined using real-time PCR. Immunohistochemistry was performed on paraffin sections of rat skin to determine VEGF-A, FGF-2 CD34 and Ki-67expression. An increase in mRNA expression in the skin of the rat's rear extremity for VEGF-A and FGF-2 in the first week of the experiment was shown in the M group compared with the control rats. The upregulation of CD34 mRNA expression was also observed in the M group. We observed positive correlations between VEGF-A mRNA expression and the expression of mRNA for FGF-2 and CD34, as well as correlation between the expression of mRNA for FGF-2 and CD34. The immunohistochemical expression of VEGF-A, FGF-2 and CD34 was at a much lower level in the skin of control rats relative to the skin of massaged animals. Moreover, significantly higher immunoreactivity was shown for nuclear protein Ki-67 in epidermal cells in the M group compared with the C group. Rat skin massage increased the expression of the main angiogenesis-stimulating factors and the proliferative activity of epidermal cells, which can stimulate skin regeneration and tissue repairing processes.

Insulinlike growth factor receptor type 1 and type 2 are downregulated in the nitrofen-induced hypoplastic lung.

PubMed

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

2010-06-01

In congenital diaphragmatic hernia (CDH), high mortality rates are attributed to severe pulmonary hypoplasia. The insulinlike growth factor receptor type 1 (IGF-1R) and type 2 (IGF-2R) play a critical role in the alveologenesis during lung development. The IGF-1R null mutation mice die after birth because of respiratory failure. The IGF-2R knockout mice showed retarded lungs with poorly formed alveoli. We hypothesized that IGF-1R and IGF-2R gene expression levels are downregulated in the nitrofen-induced CDH model. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9.5 (D9.5) of gestation. Fetuses were harvested on D18 and D21 and divided into control and nitrofen groups. Relative messenger RNA (mRNA) levels of IGF-1R and IGF-2R were determined using real time reverse transcription polymerase chain reaction. Immunohistochemistry was performed to determine protein expression. Relative levels of IGF-1R mRNA were significantly decreased in the nitrofen group (2.91 +/- 0.81) on D21 compared to controls (5.29 +/- 2.59) (P < .05). Expression levels of IGF-2R mRNA on D21 were also significantly decreased in nitrofen group (1.76 +/- 0.49) compared to controls (3.59 +/- 2.45) (P < .05). Immunohistochemistry performed on D21 showed decreased IGF-1R and also IGF-2R expression in nitrofen group. Downregulation of IGF-1R and IGF-2R gene expression may interfere with normal alveologenesis causing pulmonary hypoplasia in the nitrofen-induced CDH model. Copyright 2010 Elsevier Inc. All rights reserved.

Involvement of neuropeptide Y Y1 receptors in the regulation of neuroendocrine corticotropin-releasing hormone neuronal activity.

PubMed

Dimitrov, Eugene L; DeJoseph, M Regina; Brownfield, Mark S; Urban, Janice H

2007-08-01

The neuroendocrine parvocellular CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus are the main integrators of neural inputs that initiate hypothalamic-pituitary-adrenal (HPA) axis activation. Neuropeptide Y (NPY) expression is prominent within the PVN, and previous reports indicated that NPY stimulates CRH mRNA levels. The purpose of these studies was to examine the participation of NPY receptors in HPA axis activation and determine whether neuroendocrine CRH neurons express NPY receptor immunoreactivity. Infusion of 0.5 nmol NPY into the third ventricle increased plasma corticosterone levels in conscious rats, with the peak of hormone levels occurring 30 min after injection. This increase was prevented by pretreatment with the Y1 receptor antagonist BIBP3226. Immunohistochemistry showed that CRH-immunoreactive neurons coexpressed Y1 receptor immunoreactivity (Y1r-ir) in the PVN, and a majority of these neurons (88.8%) were neuroendocrine as determined by ip injections of FluoroGold. Bilateral infusion of the Y1/Y5 agonist, [leu(31)pro(34)]NPY (110 pmol), into the PVN increased c-Fos and phosphorylated cAMP response element-binding protein expression and elevated plasma corticosterone levels. Increased expression of c-Fos and phosphorylated cAMP response element-binding protein was observed in populations of CRH/Y1r-ir cells. The current findings present a comprehensive study of NPY Y1 receptor distribution and activation with respect to CRH neurons in the PVN. The expression of NPY Y1r-ir by neuroendocrine CRH cells suggests that alterations in NPY release and subsequent activation of NPY Y1 receptors plays an important role in the regulation of the HPA.

Oral human β-defensin 2 in HIV-infected subjects with long-term use of antiretroviral therapy

PubMed Central

Nittayananta, Wipawee; Kemapunmanus, Marisa; Amornthatree, Kornthip; Talungchit, Sineepat; Sriplung, Hutcha

2012-01-01

BACKGROUND The objectives of this study were to determine 1) oral hBD2 expression in HIV-infected subjects compared to non-HIV controls, 2) the expression of oral hBD2 in HIV-infected subjects with ART compared with those without ART, and 3) factors associated with the expression of oral hBD2. METHODS Oral examination and punched biopsy on buccal mucosa were performed in HIV-infected subjects with and without ART, and non-HIV individuals. The expression of hBD2 mRNA was determined by quantitative real-time PCR. Saliva samples of both un-stimulated and stimulated saliva were collected and analyzed for hBD2 levels using ELISA. Student’s t-test and nonparametric multi-way ANOVA test were used for comparison of measurements between or among groups. RESULTS One hundred and fifty-seven HIV-infected subjects were enrolled; 99 on ART (age range 23–57 yr, mean 39 yr), 58 not on ART (age range 20–59 yr, mean 34 yr), and 50 non-HIV controls (age range 19–59 yr, mean 36 yr). The most common ART regimen was 2 NRTIs+1 NNRTI. Salivary levels of hBD2 were significantly increased in HIV infection (p< 0.001). The levels of hBD2 in stimulated saliva were also found to be significantly different between HIV-infected subjects who were and were not on ART (p< 0.001). No significant difference was observed with the expression of hBD2 mRNA. CONCLUSION Oral innate immunity is affected by HIV infection and use of ART. Salivary hBD2 levels may be the useful biomarkers to monitor those on long-term ART who are at risk of developing oral infections and malignant transformation. PMID:22680235

Methylparaben induces malformations and alterations on apoptosis, oxidant-antioxidant status, ccnd1 and myca expressions in zebrafish embryos.

PubMed

Ateş, Perihan Seda; Ünal, İsmail; Üstündağ, Ünsal Veli; Alturfan, Ahmet Ata; Yiğitbaşı, Türkan; Emekli-Alturfan, Ebru

2018-03-01

Methylparabens (MP) are widely used as preservatives in cosmetics, pharmacy, and food industry. Although acute toxicity studies in animals indicated that parabens are not significantly toxic, the effects of chronic exposure under sublethal doses are still unknown and the number of related studies is limited. Our aim was to evaluate the effects of MP on the development of zebrafish embryos focusing on development, locomotor activity, oxidant-antioxidant status, apoptosis, and ccnd1 and myca expressions. The expressions of ccnd1 and myca were determined by RT-PCR. Lipid peroxidation (LPO), nitric oxide (NO), and glutathione-S-transferase (GST) activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Locomotor activity was measured using touch-evoked movement test. MP exposure increased malformations, LPO, apoptosis, ccnd1 and myca expressions, and decreased GST activities and NO levels compared with the control group. Our findings will lead to further understanding of the mechanism of MP toxicity, and merit further research. © 2018 Wiley Periodicals, Inc.

Oncogenic miR-181a/b affect the DNA damage response in aggressive breast cancer.

PubMed

Bisso, Andrea; Faleschini, Michela; Zampa, Federico; Capaci, Valeria; De Santa, Jacopo; Santarpia, Libero; Piazza, Silvano; Cappelletti, Vera; Daidone, Mariagrazia; Agami, Reuven; Del Sal, Giannino

2013-06-01

Breast cancer is a heterogeneous tumor type characterized by a complex spectrum of molecular aberrations, resulting in a diverse array of malignant features and clinical outcomes. Deciphering the molecular mechanisms that fuel breast cancer development and act as determinants of aggressiveness is a primary need to improve patient management. Among other alterations, aberrant expression of microRNAs has been found in breast cancer and other human tumors, where they act as either oncogenes or tumor suppressors by virtue of their ability to finely modulate gene expression at the post-transcriptional level. In this study, we describe a new role for miR-181a/b as negative regulators of the DNA damage response in breast cancer, impacting on the expression and activity of the stress-sensor kinase ataxia telangiectasia mutated (ATM). We report that miR-181a and miR-181b were overexpressed in more aggressive breast cancers, and their expression correlates inversely with ATM levels. Moreover we demonstrate that deregulated expression of miR-181a/b determines the sensitivity of triple-negative breast cancer cells to the poly-ADP-ribose-polymerase1 (PARP1) inhibition. These evidences suggest that monitoring the expression of miR-181a/b could be helpful in tailoring more effective treatments based on inhibition of PARP1 in breast and other tumor types.

The inhibitor of kappa B kinase-epsilon regulates MMP-3 expression levels and can promote lung metastasis.

PubMed

Seccareccia, E; Pinard, M; Wang, N; Li, S; Burnier, J; Dankort, D; Brodt, P

2014-08-18

The factors that determine the ability of metastatic tumor cells to expand and grow in specific secondary site(s) are not yet fully understood. Matrix metalloproteinases (MMP) were identified as potential regulators of the site-specificity of metastasis. We found that lung carcinoma cells ectopically expressing high levels of the receptor for the type I insulin like growth factor receptor (M27(R) cells) had a significant reduction in MMP-3 expression levels and this coincided with reduced metastasis to the lung. We used these cells to further investigate signaling pathways regulating MMP-3 expression and the role that MMP-3 plays in lung metastasis. We show that ectopic IκB kinase ɛ (IKKɛ) expression in these cells partly restored MMP-3 expression levels and also sensitized MMP-3 transcription to induction by phorbol 12-myristate 13-acetate (PMA). This increase in MMP-3 production was due to increased activation of several signal transduction mediators, including protein kinase C alpha, ERK2, Akt and the transcription factor p65. Furthermore, reconstitution of MMP-3 expression in M27(R) cells restored their ability to colonize the lung whereas silencing of MMP-3 in M27 cells reduced metastases. Collectively, our results implicate IKKɛ as a central regulator of PMA-induced cell signaling and MMP-3 expression and identify MMP-3 as an enabler of tumor cell expansion in the lung.Oncogenesis (2014) 3, e116; doi:10.1038/oncsis.2014.28; published online 18 August 2014.

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

PubMed

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

2014-02-01

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

SRC-like adaptor protein regulates B cell development and function.

PubMed

Dragone, Leonard L; Myers, Margaret D; White, Carmen; Sosinowski, Tomasz; Weiss, Arthur

2006-01-01

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

[Transcription factors NF-kB, HIF-1, HIF-2, growth factor VEGF, VEGFR2 and carboanhydrase IX mRNA and protein level in the development of kidney cancer metastasis].

PubMed

Spirina, L V; Usynin, Y A; Yurmazov, Z A; Slonimskaya, E M; Kolegova, E S; Kondakova, I V

2017-01-01

Here, we have investigated the participation of nuclear factors NF-kB, HIF-1 and HIF-2, VEGF, VEGFR2, and carboanhydrase IX in clear-cell renal cancer. We have determined the expression and protein level of transcription factors, VEGF, VEGFR2, and carboanhydrase IX in tumor and normal tissues of 30 patients with kidney cancer. The Real-Time PCR and ELISA were used in the study. The low levels of HIF-1 mRNA expression associated with high levels of HIF-1 protein were also associated with metastasis. The expression levels of VEGF, VEGFR2, and their protein levels are increased in primary tumors of patients with disseminated kidney cancer compared to nonmetastatic cancer. No correlation was revealed between the content of mRNA and encoded proteins in the kidney cancer tissues. The changes in the ratios of mRNA levels and the respective proteins (HIF-1α, HIF-2, NF-kB, VEGF, VEGFR2, and carboanhydrase IX) may contribute to kidney-cancer metastasis.

Hybrid assemblies of ATP-sensitive K+ channels determine their muscle-type-dependent biophysical and pharmacological properties.

PubMed

Tricarico, Domenico; Mele, Antonietta; Lundquist, Andrew L; Desai, Reshma R; George, Alfred L; Conte Camerino, Diana

2006-01-24

ATP-sensitive K(+) channels (K(ATP)) are an octameric complex of inwardly rectifying K(+) channels (Kir6.1 and Kir6.2) and sulfonylurea receptors (SUR1 and SUR2A/B), which are involved in several diseases. The tissue-selective expression of the subunits leads to different channels; however, the composition and role of the functional channel in native muscle fibers is not known. In this article, the properties of K(ATP) channels of fast-twitch and slow-twitch muscles were compared by combining patch-clamp experiments with measurements of gene expression. We found that the density of K(ATP) currents/area was muscle-type specific, being higher in fast-twitch muscles compared with the slow-twitch muscle. The density of K(ATP) currents/area was correlated with the level of Kir6.2 expression. SUR2A was the most abundant subunit expressed in all muscles, whereas the vascular SUR2B subunit was expressed but at lower levels. A significant expression of the pancreatic SUR1 was also found in fast-twitch muscles. Pharmacological experiments showed that the channel response to the SUR1 agonist diazoxide, SUR2A/B agonist cromakalim, SUR1 antagonist tolbutamide, and the SUR1/SUR2A/B-antagonist glibenclamide matched the SURs expression pattern. Muscle-specific K(ATP) subunit compositions contribute to the physiological performance of different muscle fiber types and determine the pharmacological actions of drugs modulating K(ATP) activity in muscle diseases.

Transcriptome Profile Analysis from Different Sex Types of Ginkgo biloba L.

PubMed

Du, Shuhui; Sang, Yalin; Liu, Xiaojing; Xing, Shiyan; Li, Jihong; Tang, Haixia; Sun, Limin

2016-01-01

In plants, sex determination is a comprehensive process of correlated events, which involves genes that are differentially and/or specifically expressed in distinct developmental phases. Exploring gene expression profiles from different sex types will contribute to fully understanding sex determination in plants. In this study, we conducted RNA-sequencing of female and male buds (FB and MB) as well as ovulate strobilus and staminate strobilus (OS and SS) of Ginkgo biloba to gain insights into the genes potentially related to sex determination in this species. Approximately 60 Gb of clean reads were obtained from eight cDNA libraries. De novo assembly of the clean reads generated 108,307 unigenes with an average length of 796 bp. Among these unigenes, 51,953 (47.97%) had at least one significant match with a gene sequence in the public databases searched. A total of 4709 and 9802 differentially expressed genes (DEGs) were identified in MB vs. FB and SS vs. OS, respectively. Genes involved in plant hormone signal and transduction as well as those encoding DNA methyltransferase were found to be differentially expressed between different sex types. Their potential roles in sex determination of G. biloba were discussed. Pistil-related genes were expressed in male buds while anther-specific genes were identified in female buds, suggesting that dioecism in G. biloba was resulted from the selective arrest of reproductive primordia. High correlation of expression level was found between the RNA-Seq and quantitative real-time PCR results. The transcriptome resources that we generated allowed us to characterize gene expression profiles and examine differential expression profiles, which provided foundations for identifying functional genes associated with sex determination in G. biloba.

Transcriptome Profile Analysis from Different Sex Types of Ginkgo biloba L.

PubMed Central

Du, Shuhui; Sang, Yalin; Liu, Xiaojing; Xing, Shiyan; Li, Jihong; Tang, Haixia; Sun, Limin

2016-01-01

In plants, sex determination is a comprehensive process of correlated events, which involves genes that are differentially and/or specifically expressed in distinct developmental phases. Exploring gene expression profiles from different sex types will contribute to fully understanding sex determination in plants. In this study, we conducted RNA-sequencing of female and male buds (FB and MB) as well as ovulate strobilus and staminate strobilus (OS and SS) of Ginkgo biloba to gain insights into the genes potentially related to sex determination in this species. Approximately 60 Gb of clean reads were obtained from eight cDNA libraries. De novo assembly of the clean reads generated 108,307 unigenes with an average length of 796 bp. Among these unigenes, 51,953 (47.97%) had at least one significant match with a gene sequence in the public databases searched. A total of 4709 and 9802 differentially expressed genes (DEGs) were identified in MB vs. FB and SS vs. OS, respectively. Genes involved in plant hormone signal and transduction as well as those encoding DNA methyltransferase were found to be differentially expressed between different sex types. Their potential roles in sex determination of G. biloba were discussed. Pistil-related genes were expressed in male buds while anther-specific genes were identified in female buds, suggesting that dioecism in G. biloba was resulted from the selective arrest of reproductive primordia. High correlation of expression level was found between the RNA-Seq and quantitative real-time PCR results. The transcriptome resources that we generated allowed us to characterize gene expression profiles and examine differential expression profiles, which provided foundations for identifying functional genes associated with sex determination in G. biloba. PMID:27379148

Transcriptome analysis identifies genes involved in sex determination and development of Xenopus laevis gonads.

PubMed

Piprek, Rafal P; Damulewicz, Milena; Kloc, Malgorzata; Kubiak, Jacek Z

Development of the gonads is a complex process, which starts with a period of undifferentiated, bipotential gonads. During this period the expression of sex-determining genes is initiated. Sex determination is a process triggering differentiation of the gonads into the testis or ovary. Sex determination period is followed by sexual differentiation, i.e. appearance of the first testis- and ovary-specific features. In Xenopus laevis W-linked DM-domain gene (DM-W) had been described as a master determinant of the gonadal female sex. However, the data on the expression and function of other genes participating in gonad development in X. laevis, and in anurans, in general, are very limited. We applied microarray technique to analyze the expression pattern of a subset of X. laevis genes previously identified to be involved in gonad development in several vertebrate species. We also analyzed the localization and the expression level of proteins encoded by these genes in developing X. laevis gonads. These analyses pointed to the set of genes differentially expressed in developing testes and ovaries. Gata4, Sox9, Dmrt1, Amh, Fgf9, Ptgds, Pdgf, Fshr, and Cyp17a1 expression was upregulated in developing testes, while DM-W, Fst, Foxl2, and Cyp19a1 were upregulated in developing ovaries. We discuss the possible roles of these genes in development of X. laevis gonads. Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. 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.

Perceiving emotional expressions in others: Activation likelihood estimation meta-analyses of explicit evaluation, passive perception and incidental perception of emotions.

PubMed

Dricu, Mihai; Frühholz, Sascha

2016-12-01

We conducted a series of activation likelihood estimation (ALE) meta-analyses to determine the commonalities and distinctions between separate levels of emotion perception, namely incidental perception, passive perception, and explicit evaluation of emotional expressions. Pooling together more than 180 neuroimaging experiments using facial, vocal or body expressions, our results are threefold. First, explicitly evaluating the emotions of others recruits brain regions associated with the sensory processing of expressions, such as the inferior occipital gyrus, middle fusiform gyrus and the superior temporal gyrus, and brain regions involved in low-level and high-level mindreading, namely the posterior superior temporal sulcus, the inferior frontal cortex and dorsomedial frontal cortex. Second, we show that only the sensory regions were also consistently active during the passive perception of emotional expressions. Third, we show that the brain regions involved in mindreading were active during the explicit evaluation of both facial and vocal expressions. We discuss these results in light of the existing literature and conclude by proposing a cognitive model for perceiving and evaluating the emotions of others. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression of FSH receptor in the hamster ovary during perinatal development

PubMed Central

Chakraborty, Prabuddha; Roy, Shyamal K.

2014-01-01

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

Gestational choline supplementation normalized fetal alcohol-induced alterations in histone modifications, DNA methylation and POMC gene expression in β-endorphin-producing POMC neurons of the hypothalamus

PubMed Central

Bekdash, Rola A.; Zhang, Changqing; Sarkar, Dipak K.

2013-01-01

Background Prenatal exposure to ethanol reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, since these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of ethanol on these neurons. Methods Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes, and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunoabsorbent assay. Results Prenatal ethanol treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10) increased the repressive marks (H3K9me2, G9a, Setdb1) and DNA methylating enzyme (Dnmt1) and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and β-EP, and elevated corticosterone response to LPS. Gestational choline normalized the ethanol-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1 and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, β-EP production and the corticosterone response to LPS. Conclusions These data suggest that prenatal ethanol modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction of POMC gene expression. Gestational choline supplementation prevents the adverse effects of ethanol on these neurons. PMID:23413810

Testosterone regulates erectile function and Vcsa1 expression in the corpora of rats.

PubMed

Chua, Rowena G; Calenda, Giulia; Zhang, Xinhua; Siragusa, Joseph; Tong, Yuehong; Tar, Moses; Aydin, Memduh; DiSanto, Michael E; Melman, Arnold; Davies, Kelvin P

2009-05-06

Vcsa1 plays an important role in the erectile physiology of the rat. We conducted experiments to determine if erectile function, testosterone levels and Vcsa1 expression were correlated. In orchiectomized rats, total testosterone in blood fell from an average of 4 ng/ml to <0.04 ng/ml. Erectile function was significantly lower compared to controls and Vcsa1 expression was significantly (>6-fold) decreased. Injection of orchiectomized animals with testosterone (2 mg in 100ml sesame oil every 4 days for 2 weeks) restored average levels of testosterone to 2 ng/ml, increased erectile function and significantly increased Vcsa1 expression. In isolated corporal cells there was testosterone dependent Vcsa1 expression. However, intracorporal injection of orchiectomized animals with a plasmid expressing Vcsa1 or its gene product Sialorphin (previously demonstrated to improve erectile function in old animals) gave no significant improvement in erectile function. Also, the ability of Sialorphin to reduce tension in corporal smooth muscle strips isolated from orchiectomized animals was impaired compared to controls.

The effects of nongenetic memory on population level sensitivity to stress

NASA Astrophysics Data System (ADS)

Adams, Rhys; Nevozhay, Dmitry; van Itallie, Elizabeth; Bennett, Matthew; Balazsi, Gabor

2011-03-01

While gene expression is often thought of as a unidirectional determinant of cellular fitness, recent studies have shown how growth retardation due to protein expression can affect gene expression levels in single cells. We developed two yeast strains carrying a drug resistance protein under the control of different synthetic gene constructs, one of which was monostable, while the other was bistable. The gene expression of these cell populations was tuned using a molecular inducer so that their respective means and noises were identical, while their nongenetic memory properties were different. We tested the sensitivity of these two cell population distributions to the antibiotic zeocin. We found that the gene expression distributions of bistable cell populations were sensitive to stressful environments, while the gene expression distribution of monostable cells were nearly unchanged by stress. We conclude that cell populations with high nongenetic memory are more adaptable to their environment. This work was funded by the National Institutes of Health through the NIH Director's New Innovator Award Program, 1-DP2- OD006481-01.

Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

PubMed Central

2017-01-01

Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture. PMID:28540297

Cyclic strain is a weak inducer of prostacyclin synthase expression in bovine aortic endothelial cells

NASA Technical Reports Server (NTRS)

Segurola, R. J. Jr; Oluwole, B.; Mills, I.; Yokoyama, C.; Tanabe, T.; Kito, H.; Nakajima, N.; Sumpio, B. E.

1997-01-01

Recent studies indicate that hemodynamic forces such as cyclic strain and shear stress can increase prostacyclin (PGI2) secretion by endothelial cells (EC) but the effect of these forces on prostacyclin synthase (PGIS) gene expression remains unclear and is the focus of this study. Bovine aortic EC were seeded onto type I collagen coated flexible membranes and grown to confluence. The membranes and attached EC were subjected to 10% average strain at 60 cpm (0.5 sec deformation alternating with 0.5 sec relaxation) for up to 5 days. PGIS gene expression was determined by Northern blot analysis and protein level by Western blot analysis. The effect of cyclic strain on the PGIS promoter was determined by the transfection of a 1-kb human PGIS gene promoter construct coupled to a luciferase reporter gene into EC, followed by determination of luciferase activity. PGIS gene expression increased 1.7-fold in EC subjected to cyclic strain for 24 hr. Likewise, EC transfected with a pGL3B-PGIS (-1070/-10) construct showed an approximate 1.3-fold elevation in luciferase activity in EC subjected to cyclic strain for 3, 4, 8, and 12 hr. The weak stimulation of PGIS gene expression by cyclic strain was reflected in an inability to detect alterations in PGIS protein levels in EC subjected to cyclic strain for as long as 5 days. These data suggest that strain-induced stimulation of PGIS gene expression plays only a minor role in the ability of cyclic strain to stimulate PGI2 release in EC. These findings coupled with our earlier demonstration of a requisite addition of exogenous arachidonate in order to observe strain-induced PGI2 release, implicates a mechanism that more likely involves strain-induced stimulation of PGIS activity.

Structure and expression of dna methyltransferase genes from apomictic and sexual Boechera species.

PubMed

Taşkin, Kemal Melik; Özbilen, Aslıhan; Sezer, Fatih; Hürkan, Kaan; Güneş, Şebnem

2017-04-01

In this study, we determined the structure of DNA methyltransferase (DNMT) genes in apomict and sexual Boechera species and investigated the expression levels during seed development. Protein and DNA sequences of diploid sexual Boechera stricta DNMT genes obtained from Phytozome 10.3 were used to identify the homologues in apomicts, Boechera holboellii and Boechera divaricarpa. Geneious R8 software was used to map the short-paired reads library of B. holboellii whole genome or B. divaricarpa transcriptome reads to the reference gene sequences. We determined three DNMT genes; for Boechera spp. METHYLTRANSFERASE1 (MET1), CHROMOMETHYLASE 3 (CMT3) and DOMAINS REARRANGED METHYLTRANSFERASE 1/2 (DRM2). We examined the structure of these genes with bioinformatic tools and compared with other DNMT genes in plants. We also examined the levels of expression in silique tissues after fertilization by semi-quantitative PCR. The structure of DNMT proteins in apomict and sexual Boechera species share common features. However, the expression levels of DNMT genes were different in apomict and sexual Boechera species. We found that DRM2 was upregulated in apomictic Boechera species after fertilization. Phylogenetic trees showed that three genes are conserved among green algae, monocotyledons and dicotyledons. Our results indicated a deregulation of DNA methylation machinery during seed development in apomicts. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modulation of the response of prostate cancer cell lines to cisplatin treatment using small interfering RNA.

PubMed

Parra, Eduardo; Ferreira, Jorge

2013-10-01

Cisplatin is one of the most effective and widely used chemotherapeutic agents against several types of human cancers. However, the underlying mechanisms of action are not fully understood. We aimed to investigate the possible molecular mechanism(s) of acquired chemoresistance observed in prostate cancer cells treated with cisplatin. Human LNCaP cells (bearing wild-type p53) and PC-3 cells (lacking p53) were used. The expression levels of protein were determined by western blotting, and the mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cell viability was measured by MTT assay, and the transcriptional effect of small interfering RNA (siRNA) was measured by luciferase reporter gene. We showed that cisplatin treatment increased JNK-1 and JNK-2 activity and expression in both LNCaP and PC-3 cells. In addition, the knockdown of JNK-1 expression by siRNA-JNK-1 or siRNA-JNK-2 significantly impaired the upregulation of AP-1 luciferase reporter gene, but failed to decrease the levels of AP-1 reporter gene expression induced by TPA treatment. Our observations indicate that JNK-1 and JNK-2 may be involved in the chemoresistance observed in prostate cancer cells treated with cisplatin and that blocking the stimulation of Jun kinase (JNK) signaling may be important for regulating the susceptibility to cisplatin of prostate cancer.

Depigmenting effect of argan press-cake extract through the down-regulation of Mitf and melanogenic enzymes expression in B16 murine melanoma cells.

PubMed

Bourhim, Thouria; Villareal, Myra O; Gadhi, Chemseddoha; Hafidi, Abdellatif; Isoda, Hiroko

2018-06-26

Oil extraction from the kernels of Argania spinosa (L.) Skeels (Sapotaceae), an endemic tree of Morocco, produces argan press-cake (APC) used as a shampoo and to treat sprains, scabies, and for healing wounds. We have previously reported that argan oil has antimelanogenesis effect. Here, we determined if the by-product, APC, has melanogenesis regulatory effect using B16 murine melanoma cells. The effect of APC ethanol extract on cell proliferation and melanin content of B16 cells were measured, and to elucidate the mechanism involved, the expression level of melanogenic enzymes tyrosinase (TYR), dopachrome tautomerase (DCT), and tyrosinase-related protein 1 (TRP1) were determined and mRNA expression level of microphthalmia- associated transcription factor (Mitf) and Tyr genes were quantified. APC ethanol extract showed a significant melanin biosynthesis inhibitory effect on B16 cells in a time-dependent manner without cytotoxicity, which could be due to the decreased expression of TYR, TRP1, and DCT in a time-dependent manner. APC extract down regulated Mitf and Tyr. Decreased TRP1 and DCT levels could be due to post-translational modifications. These results suggest that APC extract may be used as a new source of natural whitening products and may be introduced as an important pharmacological agent for the treatment of hyperpigmentation disorders.

Inhibition of biofilm in Bacillus amyloliquefaciens Q-426 by diketopiperazines.

PubMed

Wang, Jian-Hua; Yang, Cui-Yun; Fang, Sheng-Tao; Lu, Jian; Quan, Chun-Shan

2016-09-01

Biofilm formation can make significant effects on bacteria habits and biological functions. In this study, diketopiperazines (DKPs) produced by strain of Bacillus amyloliquefaciens Q-426 was found to inhibit biofilm formed in the gas-liquid interface. Four kinds of DKPs were extracted from B. amyloliquefaciens Q-426, and we found that 0.04 mg ml(-1) DKPs could obviously inhibit the biofilm formation of the strain. DKPs produced by B. amyloliquefaciens Q-426 made a reduction on extracellular polymeric substance (EPS) components, polysaccharides, proteins, DNAs, etc. Real-time PCR was performed to determine that whether DKPs could make an obvious effect on the expression level for genes related to biofilm formation in the strain. The relative expression level of genes tasA, epsH, epsG and remB which related to proteins, extracellular matrix, and polysaccharides, were downregulated with 0.04 mg ml(-1) DKPs, while the expression level of nuclease gene nuc was significantly upregulated. The quantitative results of the mRNA expression level for these genes concerted with the quantitative results on EPS levels. All of the experimental results ultimately indicated that DKPs could inhibit the biofilm formation of the strain B. amyloliquefaciens Q-426.

Enhancing the efficacy of AREDS antioxidants in light-induced retinal degeneration

PubMed Central

Wong, Paul; Markey, M.; Rapp, C. M.; Darrow, R. M.; Ziesel, A.

2017-01-01

Purpose Light-induced photoreceptor cell degeneration and disease progression in age-related macular degeneration (AMD) involve oxidative stress and visual cell loss, which can be prevented, or slowed, by antioxidants. Our goal was to test the protective efficacy of a traditional Age-related Eye Disease Study antioxidant formulation (AREDS) and AREDS combined with non-traditional antioxidants in a preclinical animal model of photooxidative retinal damage. Methods Male Sprague-Dawley rats were reared in a low-intensity (20 lux) or high-intensity (200 lux) cyclic light environment for 6 weeks. Some animals received a daily dietary supplement consisting of a small cracker infused with an AREDS antioxidant mineral mixture, AREDS antioxidants minus zinc, or zinc oxide alone. Other rats received AREDS combined with a detergent extract of the common herb rosemary, AREDS plus carnosic acid, zinc oxide plus rosemary, or rosemary alone. Antioxidant efficacy was determined by measuring retinal DNA levels 2 weeks after 6 h of intense exposure to white light (9,000 lux). Western blotting was used to determine visual cell opsin and arrestin levels following intense light treatment. Rhodopsin regeneration was determined after 1 h of exposure to light. Gene array analysis was used to determine changes in the expression of retinal genes resulting from light rearing environment or from antioxidant supplementation. Results Chronic high-intensity cyclic light rearing resulted in lower levels of rod and cone opsins, retinal S-antigen (S-ag), and medium wavelength cone arrestin (mCAR) than found for rats maintained in low cyclic light. However, as determined by retinal DNA, and by residual opsin and arrestin levels, 2 weeks after acute photooxidative damage, visual cell loss was greater in rats reared in low cyclic light. Retinal damage decreased with AREDS plus rosemary, or with zinc oxide plus rosemary whereas AREDS alone and zinc oxide alone (at their daily recommended levels) were both ineffective. One week of supplemental AREDS plus carnosic acid resulted in higher levels of rod and cone cell proteins, and higher levels of retinal DNA than for AREDS alone. Rhodopsin regeneration was unaffected by the rosemary treatment. Retinal gene array analysis showed reduced expression of medium- wavelength opsin 1 and arrestin C in the high-light reared rats versus the low-light rats. The transition of rats from low cyclic light to a high cyclic light environment resulted in the differential expression of 280 gene markers, enriched for genes related to inflammation, apoptosis, cytokine, innate immune response, and receptors. Rosemary, zinc oxide plus rosemary, and AREDS plus rosemary suppressed 131, 241, and 266 of these genes (respectively) in high-light versus low-light animals and induced a small subset of changes in gene expression that were independent of light rearing conditions. Conclusions Long-term environmental light intensity is a major determinant of retinal gene and protein expression, and of visual cell survival following acute photooxidative insult. Rats preconditioned by high-light rearing exhibit lower levels of cone opsin mRNA and protein, and lower mCAR protein, than low-light reared animals, but greater retention of retinal DNA and proteins following photooxidative damage. Rosemary enhanced the protective efficacy of AREDS and led to the greatest effect on the retinal genome in animals reared in high environmental light. Chronic administration of rosemary antioxidants may be a useful adjunct to the therapeutic benefit of AREDS in slowing disease progression in AMD. PMID:29062223

Antihyperglycemic drug Gymnema sylvestre also shows anticancer potentials in human melanoma A375 cells via reactive oxygen species generation and mitochondria-dependent caspase pathway.

PubMed

Chakraborty, Debrup; Ghosh, Samrat; Bishayee, Kausik; Mukherjee, Avinaba; Sikdar, Sourav; Khuda-Bukhsh, Anisur Rahman

2013-09-01

Ethanolic extract of Gymnema sylvestre (GS) leaves is used as a potent antidiabetic drug in various systems of alternative medicine, including homeopathy. The present study was aimed at examining if GS also had anticancer potentials, and if it had, to elucidate its possible mechanism of action. We initially tested possible anticancer potential of GS on A375 cells (human skin melanoma) through MTT assay and determined cytotoxicity levels in A375 and normal liver cells; we then thoroughly studied its apoptotic effects on A375 cells through protocols such as Hoechst 33258, H2DCFDA, and rhodamine 123 staining and conducted ELISA for cytochrome c, caspase 3, and PARP activity levels; we determined the mRNA level expression of cytochrome c, caspase 3, Bcl2, Bax, PARP, ICAD, and EGFR signaling genes through semiquantitative reverse transcriptase polymerase chain reaction and conducted Western blot analysis of caspase 3 and PARP. We also analyzed cell cycle events, determined reactive oxygen species accumulation, measured annexin V-FITC/PI and rhodamine 123 intensity by flow cytometry. Compared with both normal liver cells and drug-untreated A375, the mortality of GS-treated A375 cells increased in a dose-dependent manner. Additionally, GS induced nuclear DNA fragmentation and showed an increased level of mRNA expression of apoptotic signal related genes cytochrome c, caspase 3, PARP, Bax, and reduced expression level of ICAD, EGFR, and the anti-apoptotic gene Bcl2. Overall results indicate GS to have significant anticancer effect on A375 cells apart from its reported antidiabetic effect, indicating possibility of its palliative use in patients with symptoms of both the diseases.

