Connecting rules from paired miRNA and mRNA expression data sets of HCV patients to detect both inverse and positive regulatory relationships

PubMed Central

2015-01-01

Background Intensive research based on the inverse expression relationship has been undertaken to discover the miRNA-mRNA regulatory modules involved in the infection of Hepatitis C virus (HCV), the leading cause of chronic liver diseases. However, biological studies in other fields have found that inverse expression relationship is not the only regulatory relationship between miRNAs and their targets, and some miRNAs can positively regulate a mRNA by binding at the 5' UTR of the mRNA. Results This work focuses on the detection of both inverse and positive regulatory relationships from a paired miRNA and mRNA expression data set of HCV patients through a 'change-to-change' method which can derive connected discriminatory rules. Our study uncovered many novel miRNA-mRNA regulatory modules. In particular, it was revealed that GFRA2 is positively regulated by miR-557, miR-765 and miR-17-3p that probably bind at different locations of the 5' UTR of this mRNA. The expression relationship between GFRA2 and any of these three miRNAs has not been studied before, although separate research for this gene and these miRNAs have all drawn conclusions linked to hepatocellular carcinoma. This suggests that the binding of mRNA GFRA2 with miR-557, miR-765, or miR-17-3p, or their combinations, is worthy of further investigation by experimentation. We also report another mRNA QKI which has a strong inverse expression relationship with miR-129 and miR-493-3p which may bind at the 3' UTR of QKI with a perfect sequence match. Furthermore, the interaction between hsa-miR-129-5p (previous ID: hsa-miR-129) and QKI is supported with CLIP-Seq data from starBase. Our method can be easily extended for the expression data analysis of other diseases. Conclusion Our rule discovery method is useful for integrating binding information and expression profile for identifying HCV miRNA-mRNA regulatory modules and can be applied to the study of the expression profiles of other complex human diseases. PMID:25707620

Expression of growth hormone gene during early development of Siberian sturgeon (Acipenser baerii)

PubMed Central

Abdolahnejad, Zeinab; Pourkazemi, Mohammad; Khoshkholgh, Majid Reza; Yarmohammadi, Mahtab

2015-01-01

The mRNA expression of growth hormone (GH) gene in early development stages of Siberian sturgeon was investigated using RT-PCR method. Samples were collected from unfertilized eggs up to 50 days post hatched (dph) larvae in 11 different times. Ribosomal protein L6 (RPL6) transcripts were used as the internal standard during quantification of GH mRNA expression. The results showed that the GH mRNA could be observed in the eyed eggs and even at unfertilized eggs of Siberian sturgeon. The highest amounts of GH mRNA were found at 25 and 50 dph larvae, while the lowest levels were detected at 1 and 3 dph larvae stage. These findings suggest that, the GH mRNA play a key role during developmental stages of Siberian sturgeon. PMID:27844010

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

PubMed

Trivedi, Vikas; Choi, Harry M T; Fraser, Scott E; Pierce, Niles A

2018-01-08

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization. © 2018. Published by The Company of Biologists Ltd.

Expression of pituitary adenylate cyclase-activating polypeptide 1 and 2 receptor mRNA in gallbladder tissue of patients with gallstone or gallbladder polyps

PubMed Central

Zhang, Zhen-Hai; Wu, Shuo-Dong; Gao, Hong; Shi, Gang; Jin, Jun-Zhe; Kong, Jing; Tian, Zhong; Su, Yang

2006-01-01

AIM: To detect the expression of pituitary adenylate cyclase-activating polypeptide receptor 1 (VPCAP1-R) and VPCAP2-R mRNA in gallbladder tissues of patients with gallstone or gallbladder polyps. METHODS: The expression of VPCAP1-R and VPCAP2-R mRNA in gallbladder tissues was detected in 25 patients with gallstone, 8 patients with gallbladder polyps and 7 donors of liver transplantation by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The VPCAP2-R mRNA expression level in the control group (1.09±0.58) was lower than that in the gallbladder polyp group (1.64 ± 0.56) and the gallstone group (1.55±0.45) (P < 0.05) while the VPCAP1-R mRNA expression level in the control group (1.15 ± 0.23) was not apparently different from that in the gallbladder polyp group (1.28±0.56) and the gallstone group (1.27 ± 0.38). CONCLUSION: The abnormal expression of VPCAP2-R mRNA in gallbladder tissue may play a role in the formation of gallbladder stone and gallbladder polyps. PMID:16552823

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

PubMed Central

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

2010-01-01

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

Cloning and mRNA expression of guanylin, uroguanylin, and guanylyl cyclase C in the Spinifex hopping mouse, Notomys alexis.

PubMed

Donald, John A; Bartolo, Ray C

2003-06-01

Guanylin and uroguanylin are peptides that activate guanylyl cyclase C (GC-C) receptors in the intestine and kidney, which causes an increase in the excretion of salt and water. The Spinifex hopping mouse, Notomys alexis, is a desert rodent that can survive for extended periods without free access to water and it was hypothesised that to conserve water, the expression of guanylin, uroguanylin, and GC-C would be down-regulated to reduce the excretion of water in urine and faeces. Accordingly, this study examined the expression of guanylin, uroguanylin, and GC-C mRNA in Notomys under normal (access to water) and water-deprived conditions. Initially, guanylin and uroguanylin cDNAs encoding the full open reading frame were cloned and sequenced. A PCR analysis showed guanylin and uroguanylin mRNA expression in the small intestine, caecum, proximal and distal colon, heart, and kidney. In addition, a partial GC-C cDNA was obtained and GC-C mRNA expression was demonstrated in the proximal and distal colon, but not the kidney. Subsequently, a semi-quantitative PCR method showed that water deprivation in Notomys caused a significant increase in guanylin and uroguanylin mRNA expression in the distal colon, and in guanylin and GC-C mRNA expression in the proximal colon. No significant difference in guanylin and uroguanylin mRNA expression was observed in the kidney. The results of this study indicate that there is, in fact, an up-regulation of the colonic guanylin system in Notomys after 7 days of water deprivation.

[Effects of Porphyromonas endodontalis lipopolysaccharides on the expression of matrix metalloproteinase-9 in mouse osteoblasts].

PubMed

Li, X L; Yu, Y Q; Qiu, L H; Yang, D; Wang, X M; Yu, J T

2017-08-09

Objective: To evaluate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein as well as enzyme activity in MC3T3-E1 cells and the role of nuclear factor-κB (NF-κB) in the process, so as to investigate the expression of MMP-9 dependent signaling pathways in mouse osteoblasts induced by Pe LPS. Methods: The experiment was conducted in 3 sessions: MC3T3-E1 cells were treated with various concentrations of Pe LPS (0-20 mg/L) and 10 mg/L Pe LPS for different time intervals (0-48 h). The expression of MMP-9 mRNA and protein were detected by real-time reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), while the enzyme activity was detected by gelatin zymography method. The expression of MMP-9 mRNA was also detected in 10 mg/L Pe LPS treated MC3T3-El cells after pretreated with specific NF-κB inhibitor BAY 11-7082 for l h. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. Results: The levels of MMP-9 mRNA and protein increased significantly after the treatment with various concentrations of Pe LPS (0-20 mg/L), which indicated that Pe LPS induced osteoblasts to express MMP-9 in dose dependent manners. The expression of MMP-9 protein increased from (5 395±362) ng/L (blank control group) to (12 684±375) ng/L (20 mg/L group). Maximal induction of MMP-9 mRNA expression was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 24 h. The expression of MMP-9 mRNA in the 20 mg/L group was about 7 times than that in the blank control group. After 24 h, the expression of MMP-9 mRNA decreased. Maximal expression of MMP-9 protein was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 48 h ([35 055±2 346] ng/L) showing the highest enzyme activity. The mRNA of MMP-9 decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. Conclusions: Pe LPS might induce the expression of MMP-9 in MC3T3-E1 cells through the signaling of NF-κB.

Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

PubMed

Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

2016-07-01

We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

[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.

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.

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

PubMed

Tachibana, Masatsugu; Shinagawa, Yasuhiro; Kawamata, Hitoshi; Omotehara, Fumie; Horiuchi, Hideki; Ohkura, Yasuo; Kubota, Keiichi; Imai, Yutaka; Fujibayashi, Takashi; Fujimori, Takahiro

2003-01-01

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

RNA Flow Cytometry Using the Branched DNA Technique.

PubMed

Soh, Kah Teong; Wallace, Paul K

2018-01-01

The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.

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.

Phenolic compounds increase the transcription of mouse intestinal maltase-glucoamylase and sucrase-isomaltase.

PubMed

Simsek, Meric; Quezada-Calvillo, Roberto; Nichols, Buford L; Hamaker, Bruce R

2017-05-24

Diverse natural phenolic compounds show inhibition activity of intestinal α-glucosidases, which may constitute the molecular basis for their ability to control systemic glycemia. Additionally, phenolics can modify mRNA expression for proteins involved in nutritional, metabolic or immune processes. To explore the possibility that phenolics can regulate the mRNA expression, enzymatic activity, and protein synthesis/processing of intestinal Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI), small intestinal explants from Balb/c mice were cultured for 24 h in the presence or absence of gallic acid, caffeic acid, and (+)-catechin at 0.1, 0.5, and 1 mM. We measured the levels of MGAM and SI mRNA expression by qRT-PCR, maltase and sucrase activities by a standard colorimetric method and the molecular size distribution of MGAM and SI proteins by western blotting. mRNA expression for MGAM was induced by the three phenolic compounds at 0.1 mM. mRNA expression for SI was induced by caffeic and gallic acids, but not by (+)-catechin. Caffeic acid was the most effective inducer of mRNA expression of these enzymes. Total maltase and sucrase activities were not affected by treatment with phenolics. The proportion of high molecular size forms of MGAM was significantly increased by two of the three phenolic compounds, but little effect was observed on SI proteins. Thus, changes in the protein synthesis/processing, affecting the proportions of the different molecular forms of MGAM, may account for the lack of correlation between mRNA expression and enzymatic activity.

Identification of microRNA-mRNA modules using microarray data.

PubMed

Jayaswal, Vivek; Lutherborrow, Mark; Ma, David D F; Yang, Yee H

2011-03-06

MicroRNAs (miRNAs) are post-transcriptional regulators of mRNA expression and are involved in numerous cellular processes. Consequently, miRNAs are an important component of gene regulatory networks and an improved understanding of miRNAs will further our knowledge of these networks. There is a many-to-many relationship between miRNAs and mRNAs because a single miRNA targets multiple mRNAs and a single mRNA is targeted by multiple miRNAs. However, most of the current methods for the identification of regulatory miRNAs and their target mRNAs ignore this biological observation and focus on miRNA-mRNA pairs. We propose a two-step method for the identification of many-to-many relationships between miRNAs and mRNAs. In the first step, we obtain miRNA and mRNA clusters using a combination of miRNA-target mRNA prediction algorithms and microarray expression data. In the second step, we determine the associations between miRNA clusters and mRNA clusters based on changes in miRNA and mRNA expression profiles. We consider the miRNA-mRNA clusters with statistically significant associations to be potentially regulatory and, therefore, of biological interest. Our method reduces the interactions between several hundred miRNAs and several thousand mRNAs to a few miRNA-mRNA groups, thereby facilitating a more meaningful biological analysis and a more targeted experimental validation.

The metastasis suppressor gene KISS-1 regulates osteosarcoma apoptosis and autophagy processes.

PubMed

Yin, Yiran; Tang, Lian; Shi, Lei

2017-03-01

The expression of the metastasis suppressor gene KISS-1 in osteosarcoma cells during apoptosis and autophagy was evaluated. MG-63 osteosarcoma cells were transfected with either KISS-1 overexpression or KISS-1 knockdown expression vector in vitro, and compared with cell lines transfected with empty vector. After 12, 24, 48 and 72 h of cell culture, the cell proliferation was examined. The MTT method was used to detect apoptosis by flow cytometry, and the mRNA levels of apoptosis and autophagy markers caspase-3, Bcl-2, Bax, LC3 and Beclin1 were assessed by RT-PCR. Our results showed that cells in the control and low expression group kept proliferating during the cell culture period of 72 h, while the cells in the overexpression group progressively decreased in number. Also, the proliferation rate of the low expression group was significantly higher than that of the control group. The relative mRNA expression levels of caspase-3 and Bax mRNA in the control and low expression group showed no change (the expression was lowest in the low expression group). Moreover, the mRNA level of Bcl-2 increased in both cell groups. The mRNA expression levels of caspase-3 and Bax in the overexpression group were increased, and the level of Bcl-2 was reduced significantly. At the same time, the relative expression level of LC3 and Beclin1 mRNA in the control and low expression groups remained the same, and that of the overexpression group increased. The mRNA levels of LC3 and Beclin1 in the overexpression group were the highest, and that of the low expression group the lowest. The differences were statistically significant (P<0.05). Based on these results, we showed that KISS-1 inhibited the proliferation of osteosarcoma in vitro, probably by accelerating the processes of apoptosis and autophagy in the cells.

The lower expression of gonadotropin-releasing hormone receptor associated with poor prognosis in gastric cancer

PubMed Central

Lu, Mingzhu; Zhu, Jing; Ling, Yang; Shi, Wenping; Zhang, Changsong; Wu, Haorong

2015-01-01

Aims: Expression of gonadotropin-releasing hormone receptor (GnRHR) has been demonstrated in a number of malignancies. The aim is to investigate the expression of GnRHR and prognosis in gastric cancer. Methods and materials: GnRHR mRNA was examined in tumor and non-tumor tissues from 48 gastric cancer patients by Real-time PCR. The GnRHR protein expression was performed by immunohistochemical analysis. Results: The expression of GnRHR mRNA was higher (mean ± SD, -10.06 ± 1.28) in gastric tumor tissues than matched non-tumor tissues (mean ± SD, -12.43 ± 1.33). GnRHR mRNA expression was associated with lymph node metastasis, distant metastasis, and TNM stage. We found the decreased expression of GnRHR mRNA were significantly correlated with poor overall survival (P = 0.003). Immunocytochemical staining of GnRHR in tumor tissues showed mainly weak staining (43.48%, 10/23) and moderate staining (21.74%, 5/23) in high GnRHR mRNA patients, and mainly negative staining in low GnRHR mRNA patients. And the staining of GnRHR was not detection in tumor tissues for more than half of gastric patients (52.08%, 25/48). These results implied that the loss of GnRHR protein could be a main event in gastric cancer. Conclusion: The GnRHR expression is very low in gastric cancer, and the loss of GnRHR expression could be a poor prognostic factor, which implied that GnRHR could play an important role in the development of gastric cancer. PMID:26550267

Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma

PubMed Central

2011-01-01

Background Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines. Methods Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome. Results General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome. Conclusions Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs. PMID:21767383

The histological characteristics, age-related thickness change of skin, and expression of the HSPs in the skin during hair cycle in yak (Bos grunniens)

PubMed Central

Yang, Xue; Cui, Yan; Yue, Jing; He, Honghong; Yu, Chuan; Liu, Penggang; Liu, Jun; Ren, Xiandong; Meng, Yun

2017-01-01

Objective This experiment was conducted to study the histological characteristics, age-related thickness changes, and expression of HSPs in the skin of yak. Methods A total of 20 yaks (10 males and 10 females) were used. Different regions of the normal skin of three different ages (newborn, half-year-old and adult) of yaks were harvested for histological study and thickness measurement. Biopsy samples were taken from the scapula regions of the skin from the same five approximately 1-year-old yaks during the hair cycle (telogen, anagen and catagen). RT-PCR, western blot and immunohistochemistry methods using the mRNA and protein levels were used to detect the expression of HSP27, HSP70 and HSP90. RT-PCR method was used to detect the mRNA expression of CGI-58 and KDF1. The IPP6.0 software was used to analyze the immunohistochemistry and measure the thickness of the skin. Results The general histological structure of hairy yak skin was similar to other domestic mammals. The unique features included prominent cutaneous vascular plexuses, underdeveloped sweat glands, a large number of nasolabial glands in the nasolabial plate, and hair follicle groups composed of one primary follicle and several secondary follicles. The skin, epidermis and dermis thickness did vary significantly between different body regions and different ages. The thickness of the skin, epidermis and dermis increased from newborn to adult in yaks. Yak skin thickness decreased from dorsally to ventrally on the trunk. The skin on the lateral surface was thicker than the skin on the medial surface on the limbs. HSP27, HSP70 and HSP90 showed different expression patterns during the hair cycle using RT-PCR, western blot and immunohistochemistry methods. The expression of HSP27 mRNA and protein in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of HSP70 mRNA and protein in the telogen stage was the highest, followed by the anagen stage, and the expression in the catagen stage was the lowest. The expression of HSP90 mRNA and protein in the anagen stage was the highest, followed by the telogen stage, and the expression in the catagen stage was the lowest. HSPs were mainly expressed in the outer root sheath of hair follicle during the hair cycle, also expressed in epidermis, sebaceous gland and sweat gland in the skin of Yak. The expression of CGI-58 mRNA in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of KDF1 mRNA in the telogen stage was the highest, followed by the catagen stage, and the expression in the anagen stage was the lowest. Meaning In this study, we examined and fully described the histology of normal skin in Yak and measured the skin thickness of different ages and different regions in Yak. These data may be useful to better understand and appreciate the adaptability features of yak skin. Our investigation reports the expression patterns of HSPs in yak skin for the first time. The different expression pattern of HSPs during the hair cycle suggests they may play different roles in yak hair follicle biology. PMID:28463974

The Expression and Significance of Feces Cyclooxygensae-2 mRNA in Colorectal Cancer and Colorectal Adenomas

PubMed Central

Li, Xiaofeng; Kong, Lixia; Liao, Suhuan; Lu, Jing; Ma, Lin; Long, Xiaohua

2017-01-01

Background/Aim: This study aims to explore the expression and significance of feces cyclooxygensae-2 (COX-2) mRNA in colorectal cancer and colorectal adenomas. Materials and Methods: The expression of feces COX-2 mRNA in colorectal cancer (n = 28), colorectal adenomas (n = 54), and normal control group (n = 11) were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The positive rate of fecal occult blood test (FOBT) were detected in colorectal cancer (n = 30), colorectal adenomas (n = 56), and normal control group (n = 11); the sensitivity of the two methods was also compared. Results: The positive rate of feces COX-2 mRNA in colorectal cancer was 82.1% (25/28), which was significantly higher than colorectal adenomas 59.3% (32/54), and normal tissues 18.2% (2/11), the difference being significant between the three groups (χ2= 13.842, P = 0.001). The positive rate of FOBT in colorectal cancer was 73.3% (10/30), which was significantly higher than colorectal adenomas 10.7% (6/56) and normal tissues 9.1% (1/11), the difference being significant between these three groups (χ2= 7.525, P = 0.023). There was no significant association between feces COX-2 expression and various clinical pathological features of colorectal cancer and colorectal adenomas (P > 0.05). The sensitivity of the RT-PCR method is higher than FOBT, however, the specificity of FOBT is slightly higher than RT-PCR. Conclusions: High expression of feces COX-2 mRNA in colorectal adenomas and colorectal cancer is a common event; it is an early event in the development of colorectal adenomas to colorectal cancer. Feces COX-2 mRNA has a high sensitivity for detect colorectal cancer; combination with FOBT will be the best alternative. Feces COX-2 can be potentially used in the early diagnosis and screening of colorectal cancer. PMID:28139497

Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells

PubMed Central

Fe Lanfranco, Maria; Loane, David J.; Mocchetti, Italo; Burns, Mark P.; Villapol, Sonia

2017-01-01

Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope® probes IL-1β, TNFα, TGFβ, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels. PMID:29238736

Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

PubMed

Tsuruta, Lilian Rumi; Lopes Dos Santos, Mariana; Yeda, Fernanda Perez; Okamoto, Oswaldo Keith; Moro, Ana Maria

2016-12-01

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

[Influence of fluorine on expression of androgen-binding protein and inhibin B mRNA in rat testis sertoli cells].

PubMed

Xu, Rui; Shang, Weichao; Liu, Jianmin; Duan, Liju; Ba, Yue; Zhang, Huizhen; Cheng, Xuemin; Cui, Liuxin

2010-09-01

To study the influence of fluorine on the transcription level of androgen binding protein (ABP) and inhibin B (INHB) mRNA in testis sertoli cells of Sprague Dawley rats. A method was set up the model to culture the Sertoli cells. Use a series of concentrations of NaF solutions of 2.5, 5.0, 10.0 and 20.0 mg/L to poison the cells and then, measure the relative expression amount of ABP and INHB mRNA by RT-PCR method. (1) Compare the relative expression amount of ABP mRNA of each group of different concentration with the control group. 2.5 mg/L group was higher than that in the control group, and the difference has the statistical significance (P < 0.05). The 5.0 mg/L group was also higher than that of the control group, and the difference has no statistical significance (P > 0.05). (2) Compare the relative expression amount of INH B mRNA of each group of different concentration with the control group. Both the 2.5 mg/L group and the 5.0 mg/L group were higher than that in the control group, and the difference has the statistical significance (P < 0.05). The rest 2 groups were lower than that in the control group and the difference has no statistical significance (P > 0.05). In the range of concentrations between 2.5 and 20.0 mg/L, no distinct influence of fluorine on the expression of androgen binding protein (ABP) and inhibin B (INHB) mRNA in testis sertoli cells of Sprague Dawley rats.

The association of the placental Hypoxia-inducible factor1-α polymorphisms and HIF1-α mRNA expression with preeclampsia.

PubMed

Harati-Sadegh, Mahdiyeh; Kohan, Leila; Teimoori, Batool; Mehrabani, Mehrnaz; Salimi, Saeedeh

2018-07-01

Evidence has confirmed that placental/fetal hypoxia plays a key role in both endothelial cell dysfunction and PE pathogenesis. The aim of the present study was to investigate whether maternal/placental hypoxia-inducible factor1-α (HIF1-α) C1772T (rs11549465) and/or G1790A (rs11549467) polymorphisms and HIF1-α mRNA expression are associated with PE development. The blood samples of 203 PE and 202 control women and the placenta of 86 PE and 84 control women were collected after delivery. The HIF1-α polymorphisms were genotyped using PCR- RFLP method. The mRNA expression levels were measured by Quantitative Real -Time PCR. The present study found no association between maternal HIF1-α rs11549465 and rs11549467 and placental rs11549467 polymorphisms and PE. However, the placental rs11549465 polymorphism was associated with PE in the dominant model. The CT/GG combined genotypes and TG haplotype of placental rs11549465 and rs11549467 polymorphisms were associated with higher risk of PE. The HIF1-α mRNA expression was 3-fold higher in the PE women. The rs11549465 TT genotype was associated with higher HIF1-α mRNA expression in PE women and in total population and rs11549467 GA genotype was associated with higher mRNA expression in total population. The relative mRNA expression of HIF1-α gene was higher in presence of CC/GA, TT/GG and TT/GA combined genotypes. This study found an association between placental but not maternal HIF1-α rs11549465 polymorphism and PE in the dominant model. The HIF1-α mRNA expression was higher in the placenta of PE women and was associated with rs11549465 and rs11549467 polymorphisms. Copyright © 2018. Published by Elsevier Ltd.

Inventory of high-abundance mRNAs in skeletal muscle of normal men.

PubMed

Welle, S; Bhatt, K; Thornton, C A

1999-05-01

G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

Evaluation of 5-fluorouracil metabolic enzymes as predictors of response to adjuvant chemotherapy outcomes in patients with stage II/III colorectal cancer: a decision-curve analysis.

PubMed

Shigeta, Kohei; Ishii, Yoshiyuki; Hasegawa, Hirotoshi; Okabayashi, Koji; Kitagawa, Yuko

2014-12-01

The effectiveness of 5-fluorouracil (5-FU)-based adjuvant chemotherapy is reported in patients with colorectal cancer (CRC), but the usefulness of 5-FU metabolic enzymes as predictive biomarkers of the efficacy of this chemotherapy remains unclear. This study aims to verify whether 5-FU metabolic enzymes are predictive biomarkers in the clinical setting of adjuvant chemotherapy for stage II/III CRC. In total, 179 patients with stage II/III CRC who were treated at our institute between 2000 and 2010 were enrolled. Messenger RNA (mRNA) expression of major 5-FU metabolic enzymes, namely thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase (TP), orotate phosphoribosyl transferase, and β-actin (control) was evaluated using the Danenberg Tumor Profile method. mRNA expression and other clinicopathological data were investigated with regard to CRC relapse. A total of 78 patients underwent surgery alone, while 101 underwent adjuvant chemotherapy (5-FU plus leucovorin [LV] or tegafur plus uracil /LV) following surgery. Relapse-free survival was longer and risk of recurrence was lower in association with high TP mRNA expression than in association with low TP mRNA expression in the adjuvant chemotherapy group (hazard ratio 0.66; 95 % confidence interval 0.47-0.92; p = 0.016), but not in the surgery alone group. mRNA expression of no other enzymes was associated with relapse in both groups. In decision-curve analyses, the predictive efficiency of TP mRNA expression plus clinicopathological factors was slightly better than that of clinicopathological factors only. TP mRNA expression in tumors predicted the effects of adjuvant chemotherapy for stage II/III CRC, although the beneficial effects were marginal.

[N(omega)-nitro-L-arginine methyl ester inhibits the up-regulated expression of neuronal nitric oxide synthase/NMDA receptor in the morphine analgesia tolerance rats].

PubMed

Yu, Ling; Xue, Fu-Shan; Li, Cheng-Wen; Xu, Ya-Chao; Zhang, Guo-Hua; Liu, Kun-Peng; Liu, Yi; Sun, Hai-Tao

2006-12-25

The effect of systemic administration of nonspecific nitric oxide synthase inhibitor (N(omega)-nitro-L-arginine methyl ester, L-NAME) on morphine analgesia tolerance was observed by using the thermal tail-flick method, and the roles of NO and NMDA receptors in morphine analgesia tolerance were evaluated on the basis of the expressions of nNOS mRNA, NR1A mRNA and NR2A mRNA in spinal cord and midbrain. Thirty-six healthy adult Sprague-Dawley rats were randomly divided into six groups (6 rats per group). Group 1, control group, received a subcutaneous (s.c.) injection of normal saline (1 ml); Groups 2, 3, 4, 5 and 6, the treatment groups received s.c. injection of L-NAME 10 mg/kg, L-NAME 20 mg/kg, morphine 10 mg/kg, L-NAME 10 mg/kg + morphine 10 mg/kg, and L-NAME 20 mg/kg + morphine 10 mg/kg, respectively. All rats received s.c. injections twice per day (8:00 and 17:00). The tail-flick latency (TFL) was measured in each rat before the injection as a baseline value, and then TFL at 50 min after the 1st injection every day as the measuring values. The animals (except for groups 2 and 5) were decapitated at 80 min after the last injection on the 8th day. The spinal segments and midbrain were removed for analysis of nNOS mRNA, NR1A mRNA and NR2A mRNA expressions by the RT-PCR method. The results showed that TFL remained unchangeable in group 2 compared with baseline value during the 7-day observation, while increased significantly on the 7th day in group 3. In group 4, TFL was longest on the 1st day, then decreased gradually from the 2nd day to the 6th day, and restored to the baseline value on the 6th day. In group 5, TFL showed a decreasing tendency during the 7-day observation, but was still significantly longer than the baseline value on the 7th day. The changes of TFL obtained in group 6 were similar to those in group 5. The results of RT-PCR showed that as compared with group 1, nNOS mRNA expressions in spinal cord and midbrain were significantly down-regulated in group 3, but the expressions of the NR1A mRNA and NR2A mRNA in both groups were similar, while the nNOS mRNA, NR(1A) mRNA and NR(2A) mRNA expressions were all significantly up-regulated in group 4. As compared with group 4, the expressions of nNOS mRNA, NR(1A) mRNA and NR(2A) mRNA were significantly inhibited in group 6. These results suggest that the expressions of nNOS and NMDA receptors in spinal cord and midbrain were significantly up-regulated in the rats with morphine analgesia tolerance. Chronic co-administration of L-NAME could effectively inhibit the morphine-induced overexpressions of nNOS and NMDA receptors, and postpone the development of morphine analgesia tolerance. Based on the results of this study, it is concluded that NO/NMDA receptor in spinal cord and midbrain is closely related to the development of morphine analgesia tolerance.

Arc/Arg3.1 mRNA expression reveals a subcellular trace of prior sound exposure in adult primary auditory cortex.

PubMed

Ivanova, T N; Matthews, A; Gross, C; Mappus, R C; Gollnick, C; Swanson, A; Bassell, G J; Liu, R C

2011-05-05

Acquiring the behavioral significance of sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 min (24 h separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in situ hybridization (FISH) to assess the layer-specific subcellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3-6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1-2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical subcellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Arc/Arg3.1 mRNA expression reveals a sub-cellular trace of prior sound exposure in adult primary auditory cortex

PubMed Central

Ivanova, Tamara; Matthews, Andrew; Gross, Christina; Mappus, Rudolph C.; Gollnick, Clare; Swanson, Andrew; Bassell, Gary J.; Liu, Robert C.

2011-01-01

Acquiring the behavioral significance of a sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 minute (24 hour separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in-situ hybridization (FISH) to assess the layer-specific sub-cellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3–6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1–2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical sub-cellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. PMID:21334422

Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy

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

Li Jinmei; Wang Xuefeng; Xi Zhiqin

2006-10-06

Purpose: TRAP220 (thyroid hormone receptor-associated protein) functions as a coactivator for nuclear receptors and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. Thus, TRAP220 enhances the function of thyroid/steroid hormone receptors such as thyroid hormone and oestrogen receptors. This study investigated the expression of TRAP220 mRNA and protein level in epileptic brains comparing with human control. Methods: We examined the expression of TRAP220 mRNA and protein levels in temporal lobes from patients with chronic pharmacoresistant epilepsy who have undergone surgery. Results: Expression of TRAP220 mRNA and protein was shown to be decreased significantly in themore » temporal cortex of the patients with epilepsy. Conclusions: Our work showed that a decrease in TRAP220 mRNA and protein levels may be involved in the pathophysiology of epilepsy and may be associated with impairment of the brain caused by frequent seizures.« less

Real-time quantitative reverse transcription-PCR assay for renal cell carcinoma-associated antigen G250.

PubMed

Chuanzhong, Ye; Ming, Guan; Fanglin, Zhang; Haijiao, Chen; Zhen, Lin; Shiping, Chen; YongKang, Zhang

2002-04-01

Gene amplification/expression of G250 is a major event in human renal tumorigenesis. G250-based therapeutic agents and G250-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen G250 alterations in renal cell cancer (RCC) patients and investigate the relationship between G250 mRNA expression and RCC. We developed a quantitative RT-PCR assay for the measurement of G250 mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The method has been applied to the measurement of quantitative mRNA level of G250 in 31 cases RCC and 6 normal renal tissues. The dynamic range was 10(3)-10(8). The relationship between Ct and log starting concentration was linear (r=0.99). G250 expression was present in all RCCs with G250 amplification but was absent in normal ones. G250 mRNA expression ranged from 2.9 x 10(3) to 6.5 x 10(7) copy/microg RNA, with a mean value of 3.5 x 10(6) copy/microg RNA. The expression of G250 revealed an inverse correlation to tumor grade. G250 mRNA level did not correlate with the cell types and clinical stages (P>0.05). G250 has the potential to be used as a marker of diagnosis and increasing proliferation in RCC. This new simple, rapid, semi-automated assay was a major alternative to competitive PCR and Northern blot analysis for gene alteration analysis in human tumors and might be a powerful tool for large randomized, prospective cooperative group trials and supporting future G250-based biological and gene therapy approaches.

Effect of Caloric Restriction and AMPK Activation on Hepatic Nuclear Receptor, Biotransformation Enzyme, and Transporter Expression in Lean and Obese Mice

PubMed Central

Kulkarni, Supriya R.; Xu, Jialin; Donepudi, Ajay C.; Wei, Wei

2014-01-01

Purpose Fatty liver alters liver transporter expression. Caloric restriction (CR), the recommended therapy to reverse fatty liver, increases Sirtuin1 deacetylase activity in liver. This study evaluated whether CR and CR mimetics reversed obesity-induced transporter expression in liver and hepatocytes. Methods mRNA and protein expression was determined in adult lean (lean) and leptin-deficient obese (OB) mice fed ad libitum or placed on 40% (kCal) reduced diet. Hepatocytes were isolated from lean and OB mice, treated with AMP Kinase activators, and gene expression was determined. Results CR decreased Oatp1a1, Oatp1b2, and Abcb11 mRNA expression in lean, but not OB mice. CR increased Abcc2 mRNA OB livers, whereas protein expression increased in both genotypes. CR increased Abcc3 protein expression increased in OB livers. CR did not alter Abcc1, 4 and 5 mRNA expression in lean mice but decreased expression in livers of OB mice. CR increased Abcc4 protein in lean, but not OB mice. Conclusions CR restriction reversed the expression of some, but not all transporters in livers of OB mice. Overall, these data indicate a potential for CR to restore some hepatic transporter changes in OB mice, but suggest a functional leptin axis is needed for reversal of expression for some transporters. PMID:23949303

Topographical cone photopigment gene expression in deutan-type red-green color vision defects.

PubMed

Bollinger, Kathryn; Sjoberg, Stacy A; Neitz, Maureen; Neitz, Jay

2004-01-01

Eye donors were identified who had X-chromosome photopigment gene arrays like those of living deuteranomalous men; the arrays contained two genes encoding long-wavelength sensitive (L) pigments as well as genes to encode middle-wavelength sensitive (M) photopigment. Ultrasensitive methods failed to detect the presence of M photopigment mRNA in the retinas of these deutan donors. This provides direct evidence that deuteranomaly is caused by the complete absence of M pigment mRNA. Additionally, for those eyes with mRNA corresponding to two different L-type photopigments, the ratio of mRNA from the first vs. downstream L genes was analyzed across the retinal topography. Results show that the pattern of first relative to downstream L gene expression in the deuteranomalous retina is similar to the pattern of L vs. M gene expression found in normal retinas.

Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

PubMed

Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

2016-06-01

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.

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.

Heparanase mRNA expression and point mutation in hepatocellular carcinoma

PubMed Central

Chen, Xiao-Peng; Liu, Yin-Bib; Rui, Jing; Peng, Shu-You; Peng, Cheng-Hong; Zhou, Zi-Yan; Shi, Liang-Hui; Shen, Hong-Wei; Xu, Bin

2004-01-01

AIM: To explore the expression of heparanase mRNA and point mutation in hepatocellular carcinoma (HCC). METHODS: Reverse transcription polymerase chain reaction was used to measure the expression of heparanase mRNA in the primary tumor tissues and surrounding liver tissues of 33 HCC patients. T-A cloning and sequencing were used to detect whether there was any mutation in the amplified PCR products. RESULTS: The expression of heparanase mRNA was positive in 16 primary tumor tissues of HCC, and the positive rate was 48.5%, which was significantly higher than that in the surrounding liver parenchyma (P < 0.01). The positive rate for heparanase gene in high-tendency to metastatic recurrence group (71.4%, 10/14) was obviously higher than that in low-tendency to metastatic recurrence group (31.6%, 6/19) (P = 0.023). The positive rate for heparanase gene in patients with metastatic recurrence during postoperative follow-up (78.6%, 11/14) was also significantly higher than that in those without metastatic recurrence (21.4%, 3/14) (P = 0.003). Sequence analysis of the HPA PCR products was made in 7 patients, and 2-point mutations were found in 4 patients, one of which was sense mutation, neither base insertion nor deletion was detected. The mutation rate was 57.1% (4/7). CONCLUSION: The expression rate of heparanase mRNA increases in HCC, and HPA mRNA may be one of the reliable markers for the metastatic activity gained by the liver tumor cells and could be used clinically in predicting metastatic recurrence of HCC. Point mutation may be one of the causes for enhanced heparanase mRNA expression. PMID:15334672

The influence of methylated septin 9 gene on RNA and protein level in colorectal cancer.