Immunoglubolin dynamics and cancer prevalence in Tasmanian devils (Sarcophilus harrisii)

PubMed Central

Ujvari, Beata; Hamede, Rodrigo; Peck, Sarah; Pemberton, David; Jones, Menna; Belov, Katherine; Madsen, Thomas

2016-01-01

Immunoglobulins such as IgG and IgM have been shown to induce anti-tumour cytotoxic activity. In the present study we therefore explore total serum IgG and IgM expression dynamics in 23 known-aged Tasmanian devils (Sarcophilus harrisii) of which 9 where affected by Devil Facial Tumour Disease (DFTD). DFTD is clonally transmissible cancer that has caused massive declines in devil numbers. Our analyses revealed that IgM and IgG expression levels as well as IgM/IgG ratios decreased with increasing devil age. Neither age, sex, IgM nor IgG expression levels affected devil DFTD status in our analyses. However, devils with increased IgM relative to IgG expression levels had significantly lower DFTD prevalence. Our results therefore suggest that IgM/IgG ratios may play an important role in determining devil susceptibility to DFTD. We consequently propose that our findings warrant further studies to elucidate the underpinning(s) of devil IgM/IgG ratios and DFTD status. PMID:27126067

The Olfactory Transcriptome and Progression of Sexual Maturation in Homing Chum Salmon Oncorhynchus keta.

PubMed

Palstra, Arjan P; Fukaya, Kosuke; Chiba, Hiroaki; Dirks, Ron P; Planas, Josep V; Ueda, Hiroshi

2015-01-01

Reproductive homing migration of salmonids requires accurate interaction between the reception of external olfactory cues for navigation to the spawning grounds and the regulation of sexual maturation processes. This study aimed at providing insights into the hypothesized functional link between olfactory sensing of the spawning ground and final sexual maturation. We have therefore assessed the presence and expression levels of olfactory genes by RNA sequencing (RNAseq) of the olfactory rosettes in homing chum salmon Oncorhynchus keta Walbaum from the coastal sea to 75 km upstream the rivers at the pre-spawning ground. The progression of sexual maturation along the brain-pituitary-gonadal axis was assessed through determination of plasma steroid levels by time-resolved fluoroimmunoassays (TR-FIA), pituitary gonadotropin subunit expression and salmon gonadotropin-releasing hormone (sgnrh) expression in the brain by quantitative real-time PCR. RNAseq revealed the expression of 75 known and 27 unknown salmonid olfactory genes of which 13 genes were differentially expressed between fish from the pre-spawning area and from the coastal area, suggesting an important role of these genes in homing. A clear progression towards final maturation was characterised by higher plasma 17α,20β-dihydroxy-4-pregnen-3-one (DHP) levels, increased pituitary luteinizing hormone β subunit (lhβ) expression and sgnrh expression in the post brain, and lower plasma testosterone (T) and 17β-estradiol (E2) levels. Olfactomedins and ependymin are candidates among the differentially expressed genes that may connect olfactory reception to the expression of sgnrh to regulate final maturation.

The Olfactory Transcriptome and Progression of Sexual Maturation in Homing Chum Salmon Oncorhynchus keta

PubMed Central

Palstra, Arjan P.; Fukaya, Kosuke; Chiba, Hiroaki; Dirks, Ron P.; Planas, Josep V.; Ueda, Hiroshi

2015-01-01

Reproductive homing migration of salmonids requires accurate interaction between the reception of external olfactory cues for navigation to the spawning grounds and the regulation of sexual maturation processes. This study aimed at providing insights into the hypothesized functional link between olfactory sensing of the spawning ground and final sexual maturation. We have therefore assessed the presence and expression levels of olfactory genes by RNA sequencing (RNAseq) of the olfactory rosettes in homing chum salmon Oncorhynchus keta Walbaum from the coastal sea to 75 km upstream the rivers at the pre-spawning ground. The progression of sexual maturation along the brain-pituitary-gonadal axis was assessed through determination of plasma steroid levels by time-resolved fluoroimmunoassays (TR-FIA), pituitary gonadotropin subunit expression and salmon gonadotropin-releasing hormone (sgnrh) expression in the brain by quantitative real-time PCR. RNAseq revealed the expression of 75 known and 27 unknown salmonid olfactory genes of which 13 genes were differentially expressed between fish from the pre-spawning area and from the coastal area, suggesting an important role of these genes in homing. A clear progression towards final maturation was characterised by higher plasma 17α,20β-dihydroxy-4-pregnen-3-one (DHP) levels, increased pituitary luteinizing hormone β subunit (lhβ) expression and sgnrh expression in the post brain, and lower plasma testosterone (T) and 17β-estradiol (E2) levels. Olfactomedins and ependymin are candidates among the differentially expressed genes that may connect olfactory reception to the expression of sgnrh to regulate final maturation. PMID:26397372

Inflammation-induced effects on iron-related proteins in splenic macrophages and the liver in mice.

PubMed

Sukumaran, Abitha; Venkatraman, Aparna; Jacob, Molly

2012-06-15

Anemia of inflammation is characterized by disturbances in systemic iron homeostasis. In order to better understand the events involved, we carried out a time-course study on the effects of acute and chronic inflammation on iron-related proteins in mouse splenic macrophages and the liver. Mice were sacrificed at various time points ranging from 0 h up to 4 weeks after induction of inflammation with turpentine oil. Expression levels of iron-related proteins in the splenic macrophages and liver were determined. Iron levels in the serum, spleen and liver were also measured. Hepatic hepcidin was found to be induced in response to inflammation. In the macrophages, expression levels of ferroportin and TfR1 were decreased at some of the time points. The expression of hepatic TfR1 and ferritin was significantly higher at the early time points. Ferritin levels in the liver decreased progressively thereafter; this was associated with significantly higher ferroportin expression in the liver, despite high levels of hepcidin, suggesting that hepcidin may not regulate ferroportin levels in the liver, unlike in the macrophages. The effects of hepcidin, thus, appeared to be tissue-specific. Serum iron levels were decreased initially; these then rose and were associated with decreasing iron levels in the liver and spleen. Thus, inflammation affected the expression levels of many proteins involved in iron homeostasis in splenic macrophages and the liver, with differences seen in the effects at these 2 sites. These effects are likely to contribute to the development of anemia of inflammation. Copyright © 2012 Elsevier Inc. All rights reserved.

Instructional Television: Visual Production Techniques and Learning Comprehension.

ERIC Educational Resources Information Center

Silbergleid, Michael Ian

The purpose of this study was to determine if increasing levels of complexity in visual production techniques would increase the viewer's learning comprehension and the degree of likeness expressed for a college level instructional television program. A total of 119 mass communications students at the University of Alabama participated in the…

Downregulation of CD147 expression by RNA interference inhibits HT29 cell proliferation, invasion and tumorigenicity in vitro and in vivo.

PubMed

Li, Rui; Pan, Yuqin; He, Bangshun; Xu, Yeqiong; Gao, Tianyi; Song, Guoqi; Sun, Huiling; Deng, Qiwen; Wang, Shukui

2013-12-01

We investigated the effect of CD147 silencing on HT29 cell proliferation and invasion. We constructed a novel short hairpin RNA (shRNA) expression vector pYr-mir30-shRNA. The plasmid was transferred to HT29 cells. The expression of CD147, MCT1 (lactate transporters monocarboxylate transporter 1) and MCT4 (lactate transporters monocarboxylate transporter 4) were monitored by quantitative PCR and western blotting, respectively. The MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) activities were determined by gelatin zymography assay, while the intracellular lactate concentration was determined by the lactic acid assay kit. WST-8 assay was used to determine the HT29 cell proliferation and the chemosensitivity. Invasion assay was used to determine the invasion of HT29 cells. In addition, we established a colorectal cancer model, and detected CD147 expression in vivo. The results showed that the expression of CD147 and MCT1 was significantly reduced at both mRNA and protein levels, and also the activity of MMP-2 and MMP-9 was reduced. The proliferation and invasion were decreased, but chemosensitivity to cisplatin was increased. In vivo, the CD147 expression was also significantly decreased, and reduced the tumor growth after CD147 gene silencing. The results demonstrated that silencing of CD147 expression inhibited the proliferation and invasion, suggesting CD147 silencing might be an adjuvant gene therapy strategy to chemotherapy.

Effects of vitamin D supplementation on alveolar macrophage gene expression: preliminary results of a randomized, controlled trial.

PubMed

Gerke, Alicia K; Pezzulo, Alejandro A; Tang, Fan; Cavanaugh, Joseph E; Bair, Thomas B; Phillips, Emily; Powers, Linda S; Monick, Martha M

2014-03-26

Vitamin D deficiency has been implicated as a factor in a number of infectious and inflammatory lung diseases. In the lung, alveolar macrophages play a key role in inflammation and defense of infection, but there are little data exploring the immunomodulatory effects of vitamin D on innate lung immunity in humans. The objective of this study was to determine the effects of vitamin D supplementation on gene expression of alveolar macrophages. We performed a parallel, double-blind, placebo-controlled, randomized trial to determine the effects of vitamin D on alveolar macrophage gene expression. Vitamin D3 (1000 international units/day) or placebo was administered to adults for three months. Bronchoscopy was performed pre- and post-intervention to obtain alveolar macrophages. Messenger RNA was isolated from the macrophages and subjected to whole genome exon array analysis. The primary outcome was differential gene expression of the alveolar macrophage in response to vitamin D supplementation. Specific genes underwent validation by polymerase chain reaction methods. Fifty-eight subjects were randomized to vitamin D (n = 28) or placebo (n = 30). There was a marginal overall difference between treatment group and placebo group in the change of 25-hydroxyvitaminD levels (4.43 ng/ml vs. 0.2 ng/ml, p = 0.10). Whole genome exon array analysis revealed differential gene expression associated with change in serum vitamin D levels in the treated group. CCL8/MCP-2 was the top-regulated cytokine gene and was further validated. Although only a non-significant increased trend was seen in serum vitamin D levels, subjects treated with vitamin D supplementation had immune-related differential gene expression in alveolar macrophages. ClinicalTrials.org: NCT01967628.

The sodium pump α1 sub-unit: a disease progression–related target for metastatic melanoma treatment

PubMed Central

Mathieu, Véronique; Pirker, Christine; Martin de Lassalle, Elisabeth; Vernier, Mathieu; Mijatovic, Tatjana; DeNeve, Nancy; Gaussin, Jean-François; Dehoux, Mischael; Lefranc, Florence; Berger, Walter; Kiss, Robert

2009-01-01

Melanomas remain associated with dismal prognosis because they are naturally resistant to apoptosis and they markedly metastasize. Up-regulated expression of sodium pump α sub-units has previously been demonstrated when comparing metastatic to non-metastatic melanomas. Our previous data revealed that impairing sodium pump α1 activity by means of selective ligands, that are cardiotonic steroids, markedly impairs cell migration and kills apoptosis-resistant cancer cells. The objective of this study was to determine the expression levels of sodium pump α sub-units in melanoma clinical samples and cell lines and also to characterize the role of α1 sub-units in melanoma cell biology. Quantitative RT-PCR, Western blotting and immunohistochemistry were used to determine the expression levels of sodium pump α sub-units. In vitro cytotoxicity of various cardenolides and of an anti-α1 siRNA was evaluated by means of MTT assay, quantitative videomicroscopy and through apoptosis assays. The in vivo activity of a novel cardenolide UNBS1450 was evaluated in a melanoma brain metastasis model. Our data show that all investigated human melanoma cell lines expressed high levels of the α1 sub-unit, and 33% of human melanomas displayed significant α1 sub-unit expression in correlation with the Breslow index. Furthermore, cardenolides (notably UNBS1450; currently in Phase I clinical trials) displayed marked anti-tumour effects against melanomas in vitro. This activity was closely paralleled by decreases in cMyc expression and by increases in apoptotic features. UNBS1450 also displayed marked anti-tumour activity in the aggressive human metastatic brain melanoma model in vivo. The α1 sodium pump sub-unit could represent a potential novel target for combating melanoma. PMID:19243476

Characterization and expression profile of the ovarian cytochrome P-450 aromatase (cyp19A1) gene during thermolabile sex determination in Pejerrey, Odontesthes bonariensis

USGS Publications Warehouse

Karube, M.; Fernandino, J.I.; Strobl-Mazzulla, P.; Strussmann, C.A.; Yoshizaki, G.; Somoza, G.M.; Patino, R.

2007-01-01

Cytochrome P450 aromatase (cyp19) is an enzyme that catalyzes the conversion of androgens to estrogens and may play a role in temperature- dependent sex determination (TSD) of reptiles, amphibians, and fishes. In this study, the ovarian P450 aromatase form (cyp19A1) of pejerrey Odontesthes bonariensis, a teleost with marked TSD, was cloned and its expression profile evaluated during gonadal differentiation at feminizing (17??C, 100% females), mixed-sex producing (24 and 25??C, 73.3 and 26.7% females, respectively), and masculinizing (29??C, 0% females) temperatures. The deduced cyp19A1 amino acid sequence shared high identity (>77.8%) with that from other teleosts but had low identity (<61.8%) with brain forms (cyp19A2), including that of pejerrey itself. The tissue distribution analysis of cyp19A1 mRNA in adult fish revealed high expression in the ovary. Semi-quantitative reverse transcription polymerase chain reaction analysis of the bodies of larvae revealed that cyp19A1 expression increased before the appearance of the first histological signs of ovarian differentiation at the feminizing temperature but remained low at the masculinizing temperature. The expression levels at mixed-sex producing temperatures were bimodal rather than intermediate, showing low and high modal values similar to those at the feminizing and masculinizing temperatures, respectively. The population percentages of high and low expression levels at intermediate temperatures were proportional to the percentage of females and males, respectively, and high levels were first observed at about the time of sex differentiation of females. These results suggest that cyp19A1 is involved in the process of ovarian formation and possibly also in the TSD of pejerrey. ?? 2007 Wiley-Liss, Inc.

Gene expression profiling in psoriatic scalp hair follicles: clobetasol propionate shampoo 0.05% normalizes psoriasis disease markers.

PubMed

Aubert, J; Reiniche, P; Fogel, P; Poulin, Y; Lui, H; Lynde, C; Shapiro, J; Villemagne, H; Soto, P; Voegel, J J

2010-11-01

Clobetasol propionate shampoo is effective and safe in treatment of scalp psoriasis (SP). Gene expression profiling of psoriatic skin biopsies led to the identification of numerous disease-related genes. However, it remained unknown whether the gene expression profile of hair follicles of SP patients was also affected. To determine whether psoriasis-related genes are differentially regulated in the hair follicles of SP patients and whether the modulation of these genes can be correlated with clinical severity scores. A single arm, open study was conducted in three centres. SP patients received daily treatment with clobetasol propionate shampoo. At Baseline, Weeks 2 and 4, investigators assessed clinical severity parameters and collected scalp hair follicles in anagen phase. Total RNA extracted from hair follicles was used to determine the expression level of 44 genes, which were reported previously to be upregulated in the skin of psoriasis patients. RNA of good quality and sufficient quantity was obtained from hair follicles of psoriasis patients and healthy volunteers (HV). The expression level of 10 inflammation-related genes was significantly increased in psoriatic hair follicles. The patient's exploratory transcriptomic score, defined as the mean fold modulation of these 10 genes compared with HV, correlated with clinical severity scores. Clobetasol propionate shampoo was effective in decreasing both the exploratory transcriptomics and the clinical severity scores. Hair follicles of SP patients are affected by the inflammatory process. The change in the expression level of inflammation-related genes correlates with the severity of the disease. © 2010 Galderma R&D. Journal of the European Academy of Dermatology and Venereology © 2010 European Academy of Dermatology and Venereology.

Job Satisfaction and Relocation Desire among Pediatric Dentists in Puerto Rico.

PubMed

Arévalo, Oscar; Saman, Daniel M; Tabares, Miguel; Hernández, Ana; Sanders-Ward, Rebecca

2015-12-01

To determine the levels of satisfaction, license status, and desire to relocate of pediatric dentists in Puerto Rico. Pediatric dentists in Puerto Rico were surveyed via telephone interviews. Data were collected through a 34-item questionnaire that explored satisfaction as related to income, continuing education, professional goals, and participation in the Mi Salud program. Frequencies, chi-square analysis, and Fisher's exact 2-tailed t-test were utilized to determine the relationships between satisfaction and the demographics of the pediatric dentists. Sixty pediatric dentists participated in our survey-77% of the total number of pediatric dentists practicing in Puerto Rico. Overall, 65% of the participating pediatric dentists expressed dissatisfaction. Male pediatric dentists were more dissatisfied than their female colleagues were. Most pediatric dentists participating in Mi Salud expressed dissatisfaction. When asked about whether or not they had considered migrating to the mainland, those who were dissatisfied were more likely to have considered that idea than were those who were satisfied. Overall, 57% of the pediatric dentists comprising our sample had considered relocating to the continental United States. In general, the pediatric dentists who participated in our study expressed dissatisfaction in most areas except when asked about their ability to reach professional goals. Determining the levels of satisfaction of health care providers is important in the maintaining of an adequate workforce. As current levels of dissatisfaction are high, it is important to determine what variables are related to satisfaction so that corrective measures can be taken to ensure that retention rates improve, thereby maintaining an adequate pediatric dental workforce.

Aging Field Collected Aedes aegypti to Determine Their Capacity for Dengue Transmission in the Southwestern United States

PubMed Central

Joy, Teresa K.; Jeffrey Gutierrez, Eileen H.; Ernst, Kacey; Walker, Kathleen R.; Carriere, Yves; Torabi, Mohammad; Riehle, Michael A.

2012-01-01

Aedes aegypti, the primary vector of dengue virus, is well established throughout urban areas of the Southwestern US, including Tucson, AZ. Local transmission of the dengue virus, however, has not been reported in this area. Although many factors influence the distribution of the dengue virus, we hypothesize that one contributing factor is that the lifespan of female Ae. aegypti mosquitoes in the Southwestern US is too short for the virus to complete development and be transmitted to a new host. To test this we utilized two age grading techniques. First, we determined parity by analyzing ovarian tracheation and found that only 40% of Ae. aegypti females collected in Tucson, AZ were parous. The second technique determined transcript levels of an age-associated gene, Sarcoplasmic calcium-binding protein 1 (SCP-1). SCP-1 expression decreased in a predictable manner as the age of mosquitoes increased regardless of rearing conditions and reproductive status. We developed statistical models based on parity and SCP-1 expression to determine the age of individual, field collected mosquitoes within three age brackets: nonvectors (0–5 days post-emergence), unlikely vectors (6–14 days post-emergence), and potential vectors (15+ days post-emergence). The statistical models allowed us to accurately group individual wild mosquitoes into the three age brackets with high confidence. SCP-1 expression levels of individual, field collected mosquitoes were analyzed in conjunction with parity status. Based on SCP-1 transcript levels and parity data, 9% of collected mosquitoes survived more than 15 days post emergence. PMID:23077536

Brain-Derived Neurotrophic Factor Gene Expression in Pediatric Bipolar Disorder: Effects of Treatment and Clinical Response

ERIC Educational Resources Information Center

Pandey, Ghanshyam N.; Rizavi, Hooriyah S.; Dwivedi, Yogesh; Pavuluri, Mani N.

2008-01-01

The study determines the gene expression of brain-derived neurotrophic factor (BDNF) in the lymphocytes of subjects with pediatric bipolar disorder (PBD) before and during treatment with mood stabilizers and in drug-free normal control subjects. Results indicate the potential of BDNF levels as a biomarker for PBD and as a treatment predictor and…

Beyond Sentences: Using the Expression, Reception, and Recall of Narratives Instrument to Assess Communication in School-Aged Children with Autism Spectrum Disorder

ERIC Educational Resources Information Center

Volden, Joanne; Dodd, Erin; Engel, Kathleen; Smith, Isabel M.; Szatmari, Peter; Fombonne, Eric; Zwaigenbaum, Lonnie; Mirenda, Pat; Bryson, Susan; Roberts, Wendy; Vaillancourt, Tracy; Waddell, Charlotte; Elsabbagh, Mayada; Bennett, Teresa; Georgiades, Stelios; Duku, Eric

2017-01-01

Purpose: Impairments in the social use of language are universal in autism spectrum disorder (ASD), but few standardized measures evaluate communication skills above the level of individual words or sentences. This study evaluated the Expression, Reception, and Recall of Narrative Instrument (ERRNI; Bishop, 2004) to determine its contribution to…

Localization of PPARdelta in murine central nervous system: expression in oligodendrocytes and neurons.

PubMed

Woods, John W; Tanen, Michael; Figueroa, David J; Biswas, Chhabi; Zycband, Emanuel; Moller, David E; Austin, Christopher P; Berger, Joel P

2003-06-13

The peroxisome proliferator-activated receptors (PPARs), PPARdelta, PPARgamma and PPARalpha, comprise a subclass of the supergene family of nuclear receptors. As such they are ligand-regulated transcription factors whose major effects are mediated by altering expression of target genes. PPARdelta has been shown to be ubiquitously expressed in mammals. However, its primary biological role(s) has yet to be defined. Several recent studies have demonstrated that PPARdelta is the most highly expressed PPAR isoform in the central nervous system, but ambiguity still exists as to the specific brain sub-regions and cells in which it is expressed. Here, utilizing novel, isoform-selective PPARdelta riboprobes and an anti-peptide antibody, we performed a series of in situ hybridization and immunolocalization studies to determine the distribution of PPARdelta in the central nervous system (CNS) of mice. We found that PPARdelta mRNA and protein is expressed throughout the brain, with particularly high levels in the entorhinal cortex, hypothalamus and hippocampus, and lower levels in the corpus callosum and caudate putamen. At the cellular level, PPARdelta mRNA and protein were found to be expressed in oligodendrocytes and neurons but not astrocytes. Such results suggest a role for PPARdelta in both myelination and neuronal functioning within the CNS.

Using the preschool language scale, fourth edition to characterize language in preschoolers with autism spectrum disorders.

PubMed

Volden, Joanne; Smith, Isabel M; Szatmari, Peter; Bryson, Susan; Fombonne, Eric; Mirenda, Pat; Roberts, Wendy; Vaillancourt, Tracy; Waddell, Charlotte; Zwaigenbaum, Lonnie; Georgiades, Stelios; Duku, Eric; Thompson, Ann

2011-08-01

The Preschool Language Scale, Fourth Edition (PLS-4; Zimmerman, Steiner, & Pond, 2002) was used to examine syntactic and semantic language skills in preschool children with autism spectrum disorders (ASD) to determine its suitability for use with this population. We expected that PLS-4 performance would be better in more intellectually able children and that receptive skills would be relatively more impaired than expressive abilities, consistent with previous findings in the area of vocabulary. Our sample consisted of 294 newly diagnosed preschool children with ASD. Children were assessed via a battery of developmental measures, including the PLS-4. As expected, PLS-4 scores were higher in more intellectually able children with ASD, and overall, expressive communication was higher than auditory comprehension. However, this overall advantage was not stable across nonverbal developmental levels. Expressive skills were significantly better than receptive skills at the youngest developmental levels, whereas the converse applied in children with more advanced development. The PLS-4 can be used to obtain a general index of early syntax and semantic skill in young children with ASD. Longitudinal data will be necessary to determine how the developmental relationship between receptive and expressive language skills unfolds in children with ASD.

Validation of endogenous internal real-time PCR controls in renal tissues.

PubMed

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R; Mrug, Michal

2009-01-01

Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means. Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. Copyright 2009 S. Karger AG, Basel.

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

PubMed

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

2016-06-01

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

Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

PubMed

Lu, B; Yang, X J; Chen, K; Yang, D J; Yan, J Q

2009-12-15

Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

CD30 Expression by B and T Cells: A Frequent Finding in Angioimmunoblastic T-Cell Lymphoma and Peripheral T-Cell Lymphoma-Not Otherwise Specified.

PubMed

Onaindia, Arantza; Martínez, Nerea; Montes-Moreno, Santiago; Almaraz, Carmen; Rodríguez-Pinilla, Socorro M; Cereceda, Laura; Revert, Jose B; Ortega, César; Tardio, Antoni; González, Lucía; García, Sonia; Camacho, Francisca I; González-Vela, Carmen; Piris, Miguel A

2016-03-01

CD30 expression in peripheral T-cell lymphoma (PTCL) and angioimmunoblastic T-cell lymphoma (AITL) is currently of great interest because therapy targeting CD30 is of clinical benefit, but the clinical and therapeutic relevance of CD30 expression in these neoplasms still remains uncertain. The aim of this study was to better quantify CD30 expression in AITL and PTCL-not otherwise specified (NOS). The secondary objective was to determine whether CD30 cells exhibit a B-cell or a T-cell phenotype. Gene expression profiling was studied in a series of 37 PTCL cases demonstrating a continuous spectrum of TNFRSF8 expression. This prompted us to study CD30 immunohistochemical (IHC) expression and mRNA levels by reverse transcription polymerase chain reaction (RT-PCR) in a different series of 51 cases (43 AITLs and 8 PTCL-NOSs) in routine samples. Double stainings with PAX5/CD30, CD3/CD30, and LEF1/CD30 were performed to study the phenotype of CD30 cells. Most (90%) of the cases showed some level of CD30 expression by IHC (1% to 95%); these levels were high (>50% of tumoral cells) in 14% of cases. CD30 expression was not detected in 10% of the cases. Quantitative RT-PCR results largely confirmed these findings, demonstrating a moderately strong correlation between global CD30 IHC and mRNA levels (r=0.65, P=1.75e-7). Forty-four of the positive cases (98%) contained CD30-positive B cells (PAX5), whereas atypical CD30-positive T cells were detected in 42 cases (93%). In conclusion, our data show that most AITL and PTCL-NOS cases express CD30, exhibiting very variable levels of CD30 expression that may be measured by IHC or RT-PCR techniques.

Identification of nuclear genes controlling chlorophyll synthesis in barley by RNA-seq.

PubMed

Shmakov, Nickolay A; Vasiliev, Gennadiy V; Shatskaya, Natalya V; Doroshkov, Alexey V; Gordeeva, Elena I; Afonnikov, Dmitry A; Khlestkina, Elena K

2016-11-16

Albinism in plants is characterized by lack of chlorophyll and results in photosynthesis impairment, abnormal plant development and premature death. These abnormalities are frequently encountered in interspecific crosses and tissue culture experiments. Analysis of albino mutant phenotypes with full or partial chlorophyll deficiency can shed light on genetic determinants and molecular mechanisms of albinism. Here we report analysis of RNA-seq transcription profiling of barley (Hordeum vulgare L.) near-isogenic lines, one of which is a carrier of mutant allele of the Alm gene for albino lemma and pericarp phenotype (line i:BwAlm). 1221 genome fragments have statistically significant changes in expression levels between lines i:BwAlm and Bowman, with 148 fragments having increased expression levels in line i:BwAlm, and 1073 genome fragments, including 42 plastid operons, having decreased levels of expression in line i:BwAlm. We detected functional dissimilarity between genes with higher and lower levels of expression in i:BwAlm line. Genes with lower level of expression in the i:BwAlm line are mostly associated with photosynthesis and chlorophyll synthesis, while genes with higher expression level are functionally associated with vesicle transport. Differentially expressed genes are shown to be involved in several metabolic pathways; the largest fraction of such genes was observed for the Calvin-Benson-Bassham cycle. Finally, de novo assembly of transcriptome contains several transcripts, not annotated in current H. vulgare genome version. Our results provide the new information about genes which could be involved in formation of albino lemma and pericarp phenotype. They demonstrate the interplay between nuclear and chloroplast genomes in this physiological process.

Effects of Different Levels of Calcium Intake on Brain Cell Apoptosis in Fluorosis Rat Offspring and Its Molecular Mechanism.

PubMed

Sun, Yan; Ke, Lulu; Zheng, Xiangren; Li, Tao; Ouyang, Wei; Zhang, Zigui

2017-04-01

The purpose of the investigation is to reveal the influence of dietary calcium on fluorosis-induced brain cell apoptosis in rat offspring, as well as the underlying molecular mechanism. Sprague-Dawley (SD) female rats were randomly divided into five groups: control group, fluoride group, low calcium, low calcium fluoride group, and high calcium fluoride group. SD male rats were used for breeding only. After 3 months, male and female rats were mated in a 1:1 ratio. Subsequently, 18-day-old gestation rats and 14- and 28-day-old rats were used as experimental subjects. We determined the blood/urine fluoride, the blood/urine calcium, the apoptosis in the hippocampus, and the expression levels of apoptosis-related genes, namely Bcl-2, caspase 12, and JNK. Blood or blood/urine fluoride levels and apoptotic cells were found significantly increased in fluorosis rat offspring as compared to controls. Furthermore, the Bcl-2 messenger RNA (mRNA) expression levels significantly decreased, and caspase 12 mRNA levels significantly increased in each age group as compared to controls. Compared with the fluoride group, the blood/urine fluoride content and apoptotic cells evidently decreased in the high calcium fluoride group, Bcl-2 mRNA expression significantly increased and caspase 12 mRNA expression significantly decreased in each age group. All results showed no gender difference. Based on these results, the molecular mechanisms of fluorosis-induced brain cell apoptosis in rat offspring may include the decrease in Bcl-2 mRNA expression level and increase in caspase 12 mRNA expression signaling pathways. High calcium intake could reverse these gene expression trends. By contrast, low calcium intake intensified the toxic effects of fluoride on brain cells.

Research on Grid Size Suitability of Gridded Population Distribution in Urban Area: A Case Study in Urban Area of Xuanzhou District, China.

PubMed

Dong, Nan; Yang, Xiaohuan; Cai, Hongyan; Xu, Fengjiao

2017-01-01

The research on the grid size suitability is important to provide improvement in accuracies of gridded population distribution. It contributes to reveal the actual spatial distribution of population. However, currently little research has been done in this area. Many well-modeled gridded population dataset are basically built at a single grid scale. If the grid cell size is not appropriate, it will result in spatial information loss or data redundancy. Therefore, in order to capture the desired spatial variation of population within the area of interest, it is necessary to conduct research on grid size suitability. This study summarized three expressed levels to analyze grid size suitability, which include location expressed level, numeric information expressed level, and spatial relationship expressed level. This study elaborated the reasons for choosing the five indexes to explore expression suitability. These five indexes are consistency measure, shape index rate, standard deviation of population density, patches diversity index, and the average local variance. The suitable grid size was determined by constructing grid size-indicator value curves and suitable grid size scheme. Results revealed that the three expressed levels on 10m grid scale are satisfying. And the population distribution raster data with 10m grid size provide excellent accuracy without loss. The 10m grid size is recommended as the appropriate scale for generating a high-quality gridded population distribution in our study area. Based on this preliminary study, it indicates the five indexes are coordinated with each other and reasonable and effective to assess grid size suitability. We also suggest choosing these five indexes in three perspectives of expressed level to carry out the research on grid size suitability of gridded population distribution.

Research on Grid Size Suitability of Gridded Population Distribution in Urban Area: A Case Study in Urban Area of Xuanzhou District, China

PubMed Central

Dong, Nan; Yang, Xiaohuan; Cai, Hongyan; Xu, Fengjiao

2017-01-01

The research on the grid size suitability is important to provide improvement in accuracies of gridded population distribution. It contributes to reveal the actual spatial distribution of population. However, currently little research has been done in this area. Many well-modeled gridded population dataset are basically built at a single grid scale. If the grid cell size is not appropriate, it will result in spatial information loss or data redundancy. Therefore, in order to capture the desired spatial variation of population within the area of interest, it is necessary to conduct research on grid size suitability. This study summarized three expressed levels to analyze grid size suitability, which include location expressed level, numeric information expressed level, and spatial relationship expressed level. This study elaborated the reasons for choosing the five indexes to explore expression suitability. These five indexes are consistency measure, shape index rate, standard deviation of population density, patches diversity index, and the average local variance. The suitable grid size was determined by constructing grid size-indicator value curves and suitable grid size scheme. Results revealed that the three expressed levels on 10m grid scale are satisfying. And the population distribution raster data with 10m grid size provide excellent accuracy without loss. The 10m grid size is recommended as the appropriate scale for generating a high-quality gridded population distribution in our study area. Based on this preliminary study, it indicates the five indexes are coordinated with each other and reasonable and effective to assess grid size suitability. We also suggest choosing these five indexes in three perspectives of expressed level to carry out the research on grid size suitability of gridded population distribution. PMID:28122050

High p-Smad2 expression in stromal fibroblasts predicts poor survival in patients with clinical stage I to IIIA non-small cell lung cancer.

PubMed

Chen, Yongbing; Xing, Pengfei; Chen, Yuanyuan; Zou, Li; Zhang, Yongsheng; Li, Feng; Lu, Xueguan

2014-11-05

Increasing evidence indicates that the TGFβ/Smad signaling pathway plays a prominent role in tumor initiation, progression, and metastasis. Therefore, we investigate the expression of p-Smad2 in surgical resection specimens from non-small cell lung cancer, and evaluate the prognostic significance of p-Smad2 expression in stromal fibroblasts and cancer cells for patients with clinical stage I to IIIA non-small cell lung cancer. The immunohistochemical expression of p-Smad2 was evaluated in 78 formalin-fixed paraffin-embedded surgical resection specimens from clinical stage I to IIIA non-small cell lung cancer. Correlations between p-Smad2 expression and clinicopathologic characteristics were determined by Chi-square test. The prognostic significance of p-Smad2 expression in stromal fibroblasts and cancer cells with regard to overall survival was determined by Kaplan-Meier. There were 38.5% (30/78) and 92.3% (72/78) patients with high p-Smad2 expression in stromal fibroblasts and cancer cells, respectively. There was a positive correlation between the p-Smad2 expression level in stromal fibroblasts and the p-Smad2 expression level in cancer cells (χ2=4.176, P=0.045). No significant correlation of p-Smad2 expression in stromal fibroblasts or cancer cells with any of clinicopathologic characteristics was found. The 3-year overall survival rates with low and high p-Smad2 expression in stromal fibroblasts were 53.7% and 37.7%, respectively (χ2=3.86, P=0.049). No significant association was found between low and high p-Smad2 expression in cancer cells with respect to overall survival, respectively (χ2=0.34, P=0.562). The results suggested that high p-Smad2 expression in stromal fibroblasts predicted poor survival in patients with clinical stage I to IIIA non-small cell lung cancer.

Vitamin E and vitamin E-quinone levels in red blood cells and plasma of newborn infants and their mothers.

PubMed

Jain, S K; Wise, R; Bocchini, J J

1996-02-01

Vitamin E is a physiological antioxidant and protects cell membranes from oxidative damage. This study has determined whether vitamin E level in RBC of newborns has any relationship with its level in their mothers. We have also examined levels of vitamin E and vitamin E-quinone, an oxidized product of vitamin E, in paired samples of red blood cells (RBC) and plasma of newborns and their mothers. Blood was collected from 26 mothers and their full-term placental cords at delivery. Vitamin E and vitamin E-quinone levels were determined in RBC and plasma by HPLC. Newborn-plasma had significantly lower vitamin E levels compared with maternal-plasma both when expressed as nmole/ml (5.5+/-0.4 vs 26.1+/-1.1, p = 0.0001) or nmole/mumole total lipids (1.9+/-0.1 vs 2.6+/-0.1, p = 0.0001). Vitamin E level in the newborn-RBC was similar to that of maternal-RBC when expressed as nmole/ml packed cells (2.77+/-0.14 vs 2.95+/-0.13), but was significantly lower when expressed as nmole/mumole total lipids (0.56+/-0.03 vs 0.64+/-0.04, p = 0.03) from that of maternal-RBC. Vitamin E-quinone levels are significantly elevated in newborns compared with their mothers both in RBC (29.4+/-2.1 vs 24.1+/-1.2, p = 0.04) and plasma (39.9+/-5.3 vs 25.3+/-4.2, p = 0.006) when expressed as nmole/mmole total lipids but not when expressed as nmole/ml. There was a significant correlation of vitamin E between newborn-plasma and newborn-RBC (r = 0.65, p = 0.0002 for nmole per ml packed RBC;r = 0.63, p = 0.0007 for nmole per mumole total lipids). The relationship between maternal plasma and newborn plasma was significant when vitamin E was normalized with nmole/mumole total lipid (r = 0.54, p = 0.007 but not when expressed as nmole/ml (r = 0.09, p = 0.64). However, vitamin E in the RBC of maternal and newborn had significant correlation when expressed as per ml packed cells (r = 0.61, p = 0.001) and per total lipid (r = 0.46, p = 0.02). There was no relationship of vitamin E-quinone levels between RBC and plasma of newborns and their mothers. Elevated blood levels of vitamin E-quinone suggest increased oxidative stress and utilization of vitamin E in newborns compared to their mothers. Because vitamin E levels in RBC of newborns are lower and significantly related to vitamin E levels in RBC of their mothers, an increase in vitamin E supplementation to mothers during pregnancy may increase vitamin E levels in the newborn and help impede the effect of extrauterine oxygen toxicity.

Effect of Anger Patterns and Depression on Serum IgA and NK Cell Frequency.

PubMed

Farnam, Alireza; Majidi, Jafar; Nourazar, Seyyed Gholamreza; Ghojazadeh, Morteza; Movassaghpour, Aliakbar; Zolbanin, Saeedeh Majidi

2016-03-01

There are conflicting findings about relationship between depression and anger with immunological parameters. To investigate the relationship between anger patterns and immune system in depressed patients. Thirty-five patients with major depressive disorder were selected according to DSM-IV criteria. The Hamilton Depression Scale and Spielberger Anger questionnaires were used to determine severity of depression and "anger expression pattern", respectively. The control group without a previous history of mental illness was also selected. In the group of patients with moderate depression, serum IgA levels and NK cell percentage were measured. Mean differences of all types of "anger expression pattern", including; "state-trait anger", "anger expression out", "anger expression in", "anger control out" and "anger control in", between study and control groups, were statistically significant (p<0.05). Difference in mean serum levels of IgA in either group was not significant (p=0.9), but the mean difference was significant in terms of NK-cell percentage in both groups (p=0.04). There was no significant relationship between IgA levels and percentage of NK- cell with all types of "anger expression pattern" in both groups. Only in the control group, IgA had significant correlation with anger control out (p=0.04). Moderately depressed patients versus control group had higher Spielberger scores in all types of anger expression pattern except anger control-out and anger control-in. We found no evidence supporting the relationship between" anger expression pattern" and IgA levels and NK cell percentage; however, it seems that depression itself causes reduced number of NK cells and increased IgA levels.

Upregulation of High Mobility Group Box 1 May Contribute to the Pathogenesis of Biliary Atresia.

PubMed

Ye, Chun Jing; Wang, Jiang; Yang, Yi Fan; Shen, Zhen; Chen, Gong; Huang, Yan Lei; Zheng, Yi Jie; Dong, Rui; Zheng, Shan

2018-06-17

 Biliary atresia (BA) is a progressive inflammatory obstructive cholangiopathy in infants. High mobility group box 1 (HMGB1) is known to play an important role as a late mediator of inflammation. However, it is not clear whether HMGB1 levels are of clinical significance in patients with BA. The aim of this study was to determine correlations between serum HMGB1 levels and the clinicopathologic features of BA.  Serum samples were collected from 19 infants with BA, 7 infants with anicteric choledochal cysts (CC) and normal liver function, and 8 healthy controls. Serum HMGB1 levels were measured with an enzyme-linked immunosorbent assay. Routine liver function tests were performed on serum samples. Quantitative real-time polymerase chain reaction and western blot analyses were used to detect HMGB1 expression in BA liver biopsy tissues. Localization of HMGB1 expression in the hepatic lobule was determined by immunohistochemical analysis.  HMGB1 levels in serum collected from BA infants were significantly elevated compared with CC and healthy control patients. Furthermore, elevated serum levels of HMGB1 in BA infants positively correlated with gamma-glutamyl transferase levels. HMGB1 messenger ribonucleic acid and protein expression levels were upregulated in BA liver biopsy tissues compared with CC patients. Immunohistochemical analysis also revealed increased positive immunostaining for HMGB1 in BA liver tissues as compared with CC tissues.  HMGB1 may play a crucial role in the pathogenesis of BA. Additionally, the correlation of serum HMGB1 levels with gamma-glutamyl transferase levels may provide a novel marker for the diagnosis of BA. Georg Thieme Verlag KG Stuttgart · New York.

TNF-α, IL-6 and IL-10 expressions, responsible for disparity in action of curcumin against cisplatin-induced nephrotoxicity in rats.

PubMed

Kumar, Parveen; Sulakhiya, Kunjbihari; Barua, Chandana C; Mundhe, Nitin

2017-07-01

Cisplatin is a regularly employed effective chemotherapeutic agent for the treatment of many types of cancer. The main drawback of cisplatin treatment is kidney toxicity which affects 25-35% of treated patients. Many mechanisms are believed to be involved in this kidney damage, but inflammation plays a significant role in this event. Curcumin is a polyphenol and has antioxidant and anti-inflammatory effects. The purpose of this study was to determine the protective effects of curcumin on cisplatin-induced nephrotoxicity. Female rats were randomly divided into 5 groups: control, curcumin, cisplatin, curcumin plus cisplatin (pre-treatment group) and cisplatin plus curcumin (post-treatment group). Rats were given cisplatin (7.5 mg/kg body weight) with or without curcumin treatment (120 mg/kg body weight). Blood urea nitrogen (BUN), creatinine, albumin, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-10 expressions and histological changes were determined on the 5th day after cisplatin injection. Acute kidney damage was evident by increased BUN and creatinine levels. In addition, cisplatin showed a marked pro-inflammatory response as revealed by a significant increase in the tissue levels of TNF-α, IL-6, IL-8 and decrease in the IL-10 level. Pre-treatment of curcumin reduced cisplatin-induced nephrotoxicity which was clearly evident from the reduced BUN, creatinine, TNF-α, IL-6 and IL-8 levels and increased albumin and IL-10 levels. Additionally, these findings were also supported by histopathology of the kidneys. In contrast, post-treatment of curcumin failed to cut down the expression of inflammatory markers substantially and also neglected to increase the expression of IL-10. The disparity in the action of curcumin after pre- and post-treatment with cisplatin-induced nephrotoxicity was due to the inability of post-treatment to reduce TNF-α & IL-6, besides to show a concurrent rise in IL-10 expression in renal tissues.

[Study of the role of miRNA in mesenchymal stem cells isolated from osteoarthritis patients].

PubMed

Tornero-Esteban, P; Hoyas, J A; Villafuertes, E; Garcia-Bullón, I; Moro, E; Fernández-Gutiérrez, B; Marco, F

2014-01-01

MiRNAs act as gene silencers that are involved in the regulation of essential cell functions. miR-335 is involved in regulating cell differentiation processes in progenitor cells. Mesenchymal stem cells (MSCs) are progenitor cells of chondrocytes and osteoblasts responsible for homeostatic maintenance of cartilage and bone. The aim of this study was to determine a possible relationship between the expression of miR-335 and osteoarthritis. MSCs obtained from the bone marrow of 3 osteoarthritic patients and 3 controls with no clinical signs of osteoarthritis or osteoporosis were cultured and phenotypically and functionally characterised in a 3-step culture. Expression levels of miR-335 and the mesoderm-specific transcript gene -MEST- that controls its expression were determined by quantitative PCR. Differences in the expression levels of miR-335 and MEST (median [interquartile range]: 1.69 [0.85-1.74], and 3.85 [3.20-5.67] were detected between MSCs isolated from patients with osteoarthritis and controls. Although the differences detected did not reach statistical significance (P=.1), a clear trend towards lower expression of miR-335 in osteoarthritis MSCs was observed. Given that miR-335 has the different genes involved in the Wnt signalling pathway as potential targets, the observed trend may help to ascertain, at least partially, some of the alterations which determine the onset or progression of osteoarthritis, and can therefore serve for the design of future therapeutic targets for the treatment of this disease. Copyright © 2013 SECOT. Published by Elsevier Espana. All rights reserved.

Hybrid incompatibility arises in a sequence-based bioenergetic model of transcription factor binding.

PubMed

Tulchinsky, Alexander Y; Johnson, Norman A; Watt, Ward B; Porter, Adam H

2014-11-01

Postzygotic isolation between incipient species results from the accumulation of incompatibilities that arise as a consequence of genetic divergence. When phenotypes are determined by regulatory interactions, hybrid incompatibility can evolve even as a consequence of parallel adaptation in parental populations because interacting genes can produce the same phenotype through incompatible allelic combinations. We explore the evolutionary conditions that promote and constrain hybrid incompatibility in regulatory networks using a bioenergetic model (combining thermodynamics and kinetics) of transcriptional regulation, considering the bioenergetic basis of molecular interactions between transcription factors (TFs) and their binding sites. The bioenergetic parameters consider the free energy of formation of the bond between the TF and its binding site and the availability of TFs in the intracellular environment. Together these determine fractional occupancy of the TF on the promoter site, the degree of subsequent gene expression and in diploids, and the degree of dominance among allelic interactions. This results in a sigmoid genotype-phenotype map and fitness landscape, with the details of the shape determining the degree of bioenergetic evolutionary constraint on hybrid incompatibility. Using individual-based simulations, we subjected two allopatric populations to parallel directional or stabilizing selection. Misregulation of hybrid gene expression occurred under either type of selection, although it evolved faster under directional selection. Under directional selection, the extent of hybrid incompatibility increased with the slope of the genotype-phenotype map near the derived parental expression level. Under stabilizing selection, hybrid incompatibility arose from compensatory mutations and was greater when the bioenergetic properties of the interaction caused the space of nearly neutral genotypes around the stable expression level to be wide. F2's showed higher hybrid incompatibility than F1's to the extent that the bioenergetic properties favored dominant regulatory interactions. The present model is a mechanistically explicit case of the Bateson-Dobzhansky-Muller model, connecting environmental selective pressure to hybrid incompatibility through the molecular mechanism of regulatory divergence. The bioenergetic parameters that determine expression represent measurable properties of transcriptional regulation, providing a predictive framework for empirical studies of how phenotypic evolution results in epistatic incompatibility at the molecular level in hybrids. Copyright © 2014 by the Genetics Society of America.

[Clinical and prognostic significance of preoperative serum CA153, CEA and TPS levels in patients with primary breast cancer].

PubMed

Chen, Yan; Zheng, Yu-hong; Lin, Ying-ying; Hu, Min-hua; Chen, Yan-song

2011-11-01

To investigate the clinical and prognostic values of preoperative serum CA153, CEA and TPS levels in patients with primary breast cancer. A total of 386 hospitalized patients with stage I ∼ IV breast cancer from Nov 1998 to Feb 2009 were followed up, and their clinicopathological data were analyzed retrospectively to determine the factors affecting their prognosis. First, preoperative serum CA153 expression level was significantly associated with the age of onset and tumor size (P < 0.05), the expression of serum CEA was correlated with tumor size (P < 0.05), and the expression of serum tissue polypeptide specific antigen (TPS) was correlated with tumor size and lymph node metastases (P < 0.05). Second, the overall survival was significantly shorter among patients with elevated serum CA153, CEA or TPS, respectively (P < 0.05 for overall). Finally, multivariate Cox regression analysis indicated that estrogen receptor status (ER) and elevated preoperative values of CA 153 are independent prognostic factors for overall survival (P < 0.05), and CA 153 is a risk factor but estrogen receptor status is a protective factor for overall survival. Higher preoperative expression of serum CA153, CEA or TPS is closely correlated with clinicopathological characteristics and overall survival. The prognosis is poorer in primary breast cancer patients with higher CA15-3 expression level, and pre-treatment CA153 expression level can be used as an independent prognostic parameter in patients with primarily breast cancer.

Progesterone receptor expression during prostate cancer progression suggests a role of this receptor in stromal cell differentiation.

PubMed

Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen

2015-07-01

The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.

The ESR1 and GPX1 gene expression level in human malignant and non-malignant breast tissues.

PubMed

Król, Magdalena B; Galicki, Michał; Grešner, Peter; Wieczorek, Edyta; Jabłońska, Ewa; Reszka, Edyta; Morawiec, Zbigniew; Wąsowicz, Wojciech; Gromadzińska, Jolanta

2018-01-01

The aim of this study was to establish whether the gene expression of estrogen receptor alpha (encoded by ESR1) correlates with the expression of glutathione peroxidase 1 (encoded by GPX1) in the tumor and adjacent tumor-free breast tissue, and whether this correlation is affected by breast cancer. Such relationships may give further insights into breast cancer pathology with respect to the status of estrogen receptor. We used the quantitative real-time PCR technique to analyze differences in the expression levels of the ESR1 and GPX1 genes in paired malignant and non-malignant tissues from breast cancer patients. ESR1 and GPX1 expression levels were found to be significantly down-regulated by 14.7% and 7.4% (respectively) in the tumorous breast tissue when compared to the non-malignant one. Down-regulation of these genes was independent of the tumor histopathology classification and clinicopathological factors, while the ESR1 mRNA level was reduced with increasing tumor grade (G1: 103% vs. G2: 85.8% vs. G3: 84.5%; p<0.05). In the non-malignant and malignant breast tissues, the expression levels of ESR1 and GPX1 were significantly correlated with each other (Rs=0.450 and Rs=0.360; respectively). Our data suggest that down-regulation of ESR1 and GPX1 was independent of clinicopathological factors. Down-regulation of ESR1 gene expression was enhanced by the development of the disease. Moreover, GPX1 and ESR1 gene expression was interdependent in the malignant breast tissue and further work is needed to determine the mechanism underlying this relationship.

Perturbation of Staphylococcus aureus Gene Expression by the Enoyl-Acyl Carrier Protein Reductase Inhibitor AFN-1252

PubMed Central

Parsons, Joshua B.; Kukula, Maciej; Jackson, Pamela; Pulse, Mark; Simecka, Jerry W.; Valtierra, David; Weiss, William J.; Kaplan, Nachum

2013-01-01

This study examines the alteration in Staphylococcus aureus gene expression following treatment with the type 2 fatty acid synthesis inhibitor AFN-1252. An Affymetrix array study showed that AFN-1252 rapidly increased the expression of fatty acid synthetic genes and repressed the expression of virulence genes controlled by the SaeRS 2-component regulator in exponentially growing cells. AFN-1252 did not alter virulence mRNA levels in a saeR deletion strain or in strain Newman expressing a constitutively active SaeS kinase. AFN-1252 caused a more pronounced increase in fabH mRNA levels in cells entering stationary phase, whereas the depression of virulence factor transcription was attenuated. The effect of AFN-1252 on gene expression in vivo was determined using a mouse subcutaneous granuloma infection model. AFN-1252 was therapeutically effective, and the exposure (area under the concentration-time curve from 0 to 48 h [AUC0–48]) of AFN-1252 in the pouch fluid was comparable to the plasma levels in orally dosed animals. The inhibition of fatty acid biosynthesis by AFN-1252 in the infected pouches was signified by the substantial and sustained increase in fabH mRNA levels in pouch-associated bacteria, whereas depression of virulence factor mRNA levels in the AFN-1252-treated pouch bacteria was not as evident as it was in exponentially growing cells in vitro. The trends in fabH and virulence factor gene expression in the animal were similar to those in slower-growing bacteria in vitro. These data indicate that the effects of AFN-1252 on virulence factor gene expression depend on the physiological state of the bacteria. PMID:23459481

Increased expression of Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 correlated with poor prognosis of hepatocellular carcinoma.

PubMed

Yang, Lian-Yue; Tao, Yi-Ming; Ou, Di-Peng; Wang, Wei; Chang, Zhi-Gang; Wu, Fan

2006-10-01

Because of its role in cell migration, the Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 has been implicated in cancer metastasis. Evidence to support such a role of WAVE2 in human cancer, however, is lacking. We thus examined the expression of WAVE2 in hepatocellular carcinoma (HCC) tissues to test whether the levels of WAVE2 expression correlated to the progression of HCC. Samples of 112 HCC patients were determined immunohistochemically for WAVE2 expression and the correlation of WAVE2 levels with prognosis was analyzed. Among the 112 cases, 31 paired HCC and paracarcinomatous liver tissue specimens were analyzed for WAVE2 levels by reverse transcription-PCR and Western blotting, respectively. Among 112 cases of HCCs, the immunohistochemistry data indicated significant increase of WAVE2 expression levels in 71 cases. Importantly, the increased WAVE2 expression correlated with the multiple tumor nodules (P = 0.008), the absence of capsular formation (P = 0.035), Edmondson-Steiner grade (P = 0.009), vein invasion (P = 0.023), and a shortened median survival time (326 versus 512 days; P = 0.003). Multivariable Cox regression analysis revealed the WAVE2 expression level was an independent factor for prognosis. The immunohistochemistry data were further confirmed by results of reverse transcription-PCR and Western analysis of 31 HCC cases, in which the WAVE2 mRNA and protein in HCC tissues were significantly elevated when compared with paracarcinomatous liver tissue (P < 0.001). WAVE2 expression is elevated in HCC tissues, which correlates with a poor prognosis, suggesting WAVE2 as a candidate prognostic marker of HCC.

Deregulation of PAX2 expression in renal cell tumours: mechanisms and potential use in differential diagnosis

PubMed Central

Patrício, Patrícia; Ramalho-Carvalho, João; Costa-Pinheiro, Pedro; Almeida, Mafalda; Barros-Silva, João Diogo; Vieira, Joana; Dias, Paula Cristina; Lobo, Francisco; Oliveira, Jorge; Teixeira, Manuel R; Henrique, Rui; Jeronimo, Carmen

2013-01-01

Expression of PAX2 (Paired-box 2) is suppressed through promoter methylation at the later stages of embryonic development, but eventually reactivated during carcinogenesis. Pax-2 is commonly expressed in the most prevalent renal cell tumour (RCT) subtypes—clear cell RCC (ccRCC), papillary RCC (pRCC) and oncocytoma—but not in chromophobe RCC (chrRCC), which frequently displays chromosome 10 loss (to which PAX2 is mapped). Herein, we assessed the epigenetic and/or genetic alterations affecting PAX2 expression in RCTs and evaluated its potential as biomarker. We tested 120 RCTs (30 of each main subtype) and four normal kidney tissues. Pax-2 expression was assessed by immunohistochemistry and PAX2 mRNA expression levels were determined by quantitative RT-PCR. PAX2 promoter methylation status was assessed by methylation-specific PCR and bisulfite sequencing. Chromosome 10 and PAX2 copy number alterations were determined by FISH. Pax-2 immunoexpression was significantly lower in chrRCC compared to other RCT subtypes. Using a 10% immunoexpression cut-off, Pax-2 immunoreactivity discriminated chrRCC from oncocytoma with 67% sensitivity and 90% specificity. PAX2 mRNA expression was significantly lower in chrRCC, compared to ccRCC, pRCC and oncocytoma, and transcript levels correlated with immunoexpression. Whereas no promoter methylation was found in RCTs or normal kidney, 69% of chrRCC displayed chromosome 10 monosomy, correlating with Pax-2 immunoexpression. We concluded that Pax-2 expression might be used as an ancillary tool to discriminate chrRCC from oncocytomas with overlapping morphological features. The biological rationale lies on the causal relation between Pax-2 expression and chromosome 10 monosomy, but not PAX2 promoter methylation, in chrRCC. PMID:23890189

Expression of cytochrome P-450 4 enzymes in the kidney and liver: regulation by PPAR and species-difference between rat and human.

PubMed

Ito, Osamu; Nakamura, Yasuhiro; Tan, Liping; Ishizuka, Tsuneo; Sasaki, Yuko; Minami, Naoyoshi; Kanazawa, Masayuki; Ito, Sadayoshi; Sasano, Hironobu; Kohzuki, Masahiro

2006-03-01

Members of the cytochrome P-450 4 (CYP4) family catalyze the omega-hydroxylation of fatty acids, and some of them have the PPAR response element in the promoter area of the genes. The localization of CYP4A and PPAR isoforms and the effect of PPAR agonists on CYP4A protein level and activity were determined in rat kidney and liver. Immunoblot analysis showed that CYP4A was expressed in the liver and proximal tubule, with lower expression in the preglomerular microvessel, glomerulus and thick ascending limb (TAL), but the expression was not detected in the collecting duct. PPARalpha was expressed in the liver, proximal tubule and TAL. PPARgamma was expressed in the collecting duct, with lower expression in the TAL, but no expression in the proximal tubule and liver. The PPARalpha agonist clofibrate induced CYP4A protein levels and activity in the renal cortex and liver. The PPARgamma agonist pioglitazone did not modulate them in these tissues. The localization of CYP4A and CYP4F were further determined in human kidney and liver by immunohistochemical technique. Immunostainings for CYP4A and CYP4F were observed in the hepatocytes of the liver lobule and the proximal tubules, with lower stainings in the TALs and collecting ducts, but no staining in the glomeruli or renal vasculatures. These results indicate that the inducibility of CYP4A by PPAR agonists in the rat tissues correlates with the expression of the respective PPAR isoforms, and that the localization of CYP4 in the kidney has a species-difference between rat and human.

Apoptosis gene expression and death receptor signaling in mitomycin-C-treated human tenon capsule fibroblasts.

PubMed

Crowston, Jonathan G; Chang, Lydia H; Constable, Peter H; Daniels, Julie T; Akbar, Arne N; Khaw, Peng T

2002-03-01

To examine the effect of mitomycin-C on the expression of apoptosis genes in human Tenon capsule fibroblasts and to evaluate whether death receptor signaling modulates mitomycin-C cytotoxicity. Bcl-2, Bax, Bcl-x, Fas (CD95) and tumor necrosis factor (TNF) receptor expression was determined by flow cytometry in control and mitomycin-C-treated Tenon fibroblasts. Fibroblast death was quantified using a lactate dehydrogenase release assay. The effect of Fas and TNF-receptor signaling was evaluated using Fas-specific antibodies and soluble TNF-alpha. Tenon fibroblasts constitutively express Bcl-2, Bax, and Bcl-x in culture. Mitomycin-C (0.4 mg/mL) induced a small but consistent increase in the expression of all three proteins. Tenon fibroblasts express low levels of Fas but are resistant to the effects of Fas-receptor ligation. Mitomycin-C (0.01-1.0 mg/mL) led to a significant increase in Fas expression at all concentrations tested (P < 0.01). Pretreatment with mitomycin-C (0.4 mg/mL) rendered fibroblasts susceptible to agonistic anti-Fas monoclonal IgM antibodies (50-500 ng/mL) and led to a further 50% reduction in viable fibroblasts at 48 hours, compared with mitomycin-C alone (P < 0.05). Antibodies that block the Fas receptor did not inhibit mitomycin-C-induced apoptosis. Mitomycin-C alters apoptosis gene expression and primes fibroblasts to the effects of Fas receptor ligation. Factors other than the level of Fas receptor expression modulate the response to Fas receptor signaling. Determining the signals that regulate fibroblast apoptosis may help to refine therapeutic strategies for switching off the subconjunctival healing response and maintaining intraocular pressure control.

A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

PubMed

Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

2013-06-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting. Copyright © 2013 International Society for Advancement of Cytometry.

Postnatal expression and androgen regulation of HOXBES2 homeoprotein in rat epididymis.

PubMed

Prabagaran, Esakki; Hegde, Uma C; Moodbidri, Sudhir B; Bandivdekar, Atmaram H; Raghavan, Vijaya P

2007-01-01

The multifunctional and androgen-regulated epididymis is known to provide a conducive microenvironment for the maturation and storage of mature spermatozoa. HOXB2 homeodomain-containing epididymis-specific sperm protein (HOXBES2), a molecule first reported by our group, exhibits cell- and region-specific expression. It was found in the cytoplasm of the principal epithelial cells with maximum in the distal segments of the rat epididymis. The present study was undertaken to determine whether HOXBES2 expression is regulated by androgens and postnatal epididymal development. Toward this, the epididymis was disallowed access to circulating androgens either by chemical or biologic castration. In bilaterally orchidectomized animals, the levels of immunoreactive HOXBES2 declined to <5 % of those seen in sham-operated animals. Exogenous dihydrotestosterone (DHT) supplementation (250 microg/kg body weight) for 7 days restored the expression levels to >or= 90 % of that observed in intact animals. Ethylene dimethane sulfonate (EDS) administration completely abolished HOXBES2 expression in the epididymis, and supplementation with DHT or DHT + estradiol for 10 days re-established HOXBES2 expression to near normalcy. However, in the estradiol alone-supplemented EDS-treated group, HOXBES2 remained undetected. The unaltered HOXBES2 expression following efferent duct ligation suggested that HOXBES2 is not critically dependent on testicular factors. During postnatal development, protein expression in the epididymis begins to appear from day 40 and 50 and increased from day 60 onward, coinciding with the mature levels of circulating androgens and the well-differentiated epididymis. Thus, the data obtained from this study suggests that HOXBES2 expression could be regulated by androgens, and its expression level is closely associated with the postnatal development of the epididymis.

Effects of Added Zinc on Skeletal Muscle Morphometrics and Gene Expression of Finishing Pigs Fed Ractopamine-HCL.

PubMed

Burnett, D D; Paulk, C B; Tokach, M D; Nelssen, J L; Vaughn, M A; Phelps, K J; Dritz, S S; DeRouchey, J M; Goodband, R D; Haydon, K D; Gonzalez, J M

2016-01-01

Finishing pigs (n = 320) were used in a 35-day study to determine the effects of ractopamine-HCl (RAC) and supplemental Zinc (Zn) level on loin eye area (LEA) and gene expression. Pens were randomly allotted to the following treatments for the final 35 days on feed: a corn-soybean meal diet (CON), a diet with 10 ppm RAC (RAC+), and RAC diet plus added Zn at 75, 150, or 225 ppm. Sixteen pigs per treatment were randomly selected for collection of serial muscle biopsies and carcass data on day 0, 8, 18, and 32 of the treatment phase. Compared to CON carcasses, RAC+ carcasses had 12.6% larger (P = 0.03) LEA. Carcasses from RAC diets with added Zn had a tendency for increased (quadratic, P < 0.10) LEA compared to the RAC+ carcasses. Compared to RAC+ pigs, relative expression of IGF1 decreased with increasing levels of Zn on day 8 and 18 of treatment, but expression levels were similar on day 32 due to Zn treatments increasing in expression while the RAC+ treatment decreased (Zn quadratic × day quadratic, P = 0.04). A similar trend was detected for the expression of β1-receptor where expression levels in the RAC+ pigs were greater than Zn supplemented pigs on day 8 and 18 of the experiment, but the magnitude of difference between the treatments was reduced on day 32 due to a decrease in expression by RAC+ pigs and an increase in expression by the Zn pigs (Zn quadratic × day quadratic, P = 0.01). The ability of Zn to prolong the expression of these two genes may be responsible for the tendency of Zn to increase LEA in RAC supplemented pigs.

Crystallization of Membrane Proteins by Vapor Diffusion

PubMed Central

Delmar, Jared A.; Bolla, Jani Reddy; Su, Chih-Chia; Yu, Edward W.

2016-01-01

X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization. PMID:25950974

Knockdown of Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 inhibits cell division and increases apoptosis in gastric cancer.

PubMed

Tang, Ze; He, Gan; Xu, Jie; Zhongfu, Li

2017-05-01

Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a pleiotropic protein associated with numerous cell functions, including transcription and differentiation. The role of CITED2 has been investigated in a number of malignancies; however, the roles of this protein in gastric cancers remain unclear. Therefore, we determined the role of CITED2 in gastric cancers. Gastric cancer cell lines (MKN74, MKN28, 7901, and AGS) were used to assess CITED2 transcript levels. Messenger RNA levels were determined using quantitative polymerase chain reaction. Lentiviral vectors containing CITED2 small interfering RNA were used to knockdown CITED2 expression. Cell proliferation was assessed with fluorescent imaging and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assays. Apoptosis and cell cycle stages were assessed through flow cytometry, and formation of colonies was determined using a fluorescent microscope. All cell lines tested in this study expressed CITED2. The cell line expressing the highest levels of CITED2 (MKN74) showed significant knockdown of endogenous CITED2 expression on lentiviral infection. Cell proliferation was shown to be lower in CITED2 knockdown MKN74 cells. G1/S-phase cell cycle arrest was observed on silencing of CITED2 in MKN74 cells. A significant increase in apoptosis was observed on CITED2 knock down in MKN74 cells, while colony forming ability was significantly inhibited after knock down of CITED2. CITED2 supports gastric cancer cell colony formation and proliferation while inhibiting apoptosis making it a potential gene therapy target for gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Matrix metalloproteinase-9, a potential biological marker in invasive pituitary adenomas.

PubMed

Gong, Jian; Zhao, Yunge; Abdel-Fattah, Rana; Amos, Samson; Xiao, Aizhen; Lopes, M Beatriz S; Hussaini, Isa M; Laws, Edward R

2008-01-01

We analyzed MMP-9 expression using mRNA and protein level determinations and explored the possibility that matrix metalloproteinase-9 (MMP-9) is a potential biological marker of pituitary adenoma invasiveness and whether MMP-9 could be used to discriminate the extent of invasiveness among different hormonal subtypes, tumor sizes, growth characteristics, and primary versus recurrent tumors. 73 pituitary tumor specimens were snap frozen in liquid nitrogen immediately after surgical resection. RNA and protein were extracted. MMP-9 mRNA transcripts were analyzed by quantitative RT-PCR. MMP-9 protein activity was analyzed by gelatin zymography and validated by western blot analysis. Immunohistochemistry was performed to identify the presence and localization of MMP-9 in pituitary adenomas. Statistical differences between results were determined using Student's t-test or one way ANOVA. Comparing different hormonal subtypes of noninvasive and invasive pituitary tumors, MMP-9 mRNA expression was significantly increased in the majority of invasive adenomas. Considering the protein levels, our data also showed a significant increase in MMP-9 activity in the majority of invasive adenomas and these differences were confirmed by western blot analysis and immunohistochemistry. In addition, consistent differences in MMP-9 expression levels were found according to tumor subtype, tumor size, tumor extension and primary versus redo-surgery. MMP-9 expression can consistently distinguish invasive pituitary tumors from noninvasive pituitary tumors and would reflect the extent of invasiveness in pituitary tumors according to tumor subtype, size, tumor extension, primary and redo surgery, even at early stages of invasiveness. MMP-9 may be considered a potential biomarker to determine and predict the invasive nature of pituitary tumors.

HSV-1 infection of human corneal epithelial cells: receptor-mediated entry and trends of re-infection.

PubMed

Shah, Arpeet; Farooq, Asim V; Tiwari, Vaibhav; Kim, Min-Jung; Shukla, Deepak

2010-11-20

The human cornea is a primary target for herpes simplex virus-1 (HSV-1) infection. The goals of the study were to determine the cellular modalities of HSV-1 entry into human corneal epithelial (HCE) cells. Specific features of the study included identifying major entry receptors, assessing pH dependency, and determining trends of re-infection. A recombinant HSV-1 virus expressing beta-galactosidase was used to ascertain HSV-1 entry into HCE cells. Viral replication within cells was confirmed using a time point plaque assay. Lysosomotropic agents were used to test for pH dependency of entry. Flow cytometry and immunocytochemistry were used to determine expression of three cellular receptors--nectin-1, herpesvirus entry mediator (HVEM), and paired immunoglobulin-like 2 receptor alpha (PILR-a). The necessity of these receptors for viral entry was tested using antibody-blocking. Finally, trends of re-infection were investigated using viral entry assay and flow cytometry post-primary infection. Cultured HCE cells showed high susceptibility to HSV-1 entry and replication. Entry was demonstrated to be pH dependent as blocking vesicular acidification decreased entry. Entry receptors expressed on the cell membrane include nectin-1, HVEM, and PILR-α. Receptor-specific antibodies blocked entry receptors, reduced viral entry and indicated nectin-1 as the primary receptor used for entry. Cells re-infected with HSV-1 showed a decrease in entry, which was correlated to decreased levels of nectin-1 as demonstrated by flow cytometry. HSV-1 is capable of developing an infection in HCE cells using a pH dependent entry process that involves primarily nectin-1 but also the HVEM and PILR-α receptors. Re-infected cells show decreased levels of entry, correlated with a decreased level of nectin-1 receptor expression.

A Potential Role for Endoplasmic Reticulum Stress in Progesterone Deficiency in Obese Women.

PubMed

Takahashi, Nozomi; Harada, Miyuki; Hirota, Yasushi; Zhao, Lin; Azhary, Jerilee M K; Yoshino, Osamu; Izumi, Gentaro; Hirata, Tetsuya; Koga, Kaori; Wada-Hiraike, Osamu; Fujii, Tomoyuki; Osuga, Yutaka

2017-01-01

Obesity in reproductive-aged women is associated with a shorter luteal phase and lower progesterone levels. Lipid accumulation in follicles of obese women compromises endoplasmic reticulum (ER) function, activating ER stress in granulosa cells. We hypothesized that ER stress activation in granulosa-lutein cells (GLCs) would modulate progesterone production and contribute to obesity-associated progesterone deficiency. Pretreatment with an ER stress inducer, tunicamycin or thapsigargin, inhibited human chorionic gonadotropin (hCG)-stimulated progesterone production in cultured human GLCs. Pretreatment of human GLCs with tunicamycin inhibited hCG-stimulated expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) messenger RNAs (mRNAs) without affecting expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), as determined by real-time quantitative polymerase chain reaction. Pretreatment with tunicamycin also inhibited hCG-stimulated expression of StAR protein and 3β-HSD enzyme activity in cultured human GLCs, as determined by Western blot analysis and an enzyme immunoassay, respectively, but did not affect hCG-induced intracellular 3',5'-cyclic adenosine monophosphate accumulation. Furthermore, tunicamycin attenuated hCG-induced protein kinase A and extracellular signal-regulated kinase activation, as determined by Western blot analysis. In vivo administration of tunicamycin to pregnant mare serum gonadotropin-treated immature mice prior to hCG treatment inhibited the hCG-stimulated increase in serum progesterone levels and hCG-induced expression of StAR and 3β-HSD mRNA in the ovary without affecting serum estradiol levels or the number of corpora lutea. Our findings indicate that ER stress in the follicles of obese women contributes to progesterone deficiency by inhibiting hCG-induced progesterone production in granulosa cells. Copyright © 2017 by the Endocrine Society.