PubMed

Tóth, Kinga; Galamb, Orsolya; Spisák, Sándor; Wichmann, Barnabás; Sipos, Ferenc; Valcz, Gábor; Leiszter, Katalin; Molnár, Béla; Tulassay, Zsolt

2011-09-01

Colorectal cancer is one of the leading death causes in the world. Specificity and sensitivity of the present screening methods are unsuitable and their compliance is too low. Nowadays the most effective method is the colonoscopy, because it gives not only macroscopic diagnosis but therapeutic possibility as well, however the compliance of the patients is very low. Hence development of new diagnostic methods is needed. Altered expression of septin 9 was found in several tumor types including colorectal cancer. The aim of this study was to detect the methylation related mRNA and protein expression changes of septin 9 in colorectal adenoma-dysplasia-carcinoma sequence and to analyze its reversibility by demethylation treatment. Septin 9 protein expression showed significant difference between normal and colorectal cancer (CRC) samples (p < 0,001). According to biopsy microarray results, septin 9 mRNA expression decreased in the progression of colon neoplastic disease (p < 0,001). In laser microdissected epithelial cells, septin 9 significantly underexpressed in CRC compared to healthy controls (p < 0,001). The expression of septin9_v1 region was higher in the healthy samples, while septin9_v2, v4, v4*, v5 overexpression were detected in cancer epithelial cells compared to normal. The septin 9 mRNA and protein levels of HT29 cells increased after demethylation treatment. The increasing methylation of septin 9 gene during colorectal adenoma-dysplasia-carcinoma sequence progression is reflected in the decreasing mRNA and protein expression, especially in the epithelium. These changes can be reversed by demethylation agents converting this screening marker gene into therapeutic target.

The histological characteristics, age-related thickness change of skin, and expression of the HSPs in the skin during hair cycle in yak (Bos grunniens).

PubMed

Yang, Xue; Cui, Yan; Yue, Jing; He, Honghong; Yu, Chuan; Liu, Penggang; Liu, Jun; Ren, Xiandong; Meng, Yun

2017-01-01

This experiment was conducted to study the histological characteristics, age-related thickness changes, and expression of HSPs in the skin of yak. A total of 20 yaks (10 males and 10 females) were used. Different regions of the normal skin of three different ages (newborn, half-year-old and adult) of yaks were harvested for histological study and thickness measurement. Biopsy samples were taken from the scapula regions of the skin from the same five approximately 1-year-old yaks during the hair cycle (telogen, anagen and catagen). RT-PCR, western blot and immunohistochemistry methods using the mRNA and protein levels were used to detect the expression of HSP27, HSP70 and HSP90. RT-PCR method was used to detect the mRNA expression of CGI-58 and KDF1. The IPP6.0 software was used to analyze the immunohistochemistry and measure the thickness of the skin. The general histological structure of hairy yak skin was similar to other domestic mammals. The unique features included prominent cutaneous vascular plexuses, underdeveloped sweat glands, a large number of nasolabial glands in the nasolabial plate, and hair follicle groups composed of one primary follicle and several secondary follicles. The skin, epidermis and dermis thickness did vary significantly between different body regions and different ages. The thickness of the skin, epidermis and dermis increased from newborn to adult in yaks. Yak skin thickness decreased from dorsally to ventrally on the trunk. The skin on the lateral surface was thicker than the skin on the medial surface on the limbs. HSP27, HSP70 and HSP90 showed different expression patterns during the hair cycle using RT-PCR, western blot and immunohistochemistry methods. The expression of HSP27 mRNA and protein in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of HSP70 mRNA and protein in the telogen stage was the highest, followed by the anagen stage, and the expression in the catagen stage was the lowest. The expression of HSP90 mRNA and protein in the anagen stage was the highest, followed by the telogen stage, and the expression in the catagen stage was the lowest. HSPs were mainly expressed in the outer root sheath of hair follicle during the hair cycle, also expressed in epidermis, sebaceous gland and sweat gland in the skin of Yak. The expression of CGI-58 mRNA in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of KDF1 mRNA in the telogen stage was the highest, followed by the catagen stage, and the expression in the anagen stage was the lowest. In this study, we examined and fully described the histology of normal skin in Yak and measured the skin thickness of different ages and different regions in Yak. These data may be useful to better understand and appreciate the adaptability features of yak skin. Our investigation reports the expression patterns of HSPs in yak skin for the first time. The different expression pattern of HSPs during the hair cycle suggests they may play different roles in yak hair follicle biology.

Gene Expression, DNA Methylation and Prognostic Significance of DNA Repair Genes in Human Bladder Cancer.

PubMed

Wojtczyk-Miaskowska, Anita; Presler, Malgorzata; Michajlowski, Jerzy; Matuszewski, Marcin; Schlichtholz, Beata

2017-01-01

This study investigated the gene expression and DNA methylation of selected DNA repair genes (MBD4, TDG, MLH1, MLH3) and DNMT1 in human bladder cancer in the context of pathophysiological and prognostic significance. To determine the relationship between the gene expression pattern, global methylation and promoter methylation status, we performed real-time PCR to quantify the mRNA of selected genes in 50 samples of bladder cancer and adjacent non-cancerous tissue. The methylation status was analyzed by methylation-specific polymerase chain reaction (MSP) or digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR with gene-specific primers (MSRE-PCR). The global DNA methylation level was measured using the antibody-based 5-mC detection method. The relative levels of mRNA for MBD4, MLH3, and MLH1 were decreased in 28% (14/50), 34% (17/50) and 36% (18/50) of tumor samples, respectively. The MBD4 mRNA expression was decreased in 46% of non-muscle invasive tumors (Ta/T1) compared with 11% found in muscle invasive tumors (T2-T4) (P<0.003). Analysis of mRNA expression for TDG did not show any significant differences between Ta/T1 and T2-T4 tumors. The frequency of increased DNMT1 mRNA expression was higher in T2-T4 (52%) comparing to Ta/T1 (16%). The overall methylation rates in tumor tissue were 18% for MBD4, 25% for MLH1 and there was no evidence of MLH3 promoter methylation. High grade tumors had significantly lower levels of global DNA methylation (P=0.04). There was a significant association between shorter survival and increased expression of DNMT1 mRNA (P=0.002), decreased expression of MLH1 mRNA (P=0.032) and the presence of MLH1 promoter methylation (P=0.006). This study highlights the importance of DNA repair pathways and provides the first evidence of the role of MBD4 and MLH3 in bladder cancer. In addition, our findings suggest that DNMT1 mRNA and MLH1 mRNA expression, as well as the status of MLH1 promoter methylation, are attractive prognostic markers in this pathology. © 2017 The Author(s). Published by S. Karger AG, Basel.

The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

PubMed Central

Böttner, Martina; Barrenschee, Martina; Hellwig, Ines; Harde, Jonas; Egberts, Jan-Hendrik; Becker, Thomas; Zorenkov, Dimitri; Schäfer, Karl-Herbert; Wedel, Thilo

2013-01-01

Background & Aims Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons. Methods Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored. Results mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression. Conclusions Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system. PMID:23805210

Compositions and methods for making selenocysteine containing polypeptides

DOEpatents

Soll, Dieter; Aldag, Caroline; Hohn, Michael

2016-10-11

Non-naturally occurring tRNA.sup.Sec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNA.sup.Sec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNA.sup.Sec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNA.sup.Sec.

Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum

PubMed Central

Painter, Heather J.; Carrasquilla, Manuela; Llinás, Manuel

2017-01-01

To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite. PMID:28416533

Selection of the In Vitro Culture Media Influences mRNA Expression of Hedgehog Genes, Il-6, and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

PubMed Central

Pfeifer, N.; Baston-Büst, D. M.; Hirchenhain, J.; Friebe-Hoffmann, U.; Rein, D. T.; Krüssel, J. S.; Hess, A. P.

2012-01-01

Background. The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Methods. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. Results. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. Conclusions. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies. PMID:22919324

Glutathione S-transferase PI (GST-PI) mRNA expression and DNA methylation is involved in the pathogenesis and prognosis of NSCLC.

PubMed

Grimminger, Peter P; Maus, Martin K H; Schneider, Paul M; Metzger, Ralf; Hölscher, Arnulf H; Sugita, Hirofumi; Danenberg, Peter V; Alakus, Hakan; Brabender, Jan

2012-10-01

The aim of this study was to investigate the relevance of mRNA expression and DNA methylation of GST-PI in tumor and non-tumor lung tissue from NSCLC patients in terms of prognostic and pathogenetic value of this biomarker. Quantitative real-time PCR was used to measure mRNA expression and DNA methylation of GST-PI in paired tumor (T) and non-tumor (N) lung tissue of 91 NSCLC patients. Of all 91 patients 49% were stage I, 21% stage II and 30% stage IIIA. Forty-seven percent of the patients had squamous cell carcinoma, 36% adenocarcinoma and 17% large cell carcinoma. All patients were R0 resected. GST-PI mRNA expression could be measured in 100% in both (T and N) tissues; GST-PI DNA methylation was detected in 14% (N) and 14% (T). The median GST-PI mRNA expression in N was 7.83 (range: 0.01-19.43) and in T 13.15 (range: 0.01-116.8; p≤0.001). The median GST-PI methylation was not significantly different between T and N. No associations were seen between the mRNA expression or DNA methylation levels and clinical or histopathologic parameters such as gender, age, TNM stage, tumor histology and grading. The median survival of the investigated patients was 59.7 years (the median follow-up was 85.9 months). High GST-PI DNA methylation was significantly associated with a worse prognosis (p=0.041, log rank test). No correlation was found between the GST-PI DNA methylation levels and the correlating mRNA expression levels. GST-PI mRNA expression seems to be involved in the pathogenesis of NSCLC. High levels of GST-PI DNA methylation in tumor tissue of NSCLC patients have a potential as a biomarker identifying subpopulations with a more aggressive tumor biology. Quantitation of GST-PI DNA methylation may be a useful method to identify patients with a poor prognosis after curative resection and who will benefit from intensive adjuvant therapy. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Nitric oxide synthase expression in foetal placentas of cows with retained fetal membranes.

PubMed

Shixin, Fu; Li, Zhang; Chunhai, Luo; Chuang, Xu; Cheng, Xia; Zhe, Wang; Xiaobing, Li

2011-10-01

The objectives of this study were to investigate relationship of retained fetal membranes (RFM) to expression of NOS and NOS mRNA and to analyze pathohistological changes and the distribution of nitric oxide synthase (NOS) in foetal placentas of cows with RFM. Twenty cows were assigned to two groups, a control group (no retained fetal membranes, NRFM, n = 10) and a diseased group (RFM, n = 10). The endpoint method was used to detect the nitric oxide (NO) content and nitric oxide synthase (NOS) activity in foetal placental tissue fluid and the fluorescent quantitation PCR was used to measure the expression of NOS mRNA. Immunohistochemistry and hematoxylin-eosin staining were used to observe pathohistological changes. Tissue from RFM cows showed fibronecrosis of the chorionic villi, and a decreased number of trophoblastic cells. The majority of trophoblastic cells displayed vacuolar degeneration. Interstitium vessels were distended and congested. Expression of induced nitric oxide synthase (iNOS) protein and iNOS mRNA was significantly higher (P < 0.05) in the cytoplasm of placental villus trophoblastic cells in the RFM group. But expression of endothelial nitric oxide synthase (eNOS) protein and eNOS mRNA was significantly lower (P<0.05) in the RFM group. The NO content and NOS activity of cows with RFM were significantly higher (P < 0.05). A high expression of iNOS protein and iNOS mRNA in the cow foetal placenta could produce high content of NO, which might inhibit uterine contraction. So over expression of iNOS protein and iNOS mRNA might be an important agent of retained fetal membranes in cows, and it may be a potential diagnosis biomarker. Copyright © 2010 Elsevier Ltd. All rights reserved.

Night-time restricted feeding normalises clock genes and Pai-1 gene expression in the db/db mouse liver.

PubMed

Kudo, T; Akiyama, M; Kuriyama, K; Sudo, M; Moriya, T; Shibata, S

2004-08-01

An increase in PAI-1 activity is thought to be a key factor underlying myocardial infarction. Mouse Pai-1 (mPai-1) activity shows a daily rhythm in vivo, and its transcription seems to be controlled not only by clock genes but also by humoral factors such as insulin and triglycerides. Thus, we investigated daily clock genes and mPai-1 mRNA expression in the liver of db/db mice exhibiting high levels of glucose, insulin and triglycerides. Locomotor activity was measured using an infrared detection system. RT-PCR or in situ hybridisation methods were applied to measure gene expression. Humoral factors were measured using measurement kits. The db/ db mice showed attenuated locomotor activity rhythms. The rhythmic expression of mPer2 mRNA was severely diminished and the phase of mBmal1 oscillation was advanced in the db/db mouse liver, whereas mPai-1 mRNA was highly and constitutively expressed. Night-time restricted feeding led to a recovery not only from the diminished locomotor activity, but also from the diminished Per2 and advanced mBmal1 mRNA rhythms. Expression of mPai-1 mRNA in db/db mice was reduced to levels far below normal. Pioglitazone treatment slightly normalised glucose and insulin levels, with a slight reduction in mPai-1 gene expression. We demonstrated that Type 2 diabetes impairs the oscillation of the peripheral oscillator. Night-time restricted feeding rather than pioglitazone injection led to a recovery from the diminished locomotor activity, and altered oscillation of the peripheral clock and mPai-1 mRNA rhythm. Thus, we conclude that scheduled restricted food intake may be a useful form of treatment for diabetes.

Relationship Between the DPD and TS mRNA Expression and the Response to S-1-Based Chemotherapy and Prognosis in Patients with Advanced Gastric Cancer.

PubMed

Shen, Xiao-Ming; Zhou, Chong; Lian, Lian; Li, Li-Qun; Li, Wei; Tao, Min

2015-04-01

The aim was to determine changes in dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) mRNAs in the blood of advanced gastric cancer (AGC) patients to see whether these enzymes affected the patients' response to S-1-based chemotherapy and prognosis. For this purpose, pretreatment DPD/TS mRNA expressions were determined in 40 AGC patients using RT-PCR. The patients were then administered with S-1-based regimen (S-1 + cisplatin) and toxicities were recorded. The relationship between the DPD/TS mRNA expressions and the chemotherapy response, drug resistance, and prognosis was analyzed. The data show that DPD mRNA expression correlated significantly with Lauren type while TS mRNA expression correlated with distant metastasis. Patients with higher DPD and/or TS mRNA expression(s) showed poor response, while those with low DPD mRNA expression showed better response to the chemotherapy. Pooled analysis showed that the patients with low DPD/TS mRNA expressions had better therapeutic response. The incidence of bone marrow suppression, diarrhea, and oral mucositis was high in patients with low DPD mRNA expression. Median overall survival (OS) in 40 patients was 13.5 months. It was 17 months for low and 10 months for high DPD (P = 0.044) and TS mRNA expression (P = 0.047). Pooled analysis showed that the patients with both low DPD/TS mRNA expressions had longer OS (P = 0.001). In conclusion, the detection of DPD and/or TS mRNA expression can be used to predict the response to S-1-based chemotherapy, drug resistance, and prognosis in AGC patients as well as to help guide the individualized treatment of gastric cancer.

Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

NASA Astrophysics Data System (ADS)

Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

2018-05-01

Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

Feeder-free reprogramming of human fibroblasts with messenger RNA.

PubMed

Warren, Luigi; Wang, Jiwu

2013-11-13

This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Copyright © 2013 John Wiley & Sons, Inc.

Expression and significance of cyclooxygenase-2 mRNA in benign and malignant ascites

PubMed Central

Lu, Jing; Li, Xiao-Feng; Kong, Li-Xia; Ma, Lin; Liao, Su-Huan; Jiang, Chang-You

2013-01-01

AIM: To investigate the mRNA expression of cyclooxygensae-2 (COX-2) in benign and malignant ascites, and to explore the difference in COX-2 mRNA expression among different diseases. METHODS: A total of 36 samples were collected from the Fifth Affiliated Hospital of Sun Yat-Sen University and divided into two experimental groups: benign ascites (n = 21) and malignant ascites (n = 15). Benign ascites included cirrhotic ascites (n = 10) and tuberculous ascites (n = 5). Malignant ascites included oophoroma (n = 7), cancer of colon (n = 5), cancer of the liver (n = 6), gastric cancer (n = 2), and bladder carcinoma (n = 1). The mRNA expression of COX-2 in ascites was examined with reverse transcriptase polymerase chain reaction (RT-PCR) technology, and the positive rate of COX-2 mRNA was compared between different diseases. RESULTS: The positive rate of COX-2 mRNA in malignant ascites was 42.9% (9/21), which was significantly higher than in benign ascites, 6.7% (1/15), difference being significant between these two groups (χ2 = 4.051, P = 0.044). The proportion of the positive rate in the malignant ascites was as follows: ovarian cancers 57.1% (4/7), colon cancer 40.0% (2/5), liver cancer 33.3% (2/6), gastric cancer 50.0% (1/2), and bladder cancer 0.00% (0/1). However, there was no significant difference in COX-2 mRNA expression among various tumors with malignant ascites (χ2 = 1.614, P = 0.806). Among the benign ascites, COX-2 mRNA levels were different between the tuberculous ascites (0/5) and cirrhotic ascites (1/10), but there was no significant difference (P = 1.000). CONCLUSION: COX-2 mRNA, detected by RT-PCR, is useful in the differential diagnosis of benign and malignant ascites, which also has potential value in the clinical diagnosis of tumors. PMID:24187465

The G protein-coupled estrogen receptor 1 (GPER/GPR30) does not predict survival in patients with ovarian cancer

PubMed Central

2012-01-01

Background Even though ovarian tumors are not generally considered estrogen-sensitive, estrogens may still have an impact on ovarian tumor progression. The recently identified trans-membrane estrogen receptor GPER is involved in rapid estrogen signaling. Furthermore, it binds selective estrogen receptor modulators with agonistic effect, which could explain tamoxifen controversies. Methods GPER mRNA was assayed with quantitative real-time PCR (qPCR) in 42 primary ovarian tumors and 7 ovarian cancer cell lines. ERα and ERβ mRNA were analyzed for comparison. GPER protein was semi-quantified with densitometric scanning of Western blots and its tissue distribution analyzed with immunohistochemistry (IHC) in 40 ovarian tumors. In addition, IHC was evaluated in a tissue microarray (TMA) of 150 primary malignant ovarian tumors. Results All tumor samples contained GPER mRNA. The content of mRNA was not different between benign and malignant tumors, but one third of malignant samples over-expressed GPER mRNA. The content of ERα mRNA was higher in malignant than in benign tumors, whereas ERβ mRNA was higher in benign than in malignant tumors. GPER mRNA was detected in all seven ovarian cancer cell lines with highest levels in TOV21G and TOV112D cells. Similar expression pattern was seen for ERβ mRNA. Western blot demonstrated GPER protein in all tumor samples. Semi-quantification showed no difference between benign and malignant tumors, but about one third of malignant samples over-expressed GPER protein. GPER staining was localized mainly in epithelial cells. In the TMA study we found no correlation between GPER staining and clinical stage, histological grade or patient survival. Conclusions GPER mRNA as well as GPER protein is present in both benign and malignant ovarian tumor tissue. About one third of malignant tumors over-expressed both GPER mRNA and protein. This, however, correlated neither with histological or clinical parameters nor with patient survival. PMID:22424333

The expression and significance of feces cyclooxygensae-2 mRNA in colorectal cancer and colorectal adenomas.

PubMed

Li, Xiaofeng; Kong, Lixia; Liao, Suhuan; Lu, Jing; Ma, Lin; Long, Xiaohua

2017-01-01

This study aims to explore the expression and significance of feces cyclooxygensae-2 (COX-2) mRNA in colorectal cancer and colorectal adenomas. The expression of feces COX-2 mRNA in colorectal cancer (n = 28), colorectal adenomas (n = 54), and normal control group (n = 11) were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The positive rate of fecal occult blood test (FOBT) were detected in colorectal cancer (n = 30), colorectal adenomas (n = 56), and normal control group (n = 11); the sensitivity of the two methods was also compared. The positive rate of feces COX-2 mRNA in colorectal cancer was 82.1% (25/28), which was significantly higher than colorectal adenomas 59.3% (32/54), and normal tissues 18.2% (2/11), the difference being significant between the three groups (χ2= 13.842,P= 0.001). The positive rate of FOBT in colorectal cancer was 73.3% (10/30), which was significantly higher than colorectal adenomas 10.7% (6/56) and normal tissues 9.1% (1/11), the difference being significant between these three groups (χ2= 7.525,P= 0.023). There was no significant association between feces COX-2 expression and various clinical pathological features of colorectal cancer and colorectal adenomas (P > 0.05). The sensitivity of the RT-PCR method is higher than FOBT, however, the specificity of FOBT is slightly higher than RT-PCR. High expression of feces COX-2 mRNA in colorectal adenomas and colorectal cancer is a common event; it is an early event in the development of colorectal adenomas to colorectal cancer. Feces COX-2 mRNA has a high sensitivity for detect colorectal cancer; combination with FOBT will be the best alternative. Feces COX-2 can be potentially used in the early diagnosis and screening of colorectal cancer.

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

PubMed Central

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

2010-01-01

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

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

PubMed Central

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

2006-01-01

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

[Mechanism of TNF-α in bone defect of chronic apical periodontitis].

PubMed

Yu, Ya-Qiong; Qu, Liu; Qiu, Li-Hong; Guo, Jia-Jie; Ma, Nan; Zhu, Li

2016-08-01

To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of tumor necrosis factor-α(TNF-α) mRNA in MC3T3-E1 cells and the role of NF-κB signaling on the expression of macrophage colony stimulating factor (M-CSF) induced by TNF-α in MC3T3-El cells． METHODS: MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 10 mg/L P.e-LPS for different time (0-24 h). The expression of TNF-α mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR). MC3T3-E1 cells were treated with different concentrations of TNF-α(0-10 ng/L) for 6 h. The expression of M-CSF mRNA and protein was detected by RT-PCR and enzyme-linked immunoadsordent assay(ELISA).The expression of M-CSF protein was also detected in 10 ng/L TNF-α treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor . Statistical analysis was performed using Multi-way ANOVA and Dunnett t test with SPSS 13.0 software package. The level of TNF-α mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L)，which indicated that P.e-LPS induced osteoblasts to express TNF-α mRNA in dose dependent manners. Maximal induction of TNF-α mRNA expression was seen in the MC3T3-E1 cells treated with 10 mg/L P.e-LPS for 6 h. After 6 h, the expression of TNF-α mRNA decreased gradually .The expression of M-CSF mRNA and protein was increased in a does- dependent manner by different concentrations of TNF-α treatment（0-10 ng/L）. The expression of M-CSF protein increased from (37±2) ng/L(control group) to (301±8) ng/L(10 ng/L group).The protein of M-CSF decreased significantly after pretreatment with 10 μmol/L BAY 11-7082 for 1 h, and the expression of M-CSF proteins was reduced from (253±14) ng/L to (154±2) ng/L .BAY group had no significant difference from the control group. The expression of TNF-α mRNA was increased by P. endodontalis LPS treatment in osteoblast. TNF-α may induce the expression of M-CSF in MC3T3-E1 cells through the signaling of NF-κB. It suggests that TNF-α affect osteoblasts through autocrine way for bone destruction in chronic apical periodontitis induced by P.e-LPS.

Gene expression analysis in lymphoblasts derived from patients with autism spectrum disorder

PubMed Central

2011-01-01

Background The autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that result in severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare mutations of some genes, such as neuroligin (NLGN) 3/4, neurexin (NRXN) 1, SHANK3, MeCP2 and NHE9, have been reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD. Methods We measured mRNA expression levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction assays. Results The mRNA expression levels of NLGN3 and SHANK3 normalized by β-actin or TBP were significantly decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not quantitatively measurable in lymphoblastoid cells. Conclusions Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the possibility that decreased mRNA expression levels of these genes might be involved in the pathophysiology of ASD in a substantial population of ASD patients. PMID:21615902

Cancer survival classification using integrated data sets and intermediate information.

PubMed

Kim, Shinuk; Park, Taesung; Kon, Mark

2014-09-01

Although numerous studies related to cancer survival have been published, increasing the prediction accuracy of survival classes still remains a challenge. Integration of different data sets, such as microRNA (miRNA) and mRNA, might increase the accuracy of survival class prediction. Therefore, we suggested a machine learning (ML) approach to integrate different data sets, and developed a novel method based on feature selection with Cox proportional hazard regression model (FSCOX) to improve the prediction of cancer survival time. FSCOX provides us with intermediate survival information, which is usually discarded when separating survival into 2 groups (short- and long-term), and allows us to perform survival analysis. We used an ML-based protocol for feature selection, integrating information from miRNA and mRNA expression profiles at the feature level. To predict survival phenotypes, we used the following classifiers, first, existing ML methods, support vector machine (SVM) and random forest (RF), second, a new median-based classifier using FSCOX (FSCOX_median), and third, an SVM classifier using FSCOX (FSCOX_SVM). We compared these methods using 3 types of cancer tissue data sets: (i) miRNA expression, (ii) mRNA expression, and (iii) combined miRNA and mRNA expression. The latter data set included features selected either from the combined miRNA/mRNA profile or independently from miRNAs and mRNAs profiles (IFS). In the ovarian data set, the accuracy of survival classification using the combined miRNA/mRNA profiles with IFS was 75% using RF, 86.36% using SVM, 84.09% using FSCOX_median, and 88.64% using FSCOX_SVM with a balanced 22 short-term and 22 long-term survivor data set. These accuracies are higher than those using miRNA alone (70.45%, RF; 75%, SVM; 75%, FSCOX_median; and 75%, FSCOX_SVM) or mRNA alone (65.91%, RF; 63.64%, SVM; 72.73%, FSCOX_median; and 70.45%, FSCOX_SVM). Similarly in the glioblastoma multiforme data, the accuracy of miRNA/mRNA using IFS was 75.51% (RF), 87.76% (SVM) 85.71% (FSCOX_median), 85.71% (FSCOX_SVM). These results are higher than the results of using miRNA expression and mRNA expression alone. In addition we predict 16 hsa-miR-23b and hsa-miR-27b target genes in ovarian cancer data sets, obtained by SVM-based feature selection through integration of sequence information and gene expression profiles. Among the approaches used, the integrated miRNA and mRNA data set yielded better results than the individual data sets. The best performance was achieved using the FSCOX_SVM method with independent feature selection, which uses intermediate survival information between short-term and long-term survival time and the combination of the 2 different data sets. The results obtained using the combined data set suggest that there are some strong interactions between miRNA and mRNA features that are not detectable in the individual analyses. Copyright © 2014 Elsevier B.V. All rights reserved.

Regulation of neuropeptide Y gene expression in rat brain.

PubMed

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

1990-01-01

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

Effect of Acupotomy on FAK-PI3K Signaling Pathways in KOA Rabbit Articular Cartilages

PubMed Central

Xie, Zhan-guo; Guo, Yan; Yu, Jia-Ni; Lu, Juan; Zhang, Wei; Wang, Li-Juan; Xu, Jing; Zhao, Rui-Li; Zhou, Shuai

2017-01-01

Objective By observing the needle-knife of KOA rabbit morphology, knee joint cartilage p-FAK, p-PI3K, Aggrecan gene, and protein expression, to study the effect of needle-knife to promote cartilage cell synthesis metabolism mechanism. Method 49 male New Zealand rabbits, randomly divided into normal group (Z), model group (M), model-inhibitors (MP), needle-knife group (D), needle-knife inhibitors group (DP), electroacupuncture group (E), and electroacupuncture inhibitors (EP). RT-PCR and Western Blot were used to test each animal cartilage p-FAK, p-PI3K, and Aggrecan gene and protein expression level. Results Compared with N group, p-FAK and p-PI3K protein and mRNA expression of M group, D group, and E group increased (P < 0.05), while the protein and mRNA expression of Aggrecan reduced (P < 0.05). Compared with M group, p-FAK, p-PI3K, Aggrecan protein, and mRNA of E and D group increased (P < 0.05). Compared with E group, p-FAK, p-PI3K, Aggrecan protein, and mRNA expression of D group increased (P < 0.05); after adding inhibitors, p-FAK, p-PI3K, Aggrecan protein, and mRNA expression reduced (P < 0.05). Conclusion Needle-knife therapy can promote the repairment of cartilage cells by activating FAK-PI3K signaling pathways, promoting the synthesis of cartilage cell metabolism. PMID:29234400

[Effect of Acupoint Injection on Eosinophil Counts,Protein and mRNA Expressions of Eotaxin in Nasal Mucosa of Allergic Rhinitis Rats].

PubMed

Zhang, Yuan; Hou, Xun-Rui; Li, Li-Hong; Yang, Meng; Liang, Fei-Hong

2017-04-25

To observe the effect of acupoint injection on eosinophils (EOS) counts and expression of eotaxin in nasal mucosa of allergic rhinitis (AR) rats, so as to reveal its mechanism underlying improving AR. Twenty-four Sprague-Dawley (SD) rats were randomly divided into normal, model and acupoint injection groups ( n =8 in each group). The AR model was established by intraperitoneal injection of ovalbumin sensitization. Bilateral "Yingxiang"(LI 20) and "Yintang"(GV 29) were selected for acupoint injection of the mixture solution of lidocaine, dexamethasone, and transfer factor (0.1 mL/acupoint) on the 1 st , 5 th , 9 th , and 13 th day after AR model established, a total of four times. EOS in the nasal mucosa was counted under light microscope after HE staining. Protein and mRNA expressions of eotaxin in the nasal mucosa were detected by immunohistochemical and RT-PCR methods, respectively. Compared with the normal group, EOS counts, protein and mRNA expressions of eotaxin in the nasal mucosa were significantly higher in the model group ( P <0.05). Compared with the model group, EOS counts, protein and mRNA expressions of eotaxin in the nasal mucosa were significantly lower in the acupoint injection group ( P <0.05). Acupoint injection can reduce the nasal mucosa inflammation by suppressing the protein and mRNA expressions of eotaxin, decreasing the infiltration and gathering of EOS in the nasal mucosa.

Limb segment vibration modulates spinal reflex excitability and muscle mRNA expression after spinal cord injury

PubMed Central

Chang, Shuo-Hsiu; Tseng, Shih-Chiao; McHenry, Colleen L.; Littmann, Andrew E.; Suneja, Manish; Shields, Richard K.

2012-01-01

Objective We investigated the effect of various doses of vertical oscillation (vibration) on soleus H-reflex amplitude and post-activation depression in individuals with and without SCI. We also explored the acute effect of short-term limb vibration on skeletal muscle mRNA expression of genes associated with spinal plasticity. Methods Six healthy adults and five chronic complete SCI subjects received vibratory stimulation of their tibia over three different gravitational accelerations (0.3g, 0.6g, and 1.2g) at a fixed frequency (30 Hz). Soleus H-reflexes were measured before, during, and after vibration. Two additional chronic complete SCI subjects had soleus muscle biopsies 3 h following a single bout of vibration. Results H-reflex amplitude was depressed over 83% in both groups during vibration. This vibratory-induced inhibition lasted over 2 min in the control group, but not in the SCI group. Post-activation depression was modulated during the long-lasting vibratory inhibition. A single bout of mechanical oscillation altered mRNA expression from selected genes associated with synaptic plasticity. Conclusions Vibration of the lower leg inhibits the H-reflex amplitude, influences post-activation depression, and alters skeletal muscle mRNA expression of genes associated with synaptic plasticity. Significance Limb segment vibration may offer a long term method to reduce spinal reflex excitability after SCI. PMID:21963319

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

PubMed Central

2010-01-01

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research. PMID:21406066

Effect of Jianweiyuyang granule on gastric ulcer recurrence and expression of VEGF mRNA in the healing process of gastric ulcer in rats.

PubMed

Dai, Xing-Ping; Li, Jia-Bang; Liu, Zhao-Qian; Ding, Xiang; Huang, Cheng-Hui; Zhou, Bing

2005-09-21

To investigate the effect of Jianweiyuyang (JWYY) granule on gastric ulcer recurrence and its mechanism in the treatment of gastric ulcer in rats. Gastric ulcer in rats was induced according to Okeba's method with minor modification and the recurrence model was induced by IL-1beta. The expression of vascular endothelial growth factor mRNA (VEGF mRNA) was examined by reverse transcription polymerase chain reaction in gastric ulcer and microvessel density (MVD) adjacent to the ulcer margin was examined by immunohistochemistry. MVD was higher in the JWYY treatment group (14.0+/-2.62) compared with the normal, model and ranitidine treatment groups (2.2+/-0.84, 8.8+/-0.97, 10.4+/-0.97) in rats (P<0.01). The expression level of VEGF mRNA in gastric tissues during the healing process of JWYY treatment group rats significantly increased compared with other groups (normal group: 0.190+/-0.019, model group: 0.642+/-0.034, ranitidine group: 0.790+/-0.037, P<0.01). JWYY granules can stimulate angiogenesis and enhance the expression of VEGF mRNA in gastric ulcer rats. This might be the mechanism for JWYY accelerating the ulcer healing, and preventing the recurrence of gastric ulcer.

Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

PubMed

Li, Guang-li; Roy, Bhaskar; Li, Xing-hua; Yue, Wan-fu; Wu, Xiao-feng; Liu, Jian-mei; Zhang, Chuan-xi; Miao, Yun-gen

2009-05-01

Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins.

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

PubMed Central

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

2013-01-01

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

[Significant increase of glucose transport activity in breast cancer].

PubMed

Li, Juan; Yang, Shou-jing; Zhao, Xi-long; Zhang, Ya-qing; Li, Kai-nan; Cui, Ji-hong; Li, Jing

2008-02-01

To study the expression level and significance of glucose transporter 1 (Glut-1) in normal breast tissue, adenosis, adenoma and breast carcinoma. A total of 147 cases of female breast tissue samples, including 92 cases of invasive ductal carcinoma, 26 cases of breast fibroadenoma, 24 cases of breast adenosis and 5 cases of normal breast tissues, were collected for quantitative detection of the expression of Glut-1 protein by immunohistochemistry (EnVision method) and Western blot, and its mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). In normal breast tissue and benign lesions of the breast, Glut-1 was undetectable or only weakly detectable in cytoplasm of ductal and acinar epithelia. In contrast, the intensity of Glut-1 staining was significantly higher in invasive ductal carcinomas (P = 0.0002) with protein expression predominantly in cellular membrane and lesser in cytoplasm. Western blot and RT-PCR analyses showed that the expression of Glut-1 protein and mRNA were significantly increased in invasive ductal carcinoma than fibroadenoma (P =0.001 for protein; P <0.05 for mRNA) and adenosis (P =0.001 for protein; P < 0.05 for mRNA). There was a significant difference among groups (P = 0.0002 for protein; P = 0.0001 for mRNA). Glucose transport activity, as indicated by Glut-1 protein and its mRNA expression, significantly increases in breast carcinoma than non-cancerous lesions. The over-expression of Glut-1 in breast carcinoma is tightly coupled with tumor cell proliferation, invasion and metastasis, implying that Glut-1 may serve as a new marker in the early diagnosis and prognostication of breast malignancy as well as a new therapeutic target.