Unsupervised, statistically-based systems biology approach for unraveling the genetics of complex traits: A demonstration with ethanol metabolism.

PubMed

Lusk, Ryan; Saba, Laura M; Vanderlinden, Lauren A; Zidek, Vaclav; Silhavy, Jan; Pravenec, Michal; Hoffman, Paula L; Tabakoff, Boris

2018-04-24

A statistical pipeline was developed and used for determining candidate genes and candidate gene co-expression networks involved in two alcohol (i.e., ethanol) metabolism phenotypes, namely alcohol clearance and acetate area under the curve (AUC) in a recombinant inbred (HXB/BXH) rat panel. The approach was also used to provide an indication of how ethanol metabolism can impact the normal function of the identified networks. RNA was extracted from alcohol-naïve liver tissue of 30 strains of HXB/BXH recombinant inbred rats. The reconstructed transcripts were quantitated and data was used to construct gene co-expression modules and networks. A separate group of rats, comprising the same 30 strains, were injected with ethanol (2 gm/kg) for measurement of blood ethanol and acetate levels. These data were used for QTL analysis of the rate of ethanol disappearance and circulating acetate levels. The analysis pipeline required calculation of the module eigengene values, the correction of these values with ethanol metabolism rates and acetate levels across the rat strains and the determination of the eigengene QTLs. For a module to be considered a candidate for determining phenotype, the module eigengene values had to have significant correlation with the strain phenotypic values and the module eigengene QTLs had to overlap the phenotypic QTLs. Of the 658 transcript co-expression modules generated from liver RNA sequencing data, a single module satisfied all criteria for being a candidate for determining the alcohol clearance trait. This module contained two alcohol dehydrogenase genes, including the gene whose product was previously shown to be responsible for the majority of alcohol elimination in the rat. This module was also the only module identified as a candidate for influencing circulating acetate levels. This module was also linked to the process of generation and utilization of retinoic acid as related to the autonomous immune response. We propose that our analytical pipeline can successfully identify genetic regions and transcripts which predispose a particular phenotype and our analysis provides functional context for co-expression module components. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Effects of ageing and streptozotocin-induced diabetes on connexin43 and P2 purinoceptor expression in the rat corpora cavernosa and urinary bladder.

PubMed

Suadicani, Sylvia O; Urban-Maldonado, Marcia; Tar, Moses T; Melman, Arnold; Spray, David C

2009-06-01

To investigate whether ageing and diabetes alter the expression of the gap junction protein connexin43 (Cx43) and of particular purinoceptor (P2R) subtypes in the corpus cavernosum and urinary bladder, and determine whether changes in expression of these proteins correlate with development of erectile and bladder dysfunction in diabetic and ageing rats. Erectile and bladder function of streptozotocin (STZ)-induced diabetic, insulin-treated and age-matched control Fischer-344 rats were evaluated 2, 4 and 8 months after diabetes induction by in vivo cystometry and cavernosometry. Corporal and bladder tissue were then isolated at each of these sample times and protein expression levels of Cx43 and of various P2R subtypes were determined by Western blotting. In the corpora of control rats ageing was accompanied by a significant decrease in Cx43 and P2X(1)R, and increase in P2X(7)R expression. There was decreased Cx43 and increased P2Y(4)R expression in the ageing control rat bladder. There was a significant negative correlation between erectile capacity and P2X(1)R expression levels, and a positive correlation between bladder spontaneous activity and P2Y(4)R expression levels. There was already development of erectile dysfunction and bladder overactivity at 2 months after inducing diabetes, the earliest sample measured in the study. The development of these urogenital complications was accompanied by significant decreases in Cx43, P2Y(2)R, P2X(4)R and increase in P2X(1)R expression in the corpora, and by a doubling in Cx43 and P2Y(2)R, and significant increase in P2Y(4)R expression in the bladder. Changes in Cx43 and P2R expression were largely prevented by insulin therapy. Ageing and diabetes mellitus markedly altered the expression of the gap junction protein Cx43 and of particular P2R subtypes in the rat penile corpora and urinary bladder. These changes in Cx43 and P2R expression provide the molecular substrate for altered gap junction and purinergic signalling in these tissues, and thus probably contribute to the early development of erectile dysfunction and higher detrusor activity in ageing and in diabetic rats.

Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role.

PubMed

Takada, Tomoyuki; Sasaki, Taiyo; Sato, Ryoichi; Kikuta, Shingo; Inoue, Maki N

2018-02-01

Honey bee (Apis mellifera) workers contribute to the maintenance of colonies in various ways. The primary functions of workers are divided into two types depending on age: young workers (nurses) primarily engage in such behaviors as cleaning and food handling within the hive, whereas older workers (foragers) acquire floral nutrients beyond the colony. Concomitant with this age-dependent change in activity, physiological changes occur in the tissues and organs of workers. Nurses supply younger larvae with honey containing high levels of glucose and supply older larvae with honey containing high levels of fructose. Given that nurses must determine both the concentration and type of sugar used in honey, gustatory receptors (Gr) expressed in the chemosensory organs likely play a role in distinguishing between sugars. Glucose is recognized by Gr1 in honey bees (AmGr1); however, it remains unclear which Gr are responsible for fructose recognition. This study aimed to identify fructose receptors in honey bees and reported that AmGr3, when transiently expressed in Xenopus oocytes, responded only to fructose, and to no other sugars. We analyzed expression levels of AmGr3 to identify which tissues and organs of workers are involved in fructose recognition and determined that expression of AmGr3 was particularly high in the antennae and legs of nurses. Our results suggest that nurses use their antennae and legs to recognize fructose, and that AmGr3 functions as an accurate nutrient sensor used to maintain food quality in honey bee hives. © 2017 Wiley Periodicals, Inc.

Influence of age, sex, and strength training on human muscle gene expression determined by microarray

PubMed Central

ROTH, STEPHEN M.; FERRELL, ROBERT E.; PETERS, DAVID G.; METTER, E. JEFFREY; HURLEY, BEN F.; ROGERS, MARC A.

2010-01-01

The purpose of this study was to determine the influence of age, sex, and strength training (ST) on large-scale gene expression patterns in vastus lateralis muscle biopsies using high-density cDNA microarrays and quantitative PCR. Muscle samples from sedentary young (20–30 yr) and older (65–75 yr) men and women (5 per group) were obtained before and after a 9-wk unilateral heavy resistance ST program. RNA was hybridized to cDNA filter microarrays representing ~4,000 known human genes and comparisons were made among arrays to determine differential gene expression as a result of age and sex differences, and/or response to ST. Sex had the strongest influence on muscle gene expression, with differential expression (>1.7-fold) observed for ~200 genes between men and women (~75% with higher expression in men). Age contributed to differential expression as well, as ~50 genes were identified as differentially expressed (>1.7-fold) in relation to age, representing structural, metabolic, and regulatory gene classes. Sixty-nine genes were identified as being differentially expressed (>1.7-fold) in all groups in response to ST, and the majority of these were downregulated. Quantitative PCR was employed to validate expression levels for caldesmon, SWI/SNF (BAF60b), and four-and-a-half LIM domains 1. These significant differences suggest that in the analysis of skeletal muscle gene expression issues of sex, age, and habitual physical activity must be addressed, with sex being the most critical variable. PMID:12209020

MicroRNA-130a is highly expressed in the esophageal mucosa of achalasia patients

PubMed Central

Shoji, Hiroyuki; Isomoto, Hajime; Yoshida, Akira; Ikeda, Haruo; Minami, Hitomi; Kanda, Tsutomu; Urabe, Shigetoshi; Matsushima, Kayoko; Takeshima, Fuminao; Nakao, Kazuhiko; Inoue, Haruhiro

2017-01-01

Esophageal achalasia is considered as a risk factor of esophageal cancer. The etiologies of esophageal achalasia remain unknown. Peroral endoscopic myotomy (POEM) has recently been established as a minimally invasive method with high curability. The aims of the present study were to identify the microRNAs (miRs) specific to esophageal achalasia, to determine their potential target genes and to assess their alteration following POEM. RNA was extracted from biopsy samples from middle esophageal mucosa and analyzed using a microarray. Differentially expressed miRs in achalasia patients compared with control samples were identified and analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlations between specific miR expression levels and the patients' clinical background were also investigated. In addition, alterations of selected miR expression levels before and after POEM were analyzed. The results of RT-qPCR analysis demonstrated that the miR-130a expression levels were significantly higher in patients with achalasia (P<0.0001). In addition, miR-130a expression was significantly correlated with male sex and smoking history in patients with achalasia. However, no significant change in miR-130a expression was observed between before and after POEM. In conclusion, miR-130a is highly expressed in the esophageal mucosa of patients with achalasia and may be a biomarker of esophageal achalasia. PMID:28810541

MicroRNA-130a is highly expressed in the esophageal mucosa of achalasia patients.

PubMed

Shoji, Hiroyuki; Isomoto, Hajime; Yoshida, Akira; Ikeda, Haruo; Minami, Hitomi; Kanda, Tsutomu; Urabe, Shigetoshi; Matsushima, Kayoko; Takeshima, Fuminao; Nakao, Kazuhiko; Inoue, Haruhiro

2017-08-01

Esophageal achalasia is considered as a risk factor of esophageal cancer. The etiologies of esophageal achalasia remain unknown. Peroral endoscopic myotomy (POEM) has recently been established as a minimally invasive method with high curability. The aims of the present study were to identify the microRNAs (miRs) specific to esophageal achalasia, to determine their potential target genes and to assess their alteration following POEM. RNA was extracted from biopsy samples from middle esophageal mucosa and analyzed using a microarray. Differentially expressed miRs in achalasia patients compared with control samples were identified and analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlations between specific miR expression levels and the patients' clinical background were also investigated. In addition, alterations of selected miR expression levels before and after POEM were analyzed. The results of RT-qPCR analysis demonstrated that the miR-130a expression levels were significantly higher in patients with achalasia (P<0.0001). In addition, miR-130a expression was significantly correlated with male sex and smoking history in patients with achalasia. However, no significant change in miR-130a expression was observed between before and after POEM. In conclusion, miR-130a is highly expressed in the esophageal mucosa of patients with achalasia and may be a biomarker of esophageal achalasia.

Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells

PubMed Central

Krause, Sabrina; Boeck, Christina; Gumpp, Anja M.; Rottler, Edit; Schury, Katharina; Karabatsiakis, Alexander; Buchheim, Anna; Gündel, Harald; Kolassa, Iris-Tatjana; Waller, Christiane

2018-01-01

Background: Child maltreatment (CM) and attachment experiences are closely linked to alterations in the human oxytocin (OXT) system. However, human data about oxytocin receptor (OXTR) protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR) protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation. Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC) and plasma OXT levels were determined by radioimmunoassay (RIA) in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences. Attachment representations (secure vs. insecure) were classified using the Adult Attachment Projective Picture System (AAP) and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D). Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women. Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system. PMID:29535656

Ghrelin and NUCB2/Nesfatin-1 expression in unilateral testicular torsion-induced rats with and without N-acetylcysteine.

PubMed

Sarac, M; Bakal, U; Tartar, T; Kuloglu, T; Yardim, M; Artas, G; Aydin, S; Kazez, A

2017-08-15

Testicular torsion (TT) is a common urological problem in the field of pediatric surgery. The degree and duration of torsion determines the degree of testicular damage; however, its effects on the expression of octanoylated ghrelin and nucleobindin 2 (NUCB2) /nesfatin-1 synthetized from testicular tissue remain unclear. We explored the effects of experimentally induced unilateral TT on serum and contralateral testicular tissue ghrelin and NUCB2/nesfatin-1 levels, and determined whether N-acetyl cysteine (NAS) treatment had any effects on their expression. A total of 42 Wistar Albino strain rats were divided into 7 groups: Group (G) I control, GII sham, GIII 12-hour torsion, GIV 12-hour torsion + detorsion + 100 mg/kg NAS, GV 24-hour torsion, GVI 24-hour torsion + detorsion + 100 mg/kg NAS, and GVII 100 mg/kg NAS. Octanoylated ghrelin and NUCB2/nesfatin-1 concentrations were evaluated in serum using the ELISA method and in testicular tissue with immunohistochemical methods. Immunoreactivity of octanoylated ghrelin significantly increased in GI compared to GIII, GV, and GVI (p<0.05). NUCB2/nesfatin-1 immunoreactivity increased in GV and GVIII relative to GI (p<0.05). In the 12-hour torsion group, a significant decrease in octanoylated ghrelin levels with NAS treatment was observed; however, in the 24-hour torsion group, a significant decrease was not observed. In the 12-hour torsion + NAS treatment group, a significant change was not observed in NUCB2/nesfatin-1 expression. Following 24-hour torsion, an increase in NUCB2/nesfatin-1 levels was observed, and NAS treatment did not reverse this increase. It was determined that increases in the expression of octanoylated ghrelin and NUCB2/nesfatin-1, the latter of which was a result of TT, reflect damage in this tissue. Importantly, NAS treatment could prevent this damage. Thus, there may be a clinical application for the combined use of NAS and octanoylated ghrelin in preventing TT-related infertility.

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA

PubMed Central

Lundin, Erik; Tang, Po-Cheng; Guy, Lionel; Näsvall, Joakim; Andersson, Dan I

2018-01-01

Abstract The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients. PMID:29294020

Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium

PubMed Central

Poza-Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echavarri-Erasun, Carlos

2014-01-01

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs. PMID:24244007

Kinetics of Nif gene expression in a nitrogen-fixing bacterium.

PubMed

Poza-Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echavarri-Erasun, Carlos; Rubio, Luis M

2014-02-01

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs.

Increased expression of Apo-J and Omi/HtrA2 after Intracerebral Hemorrage in rats.

PubMed

Li, Feng; Yang, Jing; Guo, Xiaoyan; Zheng, Xiaomei; Lv, Zhiyu; Shi, Chang Qing; Li, Xiaogang

2018-03-23

To investigate the changes of Apo-J and Omi/HtrA2 protein expression in rats with intracerebral hemorrage. 150 SD adult rats were randomly divided into 3 groups: (1) Normal Control (NC) group, (2) Sham group, (3) Intracerebral Hemorrage (ICH) group. The data were collected at 6h, 12h, 1d, 2d, 3d, 5d and 7d. Apoptosis was measured by Tunel staining. The distributions of the Apo-J and Omi/HtrA2 proteins were determined by immunohistochemical staining. The levels of Apo-J mRNA and Omi/HtrA2 mRNA expressions were examined by RT-PCR. Apoptosis in ICH group was higher than Sham and NC groups (p＜0.05). Both the Apo-J and Omi/HtrA2 expression levels were increased in the peripheral region of hemorrhage, with a peak at 3d. The Apo-J mRNA level positively correlated with HtrA2 mRNA level in ICH group (r=0.883, p＜0.001). The expressions of Apo-J and Omi/HtrA2 paralelly increased in peripheral region of rat cerebral hemorrhage. Local high expressed Apo-J in the peripheral regions might play a neuroprotective role by inhibiting apoptosis via Omi/HtrA2 pathway after hemorrhage. Copyright © 2018. Published by Elsevier Inc.

Decidual β-carotene-15,15'-oxygenase-1 and 2 (BCMO1,2) expression is increased in nitrofen model of congenital diaphragmatic hernia.

PubMed

Takahashi, Hiromizu; Kutasy, Balazs; Pes, Lara; Paradisi, Francesca; Puri, Prem

2015-01-01

Retinoids are essential for fetal and lung development. Beta-carotene(BC) is the main dietary retinoid source and beta-carotene-15,15'-oxygenase-1 and 2 (Bcmo1,2) is the primary enzyme generating retinoid from BC in adult mammalian tissues. Placenta has a major role in the retinol homeostasis in fetal life: Since there is no fetal retinol synthesis, maternal retinol has to cross the placenta. It has been recently shown that BC can be converted to retinol by Bcmo1,2 in placenta for retinol transfer and moreover, BC can cross the placenta intact. The placental Bcmo1,2 expression is tightly controlled by placental retinol level. In severe retinol deficiency it has been shown that placental Bcmo1,2 expression are increased for generating retinol from dietary maternal BC even when the main retinol transfer is blocked. In recent years, low pulmonary retinol levels and disrupted retinoid signaling pathway have been implicated in the pathogenesis of pulmonary hypoplasia and congenital diaphragmatic hernia (CDH) in the nitrofen model of CDH. Recently, it has been demonstrated that the main retinol transfer in the placenta is blocked in the nitrofen model of CDH causing increased placental and decreased serum retinol level. The aim of our study was to determine maternal and fetal β-carotene levels and to investigate the hypothesis that placental expression of BCMO1 and BCMO2 is altered in nitrofen-exposed rat fetuses with CDH. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Maternal and fetal serum, placenta, liver and left lungs were harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Immunochistochemistry was performed to evaluate trophoblasts by cytokeratin expression and placental Bcmo1,2 expression. Expression levels of Bcmo1,2 genes in fetal lungs and liver were determined using RT-PCR and immunohistochemistry. BC level was measured using HPLC. Markedly increased decidual Bcmo1,2 immunoreactivity was observed in CDH group compared to controls. There was no difference neither in the trophoblastic Bcmo1,2 immunoreactivity nor in the pulmonary and liver Bcmo1,2 expression compared to controls. There was no significant difference in maternal serum BC levels between control and CDH mothers (2.14 ± 0.55 vs 2.56 ± 1.6 μM/g, p = 0.8). BC was not detectable neither in the fetal serum nor liver or lungs. Our data show that nitrofen increases maternal but not fetal Bcmo1,2 expression in the placenta in nitrofen-induced CDH group. The markedly increased decidual Bcmo1,2 expression suggests that nitrofen may trigger local, decidual retinol synthesis in the nitrofen model of CDH.

SpeCond: a method to detect condition-specific gene expression

PubMed Central

2011-01-01

Transcriptomic studies routinely measure expression levels across numerous conditions. These datasets allow identification of genes that are specifically expressed in a small number of conditions. However, there are currently no statistically robust methods for identifying such genes. Here we present SpeCond, a method to detect condition-specific genes that outperforms alternative approaches. We apply the method to a dataset of 32 human tissues to determine 2,673 specifically expressed genes. An implementation of SpeCond is freely available as a Bioconductor package at http://www.bioconductor.org/packages/release/bioc/html/SpeCond.html. PMID:22008066

Plastid Transcriptomics and Translatomics of Tomato Fruit Development and Chloroplast-to-Chromoplast Differentiation: Chromoplast Gene Expression Largely Serves the Production of a Single Protein[W][OA

PubMed Central

Kahlau, Sabine; Bock, Ralph

2008-01-01

Plastid genes are expressed at high levels in photosynthetically active chloroplasts but are generally believed to be drastically downregulated in nongreen plastids. The genome-wide changes in the expression patterns of plastid genes during the development of nongreen plastid types as well as the contributions of transcriptional versus translational regulation are largely unknown. We report here a systematic transcriptomics and translatomics analysis of the tomato (Solanum lycopersicum) plastid genome during fruit development and chloroplast-to-chromoplast conversion. At the level of RNA accumulation, most but not all plastid genes are strongly downregulated in fruits compared with leaves. By contrast, chloroplast-to-chromoplast differentiation during fruit ripening is surprisingly not accompanied by large changes in plastid RNA accumulation. However, most plastid genes are translationally downregulated during chromoplast development. Both transcriptional and translational downregulation are more pronounced for photosynthesis-related genes than for genes involved in gene expression, indicating that some low-level plastid gene expression must be sustained in chromoplasts. High-level expression during chromoplast development identifies accD, the only plastid-encoded gene involved in fatty acid biosynthesis, as the target gene for which gene expression activity in chromoplasts is maintained. In addition, we have determined the developmental patterns of plastid RNA polymerase activities, intron splicing, and RNA editing and report specific developmental changes in the splicing and editing patterns of plastid transcripts. PMID:18441214

Nrf2-related gene expression and exposure to traffic-related air pollution in elderly subjects with cardiovascular disease: An exploratory panel study

PubMed Central

Wittkopp, Sharine; Staimer, Norbert; Tjoa, Thomas; Stinchcombe, Timothy; Daher, Nancy; Schauer, James J.; Shafer, Martin M.; Sioutas, Constantinos; Gillen, Daniel L.; Delfino, Ralph J.

2015-01-01

Gene expression changes are linked to air pollutant exposures in in vitro and animal experiments. However, limited data are available on how these outcomes relate to ambient air pollutant exposures in humans. We performed an exploratory analysis testing whether gene expression levels were associated with air pollution exposures in a Los Angeles area cohort of elderly subjects with coronary artery disease. Candidate genes (35) were selected from published studies of gene expression-pollutant associations. Expression levels were measured weekly in 43 subjects (≤12 weeks) using quantitative PCR. Exposures included gaseous pollutants O3, nitrogen oxides (NOx), and CO; particulate matter (PM) pollutants elemental and black carbon (EC, BC); and size-fractionated PM mass. We measured organic compounds from PM filter extracts, including polycyclic aromatic hydrocarbons (PAHs), and determined the in vitro oxidative potential of particle extracts. Associations between exposures and gene expression levels were analyzed using mixed-effects regression models. We found positive associations of traffic-related pollutants (EC, BC, primary organic carbon, PM0.25-2.5 PAH and/or PM0.25 PAH, and NOx) with NFE2L2, Nrf2-mediated genes (HMOX1, NQO1, and SOD2), CYP1B1, IL1B, and SELP. Findings suggest that NFE2L2 gene expression links associations of traffic-related air pollution with phase I and II enzyme genes at the promoter transcription level. PMID:25564368

The effect of water deprivation on the expression of atrial natriuretic peptide and its receptors in the spinifex hopping mouse, Notomys alexis.

PubMed

Heimeier, Rachel A; Davis, Belinda J; Donald, John A

2002-08-01

This study investigated the mRNA expression of the atrial natriuretic peptide (ANP) system (peptide and receptors) during water deprivation in the spinifex hopping mouse, Notomys alexis, a native of central and western Australia that is well adapted to survive in arid environments. Initially, ANP, NPR-A and NPR-C cDNAs (partial for receptors) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. Using a semi-quantitative multiplex PCR technique, the expression of cardiac ANP mRNA and renal ANP, NPR-A, and NPR-C mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control mice (access to water). The levels of ANP mRNA expression in the heart remained unchanged, but in the kidney ANP mRNA levels were increased in the 7-day water-deprived mice, and were significantly decreased in the 14-day water-deprived mice. NPR-A mRNA levels were significantly higher in 7-day water-deprived mice while no change for NPR-A mRNA expression was observed in 14-day water-deprived mice. No variation in NPR-C mRNA levels was observed. This study shows that water deprivation differentially affects the expression of the ANP system, and that renal ANP expression is more important than cardiac ANP in the physiological adjustment to water deprivation.

Nrf2-related gene expression and exposure to traffic-related air pollution in elderly subjects with cardiovascular disease: An exploratory panel study.

PubMed

Wittkopp, Sharine; Staimer, Norbert; Tjoa, Thomas; Stinchcombe, Timothy; Daher, Nancy; Schauer, James J; Shafer, Martin M; Sioutas, Constantinos; Gillen, Daniel L; Delfino, Ralph J

2016-01-01

Gene expression changes are linked to air pollutant exposures in in vitro and animal experiments. However, limited data are available on how these outcomes relate to ambient air pollutant exposures in humans. We performed an exploratory analysis testing whether gene expression levels were associated with air pollution exposures in a Los Angeles area cohort of elderly subjects with coronary artery disease. Candidate genes (35) were selected from published studies of gene expression-pollutant associations. Expression levels were measured weekly in 43 subjects (≤ 12 weeks) using quantitative PCR. Exposures included gaseous pollutants O3, nitrogen oxides (NOx), and CO; particulate matter (PM) pollutants elemental and black carbon (EC, BC); and size-fractionated PM mass. We measured organic compounds from PM filter extracts, including polycyclic aromatic hydrocarbons (PAHs), and determined the in vitro oxidative potential of particle extracts. Associations between exposures and gene expression levels were analyzed using mixed-effects regression models. We found positive associations of traffic-related pollutants (EC, BC, primary organic carbon, PM 0.25-2.5 PAH and/or PM 0.25 PAH, and NOx) with NFE2L2, Nrf2-mediated genes (HMOX1, NQO1, and SOD2), CYP1B1, IL1B, and SELP. Findings suggest that NFE2L2 gene expression links associations of traffic-related air pollution with phase I and II enzyme genes at the promoter transcription level.

Down-regulation of long non-coding RNA RP11-708H21.4 is associated with poor prognosis for colorectal cancer and promotes tumorigenesis through regulating AKT/mTOR pathway.

PubMed

Sun, Longci; Jiang, Chunhui; Xu, Chunjie; Xue, Hanbing; Zhou, Hong; Gu, Lei; Liu, Ye; Xu, Qing

2017-04-25

Long non-coding RNAs (lncRNAs) serve critical roles in cancer development and progression. Herein, through next generation RNA sequencing and experimental validations, we determined the expression status of RP11-708H21.4 in colorectal cancer (CRC) and explored its clinical significance and biological functions in CRC. Differentially expressed lncRNAs from CRC samples and corresponding normal mucosa tissues was screened through RNA sequencing, and RP11-708H21.4 was selected for further experimental validation. The expression levels of RP11-708H21.4 in CRC tissues and cell lines were determined using qRT-PCR. Also, the relationship between the clinicopathological features and RP11-708H21.4 expression was analyzed. Cell viability was examined by CCK-8 and colony assays; cell migration and invasion were detected by transwell assays; cell cycle and cell apoptosis were analyzed by flow cytometry. The chemosensitivity of CRC cells to 5-Fluorouracil (5-FU) was also determined using CCK-8 assay. CRC xenograft tumor models were established to determine the biological functions of RP11-708H21.4 in vivo. Levels of cell cycle-related proteins and AKT/mTOR pathway-related proteins were detected by western blot assay. RP11-708H21.4 expression was aberrantly decreased in CRC, and its expression was closely associated with aggressive clinicopathologic features and unfavorable prognosis of CRC patients. Overexpressed RP11-708H21.4 suppresses CRC cell proliferation through inducing G1 arrest. Moreover, up-regulation of RP11-708H21.4 inhibits cell migration and invasion, causes cell apoptosis, and enhances 5-FU sensitivity of CRC cells. Finally, increased RP11-708H21.4 expression blocked AKT/mTOR pathway, and repressed in vivo CRC xenograft tumor growth. The results indicated that RP11-708H21.4 might have potential roles as a biomarker and a therapeutic target for CRC.

The Blood Group A Genotype Determines the Level of Expression of the Blood Group A on Platelets But Not the Anti-B Isotiter

PubMed Central

Lehner, Barbara; Eichelberger, Beate; Jungbauer, Christof; Panzer, Simon

2015-01-01

Summary Background The extent of expression of the blood group A on platelets is controversial. Further, the relation between platelets' blood group A expression and the titers of isoagglutinins has not been thoroughly investigated, so far. Methods We evaluated the relation between the genotype with platelets' blood group A and H expression estimated by flow cytometry and the titers of isoagglutinins. Results The A expression varied between genotypes and within genotypes. However, the expression in A1 was stronger than in all other genotypes (p < 0.0001). An overlap of expression levels was apparent between homozygous A1A1 and heterozygous A1 individuals. Still, The A1A1 genotype is associated with a particularly high antigen expression (p = 0.009). Platelets' A expression in A2 versus blood group O donors was also significant (p = 0.007), but there was again an overlap of expression. The secretor status had only little influence on the expression (p = 0.18). Also, isoagglutinin titers were not associated with genotypes. Conclusion: To distinguish between A1 and A2 donors may reduce incompatible platelet transfusions and therefore be favorable on platelet transfusion increment. Clinical data are needed to support this notion. PMID:26733767

GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells.

PubMed

Nazari, Fatemeh; Parham, Abbas; Maleki, Adham Fani

2015-01-01

Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, β-actin and β2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. The expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of β-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, β-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. This study demonstrated that GAPDH and especially β-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.

Losartan activates sirtuin 1 in rat reduced-size orthotopic liver transplantation

PubMed Central

Pantazi, Eirini; Bejaoui, Mohamed; Zaouali, Mohamed Amine; Folch-Puy, Emma; Pinto Rolo, Anabela; Panisello, Arnau; Palmeira, Carlos Marques; Roselló-Catafau, Joan

2015-01-01

AIM: To investigate a possible association between losartan and sirtuin 1 (SIRT1) in reduced-size orthotopic liver transplantation (ROLT) in rats. METHODS: Livers of male Sprague-Dawley rats (200-250 g) were preserved in University of Wisconsin preservation solution for 1 h at 4 °C prior to ROLT. In an additional group, an antagonist of angiotensin II type 1 receptor (AT1R), losartan, was orally administered (5 mg/kg) 24 h and 1 h before the surgical procedure to both the donors and the recipients. Transaminase (as an indicator of liver injury), SIRT1 activity, and nicotinamide adenine dinucleotide (NAD+, a co-factor necessary for SIRT1 activity) levels were determined by biochemical methods. Protein expression of SIRT1, acetylated FoxO1 (ac-FoxO1), NAMPT (the precursor of NAD+), heat shock proteins (HSP70, HO-1) expression, endoplasmic reticulum stress (GRP78, IRE1α, p-eIF2) and apoptosis (caspase 12 and caspase 3) parameters were determined by Western blot. Possible alterations in protein expression of mitogen activated protein kinases (MAPK), such as p-p38 and p-ERK, were also evaluated. Furthermore, the SIRT3 protein expression and mRNA levels were examined. RESULTS: The present study demonstrated that losartan administration led to diminished liver injury when compared to ROLT group, as evidenced by the significant decreases in alanine aminotransferase (358.3 ± 133.44 vs 206 ± 33.61, P < 0.05) and aspartate aminotransferase levels (893.57 ± 397.69 vs 500.85 ± 118.07, P < 0.05). The lessened hepatic injury in case of losartan was associated with enhanced SIRT1 protein expression and activity (5.27 ± 0.32 vs 6.08 ± 0.30, P < 0.05). This was concomitant with increased levels of NAD+ (0.87 ± 0.22 vs 1.195 ± 0.144, P < 0.05) the co-factor necessary for SIRT1 activity, as well as with decreases in ac-FoxO1 expression. Losartan treatment also provoked significant attenuation of endoplasmic reticulum stress parameters (GRP78, IRE1α, p-eIF2) which was consistent with reduced levels of both caspase 12 and caspase 3. Furthermore, losartan administration stimulated HSP70 protein expression and attenuated HO-1 expression. However, no changes were observed in protein or mRNA expression of SIRT3. Finally, the protein expression pattern of p-ERK and p-p38 were not altered upon losartan administration. CONCLUSION: The present study reports that losartan induces SIRT1 expression and activity, and that it reduces hepatic injury in a ROLT model. PMID:26185373

Losartan activates sirtuin 1 in rat reduced-size orthotopic liver transplantation.

PubMed

Pantazi, Eirini; Bejaoui, Mohamed; Zaouali, Mohamed Amine; Folch-Puy, Emma; Pinto Rolo, Anabela; Panisello, Arnau; Palmeira, Carlos Marques; Roselló-Catafau, Joan

2015-07-14

To investigate a possible association between losartan and sirtuin 1 (SIRT1) in reduced-size orthotopic liver transplantation (ROLT) in rats. Livers of male Sprague-Dawley rats (200-250 g) were preserved in University of Wisconsin preservation solution for 1 h at 4 °C prior to ROLT. In an additional group, an antagonist of angiotensin II type 1 receptor (AT1R), losartan, was orally administered (5 mg/kg) 24 h and 1 h before the surgical procedure to both the donors and the recipients. Transaminase (as an indicator of liver injury), SIRT1 activity, and nicotinamide adenine dinucleotide (NAD(+), a co-factor necessary for SIRT1 activity) levels were determined by biochemical methods. Protein expression of SIRT1, acetylated FoxO1 (ac-FoxO1), NAMPT (the precursor of NAD+), heat shock proteins (HSP70, HO-1) expression, endoplasmic reticulum stress (GRP78, IRE1α, p-eIF2) and apoptosis (caspase 12 and caspase 3) parameters were determined by Western blot. Possible alterations in protein expression of mitogen activated protein kinases (MAPK), such as p-p38 and p-ERK, were also evaluated. Furthermore, the SIRT3 protein expression and mRNA levels were examined. The present study demonstrated that losartan administration led to diminished liver injury when compared to ROLT group, as evidenced by the significant decreases in alanine aminotransferase (358.3 ± 133.44 vs 206 ± 33.61, P < 0.05) and aspartate aminotransferase levels (893.57 ± 397.69 vs 500.85 ± 118.07, P < 0.05). The lessened hepatic injury in case of losartan was associated with enhanced SIRT1 protein expression and activity (5.27 ± 0.32 vs 6.08 ± 0.30, P < 0.05). This was concomitant with increased levels of NAD(+) (0.87 ± 0.22 vs 1.195 ± 0.144, P < 0.05) the co-factor necessary for SIRT1 activity, as well as with decreases in ac-FoxO1 expression. Losartan treatment also provoked significant attenuation of endoplasmic reticulum stress parameters (GRP78, IRE1α, p-eIF2) which was consistent with reduced levels of both caspase 12 and caspase 3. Furthermore, losartan administration stimulated HSP70 protein expression and attenuated HO-1 expression. However, no changes were observed in protein or mRNA expression of SIRT3. Finally, the protein expression pattern of p-ERK and p-p38 were not altered upon losartan administration. The present study reports that losartan induces SIRT1 expression and activity, and that it reduces hepatic injury in a ROLT model.

A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

PubMed

Greenberg, N M; Reding, T V; Duffy, T; Rosen, J M

1991-10-01

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

The Study of HFE Genotypes and Its Expression Effect on Iron Status of Iranian Haemochromatosis, Iron Deficiency Anemia Patients, Iron-Taker and Non Iron-Taker Controls.

PubMed

Beiranvand, Elham; Abediankenari, Saeid; Rostamian, Mosayeb; Beiranvand, Behnoush; Naazeri, Saeed

2015-01-01

The role of HFE gene mutations or its expression in regulation of iron metabolism of hereditary haemochromatosis (HH) patients is remained controversial. Therefore here the correlation between two common HFE genotype (p.C282Y, p.H63D) and HFE gene expression with iron status in HH, iron deficiency anemia (IDA) and healthy Iranian participants was studied. For this purpose genotype determination was done by polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP). Real-Time PCR was applied for evaluation of HFE gene expression. Biochemical parameters and iron consumption were also assessed. Homozygote p.H63D mutation was seen in all HH patients and p.C282Y was not observed in any member of the population. A significant correlation was observed between serum ferritin (SF) level and gender or age of HH patients. p.H63D homozygote was seen to be able to significantly increase SF and transferrin saturation (TS) level without affecting on liver function. Our results also showed that iron consumption affects on TS level increasing. HFE gene expression level of IDA patients was significantly higher than other groups. Also the HFE gene expression was negatively correlated with TS. Finally, the main result of our study showed that loss of HFE function in HH is not derived from its gene expression inhibition and much higher HFE gene expression might lead to IDA. However we propose repeating of the study for more approval of our finding.

Gene expression profile of collagen types, osteopontin in the tympanic membrane of patients with tympanosclerosis.

PubMed

Sakowicz-Burkiewicz, Monika; Kuczkowski, Jerzy; Przybyła, Tomasz; Grdeń, Marzena; Starzyńska, Anna; Pawełczyk, Tadeusz

2017-09-01

Tympanosclerosis is a pathological process involving the middle ear. The hallmark of this disease is the formation of calcium deposits. In the submucosal layer, as well as in the right layer of the tympanic membrane, the calcium deposits result in a significant increase in the activity of fibroblasts and deposition of collagen fibers. The aim of our study was to examine the expression level of genes encoding collagen type I, II, III and IV (COL1A1, COL2A1, COL3A1, COL4A1) and osteopontin (SPP1) in the tympanic membrane of patients with tympanosclerosis. The total RNA was isolated from middle ear tissues with tympanosclerosis, received from 25 patients and from 19 normal tympanic membranes. The gene expression level was determined by real-time RT-PCR. The gene expression levels were correlated with clinical Tos classification of tympanosclerosis. We observed that in the tympanic membrane of patients with tympanosclerosis, the expression of type I collagen is decreased, while the expression of type II and IV collagen and osteopontin is increased. Moreover, mRNA levels of the investigated genes strongly correlated with the clinical stages of tympanosclerosis. The strong correlations between the expression of type I, II, IV collagen and osteopontin and the clinical stage of tympanosclerosis indicate the involvement of these proteins in excessive fibrosis and pathological remodeling of the tympanic membrane. In the future, a treatment aiming to modulate these gene expressions and/or regulation of the degradation of their protein products could be used as a new medical approach for patients with tympanosclerosis.

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

PubMed Central

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

2006-01-01

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

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

PubMed

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

2006-02-21

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

Lipocalin-2 expression and serum levels as early predictors of type 2 diabetes mellitus in obese women.

PubMed

Rashad, Nearmeen M; El-Shal, Amal S; Etewa, Rasha L; Wadea, Fady M

2017-02-01

Obesity and diabetes are increasing in epidemic proportions globally. Lipocalin-2 (LCN-2) is an inflammatory adipocytokine and obesity-related marker of low-grade inflammation. We aimed to investigate, for first time, the possible role of LCN-2 expression and serum levels in prediction of impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM) among obese Egyptian women. This study included 188 obese women and 180 controls. Obese women were subdivided into three subgroups according to their fasting blood glucose, normal glucose tolerance (NGT), IGT and T2DM. Circulating LCN-2 expression levels were determined by real time polymerase chain reaction. Serum LCN-2 concentrations were assessed by ELISA. Our findings revealed that LCN-2 expression and serum levels were higher in obese women compared to lean controls. They were higher in IGT and T2DM obese cases than in NGT obese women. Receiver operating characteristic analyses revealed that LCN-2 expression level was a useful biomarker discriminating IGT from NGT and T2DM from IGT obese women (AUC were 0.735 and 0.740, respectively). It was an independent predictor of IGT and T2DM among obese women. Serum LCN-2 level was a useful biomarker discriminating IGT from NGT and T2DM from IGT obese women (AUC were 0.705 and 0.728, respectively). It was independent predictor of T2DM without predicting IGT among obese women. The power of combined LCN-2 serum levels and expression in discriminating between IGT from NGT and T2DM from IGT obese women was high (AUC = 0.717 and 0.741, respectively). In conclusion, LCN-2 expression and serum levels could discriminate IGT from NGT and T2DM from IGT obese women and early predicting T2DM among obese women. While, LCN-2 expression level was the independent predictor of IGT in obese women. Combination of both LCN-2 expression and serum levels improved their diagnostic value in early detection of IGT and T2DM among obese women. © 2017 IUBMB Life, 69(2):88-97, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

GPR21 KO mice demonstrate no resistance to high fat diet induced obesity or improved glucose tolerance.

PubMed

Wang, Jinghong; Pan, Zheng; Baribault, Helene; Chui, Danny; Gundel, Caroline; Véniant, Murielle

2016-01-01

Gpr21 KO mice generated with Gpr21 KO ES cells obtained from Deltagen showed improved glucose tolerance and insulin sensitivity when fed a high fat diet. Further mRNA expression analysis revealed changes in Rabgap1 levels and raised the possibility that Rabgap1 gene may have been modified. To assess this hypothesis a new Gpr21 KO mouse line using TALENS technology was generated. Gpr21 gene deletion was confirmed by PCR and Gpr21 and Rabgap1 mRNA expression levels were determined by RT-PCR. The newly generated Gpr21 KO mice when fed a normal or high fat diet chow did not maintain their improved metabolic phenotype. In conclusion, Rabgap1 disturbance mRNA expression levels may have contributed to the phenotype of the originally designed Gpr21 KO mice.

Protein Expression Level of Skin Wrinkle-Related Factors in Hairless Mice Fed Hyaluronic Acid.

PubMed

Yun, Min-Kyu; Lee, Sung-Jin; Song, Hye-Jin; Yu, Heui-Jong; Rha, Chan Su; Kim, Dae-Ok; Choe, Soo-Young; Sohn, Johann

2017-04-01

The aim of this study was to evaluate the wrinkle improving effect of hyaluronic acid intakes. Wrinkles were induced by exposing the skin of hairless mice to ultraviolet B (UVB) irradiation for 14 weeks. Hyaluronic acid was administered to the mice for 14 weeks including 4 weeks before experiments. Skin tissue was assayed by enzyme-linked immunosorbent assay to determine protein expression of wrinkle-related markers. The group supplemented with high concentrations of hyaluronic acid appeared significantly better than control group for collagen, matrix metalloproteinase 1, interleukin (IL)-1β, and IL-6 assay. Transforming growth factor-β1 (TGF-β1) and hyaluronic acid synthase 2 (HAS-2) were not shown to be significantly different. In conclusion, hyaluronic acid administration regulated expression levels of proteins associated with skin integrity, and improved the wrinkle level in skin subjected to UVB irradiation.

Interferon lambda 1-3 expression in infants hospitalized for RSV or HRV associated bronchiolitis.

PubMed

Selvaggi, Carla; Pierangeli, Alessandra; Fabiani, Marco; Spano, Lucia; Nicolai, Ambra; Papoff, Paola; Moretti, Corrado; Midulla, Fabio; Antonelli, Guido; Scagnolari, Carolina

2014-05-01

The airway expression of type III interferons (IFNs) was evaluated in infants hospitalized for respiratory syncytial virus (RSV) or rhinovirus (HRV) bronchiolitis. As an additional objective we sought to determine whether a different expression of IFN lambda 1-3 was associated with different harboring viruses, the clinical course of bronchiolitis or with the levels of well established IFN stimulated genes (ISGs), such as mixovirus resistance A (MxA) and ISG56. The analysis was undertaken in 118 infants with RSV or HRV bronchiolitis. Nasopharyngeal washes were collected for virological studies and molecular analysis of type III IFN responses. RSV elicited higher levels of IFN lambda subtypes when compared with HRV. A similar expression of type III IFN was found in RSVA or RSVB infected infants and in those infected with HRVA or HRVC viruses. Results also indicate that IFN lambda 1 and IFN lambda 2-3 levels were correlated with each other and with MxA and ISG56-mRNAs. In addition, a positive correlation exists between the IFN lambda1 levels and the clinical score index during RSV infection. In particular, higher IFN lambda 1 levels are associated to an increase of respiratory rate. These findings show that differences in the IFN lambda 1-3 levels in infants with RSV or HRV infections are present and that the expression of IFN lambda 1 correlates with the severity of RSV bronchiolitis. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

PubMed

Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

1995-02-10

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Expression of Estrogen Receptors in Relation to Hormone Levels and the Nottingham Prognostic Index.

PubMed

Fahlén, Mia; Zhang, Hua; Löfgren, Lars; Masironi, Britt; VON Schoultz, Eva; VON Schoultz, B O; Sahlin, Lena

2016-06-01

Estrogen hormones have a large impact on both normal development and tumorigenesis of the breast. Breast tissue samples from 49 women undergoing surgery were included. The estrogen receptors (ERα and ERβ), ERα36 and G-coupled estrogen receptor-1 (GPER) were determined in benign and malignant breast tissue. The ERα36 and ERα mRNA levels were highest in malignant tumors. Stromal ERβ immunostaining in benign tumors was higher than in the paired normal tissue. GPER expression was lowest in benign tumors. In the malignant tumors, the Nottingham Prognostic Index (NPI) correlated positively with stromal GPER and the serum testosterone level. The serum insulin-like growth factor-1 (IGF-1) level correlated negatively with GPER mRNA and glandular ERα. The expression of ERα36 is stronger in malignant breast tissue. The strong positive correlation between NPI and GPER in malignant breast stroma indicates an important role for GPER in breast cancer prognosis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Decreased GRK3 but not GRK2 expression in frontal cortex from bipolar disorder patients

PubMed Central

Rao, Jagadeesh S; Rapoport, Stanley I; Kim, Hyung-Wook

2009-01-01

Overactivation of G-protein mediated functions and altered G-protein regulation have been reported in bipolar disorder (BD) brain. Further, drugs effective in treating BD are reported to upregulate expression of G-protein receptor kinase (GRK) 3 in rat frontal cortex. We therefore hypothesized that some G-protein subunits and GRK levels would be reduced in the brains of BD patients. We determined protein and mRNA levels of G-protein β and γ subunits, GRK2, and GRK3 in postmortem frontal cortex from 10 BD patients and 10 age-matched controls by using immunoblots and real-time RT-PCR. There were the statistically significant decreases in protein and mRNA levels of G-protein subunits β and γ and of GRK3 in the BD brains but not a significant difference in the GRK2 level. Decreased expression of G-protein subunits and of GRK3 may alter neurotransmission, leading to disturbed cognition and behavior in BD. PMID:19400979

Involvement of microRNA-181a and Bim in a rat model of retinal ischemia-reperfusion injury.

PubMed

He, Yu; Liu, Jin-Nan; Zhang, Jun-Jun; Fan, Wei

2016-01-01

To investigate the changes in the expression of microRNA-181a (miR-181a) and Bim in a rat model of retinal ischemia-reperfusion (RIR), to explore their target relationship in RIR and their involvement in regulating apoptosis of retinal ganglion cells (RGCs). Target gene prediction for miR-181a was performed with the aid of bioinformatics and Bim was identified as a potential target gene of miR-181a. A rat model of RIR was created by increasing the intraocular pressure. RGCs in the flatmounted retinas were labeled with Brn3, a marker for alive RGCs, by immunofluorescent staining. The changes in the number of RGCs after RIR were recorded. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the expression level of miR-181a in the retina. Bim/Brn3 double immunofluorescence was used to detect the localization of Bim. The expression of Bim in the retina was determined with the aids of Western blot and qRT-PCR. Compared with the negative control group, the density of RGCs was significantly lower in the ischemia/reperfusion (I/R)-24h and I/R-72h groups (P<0.001). The expression level of miR-181a started to decrease at 0h after RIR, and further decreased at 24h and 72h compared with the negative control group (P<0.001). Bim was significantly upregulated at 12h after RIR (P<0.05) and reached peak at 24, 72h compared with the negative control group (P<0.01). Pearson correlation analysis showed that the expression level of Bim was negatively correlated with the expression level of miR-181a and the density of RGCs. Bim may be a potential target gene of miR-181a. Both miR-181a and Bim are involved in RGCs death in RIR. RIR may promote RGCs apoptosis in the retina via downregulation of miR-181a and its inhibition on Bim expression.

Expression of P-gp, MRP, LRP, GST-π and TopoIIα and intrinsic resistance in human lung cancer cell lines.

PubMed

Wang, Jiarui; Zhang, Jinhui; Zhang, Lichuan; Zhao, Long; Fan, Sufang; Yang, Zhonghai; Gao, Fei; Kong, Ying; Xiao, Gary Guishan; Wang, Qi

2011-11-01

This study aimed to determine the relationship between the endogenous levels of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione-s-transferase-π (GST‑π) and topoisomerase IIα (TopoIIα) and intrinsic drug resistance in four human lung cancer cell lines, SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446, of different histological types. The expression of P-gp, MRP, LRP, GST-π and TopoIIα was measured by immunofluorescence, Western blotting and RT-PCR. Drug resistance to cisplatin, doxorubicin and VP-16 was determined using MTT assays. The correlation between expression of the resistance-related proteins and their roles in the resistance to drugs in these cancer cell lines was analyzed. We found that the endogenous levels of P-gp, MRP, LRP, GST-π and TopoIIα in the four cell lines varied. The level of GST-π in the SK-MES-1 cells was the highest, whereas the level of P-gp in the SPCA-1 cells was the lowest. The chemoresistance to cisplatin, doxorubicin and VP-16 in the four cell lines was different. The SPCA-1 cell line was most resistance to cisplatin; SK-MES-1 was most resistance to VP-16; whereas SK-MES-1 was most sensitive to doxorubicin. There was a positive correlation between GST-π expression and resistance to cisplatin, between TopoIIα expression and resistance to VP-16; and a negative correlation was noted between TopoIIα expression and resistance to doxorubicin. In summary, the endogenous expression of P-gp, MRP, LRP, GST-π and TopoIIα was different in the four human lung cancer cell lines of different histological types, and this variance may be associated with the variation in chemosensitivity to cisplatin, doxorubicin and VP-16. Among the related proteins, GST-π may be useful for the prediction of the intrinsic resistance to cisplatin, whereas TopoIIα may be useful to predict resistance to doxorubicin and VP-16 in human lung cancer cell lines.

Thrombospondin-2 Expression During Retinal Vascular Development and Neovascularization.

PubMed

Fei, Ping; Palenski, Tammy L; Wang, Shoujian; Gurel, Zafer; Hankenson, Kurt D; Sorenson, Christine M; Sheibani, Nader

2015-09-01

To determine thrombospondin-2 (TSP2) expression and its impact on postnatal retinal vascular development and retinal neovascularization. The TSP2-deficient (TSP2(-/-)) mice and a line of TSP2 reporter mice were used to assess the expression of TSP2 during postnatal retinal vascular development and neovascularization. The postnatal retinal vascularization was evaluated using immunostaining of wholemount retinas prepared at different postnatal days by collagen IV staining and/or TSP2 promoter driven green fluorescent protein (GFP) expression. The organization of astrocytes was evaluated by glial fibrillary acidic protein (GFAP) staining. Retinal vascular densities were determined using trypsin digestion preparation of wholemount retinas at 3- and 6-weeks of age. Retinal neovascularization was assessed during the oxygen-induced ischemic retinopathy (OIR). Choroidal neovascularization (CNV) was assessed using laser-induced CNV. Using the TSP2-GFP reporter mice, we observed significant expression of TSP2 mRNA in retinas of postnatal day 5 (P5) mice, which increased by P7 and remained high up to P42. Similar results were observed in retinal wholemount preparations, and western blotting for GFP with the highest level of GFP was observed at P21. In contrast to high level of mRNA at P42, the GFP fluorescence or protein level was dramatically downregulated. The primary retinal vasculature developed at a faster rate in TSP2(-/-) mice compared with TSP2(+/+) mice up to P5. However, the developing retinal vasculature in TSP2(+/+) mice caught up with that of TSP2(-/-) mice after P7. No significant differences in retinal vascular density were observed at 3- or 6-weeks of age. TSP2(-/-) mice also exhibited a similar sensitivity to the hyperoxia-mediated vessel obliteration and similar level of neovascularization during OIR as TSP2(+/+) mice. Lack of TSP2 expression minimally affected laser-induced CNV compared with TSP2(+/+) mice. Lack of TSP2 expression was associated with enhanced retinal vascularization during early postnatal days but not at late postnatal times, and minimally affected retinal and CNV. However, the utility of TSP2 as a potential therapeutic target for inhibition of ocular neovascularization awaits further investigation.

Expression of interleukine-8 as an independent prognostic factor for sporadic colon cancer dissemination.

PubMed

Nastase, A; Paslaru, L; Herlea, V; Ionescu, M; Tomescu, D; Bacalbasa, N; Dima, S; Popescu, I

2014-06-15

The aim of our study was to investigate the gene and serum protein expression profiles of IL-8 in colon cancer and associated hepatic metastasis and to correlate these results with clinicopathologic variables of the patients. IL-8 was evaluated by qPCR and ELISA in a total number of 62 colon cancer patients (n=42 by qPCR and n=20 by ELISA) in normal and tumoral tissue specimens and serum samples respectively. Additionally synchronous metastasis from 5 of these patients were also collected at the time of surgery and analyzed by qPCR. IL-8 was up regulated in all analyzed tumoral samples compared with normal tissue (P-value = 0.01) and higher expressed in metastatic tissues compared with tumoral tissues (P -value= 0.03). The median expression of IL-8 in patients over 60 years old was found to be higher compared with the median expression of IL8 in patients less than 60 years old (3.89 compared with 14.69, P -value= 0.005). According to tumor grading, we found that IL-8 in tumors with well differentiated adenocarcinoma have a median mRNA expression of 9.78 compared with a median mRNA IL8 expression of 26.63 in moderate or poor differentiated adenocarcinoma. Levels of IL-8 determined in serum were statistically significant correlated with preoperative carcinoembryonic antigen level (P -value= 0.003, R=0.57) and with distant metastasis (P-value =0.008). Serum level of IL-8 increased proportionally along with TNM tumor stage and was found to be statistically significant correlated with C-reactive protein (P -value, R=0.64). Colon cancer patients had higher IL-8 levels as determined by ELISA (median value= 29.64 pg/ml) compared with healthy controls (median value= 4.86 pg/ml). Our results provide additional support for the role of inflammation in colon cancer and indicate that IL-8 could be further validated in association with other already used markers for prognostic and diagnostic of evolutional disease in colon cancer patients.

Blocking pulmonary ICAM-1 expression ameliorates lung injury in established diet-induced pancreatitis.

PubMed

Lundberg, A H; Fukatsu, K; Gaber, L; Callicutt, S; Kotb, M; Wilcox, H; Kudsk, K; Gaber, A O

2001-02-01

To determine whether blocking the cell surface expression of intracellular adhesion molecules (ICAM-1) in established severe acute pancreatitis (AP) would ameliorate pulmonary injury. Lung injury in AP is in part mediated by infiltrating leukocytes, which are directed to lung tissue by ICAM-l. The authors' laboratory has previously demonstrated that AP results in overproduction of inflammatory cytokines, upregulation of pulmonary ICAM-1 expression, and a concomitant infiltration of neutrophils, which results in lung injury. Young female mice were fed a choline-deficient/ethionine-supplemented diet to induce AP and were treated with a blocking dose of monoclonal antibody specific to the ICAM-1 receptor. Antibody treatment was administered at 72, 96, and 120 hours after beginning the diet, and all animals were killed at 144 hours. The degree of pancreatitis was evaluated by serum biochemical and tumor necrosis factor alpha levels as well as histology. The dual radiolabeled monoclonal antibody method was used to quantitate ICAM-1 cell surface expression in pulmonary tissue. Lung injury was assessed histologically and by determining lung microvascular permeability by measuring accumulated 125I-radiolabeled albumin. Pulmonary neutrophil sequestration was determined by the myeloperoxidase assay. All mice developed severe AP, and pancreatic injury was equally severe in both treated and untreated groups. Pulmonary ICAM-1 expression was significantly upregulated in animals with AP compared with controls. Treatment with a blocking dose of anti-ICAM-1 antibody after the induction of AP resulted in inhibited ICAM-1 cell surface expression to near control levels. Compared to untreated animals with AP, mice treated with anti-ICAM-1 mice had significantly reduced histologic lung injury and neutrophil sequestration, and a decreased microvascular permeability by more than twofold. These results demonstrate for the first time that treatment targeting the cell surface expression of ICAM-1 after the induction of AP ameliorates pulmonary injury, even in the face of severe pancreatic disease.

Abnormal expression and mutation of p53 in cervical cancer--a study at protein, RNA and DNA levels.

PubMed

Ngan, H Y; Tsao, S W; Liu, S S; Stanley, M

1997-02-01

The objectives of this study are to document the status of p53 expression and mutation in cervical cancer at protein, RNA and DNA levels and to relate this to the presence of HPV. Biopsy specimens from one hundred and three squamous cell carcinoma of the cervix and histologically normal ectocervix were analysed. Fresh tissues were extracted for protein, RNA and DNA and flash frozen tissue cryostat sectioned for immunohistochemical staining. HPV DNA status was determined by PCR using L1 consensus primers and typed for HPV 16 and 18 with E6 specific primers. p53 expression was determined at the protein level by Western blotting on protein extracts and at RNA level by Northern blotting. There was no p53 overexpression or mutation detectable in the protein extracts. Three of 65 (4.6%) of the carcinomas were positive for p53 by immunostaining with the polyclonal antibody CM1. Overexpression at the RNA level was detected in 2 of 32 (6.3%) carcinomas. p53 mutation was screened for by PCR/SSCP (single strand conformation polymorphism) followed by sequencing to define the site of mutation. Two of the cervical cancers (2.0%) showed mutation in p53 in exons 7 or 8. The mutation rate in HPV positive tumours was 1.2% (1/81) and in HPV negative tumours was 5.2% (1/19). p53 overexpression or mutation does not seem to play a significant role in cervical carcinomas.

Judgment of infant cry: The roles of acoustic characteristics and sociodemographic characteristics.

PubMed

Esposito, Gianluca; Nakazawa, Jun; Venuti, Paola; Bornstein, Marc H

2015-04-01

Adult judgments of infant cry are determined by both acoustic properties of the cry and listener sociodemographic characteristics. The main purpose of this research was to investigate how these two sources shape adult judgments of infant cry. We systematically manipulated both the acoustic properties of infant cries and contrasted listener sociodemographic characteristics. Then, we asked participants to listen to several acoustic manipulations of infant cries and to judge the level of distress the infant was expressing and the level of distress participants felt when listening. Finally, as a contrasting condition, participants estimated the age of the crying infant. Using tree-based models, we found that judgments of the level of distress the infant was expressing as well as the level of distress listeners felt are mainly accounted for by select acoustic properties of infant cry (proportion of sound/pause, fundamental frequency, and number of utterances), whereas age estimates of a crying infant are determined mainly by listener sociodemographic characteristics (gender and parental status). Implications for understanding infant cry and its effects as well as early caregiver-infant interactions are discussed.

Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss.

PubMed

Hokari, Ryota; Kitagawa, Noritake; Watanabe, Chikako; Komoto, Shunsuke; Kurihara, Chie; Okada, Yoshikiyo; Kawaguchi, Atsushi; Nagao, Shigeaki; Hibi, Toshifumi; Miura, Soichiro

2008-07-01

Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE-1 are specifically expressed in the endothelium of the lymphatic systems. VEGF-C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia. Biopsy samples were obtained from duodenal biopsies in patients with intestinal lymphangiectasia complicated with protein-losing from white spot lesions in which lymphangiectasia was histologically confirmed. Immunohistochemical analysis for VEGFR3 and LYVE-1 was performed. mRNA expression of VEGF-C, VEGF-D, VEGFR3, and transcription factors was determined by the quantitative reverse transcription-polymerase chain reaction method. In the control mucosa, VEGFR3 was weakly expressed on the central lymphatic vessels in the lamina propria and LYVE-1 was expressed mainly on the lymphatic vessels in the submucosa. In intestinal lymphangiectasia, VEGFR3 and LYVE-1 expression levels were increased on the mucosal surface corresponding to widely dilated lymphatic vessels, while they were decreased in the deeper mucosa. mRNA expression study showed a significant increase in the expression level of VEGFR3 in lymphangiectasia, but the expression of VEGF-C and -D mRNA was significantly suppressed compared with that in controls despite the presence of lymphangiectasia. The mRNA expression levels of FOXC2 and SOX18 were also decreased, whereas Prox 1 was not altered. There is an altered expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa in these patients.

Lamin A expression in circulating osteoprogenitors as a potential biomarker for frailty: The Nepean Osteoporosis and Frailty (NOF) Study.

PubMed

Al Saedi, Ahmed; Gunawardene, Piumali; Bermeo, Sandra; Vogrin, Sara; Boersma, Derek; Phu, Steven; Singh, Lakshman; Suriyaarachchi, Pushpa; Duque, Gustavo

2018-02-01

Lamin A is a protein of the nuclear lamina. Low levels of lamin A expression are associated with osteosarcopenia in mice. In this study, we hypothesized that low lamin A expression is also associated with frailty in humans. We aimed to develop a non-invasive method to quantify lamin A expression in epithelial and circulating osteoprogenitor (COP) cells, and to determine the relationship between lamin A expression and frailty in older individuals. COP cells and buccal swabs were obtained from 66 subjects (median age 74; 60% female; 26 non-frail, 23 pre-frail, and 17 frail) participating at the Nepean Osteoporosis and Frailty (NOF) Study. We quantified physical performance and disability, and stratified frailty in this population. Lamin A expression in epithelial and COP cells was quantified by flow cytometry. Linear regression models estimated the relationship between lamin A expression in buccal and COP cells, and prevalent disability and frailty. Lamin A expression in buccal cells showed no association with either disability or frailty. Low lamin A expression values in COP cells were associated with frailty. Frail individuals showed 60% lower levels of lamin A compared to non-frail (95% CI -36 to -74%, p<0.001) and 62% lower levels compared to pre-frail (95%CI -40 to -76%, p<0.001). In summary, we have identified lamin A expression in COP cells as a strong indicator of frailty. Further work is needed to understand lamin A expression as a risk stratifier, biomarker, or therapeutic target in frail older persons. Copyright © 2017 Elsevier Inc. All rights reserved.

Macrophage activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression.

PubMed

Blich, Miry; Golan, Amnon; Arvatz, Gil; Sebbag, Anat; Shafat, Itay; Sabo, Edmond; Cohen-Kaplan, Victoria; Petcherski, Sirouch; Avniel-Polak, Shani; Eitan, Amnon; Hammerman, Haim; Aronson, Doron; Axelman, Elena; Ilan, Neta; Nussbaum, Gabriel; Vlodavsky, Israel

2013-02-01

Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.

Activation of RAS family genes in urothelial carcinoma.

PubMed

Boulalas, I; Zaravinos, A; Karyotis, I; Delakas, D; Spandidos, D A

2009-05-01

Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

Rats.

PubMed

Aghdam, Amir Mahmoudi; Shahabi, Parviz; Karimi-Sales, Elham; Ghiasi, Rafigheh; Sadigh-Eteghad, Saeed; Mahmoudi, Javad; Alipour, Mohammad Reza

2018-04-30

Diabetes is a common metabolic disease which leads to diabetic peripheral neuropathy. Recently, the role of microRNA-96 (miR-96) in alleviating neuropathic pain by inhibiting the expression of NaV1.3, an isoform of voltage-gated sodium channels, has been shown. Peripheral nerve injuries result in NaV1.3 elevation. Exercise has beneficial role in diabetes management and peripheral neuropathy. However, the effects of exercise on miR-96 and its target gene NaV1.3 in diabetic rats are unknown. Therefore, the present study investigated the effects of exercise training on the expression of miR-96 and NaV1.3 in diabetic rats. For this purpose, rats were randomly divided into four groups: control, exercise, diabetic and diabetic-exercise groups. Type 2 diabetes was induced by a high-fat diet and the administration of streptozotocin (STZ) (35 mg/kg, i.p.). The exercise groups were subjected to swimming exercise 5 days/week for 10 weeks. At the end of the treatment period, thermal pain threshold, determined through the tail-flick test, and the expression levels of miR-96 and its target gene NaV1.3 were determined by reverse transcription (RT)-PCR in the sciatic nerve tissues of the rats. Data of the present study indicated that diabetes diminished miR-96 expression levels, but significantly upregulated NaV1.3 expression in the sciatic nerve. On exercise training, miR-96 expression was reversed with concurrent down-regulation of the NaV1.3 expression. This study introduced a new and potential miRNA-dependent mechanism for exerciseinduced protective effects against diabetic thermal hyperalgesia.

Regulated expression of a novel TCP domain transcription factor indicates an involvement in the control of meristem activation processes in Solanum tuberosum.

PubMed

Faivre-Rampant, Odile; Bryan, Glenn J; Roberts, Alison G; Milbourne, Daniel; Viola, Roberto; Taylor, Mark A

2004-04-01

In this study, the aim was to determine whether TCP transcription factors are implicated in meristem activation in potato (Solanum tuberosum). By searching a database of potato EST sequences, with a sequence characteristically conserved in TCP domains, a potato tcp gene was identified. A BAC clone containing the tcp sequence was isolated and the genomic sequence was determined. Using a CAPS marker assay, the potato tcp gene (sttcp1) was mapped to chromosome 8. In dormant buds, relatively high levels of sttcp1-specific transcript were detected by in situ hybridization. By contrast, in sprouting buds, no expression of the sttcp1 could be detected. Furthermore, an inverse relationship between axillary bud size and the steady-state level of the sstcp1 transcript was demonstrated. In non-growing buds exhibiting correlative inhibition, sttcpI-specific transcript levels were also relatively high, but rapidly decreased when apical dominance was removed by excision of the apical bud.

A novel therapeutic effect of statins on nephrogenic diabetes insipidus

PubMed Central

Bonfrate, Leonilde; Procino, Giuseppe; Wang, David Q-H; Svelto, Maria; Portincasa, Piero

2015-01-01

Statins competitively inhibit hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase, resulting in reduced plasma total and low-density lipoprotein cholesterol levels. Recently, it has been shown that statins exert additional ‘pleiotropic’ effects by increasing expression levels of the membrane water channels aquaporin 2 (AQP2). AQP2 is localized mainly in the kidney and plays a critical role in determining cellular water content. This additional effect is independent of cholesterol homoeostasis, and depends on depletion of mevalonate-derived intermediates of sterol synthetic pathways, i.e. farnesylpyrophosphate and geranylgeranylpyrophosphate. By up-regulating the expression levels of AQP2, statins increase water reabsorption by the kidney, thus opening up a new avenue in treating patients with nephrogenic diabetes insipidus (NDI), a hereditary disease that yet lacks high-powered and limited side effects therapy. Aspects related to water balance determined by AQP2 in the kidney, as well as standard and novel therapeutic strategies of NDI are discussed. PMID:25594563

Distinct effects of thrombopoietin depending on a threshold level of activated Mpl in BaF-3 cells.

PubMed

Millot, Gaël A; Vainchenker, William; Duménil, Dominique; Svinarchuk, Fédor

2002-06-01

Thrombopoietin (TPO) plays a critical role in megakaryopoiesis through binding to its receptor Mpl. This involves activation of various intracellular signaling pathways, including phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways. Their precise role in TPO-mediated proliferation, survival and differentiation is not fully understood. In the present study, we show that TPO induces different biological responses in Mpl-transduced BaF-3 cells, depending on the cell surface density of Mpl and the resulting activation level of signaling pathways. TPO mediates cell proliferation in cells expressing high levels of Mpl but only mediates survival without proliferation in cells expressing low levels of the receptor. By using the kinase inhibitors PD98059 and LY294002, we further showed that the activation level of the PI3K and MAPK p42/44 pathways is a determining factor for the proliferative effect. In cells expressing low levels of Mpl, the survival effect was strongly dependent on the activation level of the PI3K/AKT, but not the MAPK p42/44 pathway. Moreover, this effect was correlated with the phosphorylation level of BAD but not with the expression level of Bcl-X(L). However, PI3K pathway inhibition did not increase apoptosis when BaF-3 cells proliferated in response to TPO, indicating a compensating mechanism from other Mpl signaling pathways in this case.

Effects of hypothermia and cerebral ischemia on cold-inducible RNA-binding protein mRNA expression in rat brain.

PubMed

Liu, Aijun; Zhang, Zhiwen; Li, Anmin; Xue, Jinghui

2010-08-06

CIRP (cold-inducible RNA-binding protein) mRNA is highly expressed in hypothermic conditions in mammalian cells, and the relationship between CIRP and neuroprotection for cerebral ischemia under hypothermia has been focused upon. At present, however, the expression characteristics of CIRP under hypothermia and cerebral ischemia in vivo are not clearly elucidated. In this study, CIRP mRNA expression in various regions of rat brain was examined by reverse transcriptase polymerase chain reaction (RT-PCR). CIRP expression levels were found to be similar in the hippocampus and cortex. Real-time quantitative PCR analysis revealed increasing CIRP mRNA expression in the cortex during the 24-h observation period following treatment with hypothermia or cerebral ischemia, with a greater increase in the hypothermia group. When cerebral ischemia was induced following hypothermia, CIRP mRNA expression in the cortex again showed a significant increasing tendency, but ischemia delayed the appearance of this increase. To reveal the relationship between CIRP and energy metabolism in the rat brain, lactate and pyruvate concentrations in the cortex of the rats treated with hypothermia, ischemia and ischemia after hypothermia were determined by spectrophotometric assay, and levels of phosphofructokinas-1 (PFK-1), the major regulatory enzyme of the glycolytic pathway, in the rat cortex in the three groups was also analyzed by Western blot. Using linear correlation, lactate and pyruvate concentrations, and PFK-1 levels, were each analyzed in the three groups in association with CIRP mRNA expression levels. The analysis did not reveal any correlation between the three metabolic parameters and CIRP mRNA expression induced by hypothermia, suggesting that while playing a role in neuroprotection under hypothermia, CIRP does not affect cerebral energy metabolism. Copyright 2010. Published by Elsevier B.V.

Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE)

PubMed Central

Sharon, Dror; Blackshaw, Seth; Cepko, Constance L.; Dryja, Thaddeus P.