Expression of CD30 mRNA, CD30L mRNA and a variant form of CD30 mRNA in restimulated peripheral blood mononuclear cells (PBMC) of patients with helminthic infections resembling a Th2 disease

PubMed Central

Kilwinski, J; Berger, T; Mpalaskas, J; Reuter, S; Flick, W; Kern, P

1999-01-01

It has been proposed that CD30, a member of the tumour necrosis factor (TNF) receptor superfamily, is preferentially up-regulated on Th2-type human T cells. In order to investigate a correlation between infection with Echinococcus multilocularis and CD30 expression, we analysed regulation of CD30 mRNA, a variant form of CD30 mRNA (CD30v) and CD30 ligand (CD30L) mRNA expression on PBMC from patients with alveolar echinococcosis (AE) using reverse transcriptase-polymerase chain reaction (RT-PCR). In PBMC of patients with AE as well as healthy donors, spontaneous expression of CD30L mRNA and the CD30v mRNA could be detected. However, the intact form of CD30 mRNA could be detected neither in freshly isolated PBMC of patients nor in PBMC of healthy individuals. Expression of CD30L mRNA and the variant form of CD30 mRNA was frequently detected at individual time points during 72 h of culture of PBMC stimulated with crude Echinococcus antigen. In contrast to CD30v or CD30L mRNA expression, induction of CD30 mRNA expression was detected only in three out of six (50%) healthy donors and in 10 out of 21 (48%) patients with alveolar echinococcosis after 72 h of incubation. As a control, mitogenic stimulation of PBMC of both healthy individuals and infected patients led to expression of intact CD30 mRNA within 24 h of culture. These data demonstrate the different expression of two different forms of CD30 mRNA in PBMC of human individuals. The specific induction of CD30 expression is correlated only in rare cases with the clinical status of patients with AE, indicating the lack of a general induction of CD30 mRNA in this Th2-type-dominated helminthic disease. The data provide further evidence that the CD30 receptor is not an exclusive marker for a Th2-type response. PMID:9933429

Striatal output markers do not alter in response to circling behaviour in 6-OHDA lesioned rats produced by acute or chronic administration of the monoamine uptake inhibitor BTS 74 398.

PubMed

Lane, E L; Cheetham, S; Jenner, P

2008-01-01

The monoamine uptake inhibitor BTS 74 398 induces ipsilateral circling in 6-hydroxydopamine (6-OHDA) lesioned rats without induction of abnormal motor behaviours associated with L-dopa administration. We examined whether this was reflected in the expression of peptide mRNA in the direct and indirect striatal output pathways.6-OHDA lesioning of the nigrostriatal pathway increased striatal expression of PPE-A mRNA and decreased levels of PPT mRNA with PPE-B mRNA expression remaining unchanged. Acute L-dopa administration normalised PPE-A mRNA and elevated PPT mRNA while PPE-B mRNA expression remained unchanged. Acute administration of BTS 74 398 did not alter striatal peptide mRNA levels. Following chronic treatment with L-dopa, PPE-A mRNA expression in the lesioned striatum continued to be normalised and PPT mRNA was increased compared to the intact side. PPE-B mRNA expression was also markedly increased relative to the non-lesioned striatum. Chronic BTS 74 398 administration did not alter mRNA expression in the 6-OHDA lesioned striatum although small increases in PPT mRNA expression in the intact and sham lesioned striatum were observed. The failure of BTS 74 398 to induce changes in striatal neuropeptide mRNA correlated with its failure to induce abnormal motor behaviours or behavioural sensitisation but does not explain how it produces a reversal of motor deficits. An action in another area of the brain appears likely and may explain the subsequent failure of BTS 74 398 and related compounds to exert anti-parkinsonian actions in man.

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

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

PubMed Central

2010-01-01

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

Low Incidence along with Low mRNA Levels of EGFRvIII in Prostate and Colorectal Cancers Compared to Glioblastoma

PubMed Central

Peciak, Joanna; Stec, Wojciech J; Treda, Cezary; Ksiazkiewicz, Magdalena; Janik, Karolina; Popeda, Marta; Smolarz, Maciej; Rosiak, Kamila; Hulas-Bigoszewska, Krystyna; Och, Waldemar; Rieske, Piotr; Stoczynska-Fidelus, Ewelina

2017-01-01

Background: The presence as well as the potential role of EGFRvIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFRvIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFRvIII and EGFRWT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFRvIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFRvIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFRvIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFRvIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFRvIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFRvIII importance should not be underestimated even in tumors with relatively low expression of this oncogene. PMID:28123609

[Inactivation of PMS2 gene by promoter methylation in nasopharyngeal carcinoma].

PubMed

Ni, H F; Jiang, B; Zhou, Z; Li, Y; Yuan, X Y; Cao, X L; Huang, G W

2016-11-23

Objective: To investigate the inactivation of PMS2 gene mediated by promoter methylation and its regulatory mechanism in nasopharyngeal carcinoma (NPC). Methods: Fifty-four NPC tissues, 16 normal nasopharyngeal epithelia (NNE), 5 NPC cell lines (CNE1, CNE2, TWO3, HNE1 and HONE1) and 1 normal nasopharyngeal epithelial cell line (NP69) were collected.Methylation-specific PCR (MSP) was used to detect the PMS2 promoter methylation, semi-quantitative reverse transcription PCR (qRT-PCR) was applied to determine its mRNA expression, and immunohistochemistry (IHC) was used to detect the protein expression of PMS2. The expressions of PMS2 mRNA in CNE1 and CNE2 cells before and after treated with methyltransferase inhibitor 5-aza-2-deoxycytidine were analyzed by qRT-PCR. The impact of methylation and demethylation on the mRNA expression of PMS2, and the association of mRNA and protein expression of PMS2 with clinicopathological features of nasopharyngeal cancer were analyzed. Results: Methylation of PMS2 gene was detected in all of the five NPC cell lines, but not in normal nasopharyngeal epithelial NP69 cells. The methylation rate of PMS2 gene in NPC tissues was 63% (34/54), significantly higher than that of the normal nasopharyngeal epithelia (0/16, P <0.001). The expression levels of PMS2 mRNA and protein were significantly down-regulated in the 54 NPC tissues when compared with those in the 16 NNE tissues ( P <0.001), and were also significantly lower in the 34 methylated NPC tissues than those in the 20 unmethylated NPC tissues ( P <0.001). After treatment with 5-aza-2-deoxycytidine, the expression of PMS2 mRNA was restored in the CNE1 and CNE2 cells.However, the expressions of PMS2 mRNA and protein were not significantly correlated with patients' age, gender, TNM stage, histopathologic type or lymph node metastasis ( P >0.05 for all). Conclusions: Promoter methylation-mediated inactivation of PMS2 gene participates in carcinogenesis and development of NPC. PMS2 may be a candidate tumor suppressor in the treatment for patients with inactivation of PMS2 promoter methylation.

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

PubMed Central

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

2009-01-01

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

Detection of HER2 polymorphism and expression using circulating DNA and RNA as a tool in lung adenocarcinoma patients: a case control study.

PubMed

Mirza, Masroor; Javid, Jamsheed; Yadav, Prasant; Mohan, Anant; Ray, Prakash Chandra; Saxena, Alpana

2016-06-01

Circulating DNA and RNA is an important prognostic tool for noninvasive malignant disease detection and in disease prognosis. Study aimed to evaluate the possible prognostic role of HER2 (-3444C/T) promoter polymorphism and its mRNA expression in Lung adenocarcinoma patients using circulating DNA and RNA. One hundred newly diagnosed lung adenocarcinoma patients and 100 age and sex matched healthy controls were included and allele specific (AS) polymerase chain reaction (PCR) was used for genotyping and expression was analyzed by quantitative real time PCR. Overall survival of patients was analyzed by Kaplan-Meier method. We observed a statistically significant difference in the frequency of HER2 CC, CT, and CT genotype among lung adenocarcinoma cases vs. healthy controls (P=0.001). Compared to the CC genotype, OR 2.51 (1.4-4.51), 5.97 (1.17-30.41) and RR 1.56 (1.17-2.07), 2.83 (0.82-9.73) for heterozygous CT and homozygous TT genotypes suggesting possible dominant effect on risk of lung adenocarcinoma. Cases with CC genotype showed 9.29 fold increased mRNA expression while cases with heterozygous CT and homozygous TT genotype showed 16.26, 16.72 fold increased mRNA expression (P<0.0001). We observed 13.92 fold increased HER2mRNA expression Lung adenocarcinoma patients. Patients in different TNM stages showed significant difference in HER2 mRNA expression which was found to be significantly associated (P<0.0001). Patients with distant metastases and without distant metastases had 17.44 and 11.16 fold increased HER2 mRNA expression was also found to be significantly associated (P<0.0001). It was also observed that patients with pleural effusion and without pleural effusion showed significant difference in HER2 mRNA expression (P=0.03). We also analysed patients with CC, TT, CT (P=0.02) and CT + TT (P=0.008) genotype showed 15.8, 7.9, 9.5 and 7.9 months of overall median survival time and found to be significantly associated, respectively. Patients with >13 and ≤13 fold increased HER mRNA expression also showed 7.9 and 11.5 months of overall median survival time was also found to be significantly associated (P=0.01). Our work provides evidence that circulating DNA and RNA may be a potential prognostic tool in Lung adenocarcinoma patients. Promoter polymorphism of HER2 (-3444C/T) gene had significant impact on higher HER2 mRNA expression could be a predictive factor for patients' worse overall survival and metastatic behaviour.

Lower Selenoprotein T Expression and Immune Response in the Immune Organs of Broilers with Exudative Diathesis Due to Selenium Deficiency.

PubMed

Pan, Tingru; Liu, Tianqi; Tan, Siran; Wan, Na; Zhang, Yiming; Li, Shu

2018-04-01

The objective of the present study was to investigate whether dietary selenium (Se) deficiency would affect the expression of selenoprotein T (SelT) and immune response in the immune organs of broilers. Changes in expression of inflammatory cytokines and oxidative stress response caused by Se deficiency can lead to organism damage, which in turn leads to immune response. Sixty (1-day-old) broilers were divided into the control group and Se-deficiency group. Animal models with exudative diathesis were duplicated in the broilers by feeding them Se-deficient diet for 20 days. After the Se-deficient group exhibited symptoms of exudative diathesis, all the broilers were euthanized, and their immune organs were taken for analysis. The tissues including spleen, bursa of Fabricius, and thymus were treated to determine the pathological changes (including microscopic and ultramicroscopic), the messenger RNA (mRNA) expression levels of SelT and its synthetase (SecS and SPS1), cytokine mRNA expression levels, and antioxidant status. The microscopic and ultramicroscopic analyses showed that immune tissues were obviously injured in the Se-deficient group. The mRNA expression of SelT was decreased compared with that in the control group. Meanwhile, the mRNA expression levels of SecS and SPS1 were downregulated. In the Se-deficient group, the mRNA expression levels of IL-1R and IL-1β were higher than those of three control organs. Additionally, the IL-2 and INF-γ mRNA expression levels were lower than those of the control group. The activity of CAT was decreased, and the contents of H 2 O 2 and •OH were increased due to Se deficiency. Pearson method analysis showed that the expression of SelT had a positive correlation with IL-2, INF-γ, SecS, and SPS1 and a negative correlation with IL-1R and IL-1β. In summary, these data indicated that Se-deficient diet decreased the SelT expression and its regulation of oxidative stress, and it inhibited a pleiotropic mechanism of the immune response.

[Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells].

PubMed

Xiaolan, He; Guangjie, Bao; Linglu, Sun; Xue, Zhang; Shanying, Bao; Hong, Kang

2017-08-01

Objective The effect of different oxygen tensions on the cytoskeleton remodeling of goat temporomandibular joint (TMJ) disc cells were investigated. Methods Goat TMJ disc cells were cultured under normoxia (21% O₂) and hypoxia (2%, 4%, and 8% O₂). Toluidine blue, picrosirius red, and type Ⅰ collagen immunocytochemical staining were performed to observe the changes in cell phenotype under different oxygen levels. Immunofluorescent staining and real-time reverse transcription-polymerase chain reaction analysis were then performed to identify actin, tubulin, and vimentin in the cultured disc cells. Results TMJ disc cells still displayed fibroblast characteristics under different oxygen levels and their cytoskeletons had regular arrangement. The fluorescence intensities of actin and vimentin were lowest at 4% O₂(P<0.05), whereas that of tubulin was highest at 2% O₂ (P<0.05). No significant difference among the other groups was observed (P>0.05). Actin mRNA levels were considerably decreased at 2% O₂ and 4% O₂ in hypoxic conditions, while actin mRNA expression was highest in 21% O₂. Tubulin mRNA levels considerably increased at 2% O₂, while tubulin mRNA expression was lowest in 8% O₂ (P<0.05). Vimentin mRNA expression was lowest at 4% O₂ and highest at 21% O₂, and significant differences were observed between vimentin mRNA expression levels among these oxygen levels (P<0.05). Conclusion Cytoskeletons were reconstructed in different oxygen tensions, and 2% O₂ may be the optimal oxygen level required to proliferate TMJ disc cells.

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

PubMed

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

2006-02-01

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

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.

Expression of Cytochrome P450s in the Liver of Rats Administered with Socheongryong-tang, a Traditional Herbal Formula

PubMed Central

Jin, Seong Eun; Ha, Hyekyung; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

2016-01-01

Objective: The purpose of this study was to investigate the potential influences of Socheongryong-tang (SCRT) on the messenger ribonucleic acid (mRNA) and protein expression of cytochrome P450 (CYP450) in vivo. Materials and Methods: SCRT was orally administered to either male or female Sprague-Dawley rats once daily at doses of 0, 1000, 2000, or 5000 mg/kg/day for 13 weeks. The mRNA expression of CYP450s (CYP1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2, and 4A1) in liver tissues was measured by reverse transcription polymerase chain reaction. And then, the protein expression of CYP1A1 and CYP2B1/2 in liver tissues was analyzed by the Western blot. Results: We found no significant influence in the mRNA expression of hepatic CYP1A2, 2C11, 2E1, 3A1, 3A2, and 4A1 after repeated administration of SCRT for 13 weeks. By contrast, the mRNA and protein expression of hepatic CYP1A1 was increased by repeated SCRT treatment in male rats, but not in female rats. The mRNA and protein expression of hepatic CYP2B1/2 in both genders was increased by administration of SCRT. Conclusion: A caution is needed when SCRT is co-administered with substrates of CYP2B1/2 for clinical usage. In case of male, an attention is also required when SCRT and drugs metabolized by CYP1A1 are taken together. Our findings provide information regarding the safety and effectiveness of SCRT when combined with conventional drugs. SUMMARY Oral administration of Socheongryong-tang for 13 weeks did not affect the mRNA expression of hepatic CYP1A2, 2C11, 2E1, 3A1, 3A2, and 4A1In male rats, oral administration of Socheongryong-tang for 13 weeks induced the mRNA and protein expression of hepatic CYP1A1 and CYP2B1/2In female rats, oral administration of Socheongryong-tang for 13 weeks induced the mRNA and protein expression of hepatic CYP2B1/2. Abbreviations used: SCRT: Socheongryong-tang, CYP450: Cytochrome P450, HPLC: High performance liquid chromatography, RT-PCR: Reverse transcription polymerase chain reaction. PMID:27601852

[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.

Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection.

PubMed

Van Pham, Phuc; Thi-My Nguyen, Phuoc; Thai-Quynh Nguyen, Anh; Minh Pham, Vuong; Nguyen-Tu Bui, Anh; Thi-Tung Dang, Loan; Gia Nguyen, Khue; Kim Phan, Ngoc

2014-06-01

Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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

PubMed Central

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

2015-01-01

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

Short-term application of dexamethasone on stem cells derived from human gingiva reduces the expression of RUNX2 and β-catenin.

PubMed

Kim, Bo-Bae; Kim, Minji; Park, Yun-Hee; Ko, Youngkyung; Park, Jun-Beom

2017-06-01

Objective Next-generation sequencing was performed to evaluate the effects of short-term application of dexamethasone on human gingiva-derived mesenchymal stem cells. Methods Human gingiva-derived stem cells were treated with a final concentration of 10 -7  M dexamethasone and the same concentration of vehicle control. This was followed by mRNA sequencing and data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis of RUNX2 and β-catenin. Results In total, 26,364 mRNAs were differentially expressed. Comparison of the results of dexamethasone versus control at 2 hours revealed that 7 mRNAs were upregulated and 25 mRNAs were downregulated. The application of dexamethasone reduced the expression of RUNX2 and β-catenin in human gingiva-derived mesenchymal stem cells. Conclusion The effects of dexamethasone on stem cells were evaluated with mRNA sequencing, and validation of the expression was performed with qualitative real-time polymerase chain reaction and western blot analysis. The results of this study can provide new insights into the role of mRNA sequencing in maxillofacial areas.

Effects of Different Doses of Levetiracetam on Aquaporin 4 Expression in Rats with Brain Edema Following Fluid Percussion Injury

PubMed Central

Jin, Hongbo; Li, Wenling; Dong, Changzheng; Ma, Li; Wu, Jiang; Zhao, Wenqing

2016-01-01

Background This study was designed to investigate the effects of different doses of levetiracetam on aquaporin 4 (AQP4) expression in rats after fluid percussion injury. Material/Methods Sprague-Dawley rats were randomly divided into 4 groups: sham operation group, traumatic brain injury group, low-dose levetiracetam group, and high-dose levetiracetam group. Brain edema models were established by fluid percussion injury, and intervened by the administration of levetiracetam. Samples from the 4 groups were collected at 2, 6, 12, and 24 h, and at 3 and 7 days after injury. Histological observation was performed using hematoxylin-eosin staining and immunohistochemical staining. AQP4 and AQP4 mRNA expression was detected using Western blot assay and RT-PCR. Brain water content was measured by the dry-wet method. Results Compared with the traumatic brain injury group, brain water content, AQP4 expression, and AQP4 mRNA expression were lower in the levetiracetam groups at each time point and the differences were statistically significant (P<0.05). The intervention effects of high-dose levetiracetam were more apparent. Conclusions Levetiracetam can lessen brain edema from fluid percussion injury by down-regulating AQP4 and AQP4 mRNA expression. There is a dose-effect relationship in the preventive effect of levetiracetam within a certain extent. PMID:26927633

High-Throughput RT-PCR for small-molecule screening assays

PubMed Central

Bittker, Joshua A.

2012-01-01

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis. PMID:23487248

[Regulation effects of liuwei dihuang pill, jingui shenqi pill, jiangu erxian pill containing serums on adipogenic and osteogenic differentiation-related genes expressions in the differentiation process of preadipocytes to osteoblasts].

PubMed

Cheng, Zhi-An; Han, Ling; Wei, Jian-An; Sun, Jing; Duan, Xiao-Dong

2013-02-01

To study the effects of Chinese medical recipes for invigorating Shen on rat bone marrow mesenchymal stem cells (BMSCs)-derived preadipocytes' differentiation to osteoblasts. The BMSCs were cultured using whole bone marrow adherence wall method. The BMSCs were induced to preadipocytes by classic chemical method. The osteogenic differentiation process of preadipocytes was intervened by Liuwei Dihuang Pill (LDP), Jingui Shenqi Pill (JSP), or Jiangu Erxian Pill (JEP)-containing serums (with the concentRation of 10%, on behalf of tonifying Shen yin, tonifying Shen yang, and tonifying Shen essence). Reverse transcription-real time fluorescent quantitative-PCR (RT real time qPCR) was used to detect RUNX2, ALP, BGP, BMP2, BMP4, SPP1, and IGF1 mRNA expressions of osteogenic differentiation-related genes, mRNA expressions of LPL, FABP4, and PPARgamma of adipogenic differentiation-related genes on the 6th, the 12th, and the 18th day. As for the osteogenic differentiation-related gene, when compared with the control group, there was no statistical difference in the gene expression level in the experimental groups on the 6th day (2.0 > Ratio > 0.5). On the 12th day, the mRNA expressions of IGF1 and Runx2 increased more significantly in the JSP group, with their relative quantification (Ratio) being 2.97 and 1.81 respectively. On the 18th day the IGF1 mRNA expression significantly increased, being the Ratio value of 3.74, 12.60, and 8.35, respectively, in the LDP group, the JSP group, and the JEP group. The SPP1 mRNA expression also significantly increased, with the Ratio value of 2.94, 3.18, and 2.62, respectively, in the LDP group, the JSP group, and the JEP group. As for adipogenic differentiation-related genes, on the 6th day, when compared with the control group, FABP4 mRNA expression significantly decreased in the LDP group and the JSP group (with the Ratio value of 0.47 and 0.40 respectively). The expression levels of other genes were all down-regulated, but not significantly. On the 12th day and 18th day, there was no statistical change in the adipogenic differentiation-related genes expressions (2.0 > Ratio > 0.5). Up-regulation of osteogenic differentiation-related genes expression occurred in later time, while down-regulation of adipogenic differentiation-related genes expression occurred in earlier time after treatment by Chinese medical recipes for invigorating Shen. In general, above data indicated that tonifying Shen yang was more effective in promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs.

TP53 and ATM mRNA expression in skin and skeletal muscle after low-level laser exposure.

PubMed

Guedes de Almeida, Luciana; Sergio, Luiz Philippe da Silva; de Paoli, Flavia; Mencalha, Andre Luiz; da Fonseca, Adenilson de Souza

2017-08-01

Low-level lasers are widespread in regenerative medicine, but the molecular mechanisms involved in their biological effects are not fully understood, particularly those on DNA stability. Therefore, this study aimed to investigate mRNA expression of genes related to DNA genomic stability in skin and skeletal muscle tissue from Wistar rats exposed to low-level red and infrared lasers. For this, TP53 (Tumor Protein 53) and ATM (Ataxia Telangiectasia Mutated gene) mRNA expressions were evaluated by real-time quantitative PCR (RT-qPCR) technique 24 hours after low-level red and infrared laser exposure. Our data showed that relative TP53 mRNA expression was not significantly altered in both tissues exposed to lasers. For ATM, relative mRNA expression in skin tissue was not significantly altered, but in muscle tissue, laser exposure increased relative ATM mRNA expression. Low-level red and infrared laser radiations alter ATM mRNA expression related to DNA stability in skeletal muscle tissue.

[Influence of hepatocyte growth factor on iNOS, NO and IL-1β in the cerebrum during cerebral ischemia/reperfusion in rats].

PubMed

He, Fang; Ye, Bei; Chen, Jianzhen; Sun, Xiaoyan; Li, Chang

2014-01-01

To explore the effect of hepatocyte growth factor (HGF) on inducible nitric oxide synthase (iNOS), NO and interleukin-1β (IL-1β) in the cerebrum of rats subjected to cerebral ischemia/reperfusion (I/R). Sprague-Dawley rats were randomly divided into 5 groups: a sham group, an I/R group,an HGF1 group, an HGF2 group, and an HGF3 group. The latter 3 groups were respectively injected 15, 30 and 60 μg/kg HGF. The focal cerebral I/R model was established by sutureoccluded method. After 1.5 h ischemia followed by 24 h reperfusion, the iNOS activity and NO content in the ischemic cerebral tissue were assessed. The expression of iNOS mRNA and IL-1β mRNA was detected. The level of iNOS protein and IL-1β content were determined. In addition, cultured cerebral cortical neurons in vitro were exposed to I/R. Then the expression of iNOS and IL-1β protein in the neurons was detected, and NO content was assessed. The iNOS activity and NO content in the ischemic cerebral tissue were increased. The expression of iNOS mRNA and IL-1β mRNA was upregulated. The level of iNOS protein and IL- 1β content were increased. Administration of HGF decreased the iNOS activity and NO content, and downregulated the expression of iNOS mRNA, IL-1β mRNA, iNOS protein and IL-1β content in the ischemic cerebral tissue. HGF decreased the expression of IL-1β, iNOS protein and NO content in the cortical neurons exposed to I/R in vitro. HGF can inhibit the expression of IL-1β and decrease the expression of iNOS and content of NO, which is probably one of the mechanisms mediating the protection of HGF against cerebral ischemia injury.

[Study on screening differentially expressed genes in mice livers by silver staining DD-PCR].

PubMed

Luan, Xin-Hong; Hu, Zhong-Ming; Liu, Wei-Quan; Jiang, Yu; Wang, Kai; Wu, Yong-Kui; Li, Qian-Xue

2005-08-01

To screen swimming-fatigue related genes in mice and lay theoretic basis for researching the molecular mechanism of fatigue. 30 male BALB/c mice (20 +/- 2g) were divided into control group, dipping in water group and swimming-fatigue group respectively. After fatigue for swimming in swimming-fatigue group, with control group and dipping in water group, liver tissues in mice were collected. With improved silver staining mRNA differential display method, the differentially expressed genes in mice livers were screened and evaluated by reversed Northern blot. The positive segments were analyzed homology by BLAST. 7 of DD-ESTs were gained. Two of them only expressed in swimming-fatigue group, two down-regulated expressed, and three up-regulated. One of them was a novel gene and was accepted by GenBank, AY615302. Seven DD-ESTs in swimming-fatigue mice were gained by silver staining mRNA differential display method.

G-cimp status prediction of glioblastoma samples using mRNA expression data.

PubMed

Baysan, Mehmet; Bozdag, Serdar; Cam, Margaret C; Kotliarova, Svetlana; Ahn, Susie; Walling, Jennifer; Killian, Jonathan K; Stevenson, Holly; Meltzer, Paul; Fine, Howard A

2012-01-01

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.

G-Cimp Status Prediction Of Glioblastoma Samples Using mRNA Expression Data

PubMed Central

Baysan, Mehmet; Bozdag, Serdar; Cam, Margaret C.; Kotliarova, Svetlana; Ahn, Susie; Walling, Jennifer; Killian, Jonathan K.; Stevenson, Holly; Meltzer, Paul; Fine, Howard A.

2012-01-01

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data. PMID:23139755

Inhibition of small-intestinal sugar absorption mediated by sodium orthovanadate Na3VO4 in rats and its mechanisms

PubMed Central

Ai, Jing; Du, Jie; Wang, Ning; Du, Zhi-Min; Yang, Bao-Feng

2004-01-01

AIM: To investigate the inhibitory effects of sodium orthovanadate on small-intestinal glucose and maltose absorption in rats and its mechanism. METHODS: Normal Wistar rats were lavaged with sodium orthovanadate (16 mg/kg, 4 mg/kg and 1 mg/kg) for 6 d. Blood glucose values were measured after fasting and 0.5, 1, 1.5 and 2 h after glucose and maltose feeding with oxidation-enzyme method. α-glucosidase was abstracted from the upper small intestine, and its activity was examined. mRNA expression of α-glucosidase and glucose-transporter 2 (GLUT2) in epithelial cells of the small intestine was observed by in situ hybridization. RESULTS: Sodium orthovanadate could delay the increase of plasma glucose concentration after glucose and maltose loading, area under curve (AUC) in these groups was lower than that in control group. Sodium orthovanadate at dosages of 10 μmol/L, 100 μmol/L and 1000 μmol/L could suppress the activity of α-glucosidase in the small intestine of normal rats, with an inhibition rate of 68.18%, 87.22% and 91.91%, respectively. Sodium orthovanadate reduced mRNA expression of α-glucosidase and GLUT2 in epithelial cells of small intestine. CONCLUSION: Sodium orthovanadate can reduce and delay the absorption of glucose and maltose. The mechanism may be that it can inhibit the activity and mRNA expression of α-glucosidase, as well as mRNA expression of GLUT2 in small intestine. PMID:15534916

DIFFERENTIAL EFFECTS OF SINGLE VERSUS REPEATED ALCOHOL WITHDRAWAL ON THE EXPRESSION OF ENDOCANNABINOID SYSTEM-RELATED GENES IN THE RAT AMYGDALA

PubMed Central

Serrano, Antonia; Rivera, Patricia; Pavon, Francisco J.; Decara, Juan; Suárez, Juan; de Fonseca, Fernando Rodriguez; Parsons, Loren H.

2011-01-01

Background Endogenous cannabinoids such as anandamide and 2-arachidonoylglycerol (2-AG) exert important regulatory influences on neuronal signaling, participate in short- and long-term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity and affective control. Methods The consequence of long-term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT-PCR analyses of amygdala tissue. Two groups of ethanol-exposed rats were generated by maintenance on an ethanol liquid diet (10%): one group received continuous access to ethanol for 15 days, while the second group was given intermittent access to the ethanol diet (5 days/week for 3 weeks). Control subjects were maintained on an isocaloric ethanol-free liquid diet. To provide an initial profile of acute withdrawal amygdala tissue was harvested following either 6 or 24 hours of ethanol withdrawal. Results Acute ethanol withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2-AG (FAAH and MAGL, respectively) were evident, as were reductions in mRNA expression for CB1, CB2 and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent ethanol exposure, alterations in MAGL and cannabinoid receptor-related mRNA (e.g. CB1, CB2, GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal-associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2-AG biosynthesis (e.g. DAGL-α/β) were found in any condition. Conclusions These findings suggest that ethanol dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal-related dysregulation of amygdalar neurotransmission. PMID:22141465

Fiber type dependent upregulation of human skeletal muscle UCP2 and UCP3 mRNA expression by high-fat diet.

PubMed

Schrauwen, P; Hoppeler, H; Billeter, R; Bakker, A H; Pendergast, D R

2001-04-01

To test the hypothesis that consumption of a high-fat diet leads to an increase in UCP mRNA expression in human skeletal muscle. In a group of endurance athletes, with a range in fiber type distribution, we hypothesized that the effect of the high-fat diet on UCP2 and UCP3 mRNA expression is more pronounced in muscle fibers which are known to have a high capacity to shift from carbohydrate to fat oxidation (type IIA fibers). Ten healthy trained athletes (five males, five females) consumed a low-fat diet (17+/-0.9 en% of fat) and high-fat diet (41.4+/-1.4 en% fat) for 4 weeks, separated by a 4 week wash-out period. Muscle biopsies were collected at the end of both dietary periods. Using RT-PCR, levels of UCP2 and UCP3 mRNA expression were measured and the percentage of type I, IIA and IIB fibers were determined using the myofibrillar ATPase method in all subjects. UCP3L mRNA expression tended to be higher on the high-fat diet, an effect which reached significance when only males were considered (P=0.037). Furthermore, diet-induced change in mRNA expression of UCP3T (r: 0.66, P=0.037), UCP3L (r: 0.61, P=0.06) and UCP2 (r: 0.70, P=0.025), but not UCP3S, correlated significantly with percentage dietary fat on the high-fat diet. Plasma FFA levels were not different during the two diets. Finally, the percentage of type IIA fibers was positively correlated with the diet-induced change in mRNA expression for UCP2 (r: 0.7, P=0.03), UCP3L (r: 0.73, P=0.016) and UCP3T (r: 0.68, P=0.03) but not with UCP3S (r: 0.06, NS). UCP2 and UCP3 mRNAs are upregulated by a high-fat diet. This upregulation is more pronounced in humans with high proportions of type IIA fibers, suggesting a role for UCPs in lipid utilization.

Expression analysis and clinical utility of L-Dopa decarboxylase (DDC) in prostate cancer.

PubMed

Avgeris, Margaritis; Koutalellis, Georgios; Fragoulis, Emmanuel G; Scorilas, Andreas

2008-10-01

L-Dopa decarboxylase (DDC) is a pyridoxal 5'-phosphate-dependent enzyme that was found to be involved in many malignancies. The aim of this study was to investigate the mRNA expression levels of DDC in prostate tissues and to evaluate its clinical utility in prostate cancer (CaP). Total RNA was isolated from 118 tissue specimens from benign prostate hyperplasia (BPH) and CaP patients and a highly sensitive quantitative real-time RT-PCR (qRT-PCR) method for DDC mRNA quantification has been developed using the SYBR Green chemistry. LNCaP prostate cancer cell line was used as a calibrator and GAPDH as a housekeeping gene. DDC was found to be overexpressed, at the mRNA level, in the specimens from prostate cancer patients, in comparison to those from benign prostate hyperplasia patients (p<0.001). Logistic regression and ROC analysis have demonstrated that the DDC expression has significant discriminatory value between CaP and BPH (p<0.001). DDC expression status was compared with other established prognostic factors, in prostate cancer. High expression levels of DDC were found more frequently in high Gleason's score tumors (p=0.022) as well as in advanced stage patients (p=0.032). Our data reveal the potential of DDC expression, at the mRNA level, as a novel biomarker in prostate cancer.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

PubMed

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

PubMed Central

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis. PMID:25987838

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

PubMed

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

2005-04-28

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

Actions of hypoxia on catecholamine synthetic enzyme mRNA expression before and after development of adrenal innervation in the sheep fetus

PubMed Central

Adams, M B; McMillen, I C

2000-01-01

We have investigated adrenal mRNA expression of the catecholamine synthetic enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) following acute hypoxia in fetal sheep before (< 105 days gestation, n = 20) and after (> 125 days gestation, n = 20) the development of adrenal innervation and following pretreatment with the nicotinic receptor anatgonist hexamethonium (n = 12). Total RNA was extracted from fetal adrenal glands collected at specific time points at 3-20 h after the onset of either hypoxia (∼50% reduction in fetal arterial oxygen saturation (SO2) for 30 min), or normoxia. Before 105 days, there was a decrease in adrenal TH mRNA expression at 20 h after hypoxia and adrenal TH mRNA expression was directly related to the changes in arterial PO2 measured during normoxia and hypoxia. After 125 days, adrenal TH mRNA levels were suppressed for up to 12 h following hypoxia. In both age groups, adrenal PNMT mRNA expression increased at 3-5 h after hypoxia and was inversely related to the changes in fetal arterial PO2 during normoxia or hypoxia. After 125 days, the administration of hexamethonium (25 mg kg−1, I. V.) reduced TH mRNA but not PNMT mRNA expression after normoxia. After hexamethonium pretreatment, there was no significant change in either adrenal TH or PNMT mRNA expression following hypoxia. We conclude that acute hypoxia differentially regulates adrenal TH and PNMT mRNA expression in the fetal sheep both before and after the development of adrenal innervation. After the development of adrenal innervation, however, the effect of acute hypoxia upon adrenal TH and PNMT mRNA expression is dependent upon neurogenic input acting via nicotinic receptors. PMID:11118487

Expression of three isoforms of Na-K-2Cl cotransporter (NKCC2) in the kidney and regulation by dehydration.

PubMed

Itoh, Kazuko; Izumi, Yuichiro; Inoue, Takeaki; Inoue, Hideki; Nakayama, Yushi; Uematsu, Takayuki; Fukuyama, Takashi; Yamazaki, Taiga; Yasuoka, Yukiko; Makino, Takeshi; Nagaba, Yasushi; Tomita, Kimio; Kobayashi, Noritada; Kawahara, Katsumasa; Mukoyama, Masashi; Nonoguchi, Hiroshi

2014-10-24

Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b. Copyright © 2014 Elsevier Inc. All rights reserved.

Assessing mRNA nuclear export in mammalian cells by microinjection.

PubMed

Lee, Eliza S; Palazzo, Alexander F

2017-08-15

The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of survivin mRNA in healthy oral mucosa, oral leucoplakia and oral cancer.

PubMed

Lodi, G; Franchini, R; Bez, C; Sardella, A; Moneghini, L; Pellegrini, C; Bosari, S; Manfredi, M; Vescovi, P; Carrassi, A

2010-01-01

Survivin is involved in modulation of cell death and cell division processes. Survivin expression in normal adult tissues has not been fully understood, although it is markedly lower than in cancer, where it is over-expressed. To investigate survivin expression in normal, potentially malignant and cancerous oral mucosa. We measured survivin mRNA levels by real-time RT-PCR in specimens of oral mucosa (15 from normal mucosa, 17 from potentially malignant lesions, 17 from neoplasms). Scores were compared using Kruskal-Wallis test and post hoc according to Conover. Chi-squared test was used for dichotomous data. The median relative levels of survivin mRNA resulted six for normal mucosa, eight for potentially malignant lesions, 13 for cancers: differences among these three groups were statistically significant, as between cancer and potentially malignant lesions. Expression in normal mucosa and potentially lesions group showed no significant difference. Low, but not marginal expression of survivin in normal mucosa is a new finding, and it could be explained with the higher sensibility of our methods. Survivin expression in oral potentially malignant lesions might indicate a progressive deregulation of expression paralleling oncogenesis, particularly during the first stages of process, suggesting a putative predictive role for survivin.