2002-01-01

We used the serial analysis of gene expression (SAGE) technique to catalogue and measure the relative levels of expression of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium (RPE) from one or both of two humans, aged 88 and 44 years. The cone photoreceptor contribution to all transcription in the retina was found to be similar in the macula versus the retinal periphery, whereas the rod contribution was greater in the periphery versus the macula. Genes encoding structural proteins for axons were found to be expressed at higher levels in the macula versus the retinal periphery, probably reflecting the large proportion of ganglion cells in the central retina. In comparison with the younger eye, the peripheral retina of the older eye had a substantially higher proportion of mRNAs from genes encoding proteins involved in iron metabolism or protection against oxidative damage and a substantially lower proportion of mRNAs from genes encoding proteins involved in rod phototransduction. These differences may reflect the difference in age between the two donors or merely interindividual variation. The RPE library had numerous previously unencountered tags, suggesting that this cell type has a large, idiosyncratic repertoire of expressed genes. Comparison of these libraries with 100 reported nonocular SAGE libraries revealed 89 retina-specific or enriched genes expressed at substantial levels, of which 14 are known to cause a retinal disease and 53 are RPE-specific genes. We expect that these libraries will serve as a resource for understanding the relative expression levels of genes in the retina and the RPE and for identifying additional disease genes. PMID:11756676

MGMT expression levels predict disease stabilisation, progression-free and overall survival in patients with advanced melanomas treated with DTIC.

PubMed

Busch, Christian; Geisler, Jürgen; Lillehaug, Johan R; Lønning, Per Eystein

2010-07-01

Metastatic melanoma responds poorly to systemic treatment. We report the results of a prospective single institution study evaluating O(6)-methylguanine-DNA methyltransferase (MGMT) status as a potential predictive and/or prognostic marker among patients treated with dacarbazine (DTIC) 800-1000 mg/m(2) monotherapy administered as a 3-weekly schedule for advanced malignant melanomas. The study was approved by the Regional Ethical Committee. Surgical biopsies from metastatic or loco-regional deposits obtained prior to DTIC treatment were snap-frozen immediately upon removal and stored in liquid nitrogen up to processing. Median time from enrolment to end of follow-up was 67 months. MGMT expression levels evaluated by qRT-PCR correlated significantly to DTIC benefit (CR/PR/SD; p=0.005), time to progression (TTP) (p=0.005) and overall survival (OS) (p=0.003). MGMT expression also correlated to Breslow thickness in the primary tumour (p=0.014). While MGMT promoter hypermethylation correlated to MGMT expression, MGMT promoter hypermethylation did not correlate to treatment benefit, TTP or OS, suggesting that other factors may be critical in determining MGMT expression levels in melanomas. In a Cox proportional regression analysis, serum lactate dehydrogenase (LDH, p<0.001), MGMT expression (p=0.022) and p16(INK4a) expression (p=0.037) independently predicted OS, while TTP correlated to DTIC benefit after 6 weeks only (p=0.001). Our data reveal MGMT expression levels to be associated with disease stabilisation and prognosis in patients receiving DTIC monotherapy for advanced melanoma. The role of MGMT expression as a predictor to DTIC sensitivity versus a general prognostic factor in advanced melanomas warrants further evaluation. Copyright 2010 Elsevier Ltd. All rights reserved.

[Effects of Losartan on expression of heme oxygenases in volume-overloaded rats with left-to-right shunt].

PubMed

Yuan, Li-Xing; Liu, Han-Min; Li, Mi; Gao, Ju; Zhou, Tong-Fu

2005-09-01

To study the expression of heme oxygenase-1 mRNA and pulmonary remodeling before and after surgical establishment of left-to-right shunt in volume-overloaded SD rats and rats with Losartan intervention. Left-to-right shunt volume-overloaded SD rat models were established by aortocaval shunt operation. Seven rats with shunt were placed on Losartan (Losartan group), 7 rats with but not given Losartan were included in the operation group, and 4 rats after sham operation served as controls. Pulmonary pressure and right ventricular pressure were measured during catheterization. The relative weights ventricles were determined after execution of the rats. Pulmonary vascular remodeling parameters, including percentage arterial wall thickness and percentage muscularized small arteries, were assessed by morphometry. Heme oxygenase-1 (HO-1) mRNA expression and heme oxygenase-2 (HO-2) mRNA expression were detected RT-PCR method. Pulmonary artery pressure and right ventricular relative weight decreased significantly in the rats of Losartan group; in addition, the percentage arterial wall thickness and percentage of muscularized small arteries in the Losartan group were reduced as compared with those in the operation group. The level 1 mRAN expression in rats with shunt was significantly higher than that in rats without shunt. The level mRNA expression in the Losartan group decreased remarkably as compared against that in the operation The level of HO-1 mRNA expression in lungs was significantly higher than that in ventricles. There statistically significant differences in HO-2 mRNA expression levels between the three rat groups. Losartan intervention can markedly reduce pulmonary pressure, inhibit vascular remodeling in volume-overloaded left-to-right shunt rats, and result in down-regulation of HO-1 mRNA expression.

The relationship between insight and the level of expressed emotion in patients with obsessive-compulsive disorder.

PubMed

Ozkiris, Ayse; Essizoglu, Altan; Gulec, Gulcan; Aksaray, Gokay

2015-04-01

The aim of this study is firstly to compare the obsessive-compulsive disorder (OCD) patients with good insight and OCD patients with poor insight in terms of socio-demographic and clinical features; to investigate the relation between insight and the level of the expressed emotion (EE) in the patients; and lastly to specify the factors that predict level of insight. OCD patients with good insight and patients with poor insight were compared in terms of clinical features and the perceived EE level of the patients and the individuals that they live with in order to specify the factors that predict the insight level, and to investigate the relationship between insight level and EE. It was found that the total Expressed Emotion Scale, total Level of Expressed Emotion (LEE), LEE-Emotional Response and LEE-Tolerance/Expectation subscale scores of the group comprised of patients with poor insight are higher than the other group. The results also show that the duration of illness and Yale-Brown Obsessive Compulsive Scale (Y-BOCS) total score predict insight level. This study shows that the level of EE perceived by the patients with poor insight and the person that he/she lives with, is higher than the group with good insight. The studies that investigate the relationship between the factors of insight level and EE level, which are indicated to determine the level of the illness severity and its chronicity, will enable the researchers to understand the importance of the role of the family on the treatment processes of OCD.

DEHP exposure in utero disturbs sex determination and is potentially linked with precocious puberty in female mice

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

Wang, Yongan

Human's ubiquitous exposure to di (2-ethylhexyl) phthalate (DEHP) is thought to be associated with female reproductive toxicity. Previous studies found that DEHP inhibited follicle growth and decreased estradiol levels in adult female mice. However, limited information is available on the link between in utero DEHP exposure and ovarian development in female mouse offspring. The present study evaluates the disturbances in regulatory genes involved in female sex determination and the ovarian outcomes in fetal and postnatal female mice treated with in utero DEHP exposure. Pregnant mice were exposed to DEHP by gavage, with the dosage regime beginning at human relevant exposuremore » levels. After in utero DEHP exposure, increased follicular atresia was observed in the female pups at postnatal days (PND) 21. Foxl2 expression was significantly upregulated, and Fst was significantly downregulated by DEHP above 2 mg/kg/d at PND 1 and 21. This suggests that lesion of granulosa cell differentiation and disturbance of follicle development in postnatal female mice. The expression of Cyp11a1 and Star were significantly downregulated by in utero DEHP exposure, indicating effects on estradiol biosynthesis. The female sex determination pathway was disturbed in fetus by DEHP at 2 mg/kg/d and above during the critical time window of sex determination causing significant upregulation of Foxl2, Wnt4, β-catenin and Fst. Furthermore, the increased expression of Wnt4 was supported by whole-mount in situ hybridization (WISH). These results suggest a possible association between in utero DEHP exposure and precocious puberty in the postnatal life of mice offspring, where disturbance of the sex determination regulating pathway acted as an important mechanism. - Highlights: • Maternal exposure to di (2-ethylhexyl) phthalate disturbs fetus sex determination. • DEHP upregulated Foxl2 expression potentially disturbs postnatal granulosa cell differentiation. • DEHP accelerated medulla follicular atresia potentially leading to precocious puberty.« less

Overexpression of dihydrofolate reductase is a factor of poor survival in acute lymphoblastic leukemia.

PubMed

Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Illades-Aguiar, Berenice; Rivera-Ramírez, Ana Bertha; Saavedra-Herrera, Mónica Virginia; Leyva-Vázquez, Marco Antonio

2018-06-01

Dihydrofolate reductase (DHFR) has an important function in DNA synthesis and is a target of methotrexate, which is a crucial treatment option for acute lymphoblastic leukemia (ALL). However, the number of studies conducted to date on DHFR expression in childhood ALL is limited. The aim of the present study was to determine whether the expression of DHFR is associated with survival in childhood ALL. The expression of DHFR in 96 children with ALL and 100 control individuals was determined using reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that the expression of DHFR mRNA in children with ALL was significantly increased (P<0.001), compared with that in the control group. In addition, increased levels of DHFR mRNA were observed in patients with B-cell lineage, compared with patients with T-cell lineage ALL (P<0.05). The Kaplan-Meier estimator analysis revealed that children with ALL who exhibited increased levels of DHFR mRNA had a decreased overall survival time (P<0.05). It was observed that certain patient prognostic features (including age, sex, white blood cell count and high DHFR expression), are associated with poor survival (log-rank test, P<0.05). Therefore, the results of the present study indicated that DHFR upregulation is a factor for poor survival in ALL.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

PubMed

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M; Zhou, Xinchun

2014-10-01

Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes. This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue. Copyright © 2014 Elsevier Inc. All rights reserved.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues

PubMed Central

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M.; Zhou, Xinchun

2014-01-01

Aims Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. Methods We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data were then compared in benign and malignant tissues from the same organ/tissue using the student t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. Results We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (either in nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC score of any S1PR (cytoplasmic or nuclear) between benign and malignant changes. Conclusion This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue. PMID:25084322

An atlas of histone deacetylase expression in breast cancer: fluorescence methodology for comparative semi-quantitative analysis

PubMed Central

Ververis, Katherine; Karagiannis, Tom C

2012-01-01

The histone deacetylase inhibitors, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™) and depsipeptide (Romidepsin, Istodax™) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Numerous histone deacetylase inhibitors are currently undergoing clinical trials, predominantly in combination with other cancer modalities, for the treatment of various haematological and solid malignancies. Most of the traditional compounds are known as broad-spectrum or pan-histone deacetylase inhibitors, possessing activity against a number of the 11 metal-dependent enzymes. One of the main questions in the field is whether class- or isoform-specific compounds would offer a therapeutic benefit compared to broad-spectrum inhibitors. Therefore, analysis of the relative expression of the different histone deacetylase enzymes in cancer cells and tissues is important to determine whether there are specific targets. We used a panel of antibodies directed against the 11 known mammalian histone deacetylases to determine expression levels in MCF7 breast cancer cells and in tissue representative of invasive ductal cell carcinoma and ductal carcinoma in situ. Firstly, we utilized a semi-quantitative method based on immunofluorescence staining to examine expression of the different histone deacetylases in MCF7 cells. Our findings indicate high expression levels of HDAC1, 3 and 6 in accordance with findings from others using RT-PCR and immunoblotting. Following validation of our approach we examined the expression of the different isoforms in representative control and breast cancer tissue. In general, our findings indicate higher expression of class I histone deacetylases compared to class II enzymes in breast cancer tissue. Analysis of individual cancer cells in the same tissue indicated marked heterogeneity in the expression of most class I enzymes indicating potential complications with the use of class- or isoform-specific compounds. Overall, our approach can be utilized to rapidly compare, in an unbiased semi-quantitative manner, the differential levels of expression of histone deacetylase enzymes in cells and tissues using widely available imaging software. It is anticipated that such analysis will become increasingly important as class- or isoform-specific histone deacetylase inhibitors become more readily available. PMID:22347520

An atlas of histone deacetylase expression in breast cancer: fluorescence methodology for comparative semi-quantitative analysis.

PubMed

Ververis, Katherine; Karagiannis, Tom C

2012-01-01

The histone deacetylase inhibitors, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™) and depsipeptide (Romidepsin, Istodax™) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Numerous histone deacetylase inhibitors are currently undergoing clinical trials, predominantly in combination with other cancer modalities, for the treatment of various haematological and solid malignancies. Most of the traditional compounds are known as broad-spectrum or pan-histone deacetylase inhibitors, possessing activity against a number of the 11 metal-dependent enzymes. One of the main questions in the field is whether class- or isoform-specific compounds would offer a therapeutic benefit compared to broad-spectrum inhibitors. Therefore, analysis of the relative expression of the different histone deacetylase enzymes in cancer cells and tissues is important to determine whether there are specific targets. We used a panel of antibodies directed against the 11 known mammalian histone deacetylases to determine expression levels in MCF7 breast cancer cells and in tissue representative of invasive ductal cell carcinoma and ductal carcinoma in situ. Firstly, we utilized a semi-quantitative method based on immunofluorescence staining to examine expression of the different histone deacetylases in MCF7 cells. Our findings indicate high expression levels of HDAC1, 3 and 6 in accordance with findings from others using RT-PCR and immunoblotting. Following validation of our approach we examined the expression of the different isoforms in representative control and breast cancer tissue. In general, our findings indicate higher expression of class I histone deacetylases compared to class II enzymes in breast cancer tissue. Analysis of individual cancer cells in the same tissue indicated marked heterogeneity in the expression of most class I enzymes indicating potential complications with the use of class- or isoform-specific compounds. Overall, our approach can be utilized to rapidly compare, in an unbiased semi-quantitative manner, the differential levels of expression of histone deacetylase enzymes in cells and tissues using widely available imaging software. It is anticipated that such analysis will become increasingly important as class- or isoform-specific histone deacetylase inhibitors become more readily available.

Metal ions induced heat shock protein response by elevating superoxide anion level in HeLa cells transformed by HSE-SEAP reporter gene.

PubMed

Yu, Zhanjiang; Yang, Xiaoda; Wang, Kui

2006-06-01

The aim of this work is to define the relationship between heat shock protein (HSP) and reactive oxygen species (ROS) in the cells exposed to different concentrations of metal ions, and to evaluate a new method for tracing the dynamic levels of cellular reactive oxygen species using a HSE-SEAP reporter gene. The expression of heat shock protein was measured using a secreted alkaline phosphatase (SEAP) reporter gene transformed into HeLa cell strain, the levels of superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were determined by NBT reduction assay and DCFH staining flow cytometry (FCM), respectively. The experimental results demonstrated that the expression of heat shock protein induced by metal ions was linearly related to the cellular superoxide anion level before cytotoxic effects were observed, but not related to the cellular hydrogen peroxide level. The experimental results suggested that metal ions might induce heat shock protein by elevating cellular superoxide anion level, and thus the expression of heat shock protein indicated by the HSE-SEAP reporter gene can be an effective model for monitoring the dynamic level of superoxide anion and early metal-induced oxidative stress/cytotoxicity.

Expression of multiple sexual signals by fathers and sons in the East-Mediterranean barn swallow: are advertising strategies heritable?

PubMed

Vortman, Yoni; Safran, Rebecca J; Reiner Brodetzki, Tali; Dor, Roi; Lotem, Arnon

2015-01-01

The level of expression of sexually selected traits is generally determined by genes, environment and their interaction. In species that use multiple sexual signals which may be costly to produce, investing in the expression of one sexual signal may limit the expression of the other, favoring the evolution of a strategy for resource allocation among signals. As a result, even when the expression of sexual signals is condition dependent, the relative level of expression of each signal may be heritable. We tested this hypothesis in the East-Mediterranean barn swallow (Hirundo rustica transitiva), in which males have been shown to express two uncorrelated sexual signals: red-brown ventral coloration, and long tail streamers. We show that variation in both signals may partially be explained by age, as well as by paternal origin (genetic father-son regressions), but that the strongest similarity between fathers and sons is the relative allocation towards one trait or the other (relative expression index), rather than the expression of the traits themselves. These results suggest that the expression of one signal is not independent of the other, and that genetic strategies for resource allocation among sexual signals may be selected for during the evolution of multiple sexual signals.

[Expression of c-kit in North African nasopharyngeal carcinomas: correlation with age and LMP1].

PubMed

Charfi, S; Khabir, A; Ayadi, L; Mseddi, M; Makni, H; Gorbel, A; Daoud, J; Frikha, M; Jlidi, R; Busson, P; Boudawara, T S

2007-09-01

To determine the level and prognostic significance of c-kit expression in the two age groups of North African nasopharyngeal carcinomas. A retrospective study of 99 NPC specimens from Tunisian patients was investigated by immunohistochemistry. Immunohistochemical data were correlated with Epstein-Barr virus LMP1 expression and pathological, clinical and survival parameters. c-kit was detected in 79% of the cases for patients under 30 years of age (juvenile form) but in only 56% of specimens in patients over 30 years (P=0.039) and was significantly over-expressed for patients with lymph node involvement (P=0.015). LMP1 score was 5.78 (+/-1.84) for c-kit negative tumors compared to 8,23 (+/-2.39) for c-kit positive tumors (P=0.002). Multivariate analysis including age, lymph nodes involvement and LMP1 expression as co-variables, showed that only age (P=0.027) and LMP1 expression (P=0.005) were significantly correlated to the c-kit expression. c-kit is highly expressed in the juvenile form of North African nasopharyngeal carcinomas. There is a significant association between LMP1 and c-kit expression. The contrasted levels of C-kit expression in the two age groups strengthen the hypothesis that these clinical forms result from distinct oncogenic mechanisms.

Expression of Multiple Sexual Signals by Fathers and Sons in the East-Mediterranean Barn Swallow: Are Advertising Strategies Heritable?

PubMed Central

Vortman, Yoni; Safran, Rebecca J.; Reiner Brodetzki, Tali; Dor, Roi; Lotem, Arnon

2015-01-01

The level of expression of sexually selected traits is generally determined by genes, environment and their interaction. In species that use multiple sexual signals which may be costly to produce, investing in the expression of one sexual signal may limit the expression of the other, favoring the evolution of a strategy for resource allocation among signals. As a result, even when the expression of sexual signals is condition dependent, the relative level of expression of each signal may be heritable. We tested this hypothesis in the East-Mediterranean barn swallow (Hirundo rustica transitiva), in which males have been shown to express two uncorrelated sexual signals: red-brown ventral coloration, and long tail streamers. We show that variation in both signals may partially be explained by age, as well as by paternal origin (genetic father-son regressions), but that the strongest similarity between fathers and sons is the relative allocation towards one trait or the other (relative expression index), rather than the expression of the traits themselves. These results suggest that the expression of one signal is not independent of the other, and that genetic strategies for resource allocation among sexual signals may be selected for during the evolution of multiple sexual signals. PMID:25679206

Expression patterns of flowering genes in leaves of 'Pineapple' sweet orange [Citrus sinensis (L.) Osbeck] and pummelo (Citrus grandis Osbeck).

PubMed

Pajon, Melanie; Febres, Vicente J; Moore, Gloria A

2017-08-30

In citrus the transition from juvenility to mature phase is marked by the capability of a tree to flower and fruit consistently. The long period of juvenility in citrus severely impedes the use of genetic based strategies to improve fruit quality, disease resistance, and responses to abiotic environmental factors. One of the genes whose expression signals flower development in many plant species is FLOWERING LOCUS T (FT). In this study, gene expression levels of flowering genes CiFT1, CiFT2 and CiFT3 were determined using reverse-transcription quantitative real-time PCR in citrus trees over a 1 year period in Florida. Distinct genotypes of citrus trees of different ages were used. In mature trees of pummelo (Citrus grandis Osbeck) and 'Pineapple' sweet orange (Citrus sinensis (L.) Osbeck) the expression of all three CiFT genes was coordinated and significantly higher in April, after flowering was over, regardless of whether they were in the greenhouse or in the field. Interestingly, immature 'Pineapple' seedlings showed significantly high levels of CiFT3 expression in April and June, while CiFT1 and CiFT2 were highest in June, and hence their expression induction was not simultaneous as in mature plants. In mature citrus trees the induction of CiFTs expression in leaves occurs at the end of spring and after flowering has taken place suggesting it is not associated with dormancy interruption and further flower bud development but is probably involved with shoot apex differentiation and flower bud determination. CiFTs were also seasonally induced in immature seedlings, indicating that additional factors must be suppressing flowering induction and their expression has other functions.

Ebselen suppresses inflammation induced by Helicobacter pylori lipopolysaccharide via the p38 mitogen-activated protein kinase signaling pathway.

PubMed

Xu, Ling; Gong, Changguo; Li, Guangming; Wei, Jue; Wang, Ting; Meng, Wenying; Shi, Min; Wang, Yugang

2018-05-01

Ebselen is a seleno-organic compound that has been demonstrated to have antioxidant and anti-inflammatory properties. A previous study determined that ebselen inhibits airway inflammation induced by inhalational lipopolysaccharide (LPS), however, the underlying molecular mechanism remains to be elucidated. The present study investigated the effect of ebselen on the glutathione peroxidase (GPX)‑reactive oxygen species (ROS) pathway and interleukin‑8 (IL‑8) expression induced by Helicobacter pylori LPS in gastric cancer (GC) cells. Cells were treated with 200 ng/ml H. pylori‑LPS in the presence or absence of ebselen for various durations and concentrations (µmol/l). The expression of toll‑like receptor 4 (TLR4), GPX2, GPX4, p38 mitogen‑activated protein kinase (p38 MAPK), phosphorylated‑p38 MAPK, ROS production and IL‑8 expression were detected with western blotting or ELISA. The present study revealed that TLR4 expression was upregulated; however, GPX2 and GPX4 expression was reduced following treatment with H. pylori LPS, which led to increased ROS production, subsequently altering the IL‑8 expression level in GC cells. Additionally, it was determined that ebselen prevented the reduction in GPX2/4 levels induced by H. pylori LPS, however, TLR4 expression was not affected. Ebselen may also block the expression of IL‑8 by inhibiting phosphorylation of p38 MAPK. These data suggest ebselen may inhibit ROS production triggered by H. pylori LPS treatment via GPX2/4 instead of TLR4 signaling and reduce phosphorylation of p38 MAPK, resulting in altered production of IL‑8. Ebselen may, therefore, be a potential therapeutic agent to mediate H. pylori LPS-induced cell damage.

Neurotensin gene expression increases during proestrus in the rostral medial preoptic nucleus: potential for direct communication with gonadotropin-releasing hormone neurons.

PubMed

Smith, M J; Wise, P M

2001-07-01

Neurotensin (NT)-containing neurons in the rostral portion of the medial preoptic nucleus (rMPN) of the brain may play a key role in regulating the pattern of secretion of GnRH, thereby influencing the reproductive cycle in females. The major goals of this study were to determine whether NT messenger RNA (mRNA) levels in the rMPN exhibit a unique pattern of expression in temporal association with the preovulatory LH surge and to assess whether NT neurons may communicate directly with GnRH neurons. We analyzed NT gene expression in rats using in situ hybridization over the day of proestrus and compared this with diestrous day 1. We also determined whether the high-affinity NT receptor (NT1) is expressed in GnRH neurons using dual-label in situ hybridization and whether this expression varies over the estrous cycle. We found that NT mRNA levels in the rMPN increase significantly on the day of proestrus, rising before the LH surge. No such change was detected on diestrous day 1, when the LH surge does not occur. Furthermore, we observed that a significant number of GnRH neurons coexpress NT1 mRNA and that the number of GnRH neurons expressing NT1 mRNA peaks on proestrus. Together with previous findings, our results suggest that increased expression of NT in the rMPN may directly stimulate GnRH neurons on proestrus, contributing to the LH surge. In addition, our results suggest that responsiveness of GnRH neurons to NT stimulation is enhanced on proestrus due to increased expression of NT receptors within GnRH neurons.

Expression of VGLUTs contributes to degeneration and acquisition of learning and memory.

PubMed

Cheng, Xiao-Rui; Yang, Yong; Zhou, Wen-Xia; Zhang, Yong-Xiang

2011-03-01

Vesicular glutamate transporters (VGLUTs), which include VGLUT1, VGLUT2 and VGLUT3, are responsible for the uploading of L-glutamate into synaptic vesicles. The expression pattern of VGLUTs determines the level of synaptic vesicle filling (i.e., glutamate quantal size) and directly influences glutamate receptors and glutamatergic synaptic transmission; thus, VGLUTs may play a key role in learning and memory in the central nervous system. To determine whether VGLUTs contribute to the degeneration or acquisition of learning and memory, we used an animal model for the age-related impairment of learning and memory, senescence-accelerated mouse/prone 8 (SAMP8). KM mice were divided into groups based on their learning and memory performance in a shuttle-box test. The expression of VGLUTs and synaptophysin (Syp) mRNA and protein in the cerebral cortex and hippocampus were investigated with real-time fluorescence quantitative PCR and western blot, respectively. Our results demonstrate that, in the cerebral cortex, protein expression of VGLUT1, VGLUT2, VGLUT3 and Syp was decreased in SAMP8 with age and increased in KM mice, which displayed an enhanced capacity for learning and memory. The protein expression of VGLUT2 and Syp was decreased in the hippocampus of SAMP8 with aging. The expression level of VGLUT1 and VGLUT2 proteins were highest in KM mouse group with a 76-100% avoidance score in the shuttle-box test. These data demonstrate that protein expression of VGLUT1, VGLUT2 and Syp decreases age-dependently in SAMP8 and increases in a learning- and memory-dependent manner in KM mice. Correlation analysis indicated the protein expression of VGLUT1, VGLUT2 and Syp has a positive correlation with the capacity of learning and memory. Copyright © 2011 Elsevier Inc. All rights reserved.

Rat lung metallothionein and heme oxygenase gene expression following ozone and zinc oxide exposure.

PubMed

Cosma, G; Fulton, H; DeFeo, T; Gordon, T

1992-11-01

We have conducted exposures in rats to determine pulmonary responses following inhalation of two common components of welding fumes, zinc oxide and ozone. To examine their effects on target-inducible gene expression, we measured mRNA levels of two metal-responsive genes, metallothionein (MT) and heme oxygenase (HO), in lung tissue by RNA slot-blot analysis. A 3-hr exposure to ZnO fume via a combustion furnace caused a substantial elevation in lung MT mRNA at all concentrations tested. Exposures to 5 and 2.5 mg/m3 ZnO resulted in peak 8-fold increases in MT mRNA levels (compared to air-exposed control animal values) immediately after exposure, while 1 mg/m3 ZnO exposure caused a 3.5-fold elevation in MT mRNA. These levels returned to approximate control gene expression values 24 hr after exposure. In addition, ZnO exposure caused an immediate elevation in lung HO gene expression levels, with 8-, 11-, and 5-fold increases observed after the same ZnO exposure levels (p < 0.05). Like MT gene induction, HO mRNA values returned to approximate control levels 24 hr after exposure. In striking contrast to the induction of MT and HO gene expression after ZnO exposures, there was no elevation in gene expression following a 6-hr exposure to 0.5 and 1 ppm ozone, even when lungs were examined as late as 72 hr after exposure. Our results demonstrate the induction of target gene expression following the inhalation of ZnO at concentrations equal to, and below, the current recommended threshold limit value of 5 mg/m3 ZnO. Furthermore, the lack of effect of ozone exposure on MT and HO gene expression suggests no involvement of these genes in the acute respiratory response to this oxidant compound.

Metabolomic analysis of the selection response of Drosophila melanogaster to environmental stress: are there links to gene expression and phenotypic traits?

NASA Astrophysics Data System (ADS)

Malmendal, Anders; Sørensen, Jesper Givskov; Overgaard, Johannes; Holmstrup, Martin; Nielsen, Niels Chr.; Loeschcke, Volker

2013-05-01

We investigated the global metabolite response to artificial selection for tolerance to stressful conditions such as cold, heat, starvation, and desiccation, and for longevity in Drosophila melanogaster. Our findings were compared to data from other levels of biological organization, including gene expression, physiological traits, and organismal stress tolerance phenotype. Overall, we found that selection for environmental stress tolerance changes the metabolomic 1H NMR fingerprint largely in a similar manner independent of the trait selected for, indicating that experimental evolution led to a general stress selection response at the metabolomic level. Integrative analyses across data sets showed little similarity when general correlations between selection effects at the level of the metabolome and gene expression were compared. This is likely due to the fact that the changes caused by these selection regimes were rather mild and/or that the dominating determinants for gene expression and metabolite levels were different. However, expression of a number of genes was correlated with the metabolite data. Many of the identified genes were general stress response genes that are down-regulated in response to selection for some of the stresses in this study. Overall, the results illustrate that selection markedly alters the metabolite profile and that the coupling between different levels of biological organization indeed is present though not very strong for stress selection at this level. The results highlight the extreme complexity of environmental stress adaptation and the difficulty of extrapolating and interpreting responses across levels of biological organization.

Caffeoylxanthiazonoside exerts cardioprotective effects during chronic heart failure via inhibition of inflammatory responses in cardiac cells

PubMed Central

Yang, Bin; Wang, Fei; Cao, Huili; Liu, Guifang; Zhang, Yuean; Yan, Ping; Li, Bao

2017-01-01

Caffeoylxanthiazonoside (CYT) is an active constituent isolated from the fruit of the Xanthium strumarium L plant. The aim of the present study was to investigate the cardioprotective effects of oral administration of CYT on chronic heart failure (CHF) and its underlying mechanisms. A rat model of CHF was first established, and cardiac function indices, including the heart/body weight index, left heart/body weight index, fractional shortening (FS), ejection fraction (EF), cardiac output (CO) and heart rate (HR), were subsequently determined by cardiac ultrasound. Serum levels of lactate dehydrogenase (LDH) and creatine kinase (CK), and the levels of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in heart tissues and cardiac microvascular endothelial cells (CMECs) were determined using ELISA. In addition, the protein expression levels of nuclear factor-κB (NF-κB) signaling pathway members were determined by western blotting in CMECs. The results demonstrated that oral administration of 10, 20, 40 mg/kg CYT significantly reduced cardiac hypertrophy and reversed FS, EF, CO and HR when compared with CHF model rats. In addition, CYT administration significantly decreased the levels of TNF-α, IL-6 and IL-1β in heart tissues, as well as serum LDH and CK levels. Furthermore, exposure of CMECs to 20, 40 and 80 µg/ml CYT significantly decreased the production of TNF-α, IL-1β and IL-6. The protein expression levels of cytoplasmic NF-κB p65 and IκB were upregulated, while nuclear NF-κB p65 was downregulated following treatment of CMECs with 20, 40 and 80 µg/ml CYT when compared with untreated CHF model controls. In conclusion, the results of the current study suggest that CYT demonstrates cardioprotective effects in CHF model rats by suppressing the expression of pro-inflammatory cytokines and the NF-κB signaling pathway. PMID:29104638

Caffeoylxanthiazonoside exerts cardioprotective effects during chronic heart failure via inhibition of inflammatory responses in cardiac cells.

PubMed

Yang, Bin; Wang, Fei; Cao, Huili; Liu, Guifang; Zhang, Yuean; Yan, Ping; Li, Bao

2017-11-01

Caffeoylxanthiazonoside (CYT) is an active constituent isolated from the fruit of the Xanthium strumarium L plant. The aim of the present study was to investigate the cardioprotective effects of oral administration of CYT on chronic heart failure (CHF) and its underlying mechanisms. A rat model of CHF was first established, and cardiac function indices, including the heart/body weight index, left heart/body weight index, fractional shortening (FS), ejection fraction (EF), cardiac output (CO) and heart rate (HR), were subsequently determined by cardiac ultrasound. Serum levels of lactate dehydrogenase (LDH) and creatine kinase (CK), and the levels of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in heart tissues and cardiac microvascular endothelial cells (CMECs) were determined using ELISA. In addition, the protein expression levels of nuclear factor-κB (NF-κB) signaling pathway members were determined by western blotting in CMECs. The results demonstrated that oral administration of 10, 20, 40 mg/kg CYT significantly reduced cardiac hypertrophy and reversed FS, EF, CO and HR when compared with CHF model rats. In addition, CYT administration significantly decreased the levels of TNF-α, IL-6 and IL-1β in heart tissues, as well as serum LDH and CK levels. Furthermore, exposure of CMECs to 20, 40 and 80 µg/ml CYT significantly decreased the production of TNF-α, IL-1β and IL-6. The protein expression levels of cytoplasmic NF-κB p65 and IκB were upregulated, while nuclear NF-κB p65 was downregulated following treatment of CMECs with 20, 40 and 80 µg/ml CYT when compared with untreated CHF model controls. In conclusion, the results of the current study suggest that CYT demonstrates cardioprotective effects in CHF model rats by suppressing the expression of pro-inflammatory cytokines and the NF-κB signaling pathway.

Expression profile of Lgi1 gene in mouse brain during development.

PubMed

Ribeiro, Patrícia A O; Sbragia, Lourenço; Gilioli, Rovilson; Langone, Francesco; Conte, Fábio F; Lopes-Cendes, Iscia

2008-07-01

Mutations in LGI1 were described in patients with autosomal dominant partial epilepsy with auditory features (ADPEAF), and recent clinical findings have implicated LGI1 in human brain development. However, the precise role of LGI1 in epileptogenesis remains largely unknown. The objective of this study was to determine the expression pattern of Lgi1 in mice brain during development and in adult animals. Real-time polymerase chain reaction (PCR) quantification and Western blot experiments showed a relative low expression during intrauterine stages, increasing until adulthood. In addition, we did not find significant differences between left and right hemispheres. The hippocampus presented higher levels of Lgi1 expression when compared to the neocortex and the cerebellum of adult animals; however, these results did not reach statistical significance. This study was the first to determine a specific profile of Lgi1 gene expression during central nervous system development, which suggests a possible inhibitory function in latter stages of development. In addition, we did not find differences in hemispheric expression that could explain the predominance of left-sided abnormalities in patients with ADPEAF.

Developmental regulation of vascular endothelial growth/permeability factor messenger ribonucleic acid levels in and vascularization of the villous placenta during baboon pregnancy.

PubMed

Hildebrandt, V A; Babischkin, J S; Koos, R D; Pepe, G J; Albrecht, E D

2001-05-01

Vascular endothelial growth/permeability factor (VEG/PF) has an important role in angiogenesis; however, very little is known about the developmental regulation of VEG/PF and the vascular system within the placenta during human pregnancy. In the present study, therefore, a developmental approach was used in the baboon to determine the placental source of VEG/PF and its fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (KDR/flk-1) receptors, and whether the rise in estrogen with advancing pregnancy was associated with a corresponding increase in placental VEG/PF expression and vascularization. VEG/PF messenger RNA (mRNA) levels were determined by competitive RT-PCR in villous cell fractions isolated by Percoll gradient centrifugation from placentas obtained on days 45 and 54 (very early), 60 (early), 100 (mid), and 165-170 (late) of baboon pregnancy (term = 184 days). Maternal peripheral serum estradiol increased from very low concentrations early in gestation (0.15-0.20 ng/ml) to an early surge of over 2.5 ng/ml on days 60-85, and peak levels of 4-6 ng/ml late in baboon pregnancy. VEG/PF mRNA was expressed in low level in the syncytiotrophoblast (<2,000 attomol/microgram total RNA), and values in this fraction did not change significantly with advancing gestation. VEG/PF mRNA expression was slightly greater in the inner villous core cell fraction; however, levels decreased (P < 0.05) between early and late gestation. Cytotrophoblasts were a major source of VEG/PF mRNA and levels increased (P < 0.01) from 3,631 +/- 844 attomol/microgram total RNA on day 45 to 25,807 +/- 5,873 attomol/microgram total RNA on day 170. VEG/PF protein expression determined by immunocytochemistry was abundant in cytotrophoblasts and lower in the syncytiotrophoblast and inner villous core cells. The flt-1 and KDR/flk-1 receptors were expressed in the vascular endothelial cells of the baboon villous placenta. The percentage of villous placenta occupied by blood vessels and the number of vessels/mm(2) villous tissue, determined by image analysis, progressively increased (P < 0.001; r = 0.97) from 3.4 +/- 0.2% and 447 +/- 29, respectively, on day 54 to 15.9 +/- 0.9% and 1,375 +/- 71, respectively, on day 170. In summary, the present study shows that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta, and that cytotrophoblast VEG/PF mRNA levels and vascularization of the villous placenta closely paralleled the increase in estradiol concentrations of advancing pregnancy. These results are consistent with the concept that estrogen has an important role in establishing the new vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process.

Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride.

PubMed

Panneerselvam, Lakshmikanthan; Raghunath, Azhwar; Perumal, Ekambaram

2017-09-01

Acute fluoride (F - ) toxicity is known to cause severe cardiac complications and leads to sudden heart failure. Previously, we reported that increased myocardial oxidative damage, apoptosis, altered cytoskeleton and AMPK signaling proteins associated with energy deprivation in acute F - induced cardiac dysfunction. The present study was aimed to decipher the status of myocardial heat shock proteins (Hsps-Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, Hsp90) and heat shock transcription factor 1 (Hsf1) in acute F - -intoxicated rats. In order to study the expression of myocardial Hsps, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F - for 24 h. The expression levels of myocardial Hsps were determined using RT-PCR, western blotting, and immunohistochemical studies. Acute F - -intoxicated rats showed elevated levels of both the transcripts and protein expression of Hsf1, Hsp27, Hsp32, Hsp60, and Hsp70 when compared to control. In addition, the expression levels of Hsp40 and Hsp90 were significantly declined in a dose-dependent fashion in F - -treated animals. Our result suggests that differential expression of Hsps in the rat myocardium could serve as a balance between pro-survival and death signal during acute F - -induced heart failure.

Loss of Bad expression confers poor prognosis in non-small cell lung cancer.

PubMed

Huang, Yi; Liu, Dan; Chen, Bojiang; Zeng, Jing; Wang, Lei; Zhang, Shangfu; Mo, Xianming; Li, Weimin

2012-09-01

Proapoptotic BH-3-only protein Bad (Bcl-Xl/Bcl-2-associated death promoter homolog, Bad) initiates apoptosis in human cells, and contributes to tumorigenesis and chemotherapy resistant in malignancies. This study explored association between the Bad expression level and prognosis in patients with non-small cell lung cancer (NSCLC). In our study, a cohort of 88 resected primary NSCLC cases were collected and analyzed. Bad expression level was determined via immunohistochemical staining assay. The prognostic significances of Bad expression were evaluated with univariate and multivariate survival analysis. The results showed that compared with normal lung tissues, Bad expression level significantly decreased in NSCLC (P < 0.05). Bad expression was associated with adjuvant therapy status. Loss of Bad independently predicted poor prognosis in whole NSCLC cohort and early stage subjects (T1 + T2 and N0 + N1) (all P < 0.05). Overall survival time was also drastically shortened for Bad negative phenotype in NSCLC patients with smoking history, especially lung squamous cell carcinoma (all P < 0.05). In conclusion, this study provided clinical evidence that loss of Bad is an independent and powerful predictor of adverse prognosis in NSCLC. Bad protein could be a new biomarker for selecting individual therapy strategies and predicting therapeutic response in subjects with NSCLC.

Expression of green fluorescent protein in Xylella fastidiosa is affected by passage through host plants.

PubMed

Qin, Xiaoting; Hartung, John S

2004-09-01

Xylella fastidiosa, a Gram-negative bacterial plant pathogen, causes Pierce's disease of grapevine in North America. In South America the pathogen causes citrus variegated chlorosis, which is widespread in Brazil. We have introduced into Xylella fastidiosa a mini-Tn5 transposon that encodes a green fluorescent protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was expressed well and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. The transconjugants were able to colonize the plants and were subsequently isolated from points distal to the inoculation sites. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared with that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly increased the level of expression of the GFP. The increased level of expression of GFP was transient, and was lost upon further culture in vitro. Xylella fastidiosa forms biofilms in planta which are believed to represent a metabolically differentiated state. The increased expression of GFP observed after passage through plants may be accounted for by this phenomenon.

Quantitative analysis of Epstein-Barr virus (EBV)-related gene expression in patients with chronic active EBV infection.

PubMed

Iwata, Seiko; Wada, Kaoru; Tobita, Satomi; Gotoh, Kensei; Ito, Yoshinori; Demachi-Okamura, Ayako; Shimizu, Norio; Nishiyama, Yukihiro; Kimura, Hiroshi

2010-01-01

Chronic active Epstein-Barr virus (CAEBV) infection is a systemic Epstein-Barr virus (EBV)-positive lymphoproliferative disorder characterized by persistent or recurrent infectious mononucleosis-like symptoms in patients with no known immunodeficiency. The detailed pathogenesis of the disease is unknown and no standard treatment regimen has been developed. EBV gene expression was analysed in peripheral blood samples collected from 24 patients with CAEBV infection. The expression levels of six latent and two lytic EBV genes were quantified by real-time RT-PCR. EBV-encoded small RNA 1 and BamHI-A rightward transcripts were abundantly detected in all patients, and latent membrane protein (LMP) 2 was observed in most patients. EBV nuclear antigen (EBNA) 1 and LMP1 were detected less frequently and were expressed at lower levels. EBNA2 and the two lytic genes were not detected in any of the patients. The pattern of latent gene expression was determined to be latency type II. EBNA1 was detected more frequently and at higher levels in the clinically active patients. Quantifying EBV gene expression is useful in clarifying the pathogenesis of CAEBV infection and may provide information regarding a patient's disease prognosis, as well as possible therapeutic interventions.

Influence of folic acid, pyridoxal phosphate and cobalamin on plasma homocyst(e)ine levels and the susceptibility of low-density lipoprotein to ex-vivo oxidation.

PubMed

Weiss, N; Feussner, A; Hailer, S; Spengel, F A; Keller, C; Wolfram, G

1999-10-15

Mild hyperhomocyst(e)inaemia is a risk factor for atherosclerotic vascular disease. In-vitro studies have shown that autooxidation of homocyst(e)ine is accompanied by the generation of oxygen radicals. This may lead to oxidative modification of low-density lipoproteins (LDL) and promote atherosclerotic vascular lesions. In male patients with peripheral arterial occlusive disease we determined fasting and post methionine load homocyst(e)ine levels by high performance liquid chromatography and the susceptibility of their LDL particles to ex-vivo oxidation by continously measuring the conjugated diene production induced by incubation with copper ions. Oxidation resistance (expressed as lag time), maximal oxidation rate, and extent of oxidation (expressed of total diene production) of LDL from patients with normal or mildly elevated homocyst(e)ine levels did not differ significantly. Folic acid, pyridoxal phosphate and cobalamin supplementation significantly decreased plasma homocyst(e)ine levels in hyperhomocyst(e)inaemic patients. This went along with a significant decrease in the extent of LDL oxidation and additionally increased HDL-cholesterol levels. The clinical relevance of these findings for the long-term course of atherosclerotic vascular disorders has to be determined by intervention studies.

A role for GPx3 in activity of normal and leukemia stem cells

PubMed Central

Herault, Olivier; Hope, Kristin J.; Deneault, Eric; Mayotte, Nadine; Chagraoui, Jalila; Wilhelm, Brian T.; Cellot, Sonia; Sauvageau, Martin; Andrade-Navarro, Miguel A.; Hébert, Josée

2012-01-01

The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs. PMID:22508837

Polypeptide N-acetylgalactosaminyltransferase-6 expression in gastric cancer

PubMed Central

Guo, Yan; Shi, Jingjing; Zhang, Jun; Li, Haixin; Liu, Ben; Guo, Hua

2017-01-01

Gastric cancer (GC) is one of the leading causes of cancer-related deaths, with limited improvement in its clinical outcome worldwide. Aberrant mucin-type O-glycosylation is a critical event widespread in the development of GC. Polypeptide N-acetylgalactosaminyltransferases (GALNTs) regulate the initial step and determine the sites of mucin-type O-glycoprotein bio-synthesis. GALNT6 has considerable potential as a biomarker in various cancers. The roles of GALNT6 in GC were analyzed, and the results showed that GALNT6 expression markedly increased in GC tissues compared with those in adjacent gastric tissues. High intratumoral GALNT6 density was associated with the clinicopathological parameters of TNM stage and distant metastasis. GALNT6 was identified as an independent prognosticator for the poor prognosis of GC patients. Moreover, the high expression level of GALNT6 was significantly associated with the low expression levels of E-cadherin and β-catenin and the high expression levels of MMP9. These findings indicated that GALNT6 could provide new insights into the characterization of GC as well as contribute to the development of an efficient prognostic indicator and novel therapeutic modalities for GC. PMID:28744137

An Ultra-High Fluorescence Enhancement and High Throughput Assay for Revealing Expression and Internalization of Chemokine Receptor CXCR4.

PubMed

He, Hua; Wang, Xiaojuan; Cheng, Tiantian; Xia, Yongqing; Lao, Jun; Ge, Baosheng; Ren, Hao; Khan, Naseer Ullah; Huang, Fang

2016-04-18

Revealing chemokine receptor CXCR4 expression, distribution, and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy. We here describe a sensitive and high-throughput immunoassay for determining CXCR4 expression and distribution in cancer cells. The assay is accessible to a wide range of users in an ordinary lab only by dip-coating poly(styrene-co-N-isopropylacrylamide) spheres on the glass substrate. The self- assembled spheres form three-dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 by a factor of up to 1000. CXCR4 in cells is detected by using the sandwich immunoassay, where the primary antibody recognizes CXCR4 and the secondary antibody is labeled with CF647. With the newly established assay, we quantified the total expression of CXCR4, its distribution on the cell membrane and cytoplasm, and revealed their internalization level upon SDF-1α activation in various cancer cells, even for those with extremely low expression level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.

PubMed

Voynow, J A; Young, L R; Wang, Y; Horger, T; Rose, M C; Fischer, B M

1999-05-01

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

Over, and Underexpression of Endothelin 1 and TGF-Beta Family Ligands and Receptors in Lung Tissue of Broilers with Pulmonary Hypertension

PubMed Central

Dominguez-Avila, Norma; Ruiz-Castañeda, Gabriel; González-Ramírez, Javier; Fernandez-Jaramillo, Nora; Escoto, Jorge; Sánchez-Muñoz, Fausto; Marquez-Velasco, Ricardo; Bojalil, Rafael; Espinosa-Cervantes, Román; Sánchez, Fausto

2013-01-01

Transforming growth factor beta (TGFβ) is a family of genes that play a key role in mediating tissue remodeling in various forms of acute and chronic lung disease. In order to assess their role on pulmonary hypertension in broilers, we determined mRNA expression of genes of the TGFβ family and endothelin 1 in lung samples from 4-week-old chickens raised either under normal or cold temperature conditions. Both in control and cold-treated groups of broilers, endothelin 1 mRNA expression levels in lungs from ascitic chickens were higher than levels from healthy birds (P < 0.05), whereas levels in animals with cardiac failure were intermediate. Conversely, TGFβ2 and TGFβ3 gene expression in lungs were higher in healthy animals than in ascitic animals in both groups (P < 0.05). TGFβ1, TβRI, and TβRII mRNA gene expression among healthy, ascitic, and chickens with cardiac failure showed no differences (P > 0.05). BAMBI mRNA gene expression was lowest in birds with ascites only in the control group as compared with the values from healthy birds (P < 0.05). PMID:24286074

Identification of Genes in the Phenylalanine Metabolic Pathway by Ectopic Expression of a MYB Transcription Factor in Tomato Fruit[W

PubMed Central

Dal Cin, Valeriano; Tieman, Denise M.; Tohge, Takayuki; McQuinn, Ryan; de Vos, Ric C.H.; Osorio, Sonia; Schmelz, Eric A.; Taylor, Mark G.; Smits-Kroon, Miriam T.; Schuurink, Robert C.; Haring, Michel A.; Giovannoni, James; Fernie, Alisdair R.; Klee, Harry J.

2011-01-01

Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription factor, Petunia hybrida ODORANT1, to alter Phe and phenylpropanoid metabolism in tomato (Solanum lycopersicum) fruits. Despite the importance of Phe and phenylpropanoids to plant and human health, the pathway for Phe synthesis has not been unambiguously determined. Microarray analysis of ripening fruits from transgenic and control plants permitted identification of a suite of coregulated genes involved in synthesis and further metabolism of Phe. The pattern of coregulated gene expression facilitated discovery of the tomato gene encoding prephenate aminotransferase, which converts prephenate to arogenate. The expression and biochemical data establish an arogenate pathway for Phe synthesis in tomato fruits. Metabolic profiling and 13C flux analysis of ripe fruits further revealed large increases in the levels of a specific subset of phenylpropanoid compounds. However, while increased levels of these human nutrition-related phenylpropanoids may be desirable, there were no increases in levels of Phe-derived flavor volatiles. PMID:21750236

Expression and enzymatic activity of phenylalanine ammonia-lyase and p-coumarate 3-hydroxylase in mango (Mangifera indica 'Ataulfo') during ripening.

PubMed

Palafox-Carlos, H; Contreras-Vergara, C A; Muhlia-Almazán, A; Islas-Osuna, M A; González-Aguilar, G A

2014-05-16

Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.

Bisphenol A disrupts gene expression in human placental trophoblast cells.

PubMed

Rajakumar, Chandrew; Guan, Haiyan; Langlois, David; Cernea, Maria; Yang, Kaiping

2015-06-01

This study examined the effect of bisphenol A (BPA) on human placental gene expression using primary trophoblast cells as an in vitro model system. Trophoblast cells were isolated from human placentas at term, cultured and then exposed to environmentally relevant concentrations of BPA (0.1-2 μg/ml) for up to 24h, after which levels of 11β-HSD2 mRNA, protein and activity were determined by standard radiometric conversion assay, western blotting, and qRT-PCR, respectively. The mRNA levels of several other prominent placental hormones/factors were also assessed by qRT-PCR. BPA dramatically increased levels of 11β-HSD2 activity, protein and mRNA in a time- and concentration-dependent manner (> 4-fold). BPA also augmented aromatase, glucose transporter-1, CRH, and hCG mRNA levels while reducing the level of leptin mRNA. These findings demonstrate that BPA severely disrupts human placental gene expression in vitro, which suggests that exposure to BPA may contribute to altered placental function and consequent pregnancy complications. Copyright © 2015 Elsevier Inc. All rights reserved.

Losartan attenuates vascular remodeling of the aorta in spontaneously hypertensive rats and the underlying mechanism.

PubMed

Li, Fangxiong; Shi, Ruizheng; Liao, Meichun; Li, Jianzhe; Li, Shixun; Pan, Wei; Yang, Tianlun; Zhang, Guogang

2010-08-01

To determine the effect of losartan on vascular remodeling and the underlying mechanism in spontaneously hypertensive rats(SHR). SHR of 12 weeks old were given losartan orally [0, 15, 30 mg/(kg.d), n=12]. The tail arterial pressure was measured every week. Eight weeks later, the pathological changes and p22(phox) expression in the thoracic aorta, the activity of catalase (CAT), the contents of H(2)O(2) and Ang II in the plasma were evaluated. Blood pressure was increased in the SHR accompanied by the thickened wall and increased p22(phox) expression in the thoracic aorta. The plasma levels of H(2)O(2) and Ang II were elevated while the CAT level was decreased in the SHR. Administration of losartan reversed the thickened wall and increased the CAT activity concomitantly with the decreased plasma levels of H(2)O(2) and p22(phox) expression in the SHR. The plasma level of Ang II increased after the losartan treatment. Oxidative stress induces the vascular remodeling of the aorta in the SHR. Losartan can reverse the vascular remodeling through down-regulating p22(phox) expression and inhibiting the oxidative stress.

Association of vaspin gene polymorphisms with coronary artery disease in Chinese population and function study.

PubMed

Li, Hai Ling; Zhang, Hong Li; Jian, Wei Xia; Li, Qi; Peng, Wen Hui; Xu, Ya Wei

2013-01-16

Visceral adipose tissue-derived serine protease inhibitor (vaspin) is a recently identified adipokine. Studies suggest it is involved in many diseases such as obesity, diabetes and coronary artery disease (CAD). This study is to investigate the association of single nucleotide polymorphisms (SNPs) in vaspin with CAD and its potential mechanisms. A total of 1570 consecutive patients undergoing coronary angiography were enrolled and the genotypes were determined by TaqMan allelic discrimination. Serum vaspin concentrations and mRNA expression levels were determined by ELISA and RT-PCR, respectively. Reporter gene assay was performed to investigate the effect of polymorphism on vaspin promoter function. After multivariate analysis, allele A of rs2236242 was found as an independent determinant of CAD (OR=1.32, p=0.004). Rs35262691 in vaspin promoter was associated with serum vaspin concentration and mRNA expression in peripheral blood mononuclear cells (PBMC) though no association had been found with CAD. Reporter gene assay further confirmed that CC genotype of rs35262691 had 2.1±0.4-fold higher activities than TT genotype in facilitating gene expression. Our results show that the variants of vaspin gene are associated with serum vaspin levels and risk for CAD in Chinese population. Copyright © 2012 Elsevier B.V. All rights reserved.

Effects of Wenyangbushen formula on the expression of VEGF, OPG, RANK and RANKL in rabbits with steroid-induced femoral head avascular necrosis.

PubMed

Song, Hong-Mei; Wei, Ying-Chen; Li, Nan; Wu, Bin; Xie, Na; Zhang, Kun-Mu; Wang, Shi-Zhong; Wang, He-Ming

2015-12-01

The present study aimed to investigate the effects of Wenyangbushen formula on the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), osteoprotegerin (OPG), receptor activator of nuclear factor (NF)‑κβ ligand (RANK), and RANK ligand (RANKL) in a rabbit model of steroid‑induced avascular necrosis of the femoral head (SANFH). The present study also aimed to examine the potential mechanism underlying the effect of this formula on the treatment of SANFH. A total of 136 New Zealand rabbits were randomly divided into five groups: Normal group, model group, and three groups treated with the traditional Chinese medicine (TCM), Wenyangbushen decoction, at a low, moderate and high dose, respectively. The normal group and positive control group were intragastrically administered with saline. The TCM groups were treated with Wenyangbushen decoction at the indicated dosage. Following treatment for 8 weeks, the mRNA and protein expression levels of VEGF, OPG, RANK and RANKL in the femoral head tissues were determined using reverse transcription‑quantitative polymerase chain reaction and western blot analyses, respectively. The data revealed that Wenyangbushen decoction effectively promoted the growth of bone cells, osteoblasts and chondrocytes, and prevented cell apoptosis in the SANFH. The mRNA and protein expression levels of OPG and VEGF were increased, while the levels of RANK and RANKL were reduced in the necrotic tissue of the model group, compared with those in the normal rabbits. Wenyangbushen treatment prevented these changes, manifested by an upregulation in the expression levels of VEGF and OPG, and downregulation in the expression levels of RANK and RANKL in a dose‑dependent manner. It was concluded that treatment with Wenyangbushen formula alleviated necrosis of the femoral head induced by steroids. It was observed to promote bone cell, osteoblast and chondrocyte growth, as well as prevent cell apoptosis. In addition, it upregulated the expression levels of OPG and VEGF, and inhibited the expression levels of RANK and RANKL. These results suggest the potential use of Wenyangbushen formula as a possible approach for the effective treatment of SANFH.

Effects of beta-lactam Compounds on GLT1 and xCT Expression levels as well as Ethanol Intake in Alcohol-Preferring Rats

NASA Astrophysics Data System (ADS)

Hakami, Alqassem

Drug abuse is associated with deficits in glutamate uptake and impairment of glutamate homeostasis. Glutamate transporters are the key players in regulating extracellular glutamate concentrations. Considering the importance of glutamate transporters, pharmacological management of the transporter functions can be used as very promising therapeutic targets. Ceftriaxone (beta-lactam antibiotic) has been shown to attenuate ethanol consumption and cocaine-seeking behavior in part by restoring glutamate homeostasis in mesocorticolimbic regions. Furthermore, recent studies from our lab have demonstrated the effects of amoxicillin and Augmentin on upregulating GLT-1 expression level as well as reducing ethanol consumption in male P rats. Therefore, in this project, we examined the effects of amoxicillin and Augmentin on other glutamate transporters (xCT and GLAST) expression levels in the nucleus accumbens (NAc) and prefrontal cortex (PFC). Furthermore, we also investigated the effects of clavulanic acid administration on alcohol consumption as well as GLT-1 and xCT expression levels in NAc. Additionally, we also determined whether oral Augmentin have any effect in reducing alcohol intake in male P rats. Rats were exposed to free choice of ethanol (15% and 30%), water, and food for a period of five weeks. During week six, rats were given five consecutive daily i.p. injections of saline vehicle, 100 mg/kg amoxicillin injections or 100 mg/kg Augmentin injections. Both compounds significantly increased xCT expression level in NAc. Augmentin also increased xCT expression level in PFC. In the clavulanic acid study, rats were given five consecutive i.p. injections of 5 mg/kg clavulanic acid for the treatment group and the saline injections for the saline group. Clavulanic acid significantly reduced ethanol consumption and significantly upregulated GLT-1 and xCT expression levels in NAc. In oral Augmentin study, oral gavage of Augmentin (100 mg/kg) significantly attenuated alcohol consumption in male P rats as compared to the water gavage group. These findings revealed that amoxicillin, Augmentin and clavulanic acid may have a potential therapeutic action for the treatment of alcohol dependence that are mediated through upregulation of GLT-1 and xCT expression levels in the mesocorticolimbic structures.

Variation and significance of secretory immunoglobulin A, interleukin 6 and dendritic cells in oral cancer.

PubMed

Zhang, Suxin; Zhang, Xin; Yin, Ke; Li, Tianke; Bao, Yang; Chen, Zhong

2017-04-01

The present study aimed to determine changes in the concentration of secretory immunoglobulin A (SIgA) and interleukin 6 (IL-6) in the saliva of patients with oral cancer, to evaluate the abnormal expression of cluster of differentiation (CD) 1a, CD83, CD80 and CD86 on dendritic cells (DCs) of oral cancer tissues and to discuss the interaction between SIgA, IL-6 and DCs in oral cancer. A total of 40 patients between 27 and 70 years of age, median age 52 years, with primary oral cancer were enrolled in the present study, and a group of 20 healthy male and female volunteers was used as the control group. The concentration of SIgA and IL-6 in the saliva of the preoperative patients was determined by ELISA. The expression levels of CD1a, CD83, CD80 and CD86 were detected by immunohistochemistry and flow cytometry, which was performed on histopathological sections from paraffin-embedded tumor and corresponding adjacent control tissues. The specimens were assessed using the semi-quantitative immunoreactive score (IRS). The concentration of SIgA in the saliva from patients with oral cancer decreased, whereas the IL-6 level significantly increased compared with the control subjects (P<0.05). In addition, the decrease of SIgA level and increase of IL-6 level exhibited a negative correlation (r=-0.543, P<0.05). According to the IRS score, the expression levels of CD1a, CD83, CD80 and CD86 in the cancer tissue were lower than the expression levels of the control group (P<0.05). Furthermore, the expression of CD80 and CD86 exhibited no correlation with histological grade or pathological type (P>0.05), but exhibited a negative correlation with clinical stage and lymph node metastasis (P<0.05). The concentration of SIgA and IL-6 in saliva may be used as an auxiliary diagnostic indicator for oral cancer. The detection of CD80 and CD86 expressed on DCs in oral cancer tissue may be useful for the diagnosis and evaluation of the prognosis of tumors. The present study hypothesized that the use of SIgA vaccines or IL-6 inhibitors may be useful for reversing the immune deficiency associated with DCs in oral cancer.

Establishment of lysozyme gene RNA interference systemand its involvement in salinity tolerance in sea cucumber (Apostichopus japonicus).

PubMed

Tian, Yi; Jiang, Yanan; Shang, Yanpeng; Zhang, Yu-Peng; Geng, Chen-Fan; Wang, Li-Qiang; Chang, Ya-Qing

2017-06-01

The lysozyme gene was silenced using RNA interference (RNAi) to analyze the function of lysozyme in sea cucumber under salt stress. The interfering efficiency of four lysozyme RNAi oligos ranged from 0.55 to 0.70. From the four oligos, p-miR-L245 was used for further interfering experiments because it had the best silencing efficiency. Peristomial film injection of p-miR-L245 (10 μg) was used for further interfering experiments. The lowest gene expression, determined by RT-PCR assay, in muscle, coelomic fluid, and parapodium occurred 48 h after p-miR-L245 injection, while that of body wall and tube foot was 96 h and 24 h, respectively. Lysozyme activity in muscle and body wall was significantly lower than the controls. The lowest lysozyme activity in muscle, body wall and parapodium, was found at 48, 72, and 48 h, respectively, which was consistent with the transcription expression of lysozyme. The lowest point of lysozyme activity was at 96 h in coelomic fluid and tube foot, which was laid behind lysozyme expression in transcription level. The expression profile of the lysozyme transcription level and lysozyme activity in the body wall and tube foot increased at 12 h after p-miR-L245 injection before the interference effect appeared. NKA gene expression was expressed at a high level in the positive control (PC) and negative control (NC) groups at 12, 24, and 48 h, while NKA was expressed at low levels in the lysozyme RNAi injection group at 12 and 24 h. The level of NKA gene expression recovered to the level of the PC and NC group at 48, 72, and 96 h after the lysozyme RNAi injection. NKCC1 gene expression was high in the PC and NC groups at 96 h, while the NKCC1 was expressed at a low level 96 h after lysozyme RNAi injection. The results suggest that lysozyme decay involves NKA and NKCC1 gene expression under salinity 18 psμ. The K + and Cl - concentration after lysozyme RNAi injection was lower than in the PC and NC group. Copyright © 2017 Elsevier Ltd. All rights reserved.

N-acetylcysteine Protects Mice from High Fat Diet-induced Metabolic Disorders.

PubMed

Ma, Yongjie; Gao, Mingming; Liu, Dexi

2016-08-01

To study the effects of N-acetylcysteine (NAC, C5H9NO3S) on diet-induced obesity and obesity-related metabolic disorders. Six-week-old male C57BL/6 mice fed a chow or high-fat diet (HFD) were treated with NAC (2 g/L) in drinking water for 11 weeks. Its influences on body weight and food intake were manually measured, and influence on body composition were analyzed by magnetic residence imaging. Glucose meter and ELISA were used to determine serum glucose and insulin levels, as well as lipid content in the liver. The effects of NAC treatment on mRNA levels of genes involved in inflammation, thermogenesis, and lipid metabolism in various tissues were determined by real time PCR. NAC supplementation inhibited the increase of fat mass and the development of obesity when mice were fed an HFD. NAC treatment significantly lowered HFD-induced macrophage infiltration, and enhanced adiponectin gene expression, resulting in reduced hyperglycemia and hyperinsulinemia, and improvement of insulin resistance. NAC oral administration suppressed hepatic lipid accumulation, as evidenced by lower levels of triglyceride and cholesterol in the liver. The beneficial effects are associated with a decrease of hepatic Pparγ and its target gene expression, and an increase in the expression of genes responsible for lipid oxidation and activation of farnesoid X receptor. Furthermore, NAC treatment also stimulates expression of thermogenic genes. These results provide direct proof of the protective potential of NAC against HFD-induced obesity and obesity-associated metabolic disorders.

The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis.

PubMed

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

2015-01-01

The toxin-antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.

The mazEF toxin–antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis

PubMed Central

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

2015-01-01

The toxin–antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains. PMID:26005332

WNK-OSR1/SPAK-NCC signal cascade has circadian rhythm dependent on aldosterone.

PubMed

Susa, Koichiro; Sohara, Eisei; Isobe, Kiyoshi; Chiga, Motoko; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

2012-11-02

Blood pressure and renal salt excretion show circadian rhythms. Recently, it has been clarified that clock genes regulate circadian rhythms of renal transporter expression in the kidney. Since we discovered the WNK-OSR1/SPAK-NaCl cotransporter (NCC) signal cascade, which is important for regulating salt balance and blood pressure, we have sought to determine whether NCC protein expression or phosphorylation shows diurnal rhythms in the mouse kidneys. Male C57BL/6J mice were sacrificed every 4h (at 20:00, 0:00, 4:00, 8:00, 12:00, and 16:00), and the expression and phosphorylation of WNK4, OSR1, SPAK, and NCC were determined by immunoblot. (Lights were turned on at 8:00, which was the start of the rest period, and turned off at 20:00, which was the start of the active period, since mice are nocturnal.) Although expression levels of each protein did not show diurnal rhythm, the phosphorylation levels of OSR1, SPAK, and NCC were increased around the start of the active period and decreased around the start of the rest period. Oral administration of eplerenone (10mg/day) attenuated the phosphorylation levels of these proteins and also diminished the diurnal rhythm of NCC phosphorylation. Thus, the activity of the WNK4-OSR1/SPAK-NCC cascade was shown to have a diurnal rhythm in the kidney that may be governed by aldosterone. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of Dietary Nutrient Density on Small Intestinal Phosphate Transport and Bone Mineralization of Broilers during the Growing Period.

PubMed

Li, Jianhui; Yuan, Jianmin; Miao, Zhiqiang; Song, Zhigang; Yang, Yu; Tian, Wenxia; Guo, Yuming

2016-01-01

A 2 × 4 factorial experiment was conducted to determine the effects of dietary nutrient density on growth performance, small intestinal epithelial phosphate transporter expression, and bone mineralization of broiler chicks fed with diets with different nutrient densities and nonphytate phosphorus (NPP) levels. The broilers were fed with the same starter diets from 0 to 21 days of age. In the grower phase (day 22 to 42), the broilers were randomly divided into eight groups according to body weight. Relatively high dietary nutrient density (HDND) and low dietary nutrient density (LDND) diets were assigned metabolic energy (ME) values of 3,150 and 2,950 kcal/kg, respectively. Crude protein and essential amino acid levels were maintained in the same proportion as ME to prepare the two diet types. NPP levels were 0.25%, 0.30%, 0.35%, and 0.40% of the diets. Results showed that a HDND diet significantly increased the body weight gain (BWG) of broilers and significantly decreased the feed conversion ratio and NPP consumed per BWG. HDND significantly decreased tibial P content of the broilers. Conversely, mRNA expression of NaPi-IIb and protein expression of calbindin were significantly increased in the intestine of broilers fed a HDND diet. HDND also increased vitamin D receptor (VDR) expression, especially at a relatively low dietary NPP level (0.25%). The mRNA expression of NaPi-IIa in the kidneys was significantly increased at a relatively low dietary NPP level (0.25%) to maintain P balance. Tibial P, calcium, and ash content were significantly decreased, as were calbindin and VDR expression levels in the intestine at a low NPP level. Therefore, HDND improved the growth rate of broilers and increased the expression of phosphate and calcium transporter in the small intestine, but adversely affected bone mineralization.

Impaired thymic selection in mice expressing altered levels of the SLP-76 adaptor protein.

PubMed

Ramsey, Kimberley; Luckashenak, Nancy; Koretzky, Gary A; Clements, James L

2008-02-01

Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.

Chemokine-like factor 1 (CLFK1) is over-expressed in patients with atopic dermatitis.

PubMed

Yang, Gao-Yun; Chen, Xue; Sun, Ya-Chun; Ma, Chen-Li; Qian, Ge

2013-01-01

Human chemokine-like factor 1 (CKLF1), a recently discovered chemokine, has a broad spectrum of biological functions in immune-mediated diseases. It is highly expressed on Th2 lymphocytes and is a functional ligand for human CCR4. CKLF1 has a major role in the recruitment and activation of leucocytes, which plays an important role in the pathogenesis of allergic diseases. The present study was designed to determine the expression of CKLF1 in skin and serum in patients with atopic dermatitis (AD). The CKLF1 protein expression in skin lesion was analyzed by immunohistochemistry and ELISA. The mRNA expression of CKLF1 in skin lesion was detected by Real-time PCR. The serum levels of CKLF1, IgE, eotaxin, IL-4, IL-5, and IL-13 were measured by ELISA. Histopathological changes in the skin of AD patients showed local inflammation with epidermal thickening and significant inflammatory cellular infiltration. Immunohistochemistry results demonstrated that CKLF1-staining positive cells were located in the epidermal and dermis, and that the CKLF1 expression in AD patients was significantly higher than that in normal control. The CKLF1 mRNA expression in AD patients was significantly higher than that in healthy controls. Serum CKLF1 and IgE levels were significantly increased in AD patients, as were the serum levels of IL-4, IL-5, IL-13 and eotaxin. Both CKLF1 protien and mRNA levels are overexpressed in the skin lesion of AD patients, along with an increase in serum CKLF1 level, indicating that CKLF1 may play an important role in the development of atopic dermatitis.

Intratumoral gene expression of 5-fluorouracil pharmacokinetics-related enzymes in stage I and II non-small cell lung cancer patients treated with uracil-tegafur after surgery: a prospective multi-institutional study in Japan.

PubMed

Eguchi, Keisuke; Oyama, Takahiko; Tajima, Atsushi; Abiko, Tomohiro; Sawafuji, Makoto; Horio, Hirotoshi; Hashizume, Toshinori; Matsutani, Noriyuki; Kato, Ryoichi; Nakayama, Mitsuo; Kawamura, Masafumi; Kobayashi, Koichi

2015-01-01

This investigation was conducted to assess the use of the intratumoral mRNA expression levels of nucleic acid-metabolizing enzymes as biomarkers of adjuvant chemotherapy for non-small cell lung cancer (NSCLC) using uracil-tegafur in a multi-institutional prospective study. 236 patients with a completely resected NSCLC (adenocarcinoma and squamous cell carcinoma) of pathological stage IA (maximum tumor diameter of 2 cm or greater), IB, and II tumors were given a dose of 250 mg of uracil-tegafur per square meter of body surface area per day orally for two years after surgery. Intratumoral mRNA levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) genes relative to an internal standard, β-actin, were determined using laser-capture microdissection and fluorescence-based real time PCR detection systems. Among 5-FU target enzymes, TS was the only one that showed a significant difference in the level of gene expression between the high and low gene expression groups, for both disease-free survival (DFS) and overall survival (OS), when patients were divided according to median values; 5-year DFS rates in high/low TS gene expression were 60.4% and 72.6%, respectively (p=0.050), 5-year OS rates were 78.1% and 88.6%, respectively (p=0.011). Cox's proportional hazard model indicated that the pathological stage and TS gene expression level were independent values for predicting DFS. The TS gene expression level was shown to be an independent predictive factor for DFS in stage I and II NSCLC patients who were treated with uracil-tegafur following surgery. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Plasma miR-26a as a Diagnostic Biomarker Regulates Cytokine Expression in Systemic Juvenile Idiopathic Arthritis.