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

PubMed

Hollingsworth, M A; Strawhecker, J M; Caffrey, T C; Mack, D R

1994-04-15

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy

PubMed Central

2014-01-01

Background The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. Methods After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). Results MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Conclusions Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo. PMID:25132809

Visualization of RNA–protein interactions in living cells: FMRP and IMP1 interact on mRNAs

PubMed Central

Rackham, Oliver; Brown, Chris M

2004-01-01

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA–protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells. PMID:15282548

Correlation analysis of the mRNA and miRNA expression profiles in the nascent synthetic allotetraploid Raphanobrassica

PubMed Central

Ye, Bingyuan; Wang, Ruihua; Wang, Jianbo

2016-01-01

Raphanobrassica is an allopolyploid species derived from inter-generic hybridization that combines the R genome from R. sativus and the C genome from B. oleracea var. alboglabra. In the present study, we used a high-throughput sequencing method to identify the mRNA and miRNA profiles in Raphanobrassica and its parents. A total of 33,561 mRNAs and 283 miRNAs were detected, 9,209 mRNAs and 134 miRNAs were differentially expressed respectively, 7,633 mRNAs and 39 miRNAs showed ELD expression, 5,219 mRNAs and 57 miRNAs were non-additively expressed in Raphanobrassica. Remarkably, differentially expressed genes (DEGs) were up-regulated and maternal bias was detected in Raphanobrassica. In addition, a miRNA-mRNA interaction network was constructed based on reverse regulated miRNA-mRNAs, which included 75 miRNAs and 178 mRNAs, 31 miRNAs were non-additively expressed target by 13 miRNAs. The related target genes were significantly enriched in the GO term ‘metabolic processes’. Non-additive related target genes regulation is involved in a range of biological pathways, like providing a driving force for variation and adaption in this allopolyploid. The integrative analysis of mRNA and miRNA profiling provides more information to elucidate gene expression mechanism and may supply a comprehensive and corresponding method to study genetic and transcription variation of allopolyploid. PMID:27874043

[Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

PubMed

Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

2009-04-01

To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

MCT1 and MCT4 kinetic of mRNA expression in different tissues after aerobic exercise at maximal lactate steady state workload.

PubMed

de Araujo, G G; Gobatto, C A; de Barros Manchado-Gobatto, F; Teixeira, L Fm; Dos Reis, I Gm; Caperuto, L C; Papoti, M; Bordin, S; Cavaglieri, C R; Verlengia, R

2015-01-01

We evaluate the mRNA expression of monocarboxylate transporters 1 and 4 (MCT1 and MCT4) in skeletal muscle (soleus, red and white gastrocnemius), heart and liver tissues in mice submitted to a single bout of swimming exercise at the maximal lactate steady state workload (MLSSw). After 72 h of MLSS test, the animals were submitted to a swimming exercise session for 25 min at individual MLSSw. Tissues and muscle samples were obtained at rest (control, n=5), immediately (n=5), 5 h (n=5) and 10 h (n=5) after exercise for determination of the MCT1 and MCT4 mRNA expression (RT-PCR). The MCT1 mRNA expression in liver increased after 10 h in relation to the control, immediate and 5 h groups, but the MCT4 remained unchanged. The MCT1 mRNA expression in heart increased by 31 % after 10 h when compared to immediate, but no differences were observed in relation to the control group. No significant differences were observed for red gastrocnemius in MCT1 and MCT4 mRNA expression. However, white gastrocnemius increased MCT1 mRNA expression immediately when compared to rest, 5 and 10 h test groups. In soleus muscle, the MCT1 mRNA expression increased immediately, 5 and 10 h after exercise when compared to the control. In relation to MCT4 mRNA expression, the soleus increased immediately and 10 h after acute exercise when compared to the control group. The soleus, liver and heart were the main tissues that showed improved the MCT1 mRNA expression, indicating its important role in controlling MLSS concentration in mice.

Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.

PubMed

Nomura, S; Ogami, K; Kawamura, K; Tsukamoto, I; Kudo, Y; Kanakura, Y; Kitamura, Y; Miyazaki, H; Kato, T

1997-07-01

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

Detection of MMP-9 and TIMP-3 mRNA expression in the villi of patients undergoing early spontaneous abortion: A report of 30 cases.

PubMed

Jiang, Guangli; Qi, Yuxia

2015-05-01

The aim of the present study was to investigate the correlation of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase inhibitor (TIMP)-3 expression with spontaneous abortion (SA) during early pregnancy. The villus tissues of 30 SA cases and 20 requested abortion cases were collected during surgery and constituted the SA and normal abortion (NA) groups, respectively. The total villous RNA was extracted and the expression levels of MMP -9 and TIMP-3 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) assay to calculate the MMP-9/TIMP-3 mRNA ratio. The MMP-9 mRNA expression level and MMP-9/TIMP-3 mRNA ratio of the SA group were significantly higher than those of the NA group (P<0.01), while the TIMP-3 mRNA levels of the two groups were similar (P>0.05). The MMP-9 mRNA expression level of the SA group was higher than that of the NA group; thus, the MMP-9/TIMP-3 mRNA ratio was higher. These results suggest that the expression level of MMP-9 mRNA and the MMP-9/TIMP-3 mRNA ratio are associated with SA.

A Cellular High-Throughput Screening Approach for Therapeutic trans-Cleaving Ribozymes and RNAi against Arbitrary mRNA Disease Targets

PubMed Central

Yau, Edwin H.; Butler, Mark C.; Sullivan, Jack M.

2016-01-01

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N=G,C,A,U; H=C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization. PMID:27233447

Silver nanoparticles administered to chicken affect VEGFA and FGF2 gene expression in breast muscle and heart

NASA Astrophysics Data System (ADS)

Hotowy, Anna; Sawosz, Ewa; Pineda, Lane; Sawosz, Filip; Grodzik, Marta; Chwalibog, André

2012-07-01

Nanoparticles of colloidal silver (AgNano) can influence gene expression. Concerning trials of AgNano application in poultry nutrition, it is useful to reveal whether they affect the expression of genes crucial for bird development. AgNano were administered to broiler chickens as a water solution in two concentrations (10 and 20 ppm). After dissection of the birds, breast muscles and hearts were collected. Gene expression of FGF2 and VEGFA on the mRNA and protein levels were evaluated using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay methods. The results for gene expression in the breast muscle revealed changes on the mRNA level ( FGF2 was up-regulated, P < 0.05) but not on the protein level. In the heart, 20 ppm of silver nanoparticles in drinking water increased the expression of VEGFA ( P < 0.05), at the same time decreasing FGF2 expression both on the transcriptional and translational levels. Changes in the expression of these genes may lead to histological changes, but this needs to be proven using histological and immunohistochemical examination of tissues. In general, we showed that AgNano application in poultry feeding influences the expression of FGF2 and VEGFA genes on the mRNA and protein levels in growing chicken.

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.

Chitosan nanoparticle carrying small interfering RNA to platelet-derived growth factor B mRNA inhibits proliferation of smooth muscle cells in rabbit injured arteries.

PubMed

Xia, He; Jun, Ji; Wen-Ping, Ling; Yi-Feng, Pan; Xiao-Ling, Chen

2013-10-01

The purpose of this study was to elucidate the transfection of chitosan nanoparticle carrying small interfering RNA against platelet-derived growth factor B (PDGF-B) to inhibit the expression of PDGF-B mRNA and proliferation of smooth muscle cells. A rabbit iliac artery injury model was constructed. A small interfering RNA (siRNA) against PDGF-B mRNA expression vector was constructed and packaged by chitosan nanoparticle to transfect into the vascular smooth muscle cells (vSMCs) of balloon catheter-injured rabbit iliac artery wall, using a therapeutic ultrasound for the gene delivery. The experiment was divided into two groups: experimental group, denudation and nano-PDGF-B siRNA treated, and only single denudation as control. Effects of the siRNA on the expressions of proliferating cell nuclear antigen (PCNA) and PDGF-B mRNA by vSMCs and the proliferation of vSMCs were observed with the methods of routine pathological, immunohistochemical staining, in situ hybridization and morphometry. The nano siRNA against PDGF-B was successfully transfected. The nano siRNA significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal vSMCs. The local intimal thickness and area were also reduced remarkably. In conclusion, transfection of chitosan nanoparticle carrying siRNA against PDGF-B mRNA could inhibit proliferation of vSMCs in the rabbit iliac artery injury model. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Sequential changes in luminal microflora and mucosal cytokine expression during developing of colitis in HLA-B27/beta2-microglobulin transgenic rats.

PubMed

Hata, K; Andoh, A; Sato, H; Araki, Y; Tanaka, M; Tsujikawa, T; Fujiyama, Y; Bamba, T

2001-11-01

Transgenic rats expressing HLA-B27 and human beta2-microglobulin (HLA-B27 rats) spontaneously develop chronic colitis resembling human inflammatory bowel disease. We investigated the sequential changes in the luminal bacterial flora and mucosal cytokine mRNA expression in this model. HLA-B27 rats were maintained in a specific pathogen-free environment, and luminal microflora was evaluated by standard bacterial culture technique. The expression of mucosal cytokine mRNA was analysed by RT-PCR methods. Clinical symptoms of colitis appeared at 8 weeks of age. The total number of obligate anaerobes was higher than those of facultative anaerobes during the experimental period. At 6 weeks of age, the colonization of Bacteroides spp., Bifidobacterium spp. and Lactobacillus spp. was already detectable at high concentrations, whereas Clostridium spp. and Eubacterium spp. were not detected. The expression of proinflammatory cytokines (IL-Ibeta, IL-8 and TNF-alpha) appeared at 8 weeks of age, and these were detectable until 17 weeks. A similar pattern was observed in the expression of Th1 cytokines (IL-2, IL-12 and IFN-gamma). On the other hand, the expression of Th2 cytokines (IL-4, IL-10 and TGF-beta) was weak. IL-4 mRNA expression was weakly detectable only at 6 and 8 weeks of age. The expression of IL-10 and TGF-beta mRNA was scarcely detectable throughout the experimental period. The development of colitis may be mediated by both the predominant expression of Th1 cytokines and the weakness of Th2 cytokine expression in the mucosa. The colonization of anaerobic bacteria, especially Bacteroides spp., may be initiating and promoting these cytokine responses.

[Correlation between the mRNA expression of tissue inhibitor of metalloproteinase-1 and apparent diffusion coefficient on diffusion-weighted imaging in rats' liver fibrosis].

PubMed

Zhan, Yuefu; Liang, Xianwen; Han, Xiangjun; Chen, Jianqiang; Zhang, Shufang; Tan, Shun; Li, Qun; Wang, Xiong; Liu, Fan

2017-02-28

To explore the correlation between the apparent diffusion coefficient (ADC) and mRNA expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in different stages of liver fibrosis in rats.
 Methods: A model of liver fibrosis in rats was established by intraperitoneal injection of high-fat diet combined with porcine serum. After drug administration for 4 weeks, 48 rats served as a model group and 12 rats served as a control group, then they underwent diffusion weighted imaging (DWI) scanning. The value of ADC was calculated at b value=800 s/mm2. The rats were sacrificed and carried out pathologic examination after DWI scanning immediately. The mRNA expression of TIMP-1 was detected by real time-polymerase chain reaction (RT-PCR). The rats of hepatic fibrosis were also divided into a S0 group (n=4), a S1 group (n=11), a S2 group (n=12), a S3 group (n=10), and a S4 group (n=9) according to their pathological stage. The value of ADC and the expression of TIMP-1 mRNA among the different stage groups of liver fibrosis were compared, and the correlation between ADC and the TIMP-1 mRNA were analyzed.
 Results: The ADC value and the TIMP-1 mRNA expression were significantly different between the control group and the liver fibrosis group (F=46.54 and 53.87, P<0.05). There were significant differences in the value of ADC between every two groups (all P<0.05), except the control group vs the S1 group, the S1 group vs the S2 group, and the S2 group vs the S3 group (all P>0.05). For the comparison of TIMP-1 mRNA, there was no significant difference between the S1 group and the S2 group, the S3 group and the S4 group (both P>0.05). There were significant differences among the rest of the groups (all P<0.05). Rank correlation analysis showed that there was a negative correlation between the ADC value and the TIMP-1 mRNA expression (r=-0.76, P<0.01).
 Conclusion: When the value of ADC decreases in the progress of rats' liver fibrosis, the mRNA expression of TIMP-1 increases gradually, and there is a negative correlation between them.

Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression.

PubMed

Son, Seung-Myoung; Ha, Sang-Yun; Yoo, Hae-Yong; Oh, Dongryul; Kim, Seok-Jin; Kim, Won-Seog; Ko, Young-Hyeh

2017-01-01

The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells.

PubMed

Di Fulvio, Mauricio; Lauf, Peter K; Shah, Shalin; Adragna, Norma C

2003-05-01

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.

Effect of anti-sense oligodeoxynucleotides homeobox B2 on the proliferation and expression of primary human umbilical vein endothelial cells.

PubMed

Liu, Xusheng; Zhang, Xiaoqi

2002-02-01

To explore the effect of homeobox B2 (HOXB2) anti sense oligodeoxynucleotides (asodn) on the proliferation and expression of primary human umbilical vein endothelial cells (HUVECs). Various concentrations of HOXB2 asodn modified by thiophosphate transfected the induction of liposome into HUVECs. MTT a nd RT-PCR methods were employed to determine the effect of different conc ent rations of asodn on the endothelial proliferation and the expression level of HOXB2 mRNA. After the transfection of HOXB2 asodn, the endothelial proliferation was inhibited in a dose-dependent fashion. Simultaneously, the expression of HOXB2 mRNA decreased significantly. HOXB2 plays an important role in the proliferation of endothelia.

RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section using laser microdissection--a technical report.

PubMed

Kimura, Yurika; Kubo, Sachiho; Koda, Hiroko; Shigemoto, Kazuhiro; Sawabe, Motoji; Kitamura, Ken

2013-08-01

Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection. Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone. COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases. We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

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.

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

PubMed Central

2014-01-01

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

Increased expression of matrix metalloproteinase-10, nerve growth factor and substance P in the painful degenerate intervertebral disc

PubMed Central

Richardson, Stephen M; Doyle, Paul; Minogue, Ben M; Gnanalingham, Kanna; Hoyland, Judith A

2009-01-01

Introduction Matrix metalloproteinases (MMPs) are known to be involved in the degradation of the nucleus pulposus (NP) during intervertebral disc (IVD) degeneration. This study investigated MMP-10 (stromelysin-2) expression in the NP during IVD degeneration and correlated its expression with pro-inflammatory cytokines and molecules involved in innervation and nociception during degeneration which results in low back pain (LBP). Methods Human NP tissue was obtained at postmortem (PM) from patients without a history of back pain and graded as histologically normal or degenerate. Symptomatic degenerate NP samples were also obtained at surgery for LBP. Expression of MMP-10 mRNA and protein was analysed using real-time polymerase chain reaction and immunohistochemistry. Gene expression for pro-inflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-α), nerve growth factor (NGF) and the pain-associated neuropeptide substance P were also analysed. Correlations between MMP-10 and IL-1, TNF-α and NGF were assessed along with NGF with substance P. Results MMP-10 mRNA was significantly increased in surgical degenerate NP when compared to PM normal and PM degenerate samples. MMP-10 protein was also significantly higher in degenerate surgical NP samples compared to PM normal. IL-1 and MMP-10 mRNA demonstrated a significant correlation in surgical degenerate samples, while TNF-α was not correlated with MMP-10 mRNA. NGF was significantly correlated with both MMP-10 and substance P mRNA in surgical degenerate NP samples. Conclusions MMP-10 expression is increased in the symptomatic degenerate IVD, where it may contribute to matrix degradation and initiation of nociception. Importantly, this study suggests differences in the pathways involved in matrix degradation between painful and pain-free IVD degeneration. PMID:19695094

Stimulation of Synthesis and Release of Brain-Derived Neurotropic Factor (BDNF) from Intestinal Smooth Muscle Cells by Substance P and Pituitary Adenylate Cyclase-Activating Peptide (PACAP)

PubMed Central

Al-Qudah, M.; Alkahtani, R.; Akbarali, H.I.; Murthy, K.S.; Grider, J.R.

2015-01-01

Background Brain-derived neurotrophic factor (BDNF) is a neurotrophin present in the intestine where it participates in survival and growth of enteric neurons, augmentation of enteric circuits, and stimulation of intestinal peristalsis and propulsion. Previous studies largely focused on the role of neural and mucosal BDNF. The expression and release of BDNF from intestinal smooth muscle and the interaction with enteric neuropeptides has not been studied in gut. Methods The expression and secretion of BDNF from smooth muscle cultured from rabbit longitudinal intestinal muscle in response to substance P and pituitary adenylate cyclase activating peptide (PACAP) was measured by western blot and ELISA. BDNF mRNA was measured by rt-PCR. Key Results The expression of BNDF protein and mRNA was greater in smooth muscle cells from the longitudinal muscle than from circular muscle layer. PACAP and substance P increased the expression of BDNF protein and mRNA in cultured longitudinal smooth muscle cells. PACAP and substance P also stimulated the secretion of BDNF from cultured longitudinal smooth muscle cells. Chelation of intracellular calcium with BAPTA prevented substance P-induced increase in BDNF mRNA and protein expression as well as substance P-induced secretion of BDNF. Conclusions & Inferences Neuropeptides known to be present in enteric neurons innervating the longitudinal layer increase the expression of BDNF mRNA and protein in smooth muscle cells and stimulate the release of BDNF. Considering the ability of BDNF to enhance smooth muscle contraction, this autocrine loop may partially explain the characteristic hypercontractility of longitudinal muscle in inflammatory bowel disease. PMID:26088546

Induction of B7-H1 expression by human cytomegalovirus in extravillous cytotrophoblast cells and role of MAPK pathway

PubMed Central

Gong, Wenrong; Zhao, Jianhua; Chen, Zhen; Lei, Lin; Luo, Lihua; Zhao, Xuehong; Xing, Hui; Chen, Suhua; Tu, Qisheng

2014-01-01

Objective: This paper is aimed at to evaluate B7-H1 expression as induced by human cytomegalovirus (HCMV) in extravillous cytotrophoblast cell line HPT-8 and possible underlying mechanism. Method: Real time PCR and flow cytometry were used to determine B7-H1 mRNA and protein before and after HCMV infection in HPT-8 cells. Western blot analysis was used to determine the level of MAPK phosphorylation in HPT-8 cell lines infected with HCMV. Results: 100TCID50 was found to be the most effective dose, capable of stimulating B7-H1 mRNA and protein expression in HPT-8 cells. When empty control group was considered to have a B7-H1 mRNA value of 1, B7-H1 mRNA was 4.32 in 100TCID50 group. In flow cytometry study, mean fluorescence intensity (MFI) of 100TCID50 group was 16.14, while empty control group was 1.34. Both mRNA and protein expression were found to be significantly increased (P<0.05) in 100TCID50 group compared to empty control group. The result of Western blot analysis showed increase in B7-H1 expression caused by the extracellular signaling that was related to ERK activation and the ERK inhibitor U0126 was found to reverse this increase. Conclusion: HCMV upregulates B7-H1 expression in human extravillous cytotrophoblast cell line HPT-8, which is related to MAPK activation. Our result would be helpful in finding better therapies against intrauterine HCMV infection. PMID:25225522

Expression of prostaglandin E receptor subtype EP4 in conjunctival epithelium of patients with ocular surface disorders: case-control study.

PubMed

Ueta, Mayumi; Sotozono, Chie; Yamada, Keiko; Yokoi, Norihiko; Inatomi, Tsutomu; Kinoshita, Shigeru

2012-01-01

To confirm the downregulation of PTGER4 mRNA in the conjunctiva of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and ocular cicatricial pemphigoid (OCP) patients and to examine the expression of its EP4 protein in the conjunctival epithelium of patients with various ocular surface disorders. Case-control study. We performed quantitative reverse transcription-PCR (RT-PCR) analysis of PTGER4 mRNA in conjunctival tissue sections from patients with SJS/TEN and OCP to confirm the downregulation of PTGER4 mRNA expression. We also analysed EP4 immunohistologically in other ocular surface disorders. Conjunctival tissues were obtained from patients undergoing surgical reconstruction of the ocular surface due to chemical eye burns, subacute SJS/TEN or chronic SJS/TEN, chronic OCP, severe graft versus host disease (GVHD) and from patients with Mooren's ulcers treated by resection of the inflammatory conjunctiva. The expression of PTGER4 mRNA and EP4 protein assessed by quantitative RT-PCR assay and immunohistological methods. PTGER4 mRNA was significantly lower in conjunctival tissues from SJS and OCP patients than in the control conjunctivochalasis samples. EP4 protein was detected in conjunctival epithelium from patients with chemical eye burn and in control conjunctival epithelium from patients with conjunctivochalasis. Its expression varied in conjunctival epithelium from patients with Mooren's ulcer. We did not detect EP4 immunoreactivity in conjunctival epithelium from patients with subacute SJS/TEN, severe GVHD, chronic SJS/TEN or OCP. The strong downregulation of EP4 expression in conjunctival epithelium from patients with OCP or SJS/TEN may be attributable to ocular surface inflammation.

Expression of prostaglandin E receptor subtype EP4 in conjunctival epithelium of patients with ocular surface disorders: case-control study

PubMed Central

Ueta, Mayumi; Sotozono, Chie; Yamada, Keiko; Yokoi, Norihiko; Inatomi, Tsutomu; Kinoshita, Shigeru

2012-01-01

Objectives To confirm the downregulation of PTGER4 mRNA in the conjunctiva of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and ocular cicatricial pemphigoid (OCP) patients and to examine the expression of its EP4 protein in the conjunctival epithelium of patients with various ocular surface disorders. Design Case-control study. Setting and participants We performed quantitative reverse transcription-PCR (RT-PCR) analysis of PTGER4 mRNA in conjunctival tissue sections from patients with SJS/TEN and OCP to confirm the downregulation of PTGER4 mRNA expression. We also analysed EP4 immunohistologically in other ocular surface disorders. Conjunctival tissues were obtained from patients undergoing surgical reconstruction of the ocular surface due to chemical eye burns, subacute SJS/TEN or chronic SJS/TEN, chronic OCP, severe graft versus host disease (GVHD) and from patients with Mooren's ulcers treated by resection of the inflammatory conjunctiva. Primary and secondary outcome measures The expression of PTGER4 mRNA and EP4 protein assessed by quantitative RT-PCR assay and immunohistological methods. Results PTGER4 mRNA was significantly lower in conjunctival tissues from SJS and OCP patients than in the control conjunctivochalasis samples. EP4 protein was detected in conjunctival epithelium from patients with chemical eye burn and in control conjunctival epithelium from patients with conjunctivochalasis. Its expression varied in conjunctival epithelium from patients with Mooren's ulcer. We did not detect EP4 immunoreactivity in conjunctival epithelium from patients with subacute SJS/TEN, severe GVHD, chronic SJS/TEN or OCP. Conclusions The strong downregulation of EP4 expression in conjunctival epithelium from patients with OCP or SJS/TEN may be attributable to ocular surface inflammation. PMID:23065448

Inferring microRNA regulation of mRNA with partially ordered samples of paired expression data and exogenous prediction algorithms.

PubMed

Godsey, Brian; Heiser, Diane; Civin, Curt

2012-01-01

MicroRNAs (miRs) are known to play an important role in mRNA regulation, often by binding to complementary sequences in "target" mRNAs. Recently, several methods have been developed by which existing sequence-based target predictions can be combined with miR and mRNA expression data to infer true miR-mRNA targeting relationships. It has been shown that the combination of these two approaches gives more reliable results than either by itself. While a few such algorithms give excellent results, none fully addresses expression data sets with a natural ordering of the samples. If the samples in an experiment can be ordered or partially ordered by their expected similarity to one another, such as for time-series or studies of development processes, stages, or types, (e.g. cell type, disease, growth, aging), there are unique opportunities to infer miR-mRNA interactions that may be specific to the underlying processes, and existing methods do not exploit this. We propose an algorithm which specifically addresses [partially] ordered expression data and takes advantage of sample similarities based on the ordering structure. This is done within a Bayesian framework which specifies posterior distributions and therefore statistical significance for each model parameter and latent variable. We apply our model to a previously published expression data set of paired miR and mRNA arrays in five partially ordered conditions, with biological replicates, related to multiple myeloma, and we show how considering potential orderings can improve the inference of miR-mRNA interactions, as measured by existing knowledge about the involved transcripts.

Effects of intrinsic aerobic capacity and ovariectomy on voluntary wheel running and nucleus accumbens dopamine receptor gene expression

PubMed Central

Park, Young-Min; Kanaley, Jill A.; Padilla, Jaume; Zidon, Terese; Welly, Rebecca J.; Will, Matthew J.; Britton, Steven L.; Koch, Lauren G.; Ruegsegger, Gregory N.; Booth, Frank W.; Thyfault, John P.; Vieira-Potter, Victoria J.

2016-01-01

Rats selectively bred for high (HCR) and low (LCR) aerobic capacity show a stark divergence in wheel running behavior, which may be associated with dopamine (DA) system in the brain. HCR possess greater motivation for voluntary running along with greater brain DA activity compared to LCR. We recently demonstrated that HCR are not immune to ovariectomy (OVX)-associated reductions in spontaneous cage (i.e. locomotor) activity. Whether HCR and LCR rats differ in their OVX-mediated voluntary wheel running response is unknown. PURPOSE To determine whether HCR are protected from OVX-associated reduction in voluntary wheel running. METHODS Forty female HCR and LCR rats (age ~27 weeks) had either SHM or OVX operations, and given access to a running wheel for 11 weeks. Weekly wheel running distance was monitored throughout the intervention. Nucleus accumbens (NAc) was assessed for mRNA expression of DA receptors at sacrifice. RESULTS Compared to LCR, HCR ran greater distance and had greater ratio of excitatory/inhibitory DA mRNA expression (both line main effects, P<0.05). Wheel running distance was significantly, positively correlated with the ratio of excitatory/inhibitory DA mRNA expression across animals. In both lines, OVX reduced wheel running (P<0.05). Unexpectedly, although HCR started with significantly greater voluntary wheel running, they had greater OVX-induced reduction in wheel running than LCR such that no differences were found 11 weeks after OVX between HCROVX and LCROVX (interaction, P<0.05). This significant reduction in wheel running in HCR was associated with an OVX-mediated reduction in the ratio of excitatory/inhibitory DA mRNA expression. CONCLUSION DA system in the NAc region may play a significant role in motivation to run in female rats. Compared to LCR, HCR rats run significantly more, which associates with greater ratio of excitatory/inhibitory DA mRNA expression. However, despite greater inherent motivation to run and an associated brain DA mRNA expression profile, these HCR rats are not protected against OVX-induced reduction in wheel running. The impairment in wheel running in HCR rats may be partially explained by their reduced ratio of excitatory/inhibitory DA receptor mRNA expression. PMID:27297873

Delayed expression of hpS2 and prolonged expression of CIP1/WAF1/SDI1 in human tumour cells irradiated with X-rays, fission neutrons or 1 GeV/nucleon Fe ions

NASA Technical Reports Server (NTRS)

Balcer-Kubiczek, E. K.; Zhang, X. F.; Harrison, G. H.; Zhou, X. J.; Vigneulle, R. M.; Ove, R.; McCready, W. A.; Xu, J. F.

1999-01-01

PURPOSE: Differences in gene expression underlie the phenotypic differences between irradiated and unirradiated cells. The goal was to identify late-transcribed genes following irradiations differing in quality, and to determine the RBE of 1 GeV/n Fe ions. MATERIALS AND METHODS: Clonogenic assay was used to determine the RBE of Fe ions. Differential hybridization to cDNA target clones was used to detect differences in expression of corresponding genes in mRNA samples isolated from MCF7 cells irradiated with iso-survival doses of Fe ions (0 or 2.5 Gy) or fission neutrons (0 or 1.2 Gy) 7 days earlier. Northern analysis was used to confirm differential expression of cDNA-specific mRNA and to examine expression kinetics up to 2 weeks after irradiation. RESULTS: Fe ion RBE values were between 2.2 and 2.6 in the lines examined. Two of 17 differentially expressed cDNA clones were characterized. hpS2 mRNA was elevated from 1 to 14 days after irradiation, whereas CIP1/WAF1/SDI1 remained elevated from 3 h to 14 days after irradiation. Induction of hpS2 mRNA by irradiation was independent of p53, whereas induction of CIP1/WAF1/SDI1 was observed only in wild-type p53 lines. CONCLUSIONS: A set of coordinately regulated genes, some of which are independent of p53, is associated with change in gene expression during the first 2 weeks post-irradiation.

Curcumin attenuates cardiomyocyte hypertrophy induced by high glucose and insulin via the PPARγ/Akt/NO signaling pathway.

PubMed

Chen, Rongchun; Peng, Xiaofeng; Du, Weimin; Wu, Yang; Huang, Bo; Xue, Lai; Wu, Qin; Qiu, Hongmei; Jiang, Qingsong

2015-05-01

To investigate the potential effect of curcumin on cardiomyocyte hypertrophy and a possible mechanism involving the PPARγ/Akt/NO signaling pathway in diabetes. The cardiomyocyte hypertrophy induced by high glucose (25.5mmol/L) and insulin (0.1μmol/L) (HGI) and the antihypertrophic effect of curcumin were evaluated in primary culture by measuring the cell surface area, protein content and atrial natriuretic factor (ANF) mRNA expression. The mRNA and protein expressions were assayed by reverse transcription PCR and Western blotting, whereas the NO concentration and endothelial NO synthase (eNOS) activity were determined using nitrate reduction and ELISA methods, respectively. The cardiomyocyte hypertrophy induced by HGI was characterized by increasing ANF mRNA expression, total protein content, and cell surface area, with downregulated mRNA and protein expressions of both PPARγ and Akt, which paralleled the declining eNOS mRNA expression, eNOS content, and NO concentration. The effects of HGI were inhibited by curcumin (1, 3, 10μmol/L) in a concentration-dependent manner. GW9662 (10μmol/L), a selective PPARγ antagonist, could abolish the effects of curcumin. LY294002 (20μmol/L), an Akt blocker, and N(G)-nitro-l-arginine-methyl ester (100μmol/L), a NOS inhibitor, could also diminish the effects of curcumin. The results suggested that curcumin supplementation can improve HGI-induced cardiomyocytes hypertrophy in vitro through the activation of PPARγ/Akt/NO signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Local Context Finder (LCF) reveals multidimensional relationships among mRNA expression profiles of Arabidopsis responding to pathogen infection

PubMed Central

Katagiri, Fumiaki; Glazebrook, Jane

2003-01-01

A major task in computational analysis of mRNA expression profiles is definition of relationships among profiles on the basis of similarities among them. This is generally achieved by pattern recognition in the distribution of data points representing each profile in a high-dimensional space. Some drawbacks of commonly used pattern recognition algorithms stem from their use of a globally linear space and/or limited degrees of freedom. A pattern recognition method called Local Context Finder (LCF) is described here. LCF uses nonlinear dimensionality reduction for pattern recognition. Then it builds a network of profiles based on the nonlinear dimensionality reduction results. LCF was used to analyze mRNA expression profiles of the plant host Arabidopsis interacting with the bacterial pathogen Pseudomonas syringae. In one case, LCF revealed two dimensions essential to explain the effects of the NahG transgene and the ndr1 mutation on resistant and susceptible responses. In another case, plant mutants deficient in responses to pathogen infection were classified on the basis of LCF analysis of their profiles. The classification by LCF was consistent with the results of biological characterization of the mutants. Thus, LCF is a powerful method for extracting information from expression profile data. PMID:12960373

Transcriptomics of mRNA and egg quality in farmed fish: Some recent developments and future directions.

PubMed

Sullivan, Craig V; Chapman, Robert W; Reading, Benjamin J; Anderson, Paul E

2015-09-15

Maternal mRNA transcripts deposited in growing oocytes regulate early development and are under intensive investigation as determinants of egg quality. The research has evolved from single gene studies to microarray and now RNA-Seq analyses in which mRNA expression by virtually every gene can be assessed and related to gamete quality. Such studies have mainly focused on genes changing two- to several-fold in expression between biological states, and have identified scores of candidate genes and a few gene networks whose functioning is related to successful development. However, ever-increasing yields of information from high throughput methods for detecting transcript abundance have far outpaced progress in methods for analyzing the massive quantities of gene expression data, and especially for meaningful relation of whole transcriptome profiles to gamete quality. We have developed a new approach to this problem employing artificial neural networks and supervised machine learning with other novel bioinformatics procedures to discover a previously unknown level of ovarian transcriptome function at which minute changes in expression of a few hundred genes is highly predictive of egg quality. In this paper, we briefly review the progress in transcriptomics of fish egg quality and discuss some future directions for this field of study. Copyright © 2015 Elsevier Inc. All rights reserved.

Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport

PubMed Central

Day, Pricilla E.; Ntani, Georgia; Crozier, Sarah R.; Mahon, Pam A.; Inskip, Hazel M.; Cooper, Cyrus; Harvey, Nicholas C.; Godfrey, Keith M.; Hanson, Mark A.; Lewis, Rohan M.; Cleal, Jane K.