PubMed

Sun, Juan; Feng, Miao; Wu, Fengqi; Ma, Xiaolin; Lu, Jie; Kang, Min; Liu, Zhewei

2016-08-01

We sought to identify specific microRNA (miRNA) for systemic juvenile idiopathic arthritis (sJIA) and to determine the involvement of these miRNA in regulating the expression of cytokines. Microarray profiling was performed to identify differentially expressed miRNA in sJIA plasma. Levels of candidate miRNA and mRNA were assessed by real-time PCR, and cytokines were measured by ELISA. Dual-luciferase reporter assay was used to validate the direct interaction between miR-26a and interleukin 6 (IL-6). Forty-eight miRNA were differentially expressed in the plasma of patients with sJIA compared with healthy controls (HC). Five miRNA were selected for further validation. The expression level of miR-26a was exclusively elevated in the plasma of patients with sJIA as compared with 4 rheumatic diseases and 2 subtypes of JIA (oligoarticular and polyarticular). The levels of IL-6, IL-1β, and tumor necrosis factor-α in the plasma of patients with sJIA were increased, and only IL-6 presented a positive correlation with miR-26a (r = 0.539, p < 0.0001). After stimulation with IL-6, miR-26a expression was upregulated in THP-1 cells, while the supernatant level of IL-6 was downregulated by transfection of miR-26a mimics. Consistently, direct target relationship between miR-26a and IL-6 was confirmed. This study demonstrates that miR-26a is expressed specifically and highly in sJIA plasma and suggests that miR-26a may regulate the levels of cytokines in sJIA. Our findings highlight miR-26a as a potential biomarker for the diagnosis as well as differential diagnosis of sJIA.

Role of Krüppel-like factor 4 and heat shock protein 27 in cancer of the larynx

PubMed Central

Karam, Jihad; Fadous-Khalifé, Marie Claude; Tannous, Rita; Fakhreddine, Sally; Massoud, Marcel; Hadchity, Joseph; Aftimos, Georges; Hadchity, Elie

2017-01-01

Late detection and lack of standard treatment strategies in larynx cancer patients result in high levels of mortality and poor prognosis. Prognostic stratification of larynx cancer patients based on molecular prognostic tumor biomarkers may lead to more efficient clinical management. Krüppel-like factor 4 (KLF4) and Heat Shock Protein 27 (HSP27) have an important role in tumorigenesis and are considered promising candidate biomarkers for various types of cancer. However, their role in larynx carcinoma remains to be elucidated. The present study aimed to determine KLF4 and HSP27 expression profiles in laryngeal tumors. The protein and mRNA expression levels of KLF4 and HSP27 were evaluated by immunohistochemical and reverse transcription-polymerase chain reaction analyses in 44 larynx carcinoma samples and 21 normal tissue samples, and then correlated with clinical characteristics. A differential expression of KLF4 and HSP27 was observed between normal and tumor tissues. The protein and mRNA expression levels of KLF4 were significantly decreased in larynx squamous cell carcinoma (LSCC) compared with normal tissue, whereas HSP27 was significantly overexpressed in tumor tissues compared with normal tissues, at the protein and mRNA levels. KLF4 expression decreased gradually with tumor progression whereas HSP27 expression increased. A significant difference was observed between stages I and IV. KLF4 and HSP27 exhibit opposite functions and roles in the carcinogenic process of LSCC. Their role in laryngeal cancer initiation and progression emphasizes their use as potential future targets for prognosis and treatment. KLF4 and HSP27 expression levels may act as potential biomarkers in patients with cancer of the larynx. PMID:29181170

Supplementation with tocotrienol-rich fraction alters the plasma levels of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor from young and old individuals.

PubMed

Heng, Eng Chee; Karsani, Saiful Anuar; Abdul Rahman, Mariati; Abdul Hamid, Noor Aini; Hamid, Zalina; Wan Ngah, Wan Zurinah

2013-10-01

Tocotrienol possess beneficial effects not exhibited by tocopherol. In vitro studies using animal models have suggested that these effects are caused via modulation of gene and protein expression. However, human supplementation studies using tocotrienol-rich isomers are limited. This study aims to identify plasma proteins that changed in expression following tocotrienol-rich fraction (TRF) supplementation within two different age groups. Subjects were divided into two age groups-32 ± 2 (young) and 52 ± 2 (old) years old. Four subjects from each group were assigned with TRF (78% tocotrienol and 22% tocopherol, 150 mg/day) or placebo capsules for 6 months. Fasting plasma were obtained at 0, 3, and 6 months. Plasma tocopherol and tocotrienol levels were determined. Plasma proteome was resolved by 2DE, and differentially expressed proteins identified by MS. The expressions of three proteins were validated by Western blotting. Six months of TRF supplementation significantly increased plasma levels of tocopherols and tocotrienols. Proteins identified as being differentially expressed were related to cholesterol homeostasis, acute-phase response, protease inhibitor, and immune response. The expressions of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor were validated. The old groups showed more proteins changing in expression. TRF appears to not only affect plasma levels of tocopherols and tocotrienols, but also the levels of plasma proteins. The identity of these proteins may provide insights into how TRF exerts its beneficial effects. They may also be potentially developed into biomarkers for the study of the effects and effectiveness of TRF supplementation.

Expression of immune checkpoint molecules in endometrial carcinoma

PubMed Central

LIU, JIA; LIU, YULING; WANG, WULIANG; WANG, CHENYANG; CHE, YANHONG

2015-01-01

The main obstacle in the development of an effective tumor vaccine is the inherent ability of tumors to evade immune responses. Tumors often use common immune mechanisms and regulators to evade the immune system. The present study aimed to analyze the expression levels of indoleamine 2,3-dioxygenase (IDO), programmed death-ligand (PD-L) 1, PD-L2, B7-H4, galectin-1 and galectin-3 in tissue samples from patients with endometrial carcinoma, in order to detect the immunosuppressive environment of endometrial carcinomas. The levels of IDO, PD-L1, PD-L2 and B7-H4 were analyzed by immunohistochemical methods, and the levels of galectin-1 and galectin-3 in tumor lysates were determined using ELISA. PD-L2 was expressed at low levels in the majority of tumor samples. IDO expression was detected in 38, 63 and 43% of primary endometrial carcinoma, recurrent endometrial carcinoma, and metastatic endometrial carcinoma specimens, respectively. Positive expression rates for PD-L1 were 83% in primary endometrial carcinoma, 68% in recurrent endometrial carcinoma, and 100% in metastatic endometrial carcinoma, whereas B7-H4 expression was detected in 100% of both primary endometrial carcinoma and recurrent endometrial carcinoma samples, and in 96% of metastatic endometrial carcinoma specimens. The expression levels of galectin-1 and galectin-3 were not significantly different between the normal and tumor specimens. The results of the present study suggest that the interaction between PD-1/PD-L1 and B7-H4 may be a potential target for immune intervention in the treatment of endometrial carcinoma. Furthermore, the results may provide the basis for immunosuppressant therapy in the treatment of patients with uterine cancer. PMID:26640578

No preclinical rationale for IGF1R directed therapy in chondrosarcoma of bone.

PubMed

Peterse, Elisabeth F P; Cleven, Arjen H G; De Jong, Yvonne; Briaire-de Bruijn, Inge; Fletcher, Jonathan A; Danen, Erik H J; Cleton-Jansen, Anne-Marie; Bovée, Judith V M G

2016-07-14

Chondrosarcoma is a malignant cartilage forming bone tumour for which no effective systemic treatment is available. Previous studies illustrate the need for a better understanding of the role of the IGF pathway in chondrosarcoma to determine if it can be a target for therapy, which was therefore explored in this study. Expression of mediators of IGF1R signalling and phosphorylation status of IRS1 was determined in chondrosarcoma cell lines by qRT-PCR and western blot. The effect of activation and inhibition of IGF1R signalling on downstream targets was assessed by western blot. Ten chondrosarcoma cell lines were treated with OSI-906 (IGF1R and IR dual inhibitor) after which cell proliferation and migration were determined by a viability assay and the xCELLigence system, respectively. In addition, four chondrosarcoma cell lines were treated with a combination of doxorubicin and OSI-906. By immunohistochemistry, IGF1R expression levels were determined in tissue microarrays of 187 cartilage tumours and ten paraffin embedded cell lines. Mediators of IGF1R signalling are heterogeneously expressed and phosphorylated IRS1 was detected in 67 % of the tested chondrosarcoma cell lines, suggesting that IGF1R signalling is active in a subset of chondrosarcoma cell lines. In the cell lines with phosphorylated IRS1, inhibition of IGF1R signalling decreased phosphorylated Akt levels and increased IGF1R expression, but it did not influence MAPK or S6 activity. In line with these findings, treatment with IGF1R/IR inhibitors did not impact proliferation or migration in any of the chondrosarcoma cell lines, even upon stimulation with IGF1. Although synergistic effects of IGF1R/IR inhibition with doxorubicin are described for other cancers, our results demonstrate that this was not the case for chondrosarcoma. In addition, we found minimal IGF1R expression in primary tumours in contrast to the high expression detected in chondrosarcoma cell lines, even if both were derived from the same tumour, suggesting that in vitro culturing upregulates IGF1R expression. The results from this study indicate that the IGF pathway is not essential for chondrosarcoma growth, migration or chemoresistance. Furthermore, IGF1R is only minimally expressed in chondrosarcoma primary tumours. Therefore, the IGF pathway is not expected to be an effective therapeutic target for chondrosarcoma of bone.

Transfection of the IHH gene into rabbit BMSCs in a simulated microgravity environment promotes chondrogenic differentiation and inhibits cartilage aging.

PubMed

Liu, Peng-Cheng; Liu, Kuan; Liu, Jun-Feng; Xia, Kuo; Chen, Li-Yang; Wu, Xing

2016-09-27

The effect of overexpressing the Indian hedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a simulated microgravity environment. An adenovirus plasmid encoding the rabbit IHH gene was constructed in vitro and transfected into rabbit BMSCs. Two large groups were used: conventional cell culture and induction model group and simulated microgravity environment group. Each large group was further divided into blank control group, GFP transfection group, and IHH transfection group. During differentiation induction, the expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins in each group were determined. In the conventional model, the IHH transfection group expressed high levels of cartilage-related factors (Coll2 and ANCN) at the early stage of differentiation induction and expressed high levels of cartilage hypertrophy-related factors (Coll10, annexin 5, and ALP) at the late stage. Under the simulated microgravity environment, the IHH transfection group expressed high levels of cartilage-related factors and low levels of cartilage hypertrophy-related factors at all stages of differentiation induction. Under the simulated microgravity environment, transfection of the IHH gene into BMSCs effectively promoted the generation of cartilage and inhibited cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.

Vitreous Microparticle Shedding in Retinal Detachment: A Prospective Comparative Study.

PubMed

Tumahai, Perle; Saas, Philippe; Ricouard, Fanny; Biichlé, Sabéha; Puyraveau, Marc; Laheurte, Caroline; Delbosc, Bernard; Saleh, Maher

2016-01-01

Microparticles (MPs) are membrane-derived vesicles measuring less than 1 μm in diameter. They are shed from nearly every activated or preapoptotic cell and may exhibit biologic activities in inflammation or apoptosis settings. The main purpose of this study was to determine whether MP shedding was higher in the vitreous of patients with retinal detachment (RD). This was a prospective, comparative study. Levels of vitreous MPs (including phosphatidylserine [PS]-expressing MPs, photoreceptor cell-derived MPs, and photoreceptor cell-derived MPs expressing PS) and soluble proinflammatory factors (i.e., monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and IL-6) were analyzed by flow cytometry. Samples were obtained from 49 eyes undergoing RD surgery and 41 control eyes. Vitreous levels of all the MPs studied were significantly increased in the RD group. Vitreous MP levels were correlated with levels of at least one proinflammatory factor depending on MP subsets. Concerning clinical parameters, vitreous PS-expressing MP and PS-expressing photoreceptor cell-derived MP levels were higher depending on the duration of RD at surgery, the detached retina surface, and the macula status and were found more sensitive than proinflammatory factors only for the duration of RD at surgery. Vitreous concentrations of MPs (mainly derived from photoreceptor cells) are higher after rhegmatogenous RD and found to be correlated with soluble proinflammatory factors.

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder (Paralichthys olivaceus)

NASA Astrophysics Data System (ADS)

Huang, Yajuan; Hu, Nan; Si, Yufeng; Li, Siping; Wu, Shuxian; Zhang, Meizhao; Wen, Haishen; Li, Jifang; Li, Yun; He, Feng

2018-06-01

Follistatin (Fst) is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns of Fst gene at five different development stages of Japanese flounder (stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months). The muscle tissue of Japanese flounder was obtained at different development stages in this experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

Evaluation of gene expression levels for cytokines in ocular toxoplasmosis.

PubMed

Maia, M M; Meira-Strejevitch, C S; Pereira-Chioccola, V L; de Hippólito, D D C; Silva, V O; Brandão de Mattos, C C; Frederico, F B; Siqueira, R C; de Mattos, L C

2017-10-01

This study evaluated levels for mRNA expression of 7 cytokines in ocular toxoplasmosis. Peripheral blood mononuclear cells (PBMC) of patients with ocular toxoplasmosis (OT Group, n = 23) and chronic toxoplasmosis individuals (CHR Group, n = 9) were isolated and stimulated in vitro with T. gondii antigen. Negative controls (NC) were constituted of 7 PBMC samples from individuals seronegative for toxoplasmosis. mRNA expression for cytokines was determined by qPCR. Results showed a significant increase in mRNA levels from antigen stimulated PBMCs derived from OT Group for expressing IL-6 (at P < .005 and P < .0005 for CHR and NC groups, respectively), IL-10 (at P < .0005 and P < .005 for CHR and NC groups, respectively) and TGF-β (at P < .005) for NC group. mRNA levels for TNF-α and IL-12 were also upregulated in patients with OT compared to CHR and NC individuals, although without statistical significance. Additionally, mRNA levels for IL-27 and IFN-γ in PBMC of patients with OT were upregulated in comparison with NC individuals. Differences between OT and NC groups were statistically significant at P < .05 and P < .0005, respectively. © 2017 John Wiley & Sons Ltd.

Clinical value of protein expression of kallikrein-related peptidase 7 (KLK7) in ovarian cancer.

PubMed

Dorn, Julia; Gkazepis, Apostolos; Kotzsch, Matthias; Kremer, Marcus; Propping, Corinna; Mayer, Katharina; Mengele, Karin; Diamandis, Eleftherios P; Kiechle, Marion; Magdolen, Viktor; Schmitt, Manfred

2014-01-01

Expression of the kallikrein-related peptidase 7 (KLK7) is dysregulated in ovarian cancer. We assessed KLK7 expression by ELISA and quantitative immunohistochemistry and analyzed its association with clinicopathological parameters and patients' outcome. KLK7 antigen concentrations were determined in tumor tissue extracts of 98 ovarian cancer patients by ELISA. For analysis of KLK7 immunoexpression in ovarian cancer tissue microarrays, a manual quantitative scoring system as well as a software tool for quantitative high-throughput automated image analysis was used. In immunohistochemical analyses, expression levels of KLK7 were not associated with patients' outcome. However, in multivariate analyses, KLK7 antigen levels in tumor tissue extracts were significantly associated with both overall and progression-free survival: ovarian cancer patients with high KLK7 levels had a significantly, 2-fold lower risk of death [hazard ratio (HR)=0.51, 95% confidence interval (CI)=0.29-0.90, p=0.019] or relapse [HR=0.47, 95% CI=0.25-0.91, p=0.024), as compared with patients who displayed low KLK7 levels. Our results indicate that - in contrast to earlier findings - high KLK7 antigen levels in tumor tissue extracts may be associated with a better prognosis of ovarian cancer patients.

Progesterone Receptor-A and -B Have Opposite Effects on Proinflammatory Gene Expression in Human Myometrial Cells: Implications for Progesterone Actions in Human Pregnancy and Parturition

PubMed Central

Tan, Huiqing; Yi, Lijuan; Rote, Neal S.; Hurd, William W.

2012-01-01

Context: Progesterone promotes uterine relaxation during pregnancy and its withdrawal induces labor. Progesterone withdrawal in human parturition is mediated in part by changes in the relative levels of the nuclear progesterone receptor isoforms, PR-A and PR-B, in myometrial cells. Parturition also involves myometrial inflammation; however, the functional link between nuclear PR-mediated progesterone actions and inflammation in human myometrial cells is unclear. Objective: Our objective was to determine how PR-A and PR-B regulate progesterone action in human myometrial cells and specifically the expression of genes encoding contraction-associated proteins and proinflammatory mediators. Design: Effects of PR-A and PR-B on the capacity for progesterone to modulate gene expression was determined using an immortalized human myometrial cell line stably transfected with inducible PR-A and PR-B expression transgenes and conditioned to express various PR-A and PR-B levels. Gene expression was assessed by genome wide transcriptome analysis, quantitative RT-PCR and immunoblotting. Results: PR-A and PR-B were each transcriptionally active in response to progesterone and affected the expression of distinct gene cohorts. The capacity for progesterone to affect gene expression was dependent on the PR-A to PR-B ratio. This was especially apparent for the expression of proinflammatory genes. Progesterone decreased proinflammatory gene expression when the PR-A to PR-B ratio favored PR-B and increased proinflammatory gene expression when the ratio favored PR-A. Progesterone via PR-B increased expression of inhibitor-κBα, a repressor of the nuclear factor-κB transcription factor, and inhibited basal and lipopolysaccharide-induced proinflammatory gene expression. Both of those PR-B-mediated effects were inhibited by PR-A. Conclusions: Our data suggest that during most of human pregnancy, when myometrial cells are PR-B dominant, progesterone promotes myometrial quiescence through PR-B-mediated antiinflammatory actions. At parturition, the rise in PR-A expression promotes labor by inhibiting the antiinflammatory actions of PR-B and stimulating proinflammatory gene expression in response to progesterone. PMID:22419721

Relationship Between Caregiving Burden and Anger Level in Primary Caregivers of Individuals With Chronic Mental Illness.

PubMed

Bademli, Kerime; Lök, Neslihan; Kılıc, Ayten Kaya

2017-06-01

The objective of this study was answer to the question: to what extent are the anger of the caregivers of patients diagnosed with schizophrenia and their perceived level of burden are related? The study is a descriptive and correlational study. The information form prepared by the researchers which questions the socio-demographic information of the individuals along with the "Caregiving Burden Inventory" which examines the burden of the caregiver as well as "Trait Anger and Anger Expression Style Scale (TAAES)" which determines the anger levels of the caregivers were used. The caregiving burdens of the caregivers according to the score averages were determined as 11.88±9.78 for time and dependency burden, 11.93±8.46 for developmental burden, 8.47±6.63 for physical burden, 5.61±5.26 for social burden, 6.29±5.25 for emotional burden and the total burden score was determined as 44.19±26.75. According to the trait anger and anger expression style scale score averages; trait anger was determined as 15.12±5.95, anger expression as 9.70±3.43, anger-in as 15.22±4.02, anger control as 28.05±5.57 and anger total score average as 68.11±9.97. According to the results obtained from this study, caregivers of schizophrenia patients experience developmental, physical, social and emotional burdens in addition to trait anger. The caregivers of schizophrenia patients need knowledge and support in order to control the burden and the anger they experience during the caregiving process. Copyright © 2016 Elsevier Inc. All rights reserved.

Distribution and activity levels of matrix metalloproteinase 2 and 9 in canine and feline osteosarcoma

PubMed Central

Gebhard, Christiane; Fuchs-Baumgartinger, Andrea; Razzazi-Fazeli, Ebrahim; Miller, Ingrid; Walter, Ingrid

2016-01-01

Overexpression of matrix metalloproteinases (MMPs) has been associated with increased tumor aggressiveness and metastasis dissemination. We investigated whether the contrasting metastatic behavior of feline and canine osteosarcoma is related to levels and activities of MMP2 and MMP9. Zymography and immunohistochemistry were used to determine expression levels of MMP2 and MMP9 in canine and feline osteosarcoma. Using immunohistochemistry, increased MMP9 levels were identified in most canine osteosarcomas, whereas cat samples more often displayed moderate levels. High levels of pro-MMP9, pro-MMP2, and active MMP2 were detected by gelatin zymography in both species, with significantly higher values for active MMP2 in canine osteosarcoma. These findings indicate that MMP2 is probably involved in canine and feline osteosarcoma and their expression and activity could be associated with the different metastatic behavior of canine and feline osteosarcoma. PMID:26733734

Distribution and activity levels of matrix metalloproteinase 2 and 9 in canine and feline osteosarcoma.

PubMed

Gebhard, Christiane; Fuchs-Baumgartinger, Andrea; Razzazi-Fazeli, Ebrahim; Miller, Ingrid; Walter, Ingrid

2016-01-01

Overexpression of matrix metalloproteinases (MMPs) has been associated with increased tumor aggressiveness and metastasis dissemination. We investigated whether the contrasting metastatic behavior of feline and canine osteosarcoma is related to levels and activities of MMP2 and MMP9. Zymography and immunohistochemistry were used to determine expression levels of MMP2 and MMP9 in canine and feline osteosarcoma. Using immunohistochemistry, increased MMP9 levels were identified in most canine osteosarcomas, whereas cat samples more often displayed moderate levels. High levels of pro-MMP9, pro-MMP2, and active MMP2 were detected by gelatin zymography in both species, with significantly higher values for active MMP2 in canine osteosarcoma. These findings indicate that MMP2 is probably involved in canine and feline osteosarcoma and their expression and activity could be associated with the different metastatic behavior of canine and feline osteosarcoma.

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

PubMed Central

2017-01-01

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies. PMID:28591185

Apigenin Induces the Apoptosis and Regulates MAPK Signaling Pathways in Mouse Macrophage ANA-1 Cells

PubMed Central

Liao, Yuexia; Shen, Weigan; Kong, Guimei; Lv, Houning; Tao, Wenhua; Bo, Ping

2014-01-01

Apigenin is a naturally occurring plant flavonoid that possesses antioxidant, anti-cancer and anti-inflammatory properties. However, there are few reports has been done on the ability of apigenin to induce apoptosis in macrophages. In this study, mouse macrophage ANA-1 cells were incubated with different concentrations of apigenin. The cell viability was determined by an MTT assay. The cell apoptosis were analyzed by flow cytometric analysis. Apoptosis were also analyzed using a TUNEL assay and a DNA ladder. The level of intracellular ROS was detected using a dichlorofluorescein -diacetate probe. The expression levels of apoptosis-related proteins were detected by western blot analysis. The results showed that apigenin decreased the viability of ANA-1 cells and induced apoptosis in a dose- and time-dependent manner. Apigenin increased the level of intracellular ROS, downregulated the expression of Bcl-2 and upregulated the expression of caspase-3 and caspase-8 in ANA-1 cells. Furthermore, apigenin downregulated the expression of phospho-ERK and phospho-JNK, upregulated the expression of phospho-p38 and had no significant effect on the expression of Bax, ERK, JNK and p38. The results suggested that apigenin induced cell apoptosis in mouse macrophage ANA-1 cells may via increasing intracellular ROS, regulating the MAPK pathway, and then inhibiting Bcl-2 expression. PMID:24646936

5-HT1A receptor gene silencers Freud-1 and Freud-2 are differently expressed in the brain of rats with genetically determined high level of fear-induced aggression or its absence.

PubMed

Kondaurova, Elena M; Ilchibaeva, Tatiana V; Tsybko, Anton S; Kozhemyakina, Rimma V; Popova, Nina K; Naumenko, Vladimir S

2016-09-01

Serotonin 5-HT1A receptor is known to play a crucial role in the mechanisms of genetically defined aggression. In its turn, 5-HT1A receptor functional state is under control of multiple factors. Among others, transcriptional factors Freud-1 and Freud-2 are known to be involved in the repression of 5-HT1A receptor gene expression. However, implication of these factors in the regulation of behavior is unclear. Here, we investigated the expression of 5-HT1A receptor and silencers Freud-1 and Freud-2 in the brain of rats selectively bred for 85 generations for either high level of fear-induced aggression or its absence. It was shown that Freud-1 and Freud-2 levels were different in aggressive and nonaggressive animals. Freud-1 protein level was decreased in the hippocampus, whereas Freud-2 protein level was increased in the frontal cortex of highly aggressive rats. There no differences in 5-HT1A receptor gene expression were found in the brains of highly aggressive and nonaggressive rats. However, 5-HT1A receptor protein level was decreased in the midbrain and increased in the hippocampus of highly aggressive rats. These data showed the involvement of Freud-1 and Freud-2 in the regulation of genetically defined fear-induced aggression. However, these silencers do not affect transcription of the 5-HT1A receptor gene in the investigated rats. Our data indicate the implication of posttranscriptional rather than transcriptional regulation of 5-HT1A receptor functional state in the mechanisms of genetically determined aggressive behavior. On the other hand, the implication of other transcriptional regulators for 5-HT1A receptor gene in the mechanisms of genetically defined aggression could be suggested. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression and activation of STAT3 in ischemia-induced retinopathy.

PubMed

Mechoulam, Hadas; Pierce, Eric A

2005-12-01

Signal transducer and activator of transcription protein-3 (STAT3) is a transcription factor that participates in many biological processes, including tumor angiogenesis. The expression and activation of Stat3 in the mouse model of ischemia-induced retinal neovascularization was investigated to evaluate the possible role of STAT3 in retinal vascular disease. Retinal neovascularization was induced in mice pups by exposure to hyperoxia. Gene microarrays were used to identify genes whose expression in the retina is altered at postnatal day (P)12 and P18. The relative levels of Stat3 mRNA were determined by semiquantitative RT-PCR. Stat3 protein levels and the levels of the activated form of Stat3 (pStat3) at P12, P15, P18, and P22 were determined by immunoblot analysis. Stat3 and pStat3 were demonstrated by immunofluorescence in retinal sections at P12, P15, and P18. In a series of microarray experiments, increased Stat3 mRNA levels in the retina were detected at P18. This result was validated by RT-PCR and demonstrated that Stat3 and pStat3 protein levels also increase during the development of neovascularization. Stat3 partially colocalized with blood vessels at the peak of neovascularization. pStat3 colocalized completely with blood vessels in both experimental samples and age-matched controls. pStat3 staining increased notably in the neovascular vessels at P15 and P18 and was more strongly associated with the epiretinal vessels than with inner retinal vessels. It was not detected in larger blood vessels, such as those of the optic nerve. The level of Stat3 expression increased, and pStat3 was observed in association with retinal neovascularization. Activated Stat3 was preferentially localized to neovascular retinal vessels. These data suggest that STAT3 may have a role in proliferative retinopathy.

Testosterone Regulates Erectile Function and Vcsa1 Expression in the Corpora of Rats

PubMed Central

Chua, Rowena G.; Calenda, Giulia; Zhang, Xinhua; Siragusa, Joseph; Tong, Yuehong; Tar, Moses; Aydin, Memduh; DiSanto, Michael E.; Melman, Arnold; Davies, Kelvin P.

2009-01-01

Summary Vcsa1 plays an important role in the erectile physiology of the rat. We conducted experiments to determine if erectile function, testosterone levels and Vcsa1 expression were correlated. In orchiectomized rats, total testosterone in blood fell from an average of 4ng/ml to <0.04ng/ml. Erectile function was significantly lower compared to controls and Vcsa1 expression was significantly (>6-fold) decreased. Injection of orchiectomized animals with testosterone (2mg in 100ml sesame oil every 4 days for two weeks) restored average levels of testosterone to 2ng/ml, increased erectile function and significantly increased Vcsa1 expression. In isolated corporal cells there was testosterone dependent Vcsa1 expression. However, intracorporal injection of orchiectomized animals with a plasmid expressing Vcsa1 or its gene product Sialorphin (previously demonstrated to improve erectile function in old animals) gave no significant improvement in erectile function. Also, the ability of Sialorphin to reduce tension in corporal smooth muscle strips isolated from orchiectomized animals was impaired compared to controls. PMID:19428993

Expression profiles of adult T-cell leukemia–lymphoma and associations with clinical responses to zidovudine and interferon α

PubMed Central

ALIZADEH, ASH A.; BOHEN, SEAN P.; LOSSOS, CHEN; MARTINEZ-CLIMENT, JOSE A.; RAMOS, JUAN CARLOS; CUBEDO-GIL, ELENA; HARRINGTON, WILLIAM J.; LOSSOS, IZIDORE S.

2014-01-01

Adult T-cell leukemia–lymphoma (ATLL) is an HTLV-1-associated lymphoproliferative malignancy that is frequently fatal. We compared gene expression profiles (GEPs) of leukemic specimens from nine patients with ATLL at the time of diagnosis and immediately after combination therapy with zidovudine (AZT) and interferon α (IFNα). GEPs were also related to genetic aberrations determined by comparative genomic hybridization. We identified several genes anomalously over-expressed in the ATLL leukemic cells at the mRNA level, including LYN, CSPG2, and LMO2, and confirmed LMO2 expression in ATLL cells at the protein level. In vivo AZT–IFNα therapy evoked a marked induction of interferon-induced genes accompanied by repression of cell-cycle regulated genes, including those encoding ribosomal proteins. Remarkably, patients not responding to AZT–IFNα differed most from responding patients in lower expression of these same IFN-responsive genes, as well as components of the antigen processing and presentation apparatus. Demonstration of specific gene expression signatures associated with response to AZT–IFNα therapy may provide novel insights into the mechanisms of action in ATLL. PMID:20370541

Decreased TIM-3 mRNA expression in peripheral blood mononuclear cells from nephropathy patients.

PubMed

Cai, X Z; Liu, N; Qiao, Y; Du, S Y; Chen, Y; Chen, D; Yu, S; Jiang, Y

2015-06-12

Increasing evidence shows that TIM-1 and TIM-3 in-fluence chronic autoimmune diseases, and their expression levels in immune cells from nephritic patients are still unknown. Real-time transcription-polymerase chain reaction analysis was used to deter-mine expression levels of TIM-1 and TIM-3 mRNA in peripheral blood mononuclear cells (PBMCs) from 36 patients with minimal change glo-merulopathy (MCG), 65 patients with lupus nephritis (LN), 78 patients with IgA nephropathy (IgAN), 55 patients with membranous nephropa-thy (MN), 22 patients with crescentic glomerulonephritis (CGN), 26 patients with anaphylactoid purpura nephritis (APN), and 63 healthy controls. TIM-3 mRNA expression significantly decreased in PBMCs from nephritic patients (LN, P < 0.0001; MCG, P < 0.0001; MN, P = 0.0031; CGN, P = 0.0464; IgAN, P = 0.0002; APN, P = 0.0392) com-pared with healthy controls. In contrast, there was no significant differ-ence in TIM-1 mRNA expression between the patients and the healthy controls. Our results suggest that insufficient expression of TIM-3 mRNA may be involved in the pathogenesis of nephropathy.

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

PubMed

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

2012-03-01

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

Quantification of STAT3 and VEGF expression for molecular diagnosis of lymph node metastasis in breast cancer

PubMed Central

Chen, Yujuan; Liu, Ya; Wang, Yu; Li, Wen; Wang, Xiaolu; Liu, Xuejuan; Chen, Yao; Ouyang, Chibin; Wang, Jing

2017-01-01

Abstract Background: Axillary lymph node metastasis is associated with increased risk of regional recurrence, distant metastasis, and poor survival in breast malignant neoplasm. Expression of signal transducer and activator of transcription 3 (STAT3) is significantly associated with tumor formation, migration, and invasion in various cancers. In addition, vascular endothelial growth factor (VEGF) expression could promote angiogenesis and increase the risk of tumorigenesis. To determine correlations among STAT3 expression, VEGF, and clinicopathological data on lymph node involvement in breast cancer patients after surgery. Methods: The mRNA expression levels of STAT3 and VEGFs were measured in 45 breast invasive ductal carcinoma tissues, 45 peritumoral tissues, and 45 adjacent nontumor tissues by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Postoperative pathological examination revealed explicit axillary lymph node involvement in all patients. Results: Average mRNA levels of STAT3 and VEGFs were the highest in breast invasive ductal carcinoma tissues, followed by peritumoral tissues. High expression of STAT3 showed significant positive correlation with high axillary lymph node involvement and progesterone receptor (PR), VEGF-C, VEGF-D, and vascular endothelial growth factor receptor (VEGFR)-3 expression. The expression levels of STAT3, VEGF-C, and VEGFR-3 were significantly higher in the tumor tissues of patients with axillary lymph node metastasis than in those of patients without the metastasis. Expression levels of VEGF-C and VEGFR-3 were also significantly higher in peritumoral tissues of patients with axillary lymph node metastasis. Positive correlations were found between STAT3 and VEGF-C/-D mRNA levels. Conclusion: These data suggest that STAT3/VEGF-C/VEGFR-3 signaling pathway plays an important role in carcinogenesis and lymph-angiogenesis. Our findings suggest that STAT3 may be a potential molecular biomarker for predicting the involvement of axillary lymph nodes in breast cancer, and therapies targeting STAT3 may be important for preventing breast cancer metastasis. PMID:29137038

DNA methylation of amino acid transporter genes in the human placenta.

PubMed

Simner, C; Novakovic, B; Lillycrop, K A; Bell, C G; Harvey, N C; Cooper, C; Saffery, R; Lewis, R M; Cleal, J K

2017-12-01

Placental transfer of amino acids via amino acid transporters is essential for fetal growth. Little is known about the epigenetic regulation of amino acid transporters in placenta. This study investigates the DNA methylation status of amino acid transporters and their expression across gestation in human placenta. BeWo cells were treated with 5-aza-2'-deoxycytidine to inhibit methylation and assess the effects on amino acid transporter gene expression. The DNA methylation levels of amino acid transporter genes in human placenta were determined across gestation using DNA methylation array data. Placental amino acid transporter gene expression across gestation was also analysed using data from publically available Gene Expression Omnibus data sets. The expression levels of these transporters at term were established using RNA sequencing data. Inhibition of DNA methylation in BeWo cells demonstrated that expression of specific amino acid transporters can be inversely associated with DNA methylation. Amino acid transporters expressed in term placenta generally showed low levels of promoter DNA methylation. Transporters with little or no expression in term placenta tended to be more highly methylated at gene promoter regions. The transporter genes SLC1A2, SLC1A3, SLC1A4, SLC7A5, SLC7A11 and SLC7A10 had significant changes in enhancer DNA methylation across gestation, as well as gene expression changes across gestation. This study implicates DNA methylation in the regulation of amino acid transporter gene expression. However, in human placenta, DNA methylation of these genes remains low across gestation and does not always play an obvious role in regulating gene expression, despite clear evidence for differential expression as gestation proceeds. Copyright © 2017. Published by Elsevier Ltd.