2015-01-01

Introduction Maternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth. Methods RNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR. Results Increased placental LAT2 (p = 0.01), y + LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001). Conclusion A number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter and metabolic genes and maternal smoking, physical activity and diet raises the possibility that their effects are mediated in part through alterations in placental function. The observed changes in placental gene expression in relation to modifiable maternal factors are important as they could form part of interventions aimed at maintaining a healthy lifestyle for the mother and for optimal fetal development. PMID:26657885

Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells

PubMed Central

Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

2015-01-01

Background This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. Material/Methods HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. Results The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. Conclusions TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway. PMID:26551326

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

PubMed

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Stimulation of nuclear receptor REV-ERBs regulates tumor necrosis factor-induced expression of proinflammatory molecules in C6 astroglial cells

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

Morioka, Norimitsu, E-mail: mnori@hiroshima-u.ac.jp; Tomori, Mizuki; Zhang, Fang Fang

Under physiological conditions, astrocytes maintain homeostasis in the CNS. Following inflammation and injury to the CNS, however, activated astrocytes produce neurotoxic molecules such as cytokines and chemokines, amplifying the initial molecular-cellular events evoked by inflammation and injury. Nuclear receptors REV-ERBα and REV-ERBβ (REV-ERBs) are crucial in the regulation of inflammation- and metabolism-related gene transcription. The current study sought to elucidate a role of REV-ERBs in rat C6 astroglial cells on the expression of inflammatory molecules following stimulation with the neuroinflammatory cytokine tumor necrosis factor (TNF). Stimulation of C6 cells with TNF (10 ng/ml) significantly increased the mRNA expression of CCL2, interleukin-6more » (IL-6), inducible nitric oxide synthase (iNOS), and matrix metalloprotease (MMP)-9, but not fibroblast growth factor-2 (FGF-2), cyclooxygenase-2 (COX-2) and MMP-2. Treatment with either REV-ERB agonists GSK4112 or SR9009 significantly blocked TNF-induced upregulation of CCL2 mRNA and MMP-9 mRNA, but not IL-6 mRNA and iNOS mRNA expression. Furthermore, treatment with RGFP966, a selective histone deacetylase 3 (HDAC3) inhibitor, potently reversed the inhibitory effects of GSK4112 on TNF-induced expression of MMP-9 mRNA, but not CCL2 mRNA. Expression of Rev-erbs mRNA in C6 astroglial cells, primary cultured rat cortical and spinal astrocytes was confirmed by reverse transcription polymerase chain reaction. Together, the findings demonstrate an anti-inflammatory effect, downregulating of MMP-9 and CCL2 transcription, of astroglial REV-ERBs activation through HDAC3-dependent and HDAC3-independent mechanisms. - Highlights: • Rev-erbα mRNA and Rev-erbβ mRNA are expressed in C6 astroglial cells. • TNF increases the expression of CCL2, IL-6, MMP-9 and iNOS mRNA. • REV-ERB activation inhibits CCL2 mRNA and MMP-9 mRNA expression. • HDAC3 activity is involved in the inhibitory effect of REV-ERB on MMP-9 induction.« less

Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma

PubMed Central

2010-01-01

Background The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Methods Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. Results The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. Conclusions CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients. PMID:21040522

Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

PubMed

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

1989-01-01

In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

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

Hu, Zhi; Huang, Ge; Sadanandam, Anguraj

Introduction: HJURP (Holliday Junction Recognition Protein) is a newly discovered gene reported to function at centromeres and to interact with CENPA. However its role in tumor development remains largely unknown. The goal of this study was to investigate the clinical significance of HJURP in breast cancer and its correlation with radiotherapeutic outcome. Methods: We measured HJURP expression level in human breast cancer cell lines and primary breast cancers by Western blot and/or by Affymetrix Microarray; and determined its associations with clinical variables using standard statistical methods. Validation was performed with the use of published microarray data. We assessed cell growthmore » and apoptosis of breast cancer cells after radiation using high-content image analysis. Results: HJURP was expressed at higher level in breast cancer than in normal breast tissue. HJURP mRNA levels were significantly associated with estrogen receptor (ER), progesterone receptor (PR), Scarff-Bloom-Richardson (SBR) grade, age and Ki67 proliferation indices, but not with pathologic stage, ERBB2, tumor size, or lymph node status. Higher HJURP mRNA levels significantly decreased disease-free and overall survival. HJURP mRNA levels predicted the prognosis better than Ki67 proliferation indices. In a multivariate Cox proportional-hazard regression, including clinical variables as covariates, HJURP mRNA levels remained an independent prognostic factor for disease-free and overall survival. In addition HJURP mRNA levels were an independent prognostic factor over molecular subtypes (normal like, luminal, Erbb2 and basal). Poor clinical outcomes among patients with high HJURP expression werevalidated in five additional breast cancer cohorts. Furthermore, the patients with high HJURP levels were much more sensitive to radiotherapy. In vitro studies in breast cancer cell lines showed that cells with high HJURP levels were more sensitive to radiation treatment and had a higher rate of apoptosis than those with low levels. Knock down of HJURP in human breast cancer cells using shRNA reduced the sensitivity to radiation treatment. HJURP mRNA levels were significantly correlated with CENPA mRNA levels. Conclusions: HJURP mRNA level is a prognostic factor for disease-free and overall survival in patients with breast cancer and is a predictive biomarker for sensitivity to radiotherapy.« less

Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes

PubMed Central

Yu, Qin Ping; Feng, Ding Yuan; He, Xiao Jun; Wu, Fan; Xia, Min Hao; Dong, Tao; Liu, Yi Hua; Tan, Hui Ze; Zou, Shi Geng; Zheng, Tao; Ou, Xian Hua; Zuo, Jian Jun

2017-01-01

Objective This study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula’s extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes. Methods In a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction. Results The final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber I, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type I fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC I, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC I and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor γ coactivator-1α and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by 4 μg/mL and 20 μg/mL TCMF water extraction (p<0.05). Both 1 μg/mL and 5 μg/mL of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC I mRNA expression in porcine myocytes was decreased by 5 μg/mL TCMF water extraction (p<0.05). Porcine myocyte MyHC I and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by 5 μg/mL TCMF ethyl acetate extraction (p<0.05). MyHC I and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by 1 μg/mL TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by 5 μg/mL TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by 1 μg/mL in a TCMF petroleum ether extraction (p<0.05). Conclusion These results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes. PMID:28728382

Acute Stress and Chronic Stress Change Brain-Derived Neurotrophic Factor (BDNF) and Tyrosine Kinase-Coupled Receptor (TrkB) Expression in Both Young and Aged Rat Hippocampus

PubMed Central

Shi, Shou-Sen; Shao, Shu-hong; Yuan, Bang-ping; Pan, Fang

2010-01-01

Purpose The purpose of this study is to explore the dynamic change of brain-derived neurotrophic factor (BDNF) mRNA, protein, and tyrosine kinase-coupled receptor (TrkB) mRNA of the rat hippocampus under different stress conditions and to explore the influence of senescence on the productions expression. Materials and Methods By using forced-swimming in 4℃ cold ice water and 25℃ warm water, young and aged male rats were randomly divided into acute stress (AS) and chronic mild repeated stress (CMRS) subgroups, respectively. BDNF productions and TrkB mRNA in the hippocampus were detected by using Western-blotting and reverse transcription-polymerase chain reaction (RT-PCR), separately, at 15, 30, 60, 180, and 720 min after the last stress session. Results The short AS induced a significant increase in BDNF mRNA and protein in both age groups, but the changes in the young group were substantially greater than those of the aged group (p < 0.005). The CMRS resulted in a decrease in BDNF mRNA and protein, but a significant increase in TrkB mRNA in both young and age groups. The expression of BDNF mRNA and protein in the AS groups were higher than in the CMRS groups at 15, 30, and 60 min after stress. Conclusion The results indicated that the up/down-regulation of BDNF and TrkB were affected by aging and the stimulus paradigm, which might reflect important mechanisms by which the hippocampus copes with stressful stimuli. PMID:20635439

[Effect of clinical doses of Realgar-Indigo Naturalis formula and large-dose of realgar on CYP450s of rat liver].

PubMed

Xu, Huan-Hua; Wang, Mei-Xi; Tan, Hong-Ling; Wang, Yu-Guang; Tang, Xiang-Lin; Xiao, Cheng-Rong; Li, Hua; Gao, Yue; Ma, Zeng-Chun

2017-02-01

To investigate the effect of clinical dose of Realgar-Indigo Naturais formula (RIF) and large-dose of Realgar on main drug-metabolizing enzymes CYP450s of rat liver, as well as its regulatory effect on mRNA expression. Wistar rats were administrated orally with tested drugs for 14 days. A Cocktail method combined with HPLC-MS/MS was used in the determination of 4 cytochrome P450 isozymes (CYP1A2, CYP2B, CYP3A and CYP2C) in liver of the rats, and the mRNA expression levels of the above subtypes were detected by real-time fluorescent quantitative PCR. The results showed that RIF can significantly induce CYP1A2 and CYP2B enzyme activity, and inhibit CYP3A enzyme activity. This result was consistent with the mRNA expression. However, its single compound showed weaker or even contrary phenomenon. Different doses of Realgar also showed significant inconsistencies on CYP450 enzymes activity and mRNA expression. These phenomena may be relevant with RIF compatibility synergies or toxicity reduction. The results can also prompt drug interactions when RIF is combined with other medicines in application. Copyright© by the Chinese Pharmaceutical Association.

Research on apoptotic signaling pathways of recurrent spontaneous abortion caused by dysfunction of trophoblast infiltration.

PubMed

Sun, Q; Zhang, X-L

2017-07-01

To study the apoptotic signaling pathways of recurrent spontaneous abortion caused by dysfunction of trophoblast infiltration. 60 patients with recurrent spontaneous abortion and normal abortion were selected consecutively as recurrent spontaneous abortion group and abortion group, respectively. Villous tissues were obtained and cell apoptosis was observed under a microscope; terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (Tunel) method was used to test the apoptosis rate. In situ hybridization was adopted to detect expressions of Fas messenger RNA (Fas mRNA) and Fas ligand messenger RNA (FasL mRNA); expression of Fas, FasL and protein kinase C (PKC) were examined by immunohistochemistry at protein level; fluorescence spectrophotometer was used to test Ca2+ level. The apoptosis rate, expressions of Fas mRNA, and FasL mRNA, expressions of Fas and FasL proteins, as well as Ca2+ level, were significantly higher in the recurrent spontaneous abortion group than in abortion group. The level of PKC protein was significantly lower in recurrent spontaneous abortion group than in abortion group (p<0.05). Fas-FasL and PKC signaling pathways, as well as Ca2+, may mediate the dysfunction of trophoblast infiltration, which leads to recurrent spontaneous abortion.

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

Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi

In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression ofmore » GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.« less

[Piperine regulates glucose metabolism disorder in HepG2 cells of insulin resistance models via targeting upstream target of AMPK signaling pathway].

PubMed

Wan, Chun-Ping; Wei, Ya-Gai; Li, Xiao-Xue; Zhang, Li-Jun; Yang, Rui; Bao, Zhao-Ri-Ge-Tu

2017-02-01

To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and р-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway. Copyright© by the Chinese Pharmaceutical Association.

Effect of 50 Hz electric field in diacylglycerol acyltransferase mRNA expression level and plasma concentration of triacylglycerol, free fatty acid, phospholipid and total cholesterol

PubMed Central

2012-01-01

Background The effects of exposure to a 50 Hz electric field (EF) on plasma level of triacylglycerol, free fatty acids, total cholesterol and phospholipid and mRNA expression level of diacylglycerol acyltransferase (DGAT) 1 and 2 in liver and intestines from C57BL/6 J mice were studied. Methods The test was based on comparison between mice post treated with 50 Hz EF of 45 kV/m intensity for 30 min per day for 11 days or without EF. DGATs mRNA expression was analyzed by real-time quantitative polymerase chain reaction. Results There was no difference in the gene expression level of DGAT1 in liver and intestines. The DGAT2 gene expression level in liver derived from mice treated with EF was significantly lower than those in the control (P < 0.001). Both plasma total cholesterol (P < 0.01) and phospholipid (P < 0.05) in the group exposed to EF were lower than those in the control, but there was no difference in triacylglycerol or free fatty acid levels. Conclusion Exposure to 50 Hz EF decrease the plasma levels of total cholesterol and phospholipids, and downregulated DGAT2 mRNA expression in liver. The mechanisms for the effects of EF on lipid metabolism are not well understand yet, but altered DGAT2 activity may be involved. PMID:22676350

Effect of biliary drainage on inducible nitric oxide synthase, CD14 and TGR5 expression in obstructive jaundice rats

PubMed Central

Wang, Zi-Kai; Xiao, Jian-Guo; Huang, Xue-Fei; Gong, Yi-Chun; Li, Wen

2013-01-01

AIM: To investigate the effect of biliary drainage on inducible nitric oxide synthase (iNOS), CD14 and TGR5 expression in rats with obstructive jaundice (OJ). METHODS: Male adult Sprague-Dawley rats were randomly assigned to four groups: OJ, sham operation (SH), internal biliary drainage (ID) and external biliary drainage (ED). Rat models were successfully established by two operations and succumbed for extraction of Kupffer cells (KCs) and liver tissue collection on the 8th and 15th day. KCs were isolated by in situ hepatic perfusion and digested with collagen IV, density gradient centrifuged by percoll reagent and purified by cell culture attachment. The isolated KCs were cultured with the endotoxin lipopolysaccharide (LPS) with and without the addition of ursodeoxycholic acid (UDCA). The expression of iNOS, CD14 and bile acid receptor-TGR5 protein in rat liver tissues was determined by immunohistochemistry. The expression of iNOS and CD14 messenger RNA (mRNA) on the isolated KCs was detected by reverse transcription polymerase chain reaction (PCR) and the TGR5 mRNA level in KCs was measured by real-time quantitative PCR. RESULTS: The iNOS protein was markedly expressed in the liver of OJ rats, but rare expressed in SH rats. After relief of OJ, the iNOS expression was decidedly suppressed in the ID group (ID vs OJ, P < 0.01), but obviously increased in rats of ED (ED vs OJ, P = 0.004). When interfered only with LPS, the expression of iNOS mRNA by KCs was increased in the OJ group compared with the SH group (P = 0.004). After relief of biliary obstruction, the iNOS mRNA expression showed slight changes in the ED group (ED vs OJ, P = 0.71), but dropped in the ID group (ID vs OJ, P = 0.001). Compared with the simple intervention with LPS, the expressions of iNOS mRNA were significantly inhibited in all four groups after interfered with both LPS and UDCA (P < 0.01, respectively). After bile duct ligation, the CD14 protein expression in rat liver was significantly strengthened (OJ vs SH, P < 0.01), but the CD14 mRNA level by KCs was not up-regulated (OJ vs SH, P = 0.822). After relieving the OJ, the expression of CD14 protein was reduced in the ID group (ID vs OJ, P < 0.01), but not reduced in ED group (ED vs OJ, P = 0.591). And then the CD14 mRNA expression was aggravated by ED (ED vs OJ, P < 0.01), but was not significantly different between the ID group and the SH and OJ groups (ID vs SH, P = 0.944; ID vs OJ, P = 0.513, respectively). The expression of TGR5 protein and mRNA increased significantly in OJ rats (OJ vs SH, P = 0.001, respectively). After relief of OJ, ID could reduce the expression of TGR5 protein and mRNA to the levels of SH group (ID vs SH, P = 0.22 and P = 0.354, respectively), but ED could not (ED vs SH, P = 0.001, respectively). CONCLUSION: ID could be attributed to the regulatory function of activation of KCs and release of inflammatory mediators. PMID:23613625

O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

PubMed Central

Kreth, Simone; Thon, Niklas; Eigenbrod, Sabina; Lutz, Juergen; Ledderose, Carola; Egensperger, Rupert; Tonn, Joerg C.; Kretzschmar, Hans A.; Hinske, Ludwig C.; Kreth, Friedrich W.

2011-01-01

Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined. PMID:21365007

Expression and clinical significance of ATM and PUMA gene in patients with colorectal cancer.

PubMed

Xiong, Hui; Zhang, Jiangnan

2017-12-01

The expression of ataxia-telangiectasia mutated (ATM) and p53 upregulated modulator of apoptosis (PUMA) genes in patients with colorectal cancer were investigated, to explore the correlation between the expression of ATM and PUMA and tumor development, to evaluate the clinical significance of ATM and PUMA in the treatment of colorectal cancer. Quantitative real-time PCR was used to detect the expression of ATM and PUMA in tumor tissue and adjacent healthy tissue of 67 patients with colorectal cancer and in normal colorectal tissue of 33 patients with colorectal polyps at mRNA level. The expression level of ATM mRNA in colorectal cancer tissues was significantly higher than that in normal mucosa tissues and adjacent non-cancerous tissue (P≤0.05), while no significant differences in expression level of ATM mRNA were found between normal mucosa tissues and adjacent noncancerous tissue (P=0.07). There was a negative correlation between the expression of ATM mRNA and the degree of differentiation of colorectal cancer (r= -0.312, P=0.013), while expression level of ATM mRNA was not significantly correlated with the age, sex, tumor invasion, lymph node metastasis or clinical stage (P>0.05). Expression levels of PUMA mRNA in colorectal cancer tissues, adjacent noncancerous tissue and normal tissues were 0.68±0.07, 0.88±0.04 and 1.76±0.06, respectively. Expression level of PUMA mRNA in colorectal cancer tissues and adjacent noncancerous tissue was significantly lower than that in normal colorectal tissues (P<0.05). The results showed that ATM mRNA is expressed abnormally in colorectal cancer tissues. Expression of PUMA gene in colorectal carcinoma is downregulated, and is negatively correlated with the occurrence of cancer.

Comparison of PD-L1 mRNA Expression Measured with the CheckPoint Typer® Assay with PD-L1 Protein Expression Assessed with Immunohistochemistry in Non-small Cell Lung Cancer.

PubMed

Erber, Ramona; Stöhr, Robert; Herlein, Stefanie; Giedl, Claudia; Rieker, Ralf Joachim; Fuchs, Florian; Ficker, Joachim H; Hartmann, Arndt; Veltrup, Elke; Wirtz, Ralph M; Brueckl, Wolfgang M

2017-12-01

Immunohistochemical (IHC) assessment of programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) has become important since the development of anti-PD-1/-PD-L1 directed drugs. Various PD-L1 antibodies and cut-offs have been used in different trials to predict response to these drugs, thus comparison of those studies is difficult. We compared PD-L1 mRNA expression measured by RT-qPCR with PD-L1 protein expression evaluated by IHC. Moreover, we investigated the impact of different tumour tissue acquisition methods on the reliability of PD-L1 measurement techniques. NSCLC cases (N=22), including n=9 mediastinal lymph node biopsies acquired by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and n=5 metastases, were evaluated prospectively for PD-L1 protein on tumor cells (TC) and immune cells (IC) using E1L3N and 28-8 antibodies and PD-L1 mRNA using the CheckPoint TYPER® assay. In primary NSCLC tissues, agreement between PD-L1 mRNA and TC staining using the 28-8 antibody was excellent (ĸ=0.85, p=0.0002). Comparing both PD-L1 antibodies against each other showed a kappa value of 0.58 (p=0.0106). In EBUS-TBNA, PD-L1 mRNA correlated perfectly with the 28-8 antibody (ĸ=1.0, p=0.0023). PD-L1 mRNA levels significantly differed when comparing 28-8 TC staining of tumours >49% with 1-49% and 0% (p=0.0040; p=0.0081, respectively). In metastatic lesions, differences between PD-L1 mRNA and IHC became apparent (ĸ=0.2, p=0.2525). Testing of PD-L1 mRNA and 28-8 IHC showed an excellent agreement in NSCLC samples including mediastinal lymph node biopsies. Since PD-L1 expression in >50% TC detected by 28-8 IHC can be reliably detected by RT-qPCR, quantitative PD-L1 mRNA determination should be considered as an alternative to IHC as there is no interobserver variability in RNA results. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology.

PubMed

Shen, L P; Sheridan, P; Cao, W W; Dailey, P J; Salazar-Gonzalez, J F; Breen, E C; Fahey, J L; Urdea, M S; Kolberg, J A

1998-06-01

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Lack of Change in Markers of Presynaptic Terminal Abundance Alongside Subtle Reductions in Markers of Presynaptic Terminal Plasticity in Prefrontal Cortex of Schizophrenia Patients

PubMed Central

Fung, Samantha J.; Sivagnanasundaram, Sinthuja; Shannon Weickert, Cynthia

2010-01-01

Background Reduced synaptic connectivity in frontal cortex may contribute to schizophrenia symptoms. While altered mRNA and protein expression of various synaptic genes has been found, discrepancies between studies mean a generalisable synaptic pathology in schizophrenia has not been identified. Methods We determined if mRNAs encoding presynaptic proteins enriched in inhibitory [vesicular GABA transporter (VGAT) and complexin 1] and/or excitatory [vesicular glutamate transporter (VGluT1) and complexin 2] terminals are altered in the dorsolateral prefrontal cortex of subjects with schizophrenia (n=37 patients, n=37 controls). We also measured mRNA expression of markers associated with synaptic plasticity/neurite outgrowth [growth associated protein 43 (GAP43) and neuronal navigators 1 and 2 (NAV1 and NAV2)]; and mRNAs of other synaptic-associated proteins previously implicated in schizophrenia: dysbindin and vesicle-associated membrane protein (VAMP1) mRNAs using quantitative RT-PCR. Results No significant changes in complexin 1, VGAT, complexin 2, VGluT1, dysbindin, NAV2, or VAMP1 mRNA expression were found, however we observed reduced expression of mRNAs associated with plasticity/cytoskeletal modification (GAP43 and NAV1) in schizophrenia. Although dysbindin mRNA did not differ in schizophrenia compared to controls, dysbindin mRNA positively correlated with GAP-43 and NAV1 in schizophrenia, but not in controls, suggesting low levels of dysbindin may be linked to reduced plasticity in the disease state. No relationships between three dysbindin genetic polymorphisms previously associated with dysbindin mRNA levels were found. Conclusions A reduction in the plasticity of synaptic terminals supports the hypothesis that reduced modifiability of synaptic terminals may contribute to neuropathology and working memory deficits in schizophrenia. PMID:21145444

Validation of internal controls for gene expression analysis in the intestine of rats infected with Hymenolepis diminuta.

PubMed

Hoque, Tafazzal; Bhogal, Meetu; Boghal, Meetu; Webb, Rodney A

2007-12-01

The non-invasive parasitic cestode Hymenolepis diminuta induces hypertrophy, hyperplasia and other changes in cell activity in the intestine of rats which are indicated in the expression of mRNA. We have investigated various house-keeping genes (GAPDH, beta-actin, 18S and HPRT) and other internal controls (total RNA/unit biomass, total RNA/unit length of intestine) to validate gene expression in the rat intestine after cestode infection and drug-induced neuromodulation. Variation in GAPDH, beta-actin, 18S and HPRT expression was observed in rat jejunal tissue according to treatment. Total RNA/unit length of intestine was found to be the most suitable internal control for normalizing target gene mRNA expression in both infected and/or drug-induced rat intestine. This normalization method may be applied to studies of gene expression levels in intestinal tissue where hypertrophy, hyperplasia, rapid growth and cell differentiation generally occur.

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

PubMed Central

Sugihara, T; Kobori, A; Imaeda, H; Tsujikawa, T; Amagase, K; Takeuchi, K; Fujiyama, Y; Andoh, A

2010-01-01

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa α-chain linked to a 70-kDa β-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of IκBα. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17. PMID:20089077

Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

PubMed

Huerta-Rivas, A; Pérez-García, G; González-Espinosa, C; Meneses, A

2010-01-01

Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues. Copyright 2009 Elsevier Inc. All rights reserved.

Altered PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression in ejaculated spermatozoa of men with impaired sperm characteristics.

PubMed

Giebler, Maria; Greither, Thomas; Müller, Lisa; Mösinger, Carina; Behre, Hermann M

2018-01-01

In about half the cases of involuntary childlessness, a male infertility factor is involved. The PIWI-LIKE genes, a subclade of the Argonaute protein family, are involved in RNA silencing and transposon control in the germline. Knockout of murine Piwi-like 1 and 2 homologs results in complete infertility in males. The aim of this study was to analyze whether the mRNA expression of human PIWI-LIKE 1-4 genes is altered in ejaculated spermatozoa of men with impaired sperm characteristics. Ninety male participants were included in the study, among which 47 were with normozoospermia, 36 with impaired semen characteristics according to the World Health Organization (WHO) manual, 5 th edition, and 7 with azoospermia serving as negative control for the PIWI-LIKE 1-4 mRNA expression in somatic cells in the ejaculate. PIWI-LIKE 1-4 mRNA expression in the ejaculated spermatozoa of the participants was measured by quantitative real-time PCR. In nonazoospermic men, PIWI-LIKE 1-4 mRNA was measurable in ejaculated spermatozoa in different proportions. PIWI-LIKE 1 (100.0%) and PIWI-LIKE 2 (49.4%) were more frequently expressed than PIWI-LIKE 3 (9.6%) and PIWI-LIKE 4 (15.7%). Furthermore, a decreased PIWI-LIKE 2 mRNA expression showed a significant correlation with a decreased sperm count (P = 0.022) and an increased PIWI-LIKE 1 mRNA expression with a decreased progressive motility (P = 0.048). PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression exhibited a significant association with impaired sperm characteristics and may be a useful candidate for the evaluation of the impact of PIWI-LIKE 1-4 mRNA expression on male infertility.

Inactivation of parkin by promoter methylation correlated with lymph node metastasis and genomic instability in nasopharyngeal carcinoma.

PubMed

Ni, Haifeng; Zhou, Zhen; Jiang, Bo; Yuan, Xiaoyang; Cao, Xiaolin; Huang, Guangwu; Li, Yong

2017-03-01

This study aimed to investigate the inactivation of the parkin gene by promoter methylation and its relationship with genome instability in nasopharyngeal carcinoma. Parkin was considered as a tumor suppressor gene in various types of cancers. However, its role in nasopharyngeal carcinoma is unexplored. Genomic instabilities were detected in nasopharyngeal carcinoma tissues by the random amplified polymorphic DNA. The methylation-specific polymerase chain reaction, semi-quantitative reverse transcription polymerase chain reaction, and immunohistochemical analysis were used to detect methylation and mRNA and protein expression of parkin in 54 cases of nasopharyngeal carcinoma tissues and 16 cases of normal nasopharyngeal epithelia tissues, and in 5 nasopharyngeal carcinoma cell lines (CNE1, CNE2, TWO3, C666, and HONE1) and 1 normal nasopharyngeal epithelia cell line (NP69). mRNA expression of parkin in CNE1 and CNE2 was analyzed before and after methyltransferase inhibitor 5-aza-2-deoxycytidine treatment. The relationship between promoter methylation and mRNA expression, demethylation and mRNA expression, and mRNA and protein expression of the gene and clinical factors and genomic instabilities were analyzed. The mRNA and protein expression levels were significantly reduced in 54 cases of human nasopharyngeal carcinoma compared with 16 cases of normal nasopharyngeal epithelia. Parkin-methylated cases showed significantly lower mRNA and protein expression levels compared with unmethylated cases. After 5-aza-2-deoxycytidine treatment, parkin mRNA expression was restored in CNE1 and CNE2; 92.59% (50/54) of nasopharyngeal carcinoma demonstrated genomic instability. Parkin is frequently inactivated by promoter methylation, and its mRNA and protein expression correlate with lymph node metastasis and genomic instability. Parkin deficiency probably promotes tumorigenesis in nasopharyngeal carcinoma.

Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.

PubMed

Hu, Yan; Liu, Hongxiang; Song, Chi; Xu, Wenjuan; Ji, Gaige; Zhu, Chunhong; Shu, Jingting; Li, Huifang

2015-03-15

In this study, the ontogeny of body and liver weight and the pattern of related gene mRNA expression in the hypothalamus-pituitary growth axis (HPGA) of two different duck breeds (Anas platyrhynchos domestica) were compared during embryonic and post-hatch development. Duck hypothalamic growth hormone release hormone (GHRH), somatostatin (SS), pituitary growth hormone (GH), liver growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-1) mRNA were first detected on the 13th embryonic day. During early duck development, SS maintained a lower expression status, whereas the other four genes exhibited highly significant variations in an age-specific manner. Highly significant breed specificity was observed with respect to hepatic IGF-1 mRNA expression, which showed a significant breed-age interaction effect. Compared with previous studies on chickens, significant species differences were observed regarding the mRNA expression of bird embryonic HPGA-related genes. During early development, highly significant breed and age specificity were observed with respect to developmental changes in body and liver weight, and varying degrees of significant linear correlation were found between these performances and the mRNA expression of HPGA-related genes in the duck HPGA. These results suggest that different genetic backgrounds may lead to differences in duck growth and HPGA-related gene mRNA expression, and the differential mRNA expression of related genes in the duck HPGA may be particularly important in the early growth of ducks. Furthermore, hepatic IGF-1 mRNA expression presented highly significant breed specificity, and evidence suggests the involvement of hepatic IGF-1 in mediating genetic effects on embryo and offspring growth in ducks. Copyright © 2015 Elsevier B.V. All rights reserved.

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

PubMed

Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

2016-05-01

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

MYC, FBXW7 and TP53 copy number variation and expression in Gastric Cancer

PubMed Central

2013-01-01

Background MYC deregulation is a common event in gastric carcinogenesis, usually as a consequence of gene amplification, chromosomal translocations, or posttranslational mechanisms. FBXW7 is a p53-controlled tumor-suppressor that plays a role in the regulation of cell cycle exit and reentry via MYC degradation. Methods We evaluated MYC, FBXW7, and TP53 copy number, mRNA levels, and protein expression in gastric cancer and paired non-neoplastic specimens from 33 patients and also in gastric adenocarcinoma cell lines. We also determined the invasion potential of the gastric cancer cell lines. Results MYC amplification was observed in 51.5% of gastric tumor samples. Deletion of one copy of FBXW7 and TP53 was observed in 45.5% and 21.2% of gastric tumors, respectively. MYC mRNA expression was significantly higher in tumors than in non-neoplastic samples. FBXW7 and TP53 mRNA expression was markedly lower in tumors than in paired non-neoplastic specimens. Moreover, deregulated MYC and FBXW7 mRNA expression was associated with the presence of lymph node metastasis and tumor stage III-IV. Additionally, MYC immunostaining was more frequently observed in intestinal-type than diffuse-type gastric cancers and was associated with MYC mRNA expression. In vitro studies showed that increased MYC and reduced FBXW7 expression is associated with a more invasive phenotype in gastric cancer cell lines. This result encouraged us to investigate the activity of the gelatinases MMP-2 and MMP-9 in both cell lines. Both gelatinases are synthesized predominantly by stromal cells rather than cancer cells, and it has been proposed that both contribute to cancer progression. We observed a significant increase in MMP-9 activity in ACP02 compared with ACP03 cells. These results confirmed that ACP02 cells have greater invasion capability than ACP03 cells. Conclusion In conclusion, FBXW7 and MYC mRNA may play a role in aggressive biologic behavior of gastric cancer cells and may be a useful indicator of poor prognosis. Furthermore, MYC is a candidate target for new therapies against gastric cancer. PMID:24053468

Simultaneous measurement of cerebral blood flow and mRNA signals: pixel-based inter-modality correlational analysis.

PubMed

Zhao, W; Busto, R; Truettner, J; Ginsberg, M D

2001-07-30

The analysis of pixel-based relationships between local cerebral blood flow (LCBF) and mRNA expression can reveal important insights into brain function. Traditionally, LCBF and in situ hybridization studies for genes of interest have been analyzed in separate series. To overcome this limitation and to increase the power of statistical analysis, this study focused on developing a double-label method to measure local cerebral blood flow (LCBF) and gene expressions simultaneously by means of a dual-autoradiography procedure. A 14C-iodoantipyrine autoradiographic LCBF study was first performed. Serial brain sections (12 in this study) were obtained at multiple coronal levels and were processed in the conventional manner to yield quantitative LCBF images. Two replicate sections at each bregma level were then used for in situ hybridization. To eliminate the 14C-iodoantipyrine from these sections, a chloroform-washout procedure was first performed. The sections were then processed for in situ hybridization autoradiography for the probes of interest. This method was tested in Wistar rats subjected to 12 min of global forebrain ischemia by two-vessel occlusion plus hypotension, followed by 2 or 6 h of reperfusion (n=4-6 per group). LCBF and in situ hybridization images for heat shock protein 70 (HSP70) were generated for each rat, aligned by disparity analysis, and analyzed on a pixel-by-pixel basis. This method yielded detailed inter-modality correlation between LCBF and HSP70 mRNA expressions. The advantages of this method include reducing the number of experimental animals by one-half; and providing accurate pixel-based correlations between different modalities in the same animals, thus enabling paired statistical analyses. This method can be extended to permit correlation of LCBF with the expression of multiple genes of interest.

Sequential expression of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury

PubMed Central

Li, Zhiqiang; Shu, Qingming; Li, Lingzhi; Ge, Maolin; Zhang, Yongliang

2014-01-01

Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott's method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury. PMID:25206921

Integrated analysis of miRNA and mRNA expression data identifies multiple miRNAs regulatory networks for the tumorigenesis of colorectal cancer.

PubMed

Xu, Peng; Wang, Junhua; Sun, Bo; Xiao, Zhongdang

2018-06-15

Investigating the potential biological function of differential changed genes through integrating multiple omics data including miRNA and mRNA expression profiles, is always hot topic. However, how to evaluate the repression effect on target genes integrating miRNA and mRNA expression profiles are not fully solved. In this study, we provide an analyzing method by integrating both miRNAs and mRNAs expression data simultaneously. Difference analysis was adopted based on the repression score, then significantly repressed mRNAs were screened out by DEGseq. Pathway analysis for the significantly repressed mRNAs shows that multiple pathways such as MAPK signaling pathway, TGF-beta signaling pathway and so on, may correlated to the colorectal cancer(CRC). Focusing on the MAPK signaling pathway, a miRNA-mRNA network that centering the cell fate genes was constructed. Finally, the miRNA-mRNAs that potentially important in the CRC carcinogenesis were screened out and scored by impact index. Copyright © 2018 Elsevier B.V. All rights reserved.

[Modified Cheng's Juanbi Decoction downregulates expression of prostaglandin E receptor 4 in synovial tissue in rats with adjuvant arthritis].

PubMed

Xu, Xia; Cheng, Hui; Cao, Jian; DU, Huan; Meng, Qingwei; Guo, Mengyuan

2017-06-01

Objective To investigate the effect of modified Cheng's Juanbi Decoction on the expression of prostaglandin E receptor 4 (PTGER4), the T cell receptor in the synovial tissues, in rats with adjuvant arthritis (AA). Methods A rat model of AA was established by subcutaneous injection of Freund's complete adjuvant at the vola pedis combined with ice-water bath and blowing. The degree of joint swelling and arthritis index were determined in each group. The quantitative real-time PCR was performed to assess the effect of modified Cheng's Juanbi Decoction on the mRNA expression of PTGER4in the synovial tissue. Results Cheng's Juanbi Decoction significantly alleviated the damage in the joints and synovial tissues in the AA rats. High-dose (the content of crude drug: 4 g/mL) Cheng's Juanbi Decoction significantly reduced the mRNA expression of PTGER4 in the synovial tissues. Conclusion Cheng's Juanbi Decoction can reduce the level of PTGER4 mRNA in the synovial tissue in AA rats.

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

PubMed Central

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

1989-01-01

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

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

PubMed Central

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection. PMID:25664019

Adiabatic reduction of a model of stochastic gene expression with jump Markov process.

PubMed

Yvinec, Romain; Zhuge, Changjing; Lei, Jinzhi; Mackey, Michael C

2014-04-01

This paper considers adiabatic reduction in a model of stochastic gene expression with bursting transcription considered as a jump Markov process. In this model, the process of gene expression with auto-regulation is described by fast/slow dynamics. The production of mRNA is assumed to follow a compound Poisson process occurring at a rate depending on protein levels (the phenomena called bursting in molecular biology) and the production of protein is a linear function of mRNA numbers. When the dynamics of mRNA is assumed to be a fast process (due to faster mRNA degradation than that of protein) we prove that, with appropriate scalings in the burst rate, jump size or translational rate, the bursting phenomena can be transmitted to the slow variable. We show that, depending on the scaling, the reduced equation is either a stochastic differential equation with a jump Poisson process or a deterministic ordinary differential equation. These results are significant because adiabatic reduction techniques seem to have not been rigorously justified for a stochastic differential system containing a jump Markov process. We expect that the results can be generalized to adiabatic methods in more general stochastic hybrid systems.

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

PubMed

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

2001-11-30

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

Upregulation of NLRP3 Inflammasome in the Tears and Ocular Surface of Dry Eye Patients

PubMed Central

Wu, Jihong; Chen, Ling; Wang, Yan

2015-01-01

Purpose To evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye. Methods We recruited 54 patients with Sjögren’s syndrome dry eye (SSDE), 50 patients with non-Sjögren’s syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting. Results NLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p<0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p<0.05) and NSSDE (relative 1.32-fold upregulation, p<0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold (p<0.001) in SSDE and 2.13-fold (p<0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE (p=0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p=0.001) and NSSDE (relative 2.05-fold upregulation, p=0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE (p<0.001) and NSSDE (p<0.05) groups. Conclusions In the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye. PMID:25962072

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

PubMed

Bugeon, Stéphane; de Chevigny, Antoine; Boutin, Camille; Coré, Nathalie; Wild, Stefan; Bosio, Andreas; Cremer, Harold; Beclin, Christophe

2017-11-01

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. © 2017. Published by The Company of Biologists Ltd.

[ABIN1 is not involved in imatinib upregulating A20 to inhibit the activation of NF-κB pathway in Jurkat T cells].

PubMed

Chen, Qian; Wang, Senlin; Lin, Chen; Chen, Shaohua; Zhao, Xiaoling; Li, Yangqiu

2017-05-01

Objective To investigate the effect of imatinib (IM) on the expressions of A20-binding inhibitor of NF-κB1 (ABIN1) and A20 in Jurkat T cells. Methods Jurkat T cells were treated with 25, 50 and 100 nmol/L IM for 24 hours. The mRNA and protein levels of ABIN1, A20 and NF-κB were detected by real-time quantitative PCR and Western blotting. Results IM significantly inhibited both mRNA and protein levels of ABIN1 and NF-κB, but raised the mRNA and protein levels of A20; while phorbol 12-myristate 13-acetate/ionomycin increased the expression levels of ABIN1 and A20 mRNA and protein. Conclusion IM could upregulate A20 protein to inhibit the activation of NF-κB pathway in Jurkat T cells, which was independent of the ABIN1 protein.

Cytokine expression in response to root repair agents.

PubMed

Oliveira, R R; Tavares, W L F; Reis, A L; Silva, V A; Vieira, L Q; Ribeiro Sobrinho, A P

2018-05-06

To evaluate the expression of TNF-α, IL-6, IFN-γ, TGF-β, IL-4, IL-10, RANKL, RANK and OPG on mouse calvarial bone treated with MTA, Geristore ® and Emdogain ® . Bone wounds were made on the heads of C57BL/6 mice, breaking the periosteum and the cortical surface of the calvaria. Each repair agent was inserted into sectioned Eppendorf microtubes and placed on the bone wound, and soft tissues were sutured. At 14 and 21 days, animals were sacrificed and the treated region was dissected. The calvaria bone was removed, and RNA was extracted. mRNA expression of the aforementioned cytokines was assessed using real-time PCR. Data were analysed by nonparametric methods, including the Mann-Whitney and Kruskal-Wallis tests (p<0.05). Following treatment with Emdogain ® and MTA, mRNA expression of RANKL, RANK and OPG increased significantly (p <0.05) between days 14 to 21. Geristore ® did not alter the basal expression of these mediators during the same period of evaluation. While treatment with Emdogain ® did cause a significant increase in TNF-α mRNA expression between days 14 and 21 (p <0.05), treatment with MTA did not alter the basal expression of this cytokine at either experimental time point. However, TNF-α mRNA expression was down-regulated significantly at day 21 (p <0.05) when Geristore ® was applied. A significant increase in the mRNA expression of IL-6, TGF-β, IL-10, IL-4 and IFN-γ was observed with Emdogain ® and MTA treatment between days 14 to 21, whereas Geristore ® reduced significantly the expression of IL-6, TGF-β and IL-4 (p <0.05). The clinical indication of these repair agents depends on the root resorption diagnosis. While MTA and Emdogain ® induce a pro- and anti-inflammatory response early and late, respectively, Geristore ® was not associated with an inflammatory reaction when compared to both repair agents. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

THE M-RNA, EXPRESSION OF SERCA2 AND NCX1 IN THE PROCESS OF PHARMACOLOGICAL CELL PROTECTION IN EXPERIMENTAL ACUTE PANCREATITIS INDUCED BY TAUROCHOLATE.

PubMed

Vasques, Enio Rodrigues; Cunha, José Eduardo Monteiro; Kubrusly, Marcia Saldanha; Coelho, Ana Maria; Sanpietri, Sandra N; Nader, Helena B; Tersariol, Ivarne L S; Lima, Marcelo A; Chaib, Eleazar; D'Albuquerque, Luiz Augusto Carneiro

2018-06-21

Intracellular calcium overload is known to be a precipitating factor of pancreatic cell injury in acute pancreatitis (AP). Intracellular calcium homeostasis depends of Plasmatic Membrane Calcium ATPase (PMCA), Sarcoplasmic Endothelial Reticulum Calcium ATPase 2 (SERCA 2) and the Sodium Calcium Exchanger (NCX1). The antioxidant melatonin (Mel) and Trisulfate Disaccharide (TD) that accelerates NCX1 action could reduce the cell damage determined by the AP. To evaluate m-RNA expressions of SERCA2 and NCX1 in acute pancreatitis induced by sodium taurocholate in Wistar rats pre-treated with melatonin and/or TD. Wistar rats were divided in groups: 1) without AP; 2) AP without pre-treatment; 3) AP and Melatonin; 4) AP and TD; 5) AP and Melatonin associated to TD. Pancreatic tissue samples were collected for detection of SERCA2 and NCX1 m-R NA levels by polymerase chain reaction (PCR). Increased m-RNA expression of SERCA2 in the melatonin treated group, without increase of m-RNA expression of the NCX1. The TD did not affect levels of SERCA2 and NCX1 m-RNA expressions. The combined melatonin and TD treatment reduced the m-RNA expression of SERCA2. The effect of melatonin is restricted to increased m-RNA expression of SERCA2. Although TD does not affect gene expression, its action in accelerating calcium exchanger function can explain the slightest expression of SERCA2 m-RNA when associated with Melatonin, perhaps by a joint action of drugs with different and but possibly complementary mechanisms.

Molecular cloning, characterization, and expression analysis of ghrelin and cholecystokinin in the pigeon (Columba livia).

PubMed

Xie, P; Wan, X P; Bu, Z; Zou, X T

2016-11-01

Ghrelin and cholecystokinin (CCK) are multifunctional peptides. In the current study, complete sequences of ghrelin (800 bp) and CCK (739 bp) were firstly cloned in Columba livia by using rapid amplification of cDNA ends (RACE) method. The open reading frames of ghrelin (351bp) and CCK (393bp) encoded 116 amino acids and 130 amino acids, respectively. Sequence comparison indicated that pigeon ghrelin and CCK shared high identity with those reported in other avian species. Quantitative real-time PCR analysis found that ghrelin and CCK mRNAs expressed in three intestinal segments of pigeon during development. Both ghrelin and CCK showed generally higher expressions at days posthatch than embryonic periods regardless of intestinal segments. In duodenum and ileum, the expressions of ghrelin and CCK mRNA reached the peak values at 8 d posthatch. Jejunum CCK mRNA level increased linearly after hatching, and reached the highest point at posthatch 28 d. Based on documented effects of long chain fatty acids (LCFAs) on pigeon ghrelin and CCK expression were also investigated in vitro. Higher concentrations (50 μM or 250 μM) of linoleic acid, α-linolenic acid or arachidonic acid can significantly increase ghrelin mRNA level in pigeon jejunum. However, for oleic acid, the induction of ghrelin gene expressions needed a lower concentration (5 μM). 5 μM of linoleic acid, α-linolenic acid or arachidonic acid and 250 μM palmitic acid repressed CCK expression significantly. A higher concentration (250 μM) of oleic acid or α-linolenic acid can up-regulate CCK mRNA level significantly. Our results indicated that ghrelin and CCK may act key functions in pigeon intestine development and their expressions could be regulated by LCFAs. © 2016 Poultry Science Association Inc.

Neuropeptide gene expression in brain is differentially regulated by midbrain dopamine neurons.

PubMed

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

1990-01-01

In situ hybridization was used to study the expression of prepro-neuropeptide Y (NPY), preprosomatostatin (SOM), preprotachykinin (PPT) and preprocholecystokinin (CCK) mRNA in caudate-putamen and frontoparietal cortex of rat brain with unilateral lesion of midbrain dopamine neurons. Neurons expressing NPY and SOM mRNA showed a similar distribution and the expression of both NPY and SOM appears to be regulated by dopamine in a similar fashion. Following a dopamine deafferentation, the numerical density of both NPY and SOM mRNA producing neurons almost doubled in the lesioned caudate-putamen with no change in the average grain density over positive neurons. Hence, in the intact caudate-putamen dopamine appears to suppress expression of these two neuropeptide genes leading to an activation of both NPY and SOM mRNA expression in many non- or low-expressing neurons when the level of dopamine is decreased. In the fronto-parietal cortex, on the other hand, dopamine appears to stimulate NPY and SOM gene expression. Thus, in the absence of dopamine about half of the NPY positive neurons disappeared. However, for SOM the number of positive neurons did not change, but rather most positive neurons appeared to have down-regulated their SOM mRNA expression. No evidence was found for a change in CCK mRNA expression by the dopamine deafferentation, while PPT mRNA expression decreased in the deafferented caudate-putamen. Consequently, dopamine exerts dissimilar effects on the expression of different neuropeptide genes, that in turn do not respond in the same way in different brain regions.

Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes

PubMed Central

Shvedova, Anna A.; Yanamala, Naveena; Kisin, Elena R.; Khailullin, Timur O.; Birch, M. Eileen; Fatkhutdinova, Liliya M.

2016-01-01

Background As the application of carbon nanotubes (CNT) in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT) could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans. Methods In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8), were compared with expression profiles of non-exposed (n = 7) workers (e.g., professional and/or technical staff) from the same manufacturing facility. Results Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs. Conclusion This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers of MWCNT exposures in humans. PMID:26930275

Effects of Tumor Necrosis Factor α (TNF-α) and Interleukina 10 (IL-10) on Intercellular Cell Adhesion Molecule-1 (ICAM-1) and Cluster of Differentiation 31 (CD31) in Human Coronary Artery Endothelial Cells.

PubMed

Xue, Mingming; Qiqige, Chaolumen; Zhang, Qi; Zhao, Haixia; Su, Liping; Sun, Peng; Zhao, Pengwei

2018-06-27

BACKGROUND The aim of this study was to investigate the effects of TNF-α and IL-10 on the expression of ICAM-1 and CD31 in human coronary artery endothelial cells (HCAEC). MATERIAL AND METHODS HCAEC was treated with 0, 2.5 μg/l, 5 μg/l, and 10 μg/l of TNF-α for 2 h, 6 h, and 10 h, and with 0 μg/l, 10 μg/l, 100 μg/l, and 200 μg/l of IL-10 for 5 h, 10 h and 15 h, respectively. RNA inference of TNF-αR was performed with siRNA. Real-time PCR, Western blot analysis, and ELSA were used to detect the mRNA level and protein level of ICAM-1 and CD31. RESULTS TNF-α significantly increased the mRNA and protein expression of ICAM-1 (P<0.05), and 2.5 μg/l TNF-α had the most obvious effect. RNAi of TNF-aR reduced the induction of TNF-α on the mRNA and protein expression of ICAM-1 (P<0.05). TNF-α significantly decreased the CD31 in the supernatant (P<0.05), and 2.5 μg/l TNF-a had the most obvious effect. IL-10 significantly decreased the ICAM-1 protein level. IL-10 decreased the mRNA expression and the protein expression of CD31. The effect on mRNA was not significant (P>0.05), while the effect on the protein expression was significant (P<0.05). CONCLUSIONS TNF-α and IL-10 treatment can affect the expression of ICAM-1 and CD31 in HCAEC.

Alteration in cardiac uncoupling proteins and eNOS gene expression following high-intensity interval training in favor of increasing mechanical efficiency

PubMed Central

Fallahi, Ali Asghar; Shekarfroush, Shahnaz; Rahimi, Mostafa; Jalali, Amirhossain; Khoshbaten, Ali

2016-01-01

Objective(s): High-intensity interval training (HIIT) increases energy expenditure and mechanical energy efficiency. Although both uncoupling proteins (UCPs) and endothelial nitric oxide synthase (eNOS) affect the mechanical efficiency and antioxidant capacity, their effects are inverse. The aim of this study was to determine whether the alterations of cardiac UCP2, UCP3, and eNOS mRNA expression following HIIT are in favor of increased mechanical efficiency or decreased oxidative stress. Materials and Methods: Wistar rats were divided into five groups: control group (n=12), HIIT for an acute bout (AT1), short term HIIT for 3 and 5 sessions (ST3 and ST5), long-term training for 8 weeks (LT) (6 in each group). The rats of the training groups were made to run on a treadmill for 60 min in three stages: 6 min running for warm-up, 7 intervals of 7 min running on treadmill with a slope of 5° to 20° (4 min with an intensity of 80-110% VO2max and 3 min at 50-60% VO2max), and 5-min running for cool-down. The control group did not participate in any exercise program. Rats were sacrificed and the hearts were extracted to analyze the levels of UCP2, UCP3 and eNOS mRNA by RT-PCR. Results: UCP3 expression was increased significantly following an acute training bout. Repeated HIIT for 8 weeks resulted in a significant decrease in UCPs mRNA and a significant increase in eNOS expression in cardiac muscle. Conclusion: This study indicates that Long term HIIT through decreasing UCPs mRNA and increasing eNOS mRNA expression may enhance energy efficiency and physical performance. PMID:27114795

The effects of a single bout pilates exercise on mRNA expression of bone metabolic cytokines in osteopenia women

PubMed Central

Kim, Chang Sun; Kim, Ji Yeon; Kim, Hyo Jin

2014-01-01

[Purpose] The purpose of this study was to examine the effect of a single bout pilates exercise on mRNA expression of bone metabolic cytokines in elderly osteopenia women. [Methods] We selected 11 people of elderly osteopenia women and loaded a single bout pilates exercise about RPE 10-14 level. The blood samples were collected before, immediately after and 60 minute after pilates exercise, then examined calcium metabolic markers in serum and extracted peripheral blood mononuclear cell (PBMC) from whole blood and confirmed mRNA expression of bone metabolic cytokines from PBMC. To clarify the changes during exercise, we designed repeated measure ANOVA as the control group to perform blood sampling without exercise. [Results] As a result, serum P showed significant interaction effect between group and time (p<.001), the pilates exercise group decreased about 9% at immediately after exercise and 13% during recovery after exercise (p<.05), while the control group showed a tendency to increase. Serum CK also showed a significant interaction between group and time (p<.05), the pilates group significantly increased at immediately after exercise and during recovery after exercise (p<.05) but the control group didn’t have changes. TNF-α and IL-6 mRNA expression in PBMC was significantly increased in the pilates group (p<.01, p<.05), although INF-γ mRNA expression didn’t show statistically significant difference, it tended to increase in the pilates group (NS). [Conclusion] These results suggested that a single bout pilates exercise of elderly osteopenia women cause hypophosphatemia with temporary muscle damage, and it leading high turnover bone metabolic state with to activate both of bone formation and bone resorption. PMID:25566441

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes.

PubMed

Sakairi, Toru; Abe, Yoshifusa; Jat, Parmijit S; Kopp, Jeffrey B

2010-10-01

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.

[Effects of macrophages on the biological behaviors and VEGF receptor mRNA, Hoxb2 mRNA, and integrin alphavbeta3 expressions of vascular endothelial cells].

PubMed

Liu, Liang; Liu, Xu-Sheng; Zhang, Xiao-Qi; Ming, Jia; Xu, Hui; Cheng, Tian-Min

2005-02-01

To explore the mechanism by which macrophages regulate angiogenesis by co-culturing human umbilical vein endothelial cells (ECV-304) with human macrophage cells (U937) stimulated by concanavalin A (ConA). Monolayer ECV-304 cells growing to 60% confluence were co-cultured with 1 x 10(5)/ml U937 cells in the presence or absence of ConA (ConA+U937+ECV-304 and U937+ECV-304 groups, respectively), with non-treated and ConA-treated ECV-304 cells serving as the control groups (ECV-304 and ConA+ECV-304 groups, respectively). Forty-eight h later, U937 cells were removed from the cell co-culture for examining changes in DNA synthesis of ECV-304 cells with (3)H-TdR incorporation assay and for cell cycle analysis with flow cytometry. RT-PCR was employed to assess the influence of macrophages stimulated by ConA on the expression of the target genes. With immunofluorescent method, the changes in the expression of integrin receptor alphavbeta3 of ECV-304 were determined. A significant increase in S-phase ECV-304 cells with enhanced DNA synthesis was observed after co-culture of the cells with ConA-stimulated U937 cells (P<0.01), which also resulted in significant up-regulation of the expressions of KDR mRNA (0.879+/-0.003), Hoxb2 mRNA (0.947+/-0.003) and integrin receptor alphavbeta3 (10.26+/-1.73). Macrophages can accelerate the proliferation, migration and adhesion of the vascular endothelial cells to the basilar membrane matrix by affecting their cell cycle, DNA synthesis, expression of KDR mRNA, Hoxb2 mRNA and integrin alphavbeta3, so as to modulate the angiogenetic process of the latter cells.

Smad4-Mediated Signaling Inhibits Intestinal Neoplasia by Inhibiting Expression of β-Catenin

PubMed Central

Freeman, Tanner J.; Smith, J. Joshua; Chen, Xi; Washington, M. Kay; Roland, Joseph T.; Means, Anna L.; Eschrich, Steven A.; Yeatman, Timothy J.; Deane, Natasha G.; Beauchamp, R. Daniel

2012-01-01

Background & Aims Mutational inactivation of APC is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of β-catenin, a mediator of Wnt signaling. CRC progression also involves inactivation of signaling via transforming growth factor (TGF)β and bone morphenogenic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 loss on levels of β-catenin mRNA and Wnt signaling. Methods We used microarray analysis to associate levels of Smad4 and β-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APCΔ1638/+) and (APCΔ1638/+)x(K19CreERT2Smad4lox/lox) mice using laser-capture microdissection. Results In human CRC samples, reduced levels of Smad4 correlated with increased levels of β-catenin mRNA. In Smad4-depleted cell lines, levels of β-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the β-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of β-catenin mRNA. In mice with heterozygous disruption of Apc(APCΔ1638/+), Smad4-deficient intestinal adenomas had increased levels of β-catenin mRNA and expression of Wnt target genes, compared with adenomas from APCΔ1638/+mice that expressed Smad4. Conclusions Transcription of β-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among TGF-β, BMP, and Wnt signaling pathways in CRC progression. PMID:22115830

Predictive value of PD-L1 based on mRNA level in the treatment of stage IV melanoma with ipilimumab.

PubMed

Brüggemann, C; Kirchberger, M C; Goldinger, S M; Weide, B; Konrad, A; Erdmann, M; Schadendorf, D; Croner, R S; Krähenbühl, L; Kähler, K C; Hafner, C; Leisgang, W; Kiesewetter, F; Dummer, R; Schuler, G; Stürzl, M; Heinzerling, L

2017-10-01

PD-L1 is established as a predictive marker for therapy of non-small cell lung cancer with pembrolizumab. Furthermore, PD-L1 positive melanoma has shown more favorable outcomes when treated with anti-PD1 antibodies and dacarbazine compared to PD-L1 negative melanoma. However, the role of PD-L1 expression with regard to response to checkpoint inhibition with anti-CTLA-4 is not clear, yet. In addition, the lack of standardization in the immunohistochemical assessment of PD-L1 makes the comparison of results difficult. In this study, we investigated the PD-L1 gene expression with a new fully automated technique via RT-PCR and correlated the findings with the response to the anti-CTLA-4 antibody ipilimumab. Within a retrospective multi-center trial, PD-L1 gene expression was evaluated in 78 melanoma patients in a total of 111 pre-treatment tumor samples from 6 skin cancer centers and analyzed with regard to response to ipilimumab. For meaningful statistical analysis, the cohort was enriched for responders with 30 responders and 48 non-responders. Gene expression was assessed by quantitative RT-PCR after extracting mRNA from formalin-fixed paraffin embedded tumor tissue and correlated with results from immunohistochemical (IHC) stainings. The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab. The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab.

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

PubMed

Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

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

Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O.

1988-11-01

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA andmore » DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.« less

(+/-)-3-[4-(2-dimethylamino-1-methylethoxy)-phenyl]-1H-pyrazolo[3,4- B]pyridine-1-acetic acid (Y-25510) stimulates production of IL-1 beta and IL-6 at the level of messenger RNA expression in cultured human monocytes.

PubMed

Kusuhara, H; Komatsu, H; Hisadome, M; Ikeda, Y

1996-12-01

(+/-)-3-[4-(2-Dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3, 4-b]pyridine-1-acetic acid (Y-25510) stimulated the mRNA expression for interleukin-1 beta (IL-1 beta), and enhanced the expression induced by lipopolysaccharide (LPS) in cultured human peripheral blood mononuclear cells (PBMC) and THP-1 cells, a cell-line derived from human monocytic leukemia. Y-25510 also stimulated the mRNA expression for IL-6 in both types of the cells, however, the stimulation required the presence of LPS. In THP-1 cells, the stimulation of IL-1 beta mRNA expression by Y-25510 was suppressed by cycloheximide, an inhibitor of protein synthesis. This phenomenon indicates that the stimulation requires de norv protein synthesis. In contrast, the stimulation of mRNA expression for IL-6 by Y-25510 was not suppressed by cycloheximide but suppressed by N alpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of nuclear transcription factor-kappa B (NF-kappa B) activation, in the presence of LPS, suggesting that the stimulation requires NF-kappa activation. These results demonstrate that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms. Dexamethasone suppressed the LPS-induced expression of mRNA for IL-1 beta and IL-6 in THP-1 cells, whereas the drug never suppressed the mRNA expression for these cytokines in the presence of Y-25510. The result indicates that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms from those of LPS.

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.

The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

PubMed

Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

2016-09-01

Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. © 2016 Hollerer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

PubMed Central

Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P.; Forst, Christian V.; Roth, Michael G.; Levy, David E.; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A.

2012-01-01

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. PMID:22312003

Restriction of the Xenopus DEADSouth mRNA to the primordial germ cells is ensured by multiple mechanisms.

PubMed

Yamaguchi, Takeshi; Kataoka, Kensuke; Watanabe, Kenji; Orii, Hidefumi

2014-02-01

DEADSouth mRNA encoding the RNA helicase DDX25 is a component of the germ plasm in Xenopus laevis. We investigated the mechanisms underlying its specific mRNA expression in primordial germ cells (PGCs). Based on our previous findings of several microRNA miR-427 recognition elements (MREs) in the 3' untranslated region of the mRNA, we first examined whether DEADSouth mRNA was degraded by miR-427 targeting in somatic cells. Injection of antisense miR-427 oligomer and reporter mRNA for mutated MREs revealed that DEADSouth mRNA was potentially degraded in somatic cells via miR-427 targeting, but not in PGCs after the mid-blastula transition (MBT). The expression level of miR-427 was very low in PGCs, which probably resulted in the lack of miR-427-mediated degradation. In addition, the DEADSouth gene was expressed zygotically after MBT. Thus, the predominant expression of DEADSouth mRNA in the PGCs is ensured by multiple mechanisms including zygotic expression and prohibition from miR-427-mediated degradation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Preprotachykinin A mRNA expression in the rat brain during development.

PubMed

Brené, S; Lindefors, N; Friedman, W J; Persson, H

1990-12-15

Expression of preprotachykinin A (PPT-A) mRNA was analyzed by northern blots using mRNA prepared from rat brain at 12 different developmental stages ranging from embryonic day 15 (E15) to adult. A single PPT-A mRNA of 1.3 kb was detected throughout development. PPT-A mRNA was detected as early as E15 and an approximately 3-fold increase occurred at birth. This amount remained until 3 weeks of age when the level increased, reaching a peak at 5 weeks of age. Adult amounts were approximately 3-fold higher than the levels at birth. The distribution of PPT-A mRNA-expressing cells in rat brain was studied by in situ hybridization on sections from embryonic day 20, postnatal days 4 and 7 as well as adult. Cells expressing PPT-A mRNA were detected in the forebrain at all 4 ages analyzed. However, the hybridization pattern and the labeling intensity varied in different brain regions during development. In cingulate cortex, intense labeling was seen in numerous cells at embryonic day 20 and postnatal days 4 and 7, whereas in the adult cingulate cortex only a few scattered labeled cells were observed. In frontoparietal cortex labeled cells were found from postnatal day 4 to adult, with the highest density of labeled cells at P7. Developmental differences in both the distribution of PPT-A mRNA-expressing cells and the level of PPT-A mRNA expression were also found in caudate-putamen, lateral hypothalamus and amygdala. Thus, our results show several changes in PPT-A mRNA expression during ontogeny, indicating a region and time-specific regulation of PPT-A mRNA expression during brain maturation.

Airways in smooth muscle α-actin null mice experience a compensatory mechanism that modulates their contractile response.

PubMed

Shardonofsky, Felix R; Moore, Joan; Schwartz, Robert J; Boriek, Aladin M

2012-03-01

We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (μg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.

Lower FOXO3 mRNA expression in granulosa cells is involved in unexplained infertility.

PubMed

Yamamoto, Hikaru; Yamashita, Yoshiki; Saito, Natsuho; Hayashi, Atsushi; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

2017-06-01

The aim of this study was to investigate whether FOXO1 and FOXO3 mRNA expression in granulosa cells is the cause of unexplained infertility. Thirty-one patients aged <40 years (13 with unexplained infertility and 18 with male partner infertility as a control group) whose serum anti-Müllerian hormone level was >0.5 ng/μL were enrolled in the study. All patients underwent oocyte retrieval under a short protocol from June 2012 to October 2013. Real-time PCR was carried out using mRNA extracted from granulosa cells retrieved from mature follicles. We compared FOXO1 and FOXO3 mRNA expression ratios in granulosa cells between the unexplained infertility group and the male infertility group. The relation between FOXO1 and FOXO3 mRNA expression ratios in granulosa cells and assisted reproduction technology clinical outcome was also examined. FOXO3 mRNA expression ratio was significantly lower in the unexplained infertility group than in the male infertility group. Moreover, FOXO3 mRNA expression ratio showed a positive correlation with both the number of retrieved oocytes and serum anti-Müllerian hormone level. A positive correlation was also identified between FOXO1 mRNA expression and total dose of hMG. As well, the number of retrieved oocytes in the unexplained infertility group was statistically lower than that in the male infertility group. A lower FOXO3 mRNA expression in granulosa cells leads to poor oocyte development in patients with unexplained infertility undergoing controlled ovarian stimulation for in vitro fertilization-embryo transfer. © 2017 Japan Society of Obstetrics and Gynecology.

Peroxisome Proliferator-Activated Receptors Alpha, Beta, and Gamma mRNA and Protein Expression in Human Fetal Tissues

PubMed Central

Abbott, Barbara D.; Wood, Carmen R.; Watkins, Andrew M.; Das, Kaberi P.; Lau, Christopher S.

2010-01-01

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study examines expression of PPARα, β, and γ mRNA and protein in human fetal tissues. With increasing fetal age, mRNA expression of PPARα and β increased in liver, but PPARβ decreased in heart and intestine, and PPARγ decreased in adrenal. Adult and fetal mean expression of PPARα, β, and γ mRNA did not differ in intestine, but expression was lower in fetal stomach and heart. PPARα and β mRNA in kidney and spleen, and PPARγ mRNA in lung and adrenal were lower in fetal versus adult. PPARγ in liver and PPARβ mRNA in thymus were higher in fetal versus adult. PPARα protein increased with fetal age in intestine and decreased in lung, kidney, and adrenal. PPARβ protein in adrenal and PPARγ in kidney decreased with fetal age. This study provides new information on expression of PPAR subtypes during human development and will be important in evaluating the potential for the developing human to respond to PPAR environmental or pharmaceutical agonists. PMID:20706641

Molecular evolution of adiponectin in Carnivora and its mRNA expression in relation to hepatic lipidosis.

PubMed

Nieminen, Petteri; Rouvinen-Watt, Kirsti; Kapiainen, Suvi; Harris, Lora; Mustonen, Anne-Mari

2010-09-15

Adiponectin is a novel adipocyte-derived hormone with low circulating concentrations and/or mRNA expression in obesity and non-alcoholic fatty liver disease (NAFLD). The adiponectin mRNA of several Carnivora species was sequenced to enable further gene expression studies in this clade with potential experimental species to examine the connections of hypoadiponectinemia to hepatic lipidosis. In addition, adiponectin mRNA expression was studied in the retroperitoneal fat of the American mink (Neovison vison), as hepatic lipidosis with close similarities to NAFLD can be rapidly induced to the species by fasting. The mRNA expression was determined after overnight-7d of food deprivation and 28d of re-feeding and correlated to the liver fat %. The homologies between the determined carnivoran mRNA sequences and that of the domestic dog were 92.2-99.1%. As the mRNA expression was not affected by short-term fasting and did not correlate with the liver fat %, there seems to be no clear connection between adiponectin and the development of lipidosis in the American mink. In the future, the obtained sequences can be utilized in further studies of adiponectin expression in comparative endocrinology. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

PubMed

Nakashima, Yukiko; Takahashi, Satoru

2014-08-22

Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells. Copyright © 2014 Elsevier Inc. All rights reserved.

[Effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice].

PubMed

Jin, Xin; Zhang, Hui-xin; Zhang, Yan-fen; Cui, Wen-wen; Bi, Yao; He, Qi-long; Zhou, Sheng-shan

2015-03-01

To study the effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice. Eight male C57BL/6J mice were selected in the normal group (NF), 40 male ApoE -/- mice were fed for 16 weeks, divided into the model group (HF), the rosiglitazone group ( LGLT), the Jinlida low-dose group (JLDL), the Jinlida medium-dose group (JLDM), the Jinlida high-dose group (JLDH) and then orally given drugs for 8 weeks. The organization free fatty acids, BCA protein concentration determination methods were used to determine the skeletal muscle FFA content. The Real-time fluorescent quantitative reverse transcription PCR ( RT-PCR) and Western blot method were adopted to determine mRNA and protein expressions of mice fatty acids transposition enzyme (FAT/CD36), carnitine palm acyltransferase 1 (CPT1), peroxide proliferators-activated receptor α( PPAR α). Jinlida could decrease fasting blood glucose (FBG), cholesterol (TC), triglyceride (TG), free fatty acid (FFA) and fasting insulin (FIns) and raise insulin sensitive index (ISI) in mice to varying degrees. It could also up-regulate mRNA and protein expressions of CPT1 and PPARα, and down-regulate mRNA and protein levels of FAT/CD36. Jinlida can improve fat-induced insulin resistance ApoE -/- in mice by adjusting the changes in expression of skeletal muscle lipid transport enzymes.

Population density approach for discrete mRNA distributions in generalized switching models for stochastic gene expression.

PubMed

Stinchcombe, Adam R; Peskin, Charles S; Tranchina, Daniel

2012-06-01

We present a generalization of a population density approach for modeling and analysis of stochastic gene expression. In the model, the gene of interest fluctuates stochastically between an inactive state, in which transcription cannot occur, and an active state, in which discrete transcription events occur; and the individual mRNA molecules are degraded stochastically in an independent manner. This sort of model in simplest form with exponential dwell times has been used to explain experimental estimates of the discrete distribution of random mRNA copy number. In our generalization, the random dwell times in the inactive and active states, T_{0} and T_{1}, respectively, are independent random variables drawn from any specified distributions. Consequently, the probability per unit time of switching out of a state depends on the time since entering that state. Our method exploits a connection between the fully discrete random process and a related continuous process. We present numerical methods for computing steady-state mRNA distributions and an analytical derivation of the mRNA autocovariance function. We find that empirical estimates of the steady-state mRNA probability mass function from Monte Carlo simulations of laboratory data do not allow one to distinguish between underlying models with exponential and nonexponential dwell times in some relevant parameter regimes. However, in these parameter regimes and where the autocovariance function has negative lobes, the autocovariance function disambiguates the two types of models. Our results strongly suggest that temporal data beyond the autocovariance function is required in general to characterize gene switching.

Reduction in the mRNA expression of sVEGFR1 and sVEGFR2 is associated with the selection of dominant follicle in cows.

PubMed

Ortega Serrano, P V; Guzmán, A; Hernández-Coronado, C G; Castillo-Juárez, H; Rosales-Torres, A M

2016-12-01

The vascular endothelial growth factor (VEGF) is essential for follicular development by promoting follicular angiogenesis, as well as for the proliferation and survival of granulosa cells. The biological effects of VEGF are regulated by two membrane receptors, VEGFR1 and VEGFR2, and two soluble receptors, sVEGFR1 and sVEGFR2, which play an antagonistic role. Thus, the objective of this study was to identify the mRNA expression pattern of total VEGF, VEGFR1, VEGFR2, sVEGFR1 and sVEGFR2 in bovine preselected follicles (PRF) and post-selected follicles (POF). The mRNA expression of these five genes in both granulosa cells (GC) and theca cells (TC) was compared between follicles classified as PRF and POF based on their diameter and on their ratio of estradiol/progesterone (E2/P4). Results showed a lower expression of mRNA of sVEGFR1 and sVEGFR2 in POF than in PRF (p < .05). Regarding the mRNA expression of total VEGF, VEGFR1 and VEGFR2, there was no difference between POF and PRF follicles (p > .05). Our results showed that the mRNA expression of VEGFR2 and sVEGFR1 was more abundant than the expression of VEGFR1 and sVEGFR2, while GC was the main source of mRNA for total VEGF. On the other hand, TC was the follicular compartment where the receptors were most expressed. Our results suggest that non-dominant follicles maintain a greater concentration of the mRNA expression of both membrane and soluble VEGF receptors. On the other hand, follicular dominance is related to a reduction in the mRNA expression of sVEGFR1 and sVEGFR2, which may favour VEGF binding with VEGFR2 and, hence, improve the follicular health and development. © 2016 Blackwell Verlag GmbH.

AKR1C1 and SRD5A1 messenger RNA expression at term in the human myometrium and chorioamniotic membranes.

PubMed

Lee, Richard H; Stanczyk, Frank Z; Stolz, Andrew; Ji, Qing; Yang, Gloria; Goodwin, T Murphy

2008-10-01

We sought to determine relative mRNA expression of AKR1C1 and SRD5A1, which respectively encode for the key progesterone metabolizing enzymes, 20alpha-hydroxysteroid dehydrogenase and 5alpha-reductase type 1, in the myometrium and chorioamniotic membranes during human spontaneous or induced labor and nonlabor. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to compare relative mRNA expression of AKR1C1 and SRD5A1 in the myometrium and chorioamniotic membranes from 20 subjects during three different states of labor: not in labor ( N = 10), spontaneous labor ( N = 5), or induced labor ( N = 5). Labor was defined as regular uterine contractions that resulted in cervical dilation. Myometrial AKR1C1 mRNA expression was significantly greater in spontaneously laboring subjects compared with those not in labor (2.4-fold [1.97 to 2.98], P = 0.02). There was no difference in myometrial AKR1C1 mRNA expression between those with induced labor compared with those not in labor. Regardless of labor status, no differences were observed in the chorioamniotic membrane AKR1C1 mRNA expression between the groups. SRD5A1 mRNA expression was significantly lower in the membranes of both laboring groups when compared with those not in labor (spontaneous: 0.10-fold [0.06 to 0.18], P = 0.007; induced: 0.09-fold [0.03 to 0.25], P = 0.013). Regardless of labor status, there was no difference in SRD5A1 mRNA expression in the myometrium. Our study demonstrated tissue-specific changes in progesterone metabolizing enzyme mRNA expression in human intrauterine tissue at term associated with labor status. These observed changes in mRNA expression may have important implications for progesterone metabolism at those specific sites and thereby may differentially regulate the tissue-specific progesterone concentration and/or the level of specific progesterone metabolites.

The comparison of the rejuvenation effects on the skin of Wistar rats between 10600 nm CO2 fractional laser and retinoic acid.

PubMed

Qu, Y; Ma, W-Y; Sun, Q

2017-04-01

The fractional laser and topical retinoic acid treatment have been applied for skin rejuvenation; however, the possible molecular mechanism of promoting remodeling of dermis is not clearly. Here we aimed to compare the effects of 10600 nm CO2 fractional laser and topical retinoic acid formulation on the skin collagen proliferation of Wistar rats, and to further explore the possible molecular mechanism of promoting remodeling of dermis. The hair on the back of Wistar rats was removed, and the back was divided equally into four regions with the cross-streaking method: A (the control group), B (the retinoic acid group), C (retinoic acid and fractional laser combination treatment group), and D (the fractional laser group). Specimens were collected at 3rd day and in 1-8 weeks after CO2 fractional laser irradiation; then they were used for detection of the changes of dermis thickness and content of hydroxyproline in the four regions of the rats' back. Real-time PCR method was used to detect the dynamic changes of the expression level of type III procollagen mRNA and the expression levels of miR-29a, Akt and transforming growth factor-β (TGF-β) mRNA at 3rd week in the skin tissue of Wistar rats. The thickness of dermis, content of hydroxyproline and expression level of type III procollagen mRNA in the treatment groups (B, C, and D) were found all significantly increased compared with those in the control group (A) (p<0.05); at 3rd week, up-regulation of Akt and TGF-β mRNA expression and down-regulation of miR-29a mRNA expression were observed in the treatment groups (B, C, and D). The difference in the combination treatment group (C) was the most significant (p<0.05). These results demonstrate that retinoic acid formulation and CO2 fractional laser both can promote collagen proliferation and reconstruction, with the skin rejuvenation efficacy in group C > group D > group B. miR-29a/Akt/TGF-β signal pathways may play a certain role in the promotion of collagen synthesis and proliferation.

The influence of rapamycin on the early cardioprotective effect of hypoxic preconditioning on cardiomyocytes

PubMed Central

Wang, Jiang; Maimaitili, YiLiyaer; Yu, Jin; Guo, Hai; Ma, Hai-Ping; Chen, Chun-ling

2016-01-01

Introduction The purpose of this study was to examine the effects of rapamycin on the cardioprotective effect of hypoxic preconditioning (HPC) and on the mammalian target of rapamycin (mTOR)-mediated hypoxia-inducible factor 1 (HIF-1) signaling pathway. Material and methods Primary cardiomyocytes were isolated from rat pups and underwent rapamycin and/or HPC, followed by hypoxia/re-oxygenation (H/R) injury. Cell viability and cell injury were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and qRT-PCR was used to measure HIF-1α and mTOR mRNA expression. A Langendorff heart perfusion model was conducted to observe the effect of rapamycin. Results Rapamycin treatment nearly abolished the cardioprotective effect of HPC in cardiomyocytes, reduced cell viability (p = 0.007) and increased cell damage (p = 0.032). HIF-1α and mTOR mRNA expression increased in cardiomyocytes undergoing I/R injury within 2 h after HPC. After rapamycin treatment, mTOR mRNA expression and HPC-induced HIF-1α mRNA expression were both reduced (p < 0.001). A Langendorff heart perfusion model in rat hearts showed that rapamycin greatly attenuated the cardioprotective effect of HPC in terms of heart rate, LVDP, and dp/dtmax (all, p < 0.029). Conclusions Rapamycin, through inhibition of mTOR, reduces the elevated HIF-1α expression at an early stage of HPC, and attenuates the early cardioprotective effect of HPC. PMID:28721162

[Role of CRISPR/Cas systems in drugresistance and virulence and the effect of IS600 on the expression of cse2 in Shigella].

PubMed

Hong, Lijuan; Zhang, Bing; Duan, Guangcai; Liang, Wenjuan; Wang, Yingfang; Chen, Shuaiyin; Yang, Haiyan; Xi, Yuanlin

2016-12-04

To analyze the relationship between CRISPR/Cas system and drug-resistance, virulence. To investigate the effect of IS600 on the expression of CRISPR associated gene cse2 in Shigella. CRISPR loci, CRISPR associated gene cse2, drug-resistant genes and virulent genes were detected by PCR in 33 Shigella strains; Trypan Blue counting test was used to detect bacterial virulence; Real-time PCR was used to detect relative mRNA expression of cse2; susceptibilities of Shigella strains were tested by agar diffusion method. Furthermore, we analyzed the relationship between CRISPR loci and drug-resistant genes, virulent genes. The effect of the IS600 on the expression of CRISPR associated gene cse2 was investigated. The mortality of Hela cells infected by Shigella with CRISPR1 loci was significantly lower (P<0.05) than those infected by Shigella without CRISPR1. The mRNA expression level of cse2 in group with IS600 was significantly (P<0.05) lower than that in group without IS600. CRISPR loci were widely present in Shigella. Shigella without CRISPR1 has a higher pathogenicity. Due to the insertion of IS600, the mRNA expression level of cse2 was decreased in Shigella.

Long non-coding RNAs regulate effects of β-crystallin B2 on mouse ovary development.

PubMed

Gao, Qian; Ren, Hanxiao; Chen, Mingkun; Niu, Ziguang; Tao, Haibo; Jia, Yin; Zhang, Jianrong; Li, Wenjie

2016-11-01

β-crystallin B2 (CRYBB2) knockout mice exhibit morphological and functional abnormalities in the ovary. Long non‑coding RNAs (lncRNAs) regulate gene transcription and translation, and epigenetic modification of genomic DNA. The present study investigated the role of lncRNAs in mediating the effects of CRYBB2 in the regulation of ovary development in mice. In the current study, ovary tissues from wild‑type (WT) and CRYBB2 knockout mice were subjected to lncRNA and mRNA microarray profiling. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to group the differentially expressed lncRNAs into regulated gene pathways and functions. The correlation matrix method was used to establish a network of lncRNA and mRNA co‑expression. Quantitative reverse transcription-polymerase chain reaction (RT‑qPCR) was used to verify expression of a number of these differentially expressed lncRNAs and mRNAs. There were 157 differentially expressed lncRNAs and 1,085 differentially expressed mRNAs between ovary tissues from WT and CRYBB2 knockout mice. The GO and KEGG analyses indicated that these differentially expressed lncRNAs and mRNAs were important in Ca2+ signaling and ligand and receptor interactions. The correlation matrix method established an lncRNA and mRNA co‑expression network, consisting of 53 lncRNAs and 45 mRNAs with 98 nodes and 75 connections. RT‑qPCR confirmed downregulation of lncRNA A‑30‑P01019163 expression, which further downregulated its downstream gene purinergic receptor P2X, ligand‑gated ion channel, 7 (P2rx7) expression in ovary tissues from CRYBB2 knockout mice. In conclusion, CRYBB2 regulates expression of different lncRNAs to influence ovary development. lncRNA A‑30‑P01019163 may affect ovarian cell cycle and proliferation by regulating P2rx7 expression in the ovary.

Maternal Obesity Induces Sustained Inflammation in Both Fetal and Offspring Large Intestine of Sheep

PubMed Central

Yan, Xu; Huang, Yan; Wang, Hui; Du, Min; Hess, Bret W.; Ford, Stephen P.; Nathanielsz, Peter W.; Zhu, Mei-Jun

2010-01-01

Background Both maternal obesity and inflammatory bowel diseases (IBDs) are increasing. It was hypothesized that maternal obesity induces an inflammatory response in the fetal large intestine, predisposing offspring to IBDs. Methods Nonpregnant ewes were assigned to a control (Con, 100% of National Research Council [NRC] recommendations) or obesogenic (OB, 150% of NRC) diet from 60 days before conception. The large intestine was sampled from fetuses at 135 days (term 150 days) after conception and from offspring lambs at 22.5 ± 0.5 months of age. Results Maternal obesity enhanced mRNA expression tumor necrosis factor (TNF)α, interleukin (IL)1α, IL1β, IL6, IL8, and monocyte/macrophage chemotactic protein-1 (MCP1), as well as macrophage markers, CD11b, CD14, and CD68 in fetal gut. mRNA expression of Toll-like receptor (TLR) 2 and TLR4 was increased in OB versus Con fetuses; correspondingly, inflammatory NF-κB and JNK signaling pathways were also upregulated. Both mRNA expression and protein content of transforming growth factor (TGF) β was increased. The IL-17A mRNA expression and protein content was higher in OB compared to Con samples, which was associated with fibrosis in the large intestine of OB fetuses. Similar inflammatory responses and enhanced fibrosis were detected in OB compared to Con offspring. Conclusions Maternal obesity induced inflammation and enhanced expression of proinflammatory cytokines in fetal and offspring large intestine, which correlated with increased TGFβ and IL17 expression. These data show that maternal obesity may predispose offspring gut to IBDs. PMID:21674707

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients

PubMed Central

Safari, Mohammad Taghi; Chaleshi, Vahid; Tarban, Peyman; Nourian, Mahyar; Balaii, Hedieh; Shahrokh, Shabnam; Asadzadeh Aghdaei, Hamid

2017-01-01

Aim: In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Background: Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. Methods: In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. Results: mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Conclusion: Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients. PMID:29511476

Myeloperoxidase activity and its corresponding mRNA expression as well as gene polymorphism in the population living in the coal-burning endemic fluorosis area in Guizhou of China.

PubMed

Zhang, Ting; Shan, Ke-Ren; Tu, Xi; He, Yan; Pei, Jin-Jing; Guan, Zhi-Zhong

2013-06-01

The myeloperoxidase (MPO) activity and its corresponding mRNA expression as well as gene polymorphism were investigated in the population who live in the endemic fluorosis area. In the study, 150 people were selected from the coal-burning endemic fluorosis area and 150 normal persons from the non-fluorosis area in Guizhou province of China. The blood samples were collected from these people. The activity of MPO in the plasma was determined by spectrophotometer; the expression of MPO mRNA was measured by employing real-time polymerase chain reaction; DNAs were extracted from the leucocytes in blood and five SNP genotypes of MPO promoter gene detected by a multiplex genotyping method, adapter-ligation-mediated allele-specific amplification. The results showed that the MPO activity and its corresponding mRNA in blood were significantly increased in the population living in the area of fluorosis. The different genotype frequencies of MPO, including -1228G/A, -585T/C, -463G/A, and -163C/T, and the three haplotypes with higher frequencies, including -163C-463G-585T-1228G-1276T, -163C-463G-585T-1228G-1276C, and -163C-463G-585T-1228A-1276T, were significantly associated with fluorosis. The results indicated that the elevated activity of MPO induced by endemic fluorosis may be connected in mechanism to the stimulated expression of MPO mRNA and the changed gene polymorphism.

Quantification and study of the L-DOPA decarboxylase expression in gastric adenocarcinoma cells treated with chemotherapeutic substances.

PubMed

Korbakis, Dimitrios; Fragoulis, Emmanuel G; Scorilas, Andreas

2013-03-01

3,4-Dihydroxy-L-phenylalanine decarboxylase (DDC) is an enzyme implicated in the biosynthetic pathways of the neurotransmitters dopamine and probably serotonin. DDC gene expression has been studied in numerous malignancies and the corresponding data have shown remarkable alterations in the mRNA and/or protein levels encoded by the gene. The aim of this study was to examine any modulations in the DDC mRNA levels in gastric cancer cells after their treatment with the chemotherapeutic agents 5-fluorouracil, leucovorin, irinotecan, etoposide, cisplatin, and taxol. The sensitivity of the AGS gastric adenocarcinoma cells to the antineoplastic drugs was evaluated using the MTT assay. Total RNA was extracted and reverse transcribed into cDNA. A highly sensitive quantitative real-time PCR methodology was developed for the quantification of DDC mRNA. GAPDH was used as a housekeeping gene. Relative quantification analysis was carried out using the comparative C T method ((Equation is included in full-text article.)). The treatment of AGS cells with several concentrations of various broadly used anticancer drugs resulted in significant modulations of the DDC mRNA levels compared with those in the untreated cells in a time-specific and drug-specific manner. Generally, DDC expression levels appeared to decrease after three time periods of exposure to the selected chemotherapeutic agents, suggesting a characteristic DDC mRNA expression profile that is possibly related to the mechanism of each drug. Our experimental data show that the DDC gene might serve as a new potential molecular biomarker predicting treatment response in gastric cancer cells.

Programmed Cell Death Ligand 1 Expression in Primary Central Nervous System Lymphomas: A Clinicopathological Study.

PubMed

Hayano, Azusa; Komohara, Yoshihiro; Takashima, Yasuo; Takeya, Hiroto; Homma, Jumpei; Fukai, Junya; Iwadate, Yasuo; Kajiwara, Koji; Ishizawa, Shin; Hondoh, Hiroaki; Yamanaka, Ryuya

2017-10-01

Programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) have been shown to predict response to PD-L1/PD-1-targeted therapy. We analyzed PD-L1 expression in primary central nervous system lymphomas (PCNSLs). PD-L1 protein and mRNA expression were evaluated in 64 PCNSL tissue samples. IFN-γ, IL-10, CD4, and CD8 mRNA expression was also evaluated. PD-L1 protein was detected in tumor cells in 2 (4.1%) cases and in tumor microenvironments in 25 (52%) cases. PD-L1 mRNA positively correlated with IFN-γ (p=0.0024) and CD4 (p=0.0005) mRNA expression. IFN-γ mRNA positively correlated with CD8 mRNA expression (p=0.0001). Furthermore, tumor cell PD-L1 expression correlated positively with overall survival (p=0.0177), whereas microenvironmental PD-L1 expression exhibited an insignificant negative trend with overall survival (p=0.188). PD-L1 was expressed on both tumor and/or tumor-infiltrating immune cells in PCNSL. The biological roles of this marker warrant further investigation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Influence of Expression Plasmid of Connective Tissue Growth Factor and Tissue Inhibitor of Metalloproteinase-1 shRNA on Hepatic Precancerous Fibrosis in Rats.

PubMed

Zhang, Qun; Shu, Fu-Li; Jiang, Yu-Feng; Huang, Xin-En

2015-01-01

In this study, influence caused by expression plasmids of connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) short hairpin RNA (shRNA) on mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII in hepatic tissue with hepatic fibrosis, a precancerous condition, in rats is analyzed. To screen and construct shRNA expression plasimid which effectively interferes RNA targets of CTGF and TIMP-1 in rats. 50 cleaning Wistar male rats are allocated randomly at 5 different groups after precancerous fibrosis models and then injection of shRNA expression plasimids. Plasmid psiRNA-GFP-Com (CTGF and TIMP-1 included), psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA- DUO-GFPzeo of blank plasmid are injected at group A, B, C and D, respectively, and as model control group that none plasimid is injected at group E. In 2 weeks after last injection, to hepatic tissue at different groups, protein expression of CTGF, TIMP-1, procol-α1and PC III is tested by immunohistochemical method and,mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII is measured by real-time PCR. One-way ANOVA is used to comparison between-groups. Compared with model group, there is no obvious difference of mRNA expression among CTGF,TIMP-1,procol-α1,PC III and of protein expression among CTGF, TIMP-1, procol-α1, PC III in hepatic tissue at group injected with blank plasmid. Expression quantity of mRNA of CTGF, TIMP-1, procol-α1 and PCIII at group A, B and C decreases, protein expression of CTGF, TIMP-1, procol-α1, PC III in hepatic tissue is lower, where the inhibition of combination RNA interference group (group A) on procol-α1 mRNA transcription and procol-α1 protein expression is superior to that of single interference group (group B and C) (P<0.01 or P<0.05). RNA interference on CTGF and/or TIMP-1 is obviously a inhibiting factor for mRNA and protein expression of CTGF, TIMP-1, procol-α1 and PCIII. Combination RNA interference on genes of CTGF and TIMP-1 is superior to that of single RNA interference, and this could be a contribution for prevention of precancerous condition.

The Effects of Exercise on Expression of CYP19 and StAR mRNA in Steroid-Induced Polycystic Ovaries of Female Rats.

PubMed

Aghaie, Fatemeh; Khazali, Homayoun; Hedayati, Mehdi; Akbarnejad, Ali

2018-01-01

Polycystic ovarian syndrome (PCOS) is the most frequent female endocrine disorder that affects 5-10% of women. PCOS is characterized by hyperandrogenism, oligo-/anovulation, and polycystic ovaries. The aim of the present research is to evaluate the expression of steroidogenic acute regulatory protein (StAR) and aromatase (CYP19) mRNA in the ovaries of an estradiol valerate (EV)-induced PCOS rat model, and the effect of treadmill and running wheel (voluntary) exercise on these parameters. In this experimental study, we divided adult female Wistar rats that weighed approximately 220 ± 20 g initially into control (n=10) and PCOS (n=30). Subsequently, PCOS group were divided to PCOS, PCOS with treadmill exercise (P-ExT), and PCOS with running wheel exercise (P-ExR) groups (n=10 per group). The expressions of StAR and CYP19 mRNA in the ovaries were determined by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Data were analyzed by one-way ANOVA using SPSS software, version 16. The data were assessed at α=0.05. There was significantly lower mRNA expression of CYP19 in the EV-induced PCOS, running wheel and treadmill exercise rats compared to the control group (P<0.001). Treadmill exercise (P=0.972) and running wheel exercise (P=0.839) had no significant effects on CYP19 mRNA expression compared to the PCOS group. mRNA expression of StAR in the ovaries of the PCOS group indicated an increasing trend compared to the control group, however this was not statistically significant (P=0.810). We observed that 8 weeks of running wheel and treadmill exercises could not statistically decrease StAR mRNA expression compared to the PCOS group (P=0.632). EV-induced PCOS in rats decreased CYP19 mRNA expression, but had no effect on StAR mRNA expression. We demonstrated that running wheel and moderate treadmill exercise could not modify CYP19 and StAR mRNA expressions. Copyright© by Royan Institute. All rights reserved.

Expression of BMI-1 and Mel-18 in breast tissue - a diagnostic marker in patients with breast cancer

PubMed Central

2010-01-01

Background Polycomb Group (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer. Expression of Mel-18 and Bmi-1 has been studied in tumor tissue, but not in adjacent non-cancerous breast epithelium. Our study compares the expression of the two genes in normal breast epithelium of cancer patients and relates it to the level of expression in the corresponding tumors as well as in breast epithelium of healthy women. Methods A total of 79 tumors, of which 71 malignant tumors of the breast, 6 fibroadenomas, and 2 DCIS were studied and compared to the reduction mammoplastic specimens of 11 healthy women. In addition there was available adjacent cancer free tissue for 23 of the malignant tumors. The tissue samples were stored in RNAlater, RNA was isolated to create expression microarray profile. These two genes were then studied more closely first on mRNA transcription level by microarrays (Agilent 44 K) and quantitative RT-PCR (TaqMan) and then on protein expression level using immunohistochemistry. Results Bmi-1 mRNA is significantly up-regulated in adjacent normal breast tissue in breast cancer patients compared to normal breast tissue from noncancerous patients. Conversely, mRNA transcription level of Mel-18 is lower in normal breast from patients operated for breast cancer compared to breast tissue from mammoplasty. When protein expression of these two genes was evaluated, we observed that most of the epithelial cells were positive for Bmi-1 in both groups of tissue samples, although the expression intensity was stronger in normal tissue from cancer patients compared to mammoplasty tissue samples. Protein expression of Mel-18 showed inversely stronger intensity in tissue samples from mammoplasty compared to normal breast tissue from patients operated for breast cancer. Conclusion Bmi-1 mRNA level is consistently increased and Mel-18 mRNA level is consistently decreased in adjacent normal breast tissue of cancer patients as compared to normal breast tissue in women having had reduction mammoplasties. Bmi-1/Mel-18 ratio can be potentially used as a tool for stratifying women at risk of developing malignancy. PMID:21162745

Cytochrome P450-2C11 mRNA is not expressed in endothelial cells dissected from rat renal arterioles.

PubMed

Heil, Sandra G; De Vriese, An S; Kluijtmans, Leo A J; Dijkman, Henry; van Strien, Denise; Akkers, Robert; Blom, Henk J

2005-01-01

Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles. Copyright (c) 2005 S. Karger AG, Basel.

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.

Altered Left Ventricular Ion Channel Transcriptome in a High-Fat-Fed Rat Model of Obesity: Insight into Obesity-Induced Arrhythmogenesis

PubMed Central

Yon, Marianne; Pickavance, Lucy; Yanni Gerges, Joseph; Davis, Gershan; Wilding, John; Jian, Kun; Hart, George; Boyett, Mark

2016-01-01

Introduction. Obesity is increasingly common and is associated with an increased prevalence of cardiac arrhythmias. The aim of this study was to see whether in obesity there is proarrhythmic gene expression of ventricular ion channels and related molecules. Methods and Results. Rats were fed on a high-fat diet and compared to control rats on a normal diet (n = 8). After 8 weeks, rats on the high-fat diet showed significantly greater weight gain and higher adiposity. Left ventricle samples were removed at 8 weeks and mRNA expression of ion channels and other molecules was measured using qPCR. Obese rats had significant upregulation of Cav1.2, HCN4, Kir2.1, RYR2, NCX1, SERCA2a, and RYR2 mRNA and downregulation of ERG mRNA. In the case of HCN4, it was confirmed that there was a significant increase in protein expression. The potential effects of the mRNA changes on the ventricular action potential and intracellular Ca2+ transient were predicted using computer modelling. Modelling predicted prolongation of the ventricular action potential and an increase in the intracellular Ca2+ transient, both of which would be expected to be arrhythmogenic. Conclusion. High-fat diet causing obesity results in arrhythmogenic cardiac gene expression of ion channels and related molecules. PMID:27747100

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

PubMed Central

Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

1994-01-01

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

Overexpression of early growth response-1 as a metastasis-regulatory factor in gastric cancer.

PubMed

Kobayashi, Daisuke; Yamada, Mikako; Kamagata, Chinatsu; Kaneko, Reiko; Tsuji, Naoki; Nakamura, Masashi; Yagihashi, Atsuhito; Watanabe, Naoki

2002-01-01

To investigate the potential role of a nuclear transcription factor, early growth response-1 (Egr-1), in formation and progression of gastric cancer, we compared its expression in gastric cancers with that in non-cancerous tissues. Egr-1 mRNA expression was measured using TaqMan RT-PCR. The corresponding protein expression was examined immunohistochemically. Egr-1 mRNA expression was significantly higher in gastric cancer tissues than in normal mucosa (p < 0.0005). These differences were also reflected by protein product expression. Moreover, Egr-1 mRNA expression was higher in cases with metastasis to lymph nodes or remote organs. In cultured gastric cancer cells known to have a high metastatic potential, expression of this mRNA was higher than that of parental cells. It was suggested that Egr-1 has a significant role in carcinogenesis and in cancer progression, especially metastasis. Measurement of this mRNA should be useful for evaluation of the metastatic potential of gastric cancer.

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

PubMed

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

2001-04-01

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

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.

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

PubMed

Williams, C M; Coleman, J W

1995-10-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

PubMed Central

Williams, C M; Coleman, J W

1995-01-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs. Images Figure 1 Figure 2 Figure 3 PMID:7490125

Unique Temporal Expression of Triplicated Long-Wavelength Opsins in Developing Butterfly Eyes

PubMed Central

Arikawa, Kentaro; Iwanaga, Tomoyuki; Wakakuwa, Motohiro; Kinoshita, Michiyo

2017-01-01

Following gene duplication events, the expression patterns of the resulting gene copies can often diverge both spatially and temporally. Here we report on gene duplicates that are expressed in distinct but overlapping patterns, and which exhibit temporally divergent expression. Butterflies have sophisticated color vision and spectrally complex eyes, typically with three types of heterogeneous ommatidia. The eyes of the butterfly Papilio xuthus express two green- and one red-absorbing visual pigment, which came about via gene duplication events, in addition to one ultraviolet (UV)- and one blue-absorbing visual pigment. We localized mRNAs encoding opsins of these visual pigments in developing eye disks throughout the pupal stage. The mRNAs of the UV and blue opsin are expressed early in pupal development (pd), specifying the type of the ommatidium in which they appear. Red sensitive photoreceptors first express a green opsin mRNA, which is replaced later by the red opsin mRNA. Broadband photoreceptors (that coexpress the green and red opsins) first express the green opsin mRNA, later change to red opsin mRNA and finally re-express the green opsin mRNA in addition to the red mRNA. Such a unique temporal and spatial expression pattern of opsin mRNAs may reflect the evolution of visual pigments and provide clues toward understanding how the spectrally complex eyes of butterflies evolved. PMID:29238294

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.

Seasonal Relationship between Gonadotropin, Growth Hormone, and Estrogen Receptor mRNA Expression in the Pituitary Gland of Largemouth Bass

PubMed Central

Martyniuk, Christopher J; Kroll, Kevin J.; Porak, Wesley F.; Steward, Cheree; Grier, Harry J.; Denslow, Nancy D.

2011-01-01

The objectives of this study were to investigate the seasonal changes in pituitary gonadotropins, growth hormone (GH), and estrogen receptor (ER) isoform mRNA in wild female and male largemouth bass (LMB) (Micropterus salmoides) from an unpolluted habitat to better understand reproductive physiology in this ecologically important species. Female pituitary luteinizing hormone (LH) β subunit and follicle-stimulating hormone (FSH) β subunit mRNA showed significant seasonal variation with levels peaking from January to April and were lowest from May through August. Male LMB showed more variation in gonadotropin subunit expression from month to month. Females had approximately 2–3 times higher gonadotropin mRNA levels in the pituitary when compared to males. All three gonadotropin mRNAs in females were positively correlated to gonadosomatic index (GSI), but only LHβ mRNA was correlated to GSI in males. Gonadotropin mRNA expression also increased with increasing oocyte and sperm maturation. Gonadotropin β subunit mRNA expression was positively correlated to GH mRNA in both sexes. The expression of all three ER isoforms was significantly correlated to each other in both sexes. The concurrent increase in all three ER mRNA isoforms with increasing gonadotropin mRNA in females and males suggests a prominent role for E2 feedback on pituitary gonadotropin synthesis in both sexes and that each of the three ER isoforms are likely to play a role in the pituitary during teleost reproduction. PMID:19416730

Seasonal relationship between gonadotropin, growth hormone, and estrogen receptor mRNA expression in the pituitary gland of largemouth bass.

PubMed

Martyniuk, Christopher J; Kroll, Kevin J; Porak, Wesley F; Steward, Cheree; Grier, Harry J; Denslow, Nancy D

2009-09-15

The objectives of this study were to investigate the seasonal changes in pituitary gonadotropins, growth hormone (GH), and estrogen receptor (ER) isoform mRNA in wild female and male largemouth bass (LMB) (Micropterus salmoides) from an unpolluted habitat to better understand reproductive physiology in this ecologically important species. Female pituitary luteinizing hormone (LH) beta subunit and follicle stimulating hormone (FSH) beta subunit mRNA showed significant seasonal variation with levels peaking from January to April and were lowest from May to August. Male LMB showed more variation in gonadotropin subunit expression from month to month. Females had approximately 2-3 times higher gonadotropin mRNA levels in the pituitary when compared to males. All three gonadotropin mRNAs in females were positively correlated to gonadosomatic index (GSI), but only LHbeta mRNA was correlated to GSI in males. Gonadotropin mRNA expression also increased with increasing oocyte and sperm maturation. Gonadotropin beta subunit mRNA expression was positively correlated to GH mRNA in both sexes. The expression of all three ER isoforms was significantly correlated to each other in both sexes. The concurrent increase in all three ER mRNA isoforms with increasing gonadotropin mRNA in females and males suggests a prominent role for E2 feedback on pituitary gonadotropin synthesis in both sexes and that each of the three ER isoforms are likely to play a role in the pituitary during teleost reproduction.

Effect of age on the expression of Pex (Phex) in the mouse.

PubMed

Meyer, R A; Young, C G; Meyer, M H; Garges, P L; Price, D K

2000-04-01

Pex is a newly discovered gene (also called Phex) whose mutation is the cause of X-linked hypophosphatemia. Other members of this gene family encode endopeptidases that activate or inactivate endocrine and paracrine factors. Though embryonic bone expresses mRNA for the Pex gene at relatively high levels, we have found Pex expression to be widespread in adult organs and to be poorly expressed in adult bone. This led to the hypothesis that Pex mRNA expression changes with age. To test this, genetically normal mice of the B6C3H hybrid strain were studied at 0 (newborn), 2, 3, 10, and 72 weeks of age. Organs known to express Pex were collected, and RNA was extracted from them. Following reverse transcription, cDNA was amplified by the polymerase chain reaction with primers for Pex and G3PDH, a housekeeping gene. The amplimers were separated by electrophoresis, blotted onto nylon membranes, and hybridized with radioactively labeled internal oligonucleotide probes. The radioactivity was quantified, and the data were analyzed as the Pex/G3PDH ratio. The brain samples had high levels of Pex mRNA expression that rose slightly with age. Calvaria, kidney, and lung samples had the highest Pex mRNA expression at birth. In these organs Pex mRNA expression fell with age to undetectable or barely detectable levels. Thymus, heart, and skeletal muscle samples had low Pex mRNA expression at birth that did not change with age. Some organs showed a decline in G3PDH levels with age, but Pex expression decreased more, leading to a reduced Pex/G3PDH ratio. The widespread expression of mRNA for Pex suggests a role beyond that of phosphate homeostasis. The high level of expression in newborn animals suggests a role in growth and development. This seems to occur in addition to its role for the endocrine regulation of phosphate homeostasis by as yet unknown humoral agents that must occur throughout life. In summary, Pex mRNA expression is high in brain and bone at birth. Expression remains high in brain with age but falls with age in bone, kidney, and lung.

Changes in the mRNA expression of structural proteins, hormone synthesis and secretion from bovine placentome sections after DDT and DDE treatment.

PubMed

Wojciechowska, A; Mlynarczuk, J; Kotwica, J

2017-01-15

Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3β-hydroxysteroid dehydrogenase (3βHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Aromatase, steroid-5-alpha-reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny.

PubMed

Urbatzka, R; Lutz, I; Kloas, W

2007-01-01

The key enzymes involved in the production of endogenous sex steroids are steroid-5-alpha-reductase and aromatase converting testosterone (T) into dihydrotestosterone (DHT) and into estradiol (E2), respectively. To gain more insights into the molecular mechanisms of sexual differentiation of amphibians, we determined the mRNA expression of steroid-5-alpha-reductase type1 (Srd5a1), type2 (Srd5a2) and aromatase (Aro) during ontogeny starting from the egg and ending after completion of metamorphosis in Xenopus laevis. Expression of all three enzymes was measured by means of semi-quantitative RT-PCR, determining for the first time Srd5a1 and Srd5a2 mRNA expression in amphibians. mRNA was analyzed in whole body homogenates from stage 12 to 48, while brain and gonads with kidney were studied separately from stage 48 to 66. Different ontogenetic mRNA expression patterns were observed for all genes analyzed, revealing early mRNA expression of Srd5a1 already in the egg at stage 12 whereas Srd5a2 and Aro was detected at stage 39. Sex-specific mRNA expressions of Srd5a2 and of Aro were determined in the gonads with kidney but not in brain. Srd5a2 was two-fold higher expressed in testes than in ovaries while Aro mRNA was ten-fold higher in ovaries. No gender-specific mRNA expression was observed for Srd5a1 in gonads and in brain. The ontogenetic patterns of Aro, Srd5a1 and Srd5a2 suggest that these genes are involved in sexual differentiation of gonads and brain already in early developmental stages. Especially in gonads Srd5a2 seems to be important for physiological regulation of testis development while Aro is associated with the development of ovaries.

TS mRNA levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer.

PubMed

Zhang, Qun; Shen, Jie; Wang, Hao; Hu, Jing; Yu, Lixia; Xie, Li; Wei, Jia; Liu, Baorui; Guan, Wenxian; Qian, Xiaoping

2014-02-01

The purpose of the study is to analyze the relationship between tumor thymidylate synthase (TS) mRNA expression levels and raltitrexed/pemetrexed/5-FU sensitivity. We collected freshly removed colorectal tumor specimens from 50 patients. Chemosensitivities to anticancer drugs were evaluated by histoculture drug response assay. We adopted quantitative reverse transcription polymerase chain reaction for TS mRNA detection and immunohistochemical staining for assessing TS expression in tumor tissues. There is a significant relationship between TS mRNA expression levels and in vitro chemosensitivity of freshly removed colorectal tumor specimens to pemetrexed (P < 0.001)/raltitrexed (P = 0.004)/5-FU (P = 0.007). TS mRNA expression levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer.

Expression of dopamine D2 receptor and choline acetyltransferase mRNA in the dopamine deafferented rat caudate-putamen.

PubMed

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

1990-01-01

In situ hybridization was used to study dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) mRNA expression in neurons of the rat forebrain, both on control animals and after a unilateral 6-hydroxydopamine (6-OHDA) lesion of midbrain dopamine neurons. D2R mRNA expressing neurons were seen in regions which are known to be heavily innervated by midbrain dopamine fibers such as caudate-putamen, nucleus accumbens and olfactory tubercle. ChAT mRNA expressing neurons were seen in caudate-putamen, nucleus accumbens and septal regions including vertical limb of the diagonal band. In caudate-putamen, approximately 55% of the medium sized neurons, which is the predominating neuronal cell-size in this region, were specifically labeled with the D2R probe. In addition, approximately 95% of the large size neurons in caudate-putamen were specifically labeled with both the D2R and ChAT probes, suggesting that most cholinergic neurons in the caudate-putamen express D2R mRNA. After a unilateral lesion of midbrain dopamine neurons, no change in the level of either D2R or ChAT mRNA were seen in the large size intrinsic cholinergic neurons in caudate-putamen. Similarly, no evidence was obtained for altered levels of D2R mRNA in medium size neurons in medial caudate-putamen, or nucleus accumbens. However, an increase in the number of medium size neurons expressing D2R mRNA was observed in the lateral part of the dopamine deafferented caudate-putamen. Thus, it appears that midbrain dopamine deafferentation causes an increase in D2R mRNA expression in a subpopulation of medium size neurons in the lateral caudate-putamen.

[Emodin inhibits the proliferation, transdifferentiation and collagen synthesis of pulmonary fibroblasts].

PubMed

Liu, Lijing; Yin, Huiming; He, Jianbin; Xie, Maofeng; Wang, Zaiyan; Xiao, Hua

2016-07-01

Objective To explore the effect of emodin on the proliferation, differentiation into myofibroblasts and collagen synthesis of pulmonary fibroblasts and the underlying mechanisms. Methods Human pulmonary fibroblasts MRC-5 were cultured in vitro, then the cells were inoculated with dimethyl sulfoxide (DMSO) added with 0, 10, 20, 40, 80 and 160 μmol/L emodin for 24, 48 and 72 hours. Inhibitory rate of cell proliferation was analyzed by MTT assay. Based on the results of cell proliferation experiment, MRC-5 cells were treated with DMSO (control group) and 40, 80 μmol/L emodin (in DMSO) for 48 hours. Fluorescence real-time quantitative PCR was then used to measure the mRNA expressions of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1), collagen type 1 (Col1) and collagen type 3 (Col3). The protein expressions of the above mentioned factors were also measured by Western blotting. Results In a concentration- and time-dependent manner, emodin inhibited MRC-5 cell proliferation. After 48 hours of co-culture, in comparison with control group, the mRNA and protein expression levels of α-SMA, TGF-β1, Col1 and Col3 significantly decreased, while the mRNA and protein expression levels of ADAMTS-1 significantly increased in 40 and 80 μmol/L emodin-treated groups. Moreover, in comparison with 40 μmol/L emodin-treated group, the mRNA and protein expressions of α-SMA, TGF-β1, Col1 and Col3 were significantly downregulated, but ADAMTS-1 mRNA and protein expressions were significantly upregulated in 80 μmol/L emodin-treated group. Conclusion Emodin can block pulmonary fibroblast proliferation and differentiation into myofibroblasts, and reduce the synthesis of Col1 and Col3 by inhibiting TGF-β1/ADAMTS-1 signaling pathway.

Molecular interactions of natural and synthetic steroids in female hamsters' flank organs.

PubMed

Cabeza, Marisa; Naranjo, Barak; Heuze, Yvonne; Sánchez, Araceli; Hernández, Mercedes; Sainz, Teresita; Bratoeff, Eugene

2012-05-01

The initial step of steroidal action on target cells is gene activation; therefore, the quantification of mRNA is a direct method for comparing the role of different steroids in the skin. This study demonstrated the role of several steroids on the mRNA expression encoding for different enzymes involved in the lipid metabolism in hamsters' flank organs, which are a pilosebaceous complex. To determine the effect of treatments with testosterone (T) progesterone (P), levonorgestrel (LNG), 17α-p-chlorobenzoyloxy-6-chloropregn-4,6-diene-3,20-dione (5) and 17α-p-chlorobenzoyloxy-4,6-pregnadiene-3,20-dione (6); T and/or LNG; T and 5 or 6; P and/or 5 or 6 on the expression of mRNA encoding for lipid enzymes, the steroids were applied to the glands; later, the mRNAs expression for the enzymes was determined by PCR. The binding of 5 and 6 to the progesterone receptor (PR) was also evaluated. Treatments with T, LNG, T+LNG, P, T+P, 5, T+5, T+6, P, P+5 and P+6 increased the mRNA expression for glycerol 3-phosphate acyl transferase (GPAT), β-hydroxy-β-methylglutaryl-CoA synthase (HMG-CoA-S), β-hydroxy-β-methylglutaryl-CoA reductase (HMG-CoA-R), phosphatidylinositol synthase as compared to the controls. However, squalene synthase was increased with all treatments except with T+5 and 6; 6 did not significantly increase the expression for GPAT or HMG-CoA-S, however it increased the concentration of HMG-CoA-R enzyme. 5 and 6 bind to the PR, thus indicating that the effect of these steroids on the mRNA expression could be the result of their binding. The lipid metabolism is regulated by several steroids thought different mechanism of action, in flank organs. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The effects of pilates exercise on lipid metabolism and inflammatory cytokines mRNA expression in female undergraduates

PubMed Central

Kim, Hyo-Jin; Kim, Jiyeon; Kim, Chang-Sun

2014-01-01

[Purpose] The purpose of the study was to verify the effects of Pilates exercise by observing the impact of 8 weeks of Pilates exercise on lipid metabolism and inflammatory cytokine mRNA expression in female undergraduates in their 20s who had no prior experience in Pilates exercise and had not exercised in the previous 6 months. [Methods] There were 18 subjects with no prior experience in Pilates exercise. The subjects were separated into the Pilates exercise group (n = 9) and the non-exercise control group (n = 9). The former performed Pilates exercise for 60-70 minutes over 8 weeks with a gradual strength increase of 9-16 in the Rating of Perceived Exercise (RPE). The body composition, creatine kinase in the bloodstream and lipid metabolism (TC, LDL-C, HDL-C, TG) were measured before and after the experiment and Real-Time PCR was used to investigate the mRNA expression of the inflammatory cytokines IL-6 and TNF-⍺. [Results] The creatine kinase (CK) in the blood had significant differences between the groups. The test group showed significant increase compared to the control group after 8 weeks of Pilates exercise (p = 0.007). Lipid analysis showed that the level of high-density lipoprotein cholesterol (HDL-C) was significantly different in the two groups (p = 0.049), with the Pilates exercise group exhibiting significantly higher levels compared to the control group. No significant differences were observed in the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG). IL-6 mRNA expression did not show significant differences between the groups either. Timing and TNF-α mRNA expression showed significant effect in both the exercise and the control groups (p = 0.013) but no correlation. [Conclusion] It was found from the study that Pilates exercise for 8 weeks affected CK expression (the muscle damage marker) and induced positive changes in the levels of high-density lipoprotein. PMID:25566463

Differential expression of cytokeratin mRNA and protein in normal prostate, prostatic intraepithelial neoplasia, and invasive carcinoma.

PubMed Central

Yang, Y.; Hao, J.; Liu, X.; Dalkin, B.; Nagle, R. B.

1997-01-01

The expression of cytokeratin (CK) mRNA for CK5, -8, -14, -16, and -19 was investigated in normal prostate, prostatic intraepithelial neoplasia (PIN) lesions, and invasive carcinoma using in situ hybridization. Protein localization was carried out in adjacent sections using immunohistochemistry and correlated with mRNA expression. Snap-frozen human prostate samples including 22 examples of normal glands, 20 cases of PIN lesions, and 12 cases of invasive carcinoma were examined. CK5 and -14 mRNA and protein were prominently expressed only in the basal cells of normal glands and PIN lesions. CK14 mRNA was absent in the luminal cells of the most of the PIN lesions but was seen at a low level in some PIN lesions. CK14 protein was not detected in any PIN lesion, suggesting that, if the cell that makes up the PIN lesions is derived from a basal cell, CK14 translation is depressed although a low level of CK14 mRNA may persist. CK8 mRNA and protein were constitutively expressed in all epithelia of normal and abnormal prostate tissues. CK19 mRNA and protein were persistently expressed in both basal and luminal cells of the tubular portion of normal glands as well as PIN lesions, but were expressed heterogeneously in both basal and luminal cells of normal alveoli. CK16 mRNA was expressed in a similar pattern as CK19, but CK16 protein was not detected either in normal or in abnormal prostate tissues. In conclusion, the expression of CK19 in PIN lesions is similar to its tubular expression and would support an origin of PIN lesions from this structure rather than the alveolar portion of the glands. The similar cytokeratin expression between PIN lesions and invasive carcinoma further supports the concept that PIN is a precursor lesion of invasive carcinoma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9033282

[Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice].

PubMed

Liu, G Y; Gao, Z H; Li, L; Song, T T; Sheng, X G

2016-06-25

To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (P<0.01). (2) The tumor volume was (491± 68) mm(3) and tumor mass was (2.6±0.4) g in Jagged1 interference group, which were significantly lower than that in the blank plasmid group [(842±88) mm(3) and (4.4±0.8) g, respectively] and that in the control group [(851±90) mm(3) and (4.5±0.9) g, respectively; P<0.05], the tumor inhibition rate was 42.2% in Jagged1 interference group, which was significantly higher than that in the blank plasmid group and that in the control group (2.2% and 0, respectively), the differences were statistically significant (P<0.05). The relative mRNA and protein expression of Jagged1, Hes1 and Hey1 in xenograft tissues of nude micein Jagged1 interference group were lower than that in the other two groups, the differences were statistically significant (P<0.05). There were no differences of relative mRNA and protein expression of Notch1 in xenograft tissues of nude mice among the three groups (P>0.05). Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.

Expression and methylation of BDNF in the human brain in schizophrenia.

PubMed

Cheah, Sern-Yih; McLeay, Robert; Wockner, Leesa F; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

2017-08-01

To examine the combined effect of the BDNF Val66Met (rs6265) polymorphism and BDNF DNA methylation on transcriptional regulation of the BDNF gene. DNA methylation profiles were generated for CpG sites proximal to Val66Met, within BDNF promoter I and exon V for prefrontal cortex samples from 25 schizophrenia and 25 control subjects. Val66Met genotypes and BDNF mRNA expression data were generated by transcriptome sequencing. Expression, methylation and genotype data were correlated and examined for association with schizophrenia. There was 43% more of the BDNF V-VIII-IX transcript in schizophrenia samples. BDNF mRNA expression and DNA methylation of seven CpG sites were not associated with schizophrenia after accounting for age and PMI effects. BDNF mRNA expression and DNA methylation were not altered by Val66Met after accounting for age and PMI effects. DNA methylation of one CpG site had a marginally significant positive correlation with mRNA expression in schizophrenia subjects. Schizophrenia risk was not associated with differential BDNF mRNA expression and DNA methylation. A larger age-matched cohort with comprehensive clinical history is required to accurately identify the effects of genotype, mRNA expression and DNA methylation on schizophrenia risk.

Increased expression of ADAM 9 and ADAM 15 mRNA in pancreatic cancer.

PubMed

Yamada, Daisuke; Ohuchida, Kenoki; Mizumoto, Kazuhiro; Ohhashi, Seiji; Yu, Jun; Egami, Takuya; Fujita, Hayato; Nagai, Eishi; Tanaka, Masao

2007-01-01

A disintegrin and metalloproteases (ADAMs) comprise a multifunctional family of membrane-anchored proteins. ADAM 9 and ADAM 15 are involved in cell migration and invasion. Expression of ADAM 9 and ADAM 15 was reported to be altered in several types of cancer. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure the expression of ADAM 9 mRNA in bulk pancreatic tissues. Results showed no significant difference in the expression of ADAM 9 mRNA between pancreatic cancer and non-neoplastic pancreas. Primary cultured pancreatic fibroblasts also expressed ADAM 9 mRNA. Therefore, a laser microdissection and pressure catapulting technique was employed to isolate cancer cells from tumor tissues. The expression of ADAM 9 and ADAM 15 mRNA was measured in microdissected samples (cancer cells, n = 11; normal epithelial cells, n = 13 for ADAM 9; cancer cells, n = 9; normal epithelial cells, n = 9 for ADAM 15). Pancreatic cancer cells expressed significantly higher levels of ADAM 9 and ADAM 15 mRNA than did normal pancreatic epithelial cells (p = 0.016 for ADAM 9; p = 0.004 for ADAM 15). ADAM 9 and ADAM 15 are involved in pancreatic cancer. Microdissection-based analysis appears to be indispensable for the accurate analysis of the expression of certain ADAM family members in pancreatic cancer.

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

PubMed

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

2009-08-01

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

Midbrain dopamine neurons regulate preprotachykinin-A mRNA expression in the rat forebrain during development.

PubMed

Brené, S; Lindefors, N; Persson, H

1992-06-01

Intracerebroventricular 6-hydroxydopamine injections were performed at postnatal days 3 and 6 in animals pretreated with the norepinephrine uptakeblocker desimipramine in order to generate a selective lesion of dopamine neurons. In situ hybridization was then used to analyze preprotachykinin-A (PPT-A) mRNA expression in the lesioned as well as in saline-injected control animals. The midbrain dopaminergic lesion caused a 22-25% increase in the level of PPT-A mRNA in cingulate cortex and frontoparietal cortex when analysed at 2 weeks of age, compared to saline-injected control animals. In contrast, the lesion caused no change in PPT-A mRNA expression in the neonatal caudate-putamen. These results indicate that dopamine neurons downregulate the expression of PPT-A mRNA specifically in cingulate cortex and frontoparietal cortex during early postnatal brain development. In the adult rat forebrain, lesioned at P3 and P6, no change in the level of PPT-A mRNA was seen in cingulate cortex and frontoparietal cortex. However, a 29% decrease in PPT-A mRNA was seen in the lateral caudate-putamen with no significant change in neurons of medial caudate-putamen. Thus, dopamine neurons appears to exert a region specific influence on PPT-A mRNA expression during brain development.

Cytokine stimulation of MUC4 expression in human female reproductive tissue carcinoma cell lines and endometrial cancer.

PubMed

Chapela, Patricia J; Broaddus, Russell R; Hawkins, Shannon M; Lessey, Bruce A; Carson, Daniel D

2015-11-01

MUC4, a transmembrane glycoprotein, interferes with cell adhesion, and promotes EGFR signaling in cancer. Studies in rat models have demonstrated steroid hormonal regulation of endometrial MUC4 expression. In this study, qRT-PCR screening of mouse tissues determined that Muc4 mRNA also was robustly expressed in mouse uteri. Previous studies from our labs have demonstrated MUC4 mRNA was expressed at levels <1% of MUC1 mRNA in human endometrium and endometriotic tissue. Multiple human endometrial adenocarcinoma cell lines were assayed for MUC4 mRNA expression revealing extremely low basal expression in the Ishikawa, RL-95-2, AN3CA, and KLE lines. Moderate to high expression was observed in HEC50 and HEC-1A cells. MUC4 mRNA expression was not affected by progesterone and/or estrogen treatment, but was greatly stimulated at both mRNA and protein levels by proinflammatory cytokines (IFN-γ and TNF-α), particularly when used in combination. In endometrial tissue, MUC4 mRNA levels did not change significantly between normal or cancerous samples; although, a subset of patients with grade 1 and 2 tumors displayed substantially higher expression. Likewise, immunostaining of human endometrial adenocarcinoma tissues revealed little to no staining in many patients (low MUC4), but strong staining in some patients (high MUC4) independent of cancer grade. In cases where staining was observed, it was heterogeneous with some cells displaying robust MUC4 expression and others displaying little or no staining. Collectively, these observations demonstrate that while MUC4 is highly expressed in the mouse uterus, it is not a major mucin in normal human endometrium. Rather, MUC4 is a potential marker of endometrial adenocarcinoma in a subset of patients. © 2015 Wiley Periodicals, Inc.

Reversible Ca2+-induced fast-to-slow transition in primary skeletal muscle culture cells at the mRNA level

PubMed Central

Meißner, Joachim D; Kubis, Hans-Peter; Scheibe, Renate J; Gros, Gerolf

2000-01-01

The adult fast character and a Ca2+-inducible reversible transition from a fast to a slow type of rabbit myotube in a primary culture were demonstrated at the mRNA level by Northern blot analysis with probes specific for different myosin heavy chain (MyHC) isoforms and enzymes of energy metabolism. No non-adult MyHC isoform mRNA was detected after 22 days of culture. After 4 weeks of culture the fast MyHCIId mRNA was strongly expressed while MyHCI mRNA was virtually absent, indicating the fast adult character of the myotubes in the primary skeletal muscle culture. The data show that a fast-to-slow transition occurred in the myotubes at the level of MyHC isoform gene expression after treatment with the Ca2+ ionophore A23187. The effects of ionophore treatment were decreased levels of fast MyHCII mRNA and an augmented expression of the slow MyHCI gene. Changes in gene expression started very rapidly 1 day after the onset of ionophore treatment. Levels of citrate synthase mRNA increased and levels of glyceraldehyde 3-phosphate dehydrogenase mRNA decreased during ionophore treatment. This points to a shift from anaerobic to oxidative energy metabolism in the primary skeletal muscle culture cells at the level of gene expression. Withdrawal of the Ca2+ ionophore led to a return to increased levels of MyHCII mRNA and decreased levels of MyHCI mRNA, indicating a slow-to-fast transition in the myotubes and the reversibility of the effect of ionophore on MyHC isoform gene expression. PMID:10673542

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

PubMed Central

Okamura, Masumi; Yamanaka, Yasutaka; Shigemoto, Maki; Kitadani, Yuya; Kobayashi, Yuhko; Kambe, Taiho; Nagao, Masaya; Kobayashi, Issei; Okumura, Katsuzumi

2018-01-01

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. PMID:29746542

Expression profiles and associations of adiponectin and adiponectin receptors with intramuscular fat in Tibetan chicken.

PubMed

Zhang, R; Lin, Y; Zhi, L; Liao, H; Zuo, L; Li, Z; Xu, Y

2017-04-01

1. Adiponectin and its receptors (ADIPOR1 and ADIPOR2) are novel endocrine systems that act at various levels to modulate glucose and lipid metabolism. This study was designed to investigate the spatial expression of adiponectin, ADIPOR1 and ADIPOR2 genes in various tissues in Tibetan chicken. The temporal expression of adiponectin and its receptor mRNAs were also studied in adipose tissue, breast muscle and thigh muscle and the correlations of the levels of adiponectin, ADIPOR1 and ADIPOR2 mRNA with the contents of intramuscular fat in breast muscle and thigh muscle of Tibetan chicken were determined. 2. Quantitative real-time PCR detected chicken adiponectin, ADIPOR1 and ADIPOR2 mRNA transcripts in heart, liver, spleen, lung, kidney, skeletal muscle and adipose tissue. 3. Adipose tissue contained the highest amount of adiponectin mRNA followed by the kidney and liver. The expression levels of ADIPOR1 mRNA were significantly higher in adipose tissue, lung and spleen, and adipose tissue exhibited significantly higher levels of ADIPOR2 mRNA followed by the spleen and lung compared with other tissues. 4. Temporal expression profiles of adiponectin, ADIPOR1 and ADIPOR2 mRNA showed gender differences in adipose tissue and skeletal muscle at certain ages. In adipose tissue, adiponectin mRNA was higher in 154-d-old females and ADIPOR1 mRNA was higher in 154-d-old males: Adiponectin and ADIPOR2 mRNA were higher, and ADIPOR1 mRNA was lower, in thigh muscle in female compared with male chickens. 5. The correlation data showed that, except for adiponectin mRNA, the levels of ADIPOR1 and ADIPOR2 mRNA in thigh muscle of males were significantly positively correlated with IMF (r = 0.206 for the ADIPOR1 gene and r = 0.676 for the ADIPOR2 gene). 6. Taken together, it was concluded that adiponectin and the ADIPOR1 and ADIPOR2 genes are ubiquitously expressed in various tissues of Tibetan chicken and the expression of the adiponectin system is gender-dependant at certain ages in adipose tissue and skeletal muscle.

Differences in brain gene expression between sleep and waking as revealed by mRNA differential display and cDNA microarray technology.

PubMed

Cirelli, C; Tononi, G

1999-06-01

The consequences of sleep and sleep deprivation at the molecular level are largely unexplored. Knowledge of such molecular events is essential to understand the restorative processes occurring during sleep as well as the cellular mechanisms of sleep regulation. Here we review the available data about changes in neural gene expression across different behavioural states using candidate gene approaches such as in situ hybridization and immunocytochemistry. We then describe new techniques for systematic screening of gene expression in the brain, such as subtractive hybridization, mRNA differential display, and cDNA microarray technology, outlining advantages and disadvantages of these methods. Finally, we summarize our initial results of a systematic screening of gene expression in the rat brain across behavioural states using mRNA differential display and cDNA microarray technology. The expression pattern of approximately 7000 genes was analysed in the cerebral cortex of rats after 3 h of spontaneous sleep, 3 h of spontaneous waking, or 3 h of sleep deprivation. While the majority of transcripts were expressed at the same level among these three conditions, 14 mRNAs were modulated by sleep and waking. Six transcripts, four more expressed in waking and two more expressed in sleep, corresponded to novel genes. The eight known transcripts were all expressed at higher levels in waking than in sleep and included transcription factors and mitochondrial genes. A possible role for these known transcripts in mediating neural plasticity during waking is discussed.

Expression of inflammation-related genes in aldosterone-producing adenomas with KCNJ5 mutation.

PubMed

Murakami, Masanori; Yoshimoto, Takanobu; Nakano, Yujiro; Tsuchiya, Kyoichiro; Minami, Isao; Bouchi, Ryotaro; Fujii, Yasuhisa; Nakabayashi, Kazuhiko; Hashimoto, Koshi; Hata, Ken-Ichiro; Kihara, Kazunori; Ogawa, Yoshihiro

2016-08-05

The adrenocortical cells have been shown to produce various inflammatory cytokines such as TNFα and IL-6, which could modulate steroidogenesis. However, the role of inflammatory cytokines in aldosterone-producing adenomas (APAs) is not fully understood. In the present study, we examined the relationships between mRNA expression levels of the inflammation-related genes and somatic mutations in APA tissues. We evaluated mRNA expression levels of TNFA, IL6, and NFKB1 in APA tissues obtained from 44 Japanese APA patients. We revealed that mRNA expression patterns of the inflammation-related genes depended on a KCNJ5 somatic mutation. In addition, we showed that mRNA expression levels of the inflammation-related genes correlated with those of the steroidogenic enzyme CYP11B1 in the patients with APAs. The present study documented for the first time the expression of inflammation-related genes in APAs and the correlation of their expression levels with the KCNJ5 mutation status and mRNA expression levels of steroidogenic enzymes, indicating the pathophysiological relevance of inflammation-related genes in APAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Minoxidil upregulates the expression of vascular endothelial growth factor in human hair dermal papilla cells.

PubMed

Lachgar, S; Charveron, M; Gall, Y; Bonafe, J L

1998-03-01

The hair follicle dermal papilla which controls hair growth, is characterized in the anagen phase by a highly developed vascular network. We have demonstrated in a previous study that the expression of an angiogenic growth factor called vascular endothelial growth factor (VEGF) mRNA varied during the hair cycle. VEGF mRNA is strongly expressed in dermal papilla cells (DPC) in the anagen phase, but during the catagen and telogen phases. VEGF mRNA is less strongly expressed. This involvement of VEGF during the hair cycle allowed us to determine whether VEGF mRNA expression by DPC was regulated by minoxidil. In addition, the effect of minoxidil on VEGF protein synthesis in both cell extracts and DPC-conditioned medium, was investigated immunoenzymatically. Both VEGF mRNA and protein were significantly elevated in treated DPC compared with controls. DPC incubated with increasing minoxidil concentrations (0.2, 2, 6, 12 and 24 mumol/L) induced a dose-dependent expression of VEGF mRNA. Quantification of transcripts showed that DPC stimulated with 24 mumol/L minoxidil express six times more VEGF mRNA than controls. Similarly, VEGF protein production increases in cell extracts and conditioned media following minoxidil stimulation. These studies strongly support the likely involvement of minoxidil in the development of dermal papilla vascularization via a stimulation of VEGF expression, and support the hypothesis that minoxidil has a physiological role in maintaining a good vascularization of hair follicles in androgenetic alopecia.

Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination and immunomodulation.

PubMed

Fumuso, Elida; Giguère, Steeve; Wade, José; Rogan, Dragan; Videla-Dorna, Ignacio; Bowden, Raúl A

2003-11-15

Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial insemination. Endometrial biopsies were taken during consecutive cycles: (i) at estrus, when follicles reached 35 mm and at diestrus (7 +/- 1 days after ovulation); (ii) at 24 h post-AI, with dead semen (estrus) and in diestrus; (iii) at 24 h after treatment with a Mycobacterium phlei cell-wall extract (MCWE) preparation and AI (with dead semen), and at diestrus. mRNA expression was quantitated by real time PCR. Under basal conditions, SM had significantly higher mRNA expression of all cytokines in estrus and of IL-1beta and TNF-alpha in diestrus, compared to RM. After AI, there were no differences between RM and SM in estrus; however, mRNA expression for all three pro-inflammatory cytokines was higher than under basal conditions. In diestrus, RM showed significantly lower IL-1beta and TNF-alpha mRNA expression than SM. When MCWE was administered at time of AI, no differences between cytokine induction from RM and SM were found. Globally, mRNA expression for all three cytokines correlated well among themselves when expression was high. The present study showed that (i) in basal conditions RM had lower mRNA expression of pro-inflammatory cytokines than SM with no effect of estrous cycle; (ii) AI upregulated mRNA expression for all three cytokines in both RM and SM, with persistance in diestrus in the latter; (iii) treatment with MCWE at time of AI down-regulated mRNA expression of IL-1 with significant effects in SM which behaved like RM. Immunomodulation with MCWE could be of help in restoring homeostatic local inflammatory mechanisms, thus assisting in the prophylaxis of post-breeding endometritis in mares.

Effects of Electro-Acupuncture on Ovarian P450arom, P450c17α and mRNA Expression Induced by Letrozole in PCOS Rats

PubMed Central

Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

2013-01-01

Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg−1 of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and endocrine metabolic disorders in PCOS. PMID:24260211

Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats.

PubMed

Sun, Jie; Jin, Chunlan; Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

2013-01-01

Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg(-1) of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and endocrine metabolic disorders in PCOS.

Role of β1-Integrin in Colorectal Cancer: Case-Control Study

PubMed Central

Oh, Bo-Young; Kim, Kwang Ho; Chung, Soon Sup; Hong, Kyoung Sook

2014-01-01

Purpose In the metastatic process, interactions between circulating tumor cells (CTCs) and the extracellular matrix or surrounding cells are required. β1-Integrin may mediate these interactions. The aim of this study was to investigate whether β1-integrin is associated with the detection of CTCs in colorectal cancer. Methods We enrolled 30 patients with colorectal cancer (experimental group) and 30 patients with benign diseases (control group). Blood samples were obtained from each group, carcinoembryonic antigen (CEA) mRNA for CTCs marker and β1-integrin mRNA levels were estimated by using reverse transcription-polymerase chain reaction, and the results were compared between the two groups. In the experimental group, preoperative results were compared with postoperative results for each marker. In addition, we analyzed the correlation between the expressions of β1-integrin and CEA. Results CEA mRNA was detected more frequently in colorectal cancer patients than in control patients (P = 0.008). CEA mRNA was significantly reduced after surgery in the colorectal cancer patients (P = 0.032). β1-Integrin mRNA was detected more in colorectal cancer patients than in the patients with benign diseases (P < 0.001). In colorectal cancer patients, expression of β1-integrin mRNA was detected more for advanced-stage cancer than for early-stage cancer (P = 0.033) and was significantly decreased after surgery (P < 0.001). In addition, expression of β1-integrin mRNA was significantly associated with that of CEA mRNA in colorectal cancer patients (P = 0.001). Conclusion In conclusion, β1-integrin is a potential factor for forming a prognosis following surgical resection in colorectal cancer patients. β1-Integrin may be a candidate for use as a marker for early detection of micrometastatic tumor cells and for monitoring the therapeutic response in colorectal cancer patients. PMID:24851215

Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression.

PubMed

Gao, Jian; Ulekleiv, Camilla H; Halstensen, Trond S

2016-09-26

Increased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated. To examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines. The patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26 % if tumor expressed none to one EGFR-ligand, to 45 % in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did not mediate cisplatin resistance in the OSCC cell lines. Increased tumorous mRNA expression of four EGFR ligands was progressively associated with poor prognosis in HNSCC. Thus, EGFR-ligands mRNA expression pattern may be a new prognostic biomarker. The tightly regulated EGF-induced AREG mRNA expression was partly lost in the OSCC cell lines and restoring its regulation may be a new target in cancer treatment. Not applicable as the clinical data of the 498 HNSCC patients and their mRNA expression profiles were collected from the open TCGA database: http://cancergenome.nih.gov/cancersselected/headandneck .

Adverse early life experience and social stress during adulthood interact to increase serotonin transporter mRNA expression

PubMed Central

Gardner, Katherine L.; Hale, Matthew W.; Lightman, Stafford L.; Plotsky, Paul M.; Lowry, Christopher A.

2009-01-01

Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of serotonergic systems in the brain. To evaluate the effects of early life experience, adverse experiences during adulthood, and potential interactions between these factors on serotonin transporter (slc6a4) mRNA expression, we investigated in rats the effects of maternal separation (180 min/day from days 2–14 of life; MS180), neonatal handing (15 min/day from days 2–14 of life; MS15), or normal animal facility rearing control conditions (AFR) with or without subsequent exposure to adult social defeat on slc6a4 mRNA expression in the dorsal raphe nucleus (DR) and caudal linear nucleus. At the level of specific subdivisions of the DR, there were no differences in slc6a4 mRNA expression between MS15 and AFR rats. Among rats exposed to a novel cage control condition, increased slc6a4 mRNA expression was observed in the dorsal part of the DR in MS180 rats, relative to AFR control rats. In contrast, MS180 rats exposed to social defeat as adults had increased slc6a4 mRNA expression throughout the DR compared to both MS15 and AFR controls. Social defeat increased slc6a4 mRNA expression, but only in MS180 rats and only in the “lateral wings” of the DR. Overall these data demonstrate that early life experience and stressful experience during adulthood interact to determine slc6a4 mRNA expression. These data support the hypothesis that early life experience and major stressful life events contribute to dysregulation of serotonergic systems in stress-related neuropsychiatric disorders. PMID:19781533

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

PubMed

Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

2015-06-01

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

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.

Differential Expression Patterns of occ1-Related Genes in Adult Monkey Visual Cortex

PubMed Central

Takahata, Toru; Komatsu, Yusuke; Watakabe, Akiya; Hashikawa, Tsutomu; Tochitani, Shiro

2009-01-01

We have previously revealed that occ1 is preferentially expressed in the primary visual area (V1) of the monkey neocortex. In our attempt to identify more area-selective genes in the macaque neocortex, we found that testican-1, an occ1-related gene, and its family members also exhibit characteristic expression patterns along the visual pathway. The expression levels of testican-1 and testican-2 mRNAs as well as that of occ1 mRNA start of high in V1, progressively decrease along the ventral visual pathway, and end of low in the temporal areas. Complementary to them, the neuronal expression of SPARC mRNA is abundant in the association areas and scarce in V1. Whereas occ1, testican-1, and testican-2 mRNAs are preferentially distributed in thalamorecipient layers including “blobs,” SPARC mRNA expression avoids these layers. Neither SC1 nor testican-3 mRNA expression is selective to particular areas, but SC1 mRNA is abundantly observed in blobs. The expressions of occ1, testican-1, testican-2, and SC1 mRNA were downregulated after monocular tetrodotoxin injection. These results resonate with previous works on chemical and functional gradients along the primate occipitotemporal visual pathway and raise the possibility that these gradients and functional architecture may be related to the visual activity–dependent expression of these extracellular matrix glycoproteins. PMID:19073625

Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT: AFP, GPC3 mRNA for prediction of HCC.

PubMed

Wang, Yuliang; Shen, Zhongyang; Zhu, Zhijun; Han, Ruifa; Huai, Mingsheng

2011-03-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT). 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value.

Direct isolation of differentially expressed genes from a specific chromosome region of common wheat: application of the amplified fragment length polymorphism-based mRNA fingerprinting (AMF) method in combination with a deletion line of wheat.

PubMed

Kojima, T; Habu, Y; Iida, S; Ogihara, Y

2000-05-01

The amplified restriction fragment length polymorphism (AFLP)-based mRNA fingerprinting (AMF) method makes it possible systematically and conveniently to identify differentially expressed cDNAs with high reproducibility. We have applied the AMF method to the cloning of the Q gene of common wheat, which is located on the long arm of chromosome 5A and pleiotropically controls the spike morphology and the threshing character of seeds. Using the AMF method, we compared the fingerprints of mRNA samples extracted from the young spikes of Triticum aestivum cv. Chinese Spring (CS) carrying the Q gene to those of a chromosome deletion line of CS, namely, q5, which lacks 15% of 5AL including the Q gene. Approximately 12,200 fragments were produced after PCR with 256 primer combinations. Of these, 92 fragments were differentially expressed between CS and q5. Northern and Southern analyses showed that 16 fragments gave specific or relatively stronger transcript signals in CS, and these clones were present in single copy or in low copy numbers in the wheat genome. Four clones were genetically mapped to the region deleted in q5. Subsequently, one clone, pTaQ22, was mapped at the same locus as the Q gene, indicating that pTaQ22 corresponds to the Q gene or is tightly linked to it. DNA sequence data showed that pTaQ22 had no homology to any known genes, thus suggesting a novel function for this gene in flower morphogenesis. This AMF method might provide a straightforward method for isolating genes in the hexaploid background of common wheat.

[Expression of heat shock protein 70 and its mRNA in career exposure to manganese].

PubMed

Chen, Wenwen; Shao, Hua; Chi, Mingfeng; Zhang, Zhihu; Shan, Yongle; Zou, Wei

2015-10-01

To analyze the expression levels of heat shock protein70 (HSPs70) and HSPs70 mRNA in different exposure to manganese, and research the neuroprotective effect on the career exposure to manganese. From 2008 to 2009, with cross-sectional study design, and in a locomotive and rolling stock works, by stratified random sampling method, the exposed sample consisted of 180 welders from different welding shops and 100 unexposed in the last three years, non-welder controls with age-matched workers of similar socioeconomic status from the same industry. The control workers had not been exposed to neurotoxic chemicals. The mRNA expressions of four different metabolic enzyme were detected by SYBR Green I quantitative real-time polymerase chain reaction. The expression levels of the two enzymes mRNA in different exposure to manganese were analyzed. The expressions of HSPs70 were detected by Western blot. The concentration of air manganese was determined by GFAAS. The average concentration of 8 h time (8h-TWA) was used to express the level of individual exposure to manganese, according to the air manganese workplace occupational exposure limit (8h-TWA=0.15 mg/m3), the exposed group is divided into high exposed group (>0.15 mg/m3) and low exposure group (<0.15 mg/m3). The individuals exposed to manganese dose of exposed group ((0.25±0.31) mg/m3) was higher than the control group ((0.06±0.02) mg/m3) (t=6.15, P=0.001); individuals exposed to manganese dose of high exposure group for (0.42±0.34) mg/m3, which was higher than low exposure group (0.09±0.07) mg/m3 (t=9.80, P=0.001). HSPs70 mRNA and protein of exposure group (5.65±0.21, 3.26±0.15) were higher than the reference group (0.41±0.03, 1.32±0.12) (t=18.91, t=8.68, P=0.001). HSP70 mRNA and protein of high exposure group (6.48±0.37, 3.67±0.26) were higher than the low exposure group (5.15±0.23, 3.02±0.19) (t=3.24, t=2.01, P=0.003, P=0.043). The expression of peripheral blood lymphocytes HSPs70 level and HSPs70 mRNA workers exposed to manganese increased and protect nerve cells from related to Mn stimulation induced lipid peroxidation damag.

Polysome Fractionation to Analyze mRNA Distribution Profiles.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2017-02-05

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al. , 2014a and 2014b; Abdelmohsen et al. , 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al. , 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

Effect of electroacupuncture on the expression of interlukin-1beta mRNA after transient focal cerebral ischemia.

PubMed

Xu, Zhen-Feng; Wu, Gen-Cheng; Cao, Xiao-Ding

2002-01-01

It has been reported that interleukin-1beta (IL-1beta ) play a key role in the pathogenesis of cerebral ischemia. Acupuncture is an effective traditional medical therapy in China. The aim of present study was to evaluate the effect of electroacupuncture (EA) on IL-1beta mRNA expression after middle cerebral artery occlusion (MCAO) in rats. Using in situ hybridization technique, it was found that in the MCAO group the expression of IL-1beta mRNA was significantly increased at 2h, 6h, 12h after reperfusion in cerebral ischemic cortex compared with normal group. In EA+ MCAO group the expression of IL-1beta mRNA was significantly decreased at 2h, 6h and 12h in ischemic cortex compared with MCAO group. The results indicated that EA might decrease the IL-1beta protein expression by reducing the IL-beta mRNA expression in ischemic cortex.

The potential role of myocardial serotonin receptor 2B expression in canine dilated cardiomyopathy.

PubMed

Fonfara, Sonja; Hetzel, Udo; Oyama, Mark A; Kipar, Anja

2014-03-01

Serotonin signalling in the heart is mediated by receptor subtype 2B (5-HTR2B). A contribution of serotonin to valvular disease has been reported, but myocardial expression of 5-HTR2B and its role in canine dilated cardiomyopathy (DCM) is not known. The aim of the present study was to investigate myocardial 5-HTR2B mRNA expression in dogs with DCM and to correlate results with expression of markers for inflammation and remodelling. Myocardial samples from eight healthy dogs, four dogs with DCM, five with cardiac diseases other than DCM and six with systemic non-cardiac diseases were investigated for 5-HTR2B mRNA expression using quantitative PCR (qPCR). The results were compared to mRNA expression of selected cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP). Laser microdissection with subsequent qPCR and immunohistochemistry were employed to identify the cells expressing 5-HTR2B. The myocardium of control dogs showed constitutive 5-HTR2B mRNA expression. In dogs with DCM, 5-HTR2B mRNA values were significantly greater than in all other groups, with highest levels of expression in the left ventricle and right atrium. Myocytes were identified as the source of 5-HTR2B mRNA and protein. A significant positive correlation of 5-HTR2B mRNA with expression of several cytokines, MMPs and TIMPs was observed. The findings suggest that serotonin might play a role in normal cardiac structure and function and could contribute to myocardial remodelling and functional impairment in dogs with DCM. Copyright © 2013 Elsevier Ltd. All rights reserved.

Stimulatory effect of fibroblast-derived prostaglandin E₂ on keratinocyte stratification in the skin equivalent.

PubMed

Arai, Koji Y; Fujioka, Atsuko; Okamura, Ryoko; Nishiyama, Toshio

2014-01-01

Epidermal-dermal interaction plays important roles in physiological events such as wound healing. In this study, we examined a double paracrine mechanism between keratinocytes and fibroblasts through interleukin-1 (IL-1) and an IL-1-induced inflammatory mediator prostaglandin E₂ (PGE₂) using the skin equivalent. The epidermal layer of the skin equivalent expressed high levels of IL-1α mRNA (IL1A mRNA) and relatively low levels of IL-1β mRNA (IL1B mRNA). IL1A mRNA was not detected in fibroblasts. Fibroblasts also expressed low but not negligible levels of IL1B mRNA only in the presence of keratinocytes. Expression of prostaglandin-endoperoxide synthase 2 mRNA (PTGS2 mRNA) and production of PGE₂ in three-dimensionally cultured fibroblasts were noticeably stimulated by co-culture with keratinocytes, whereas PTGS2 mRNA expression in the epidermal layer was very low. In addition, hydroxyprostaglandin dehydrogenase 15-(NAD) mRNA was highly expressed in keratinocytes but not in fibroblasts, and exogenous IL-1β stimulated PTGS2 mRNA expression in the dermal equivalent. The thickness of the epidermal layer and the number of MKI67-positive keratinocytes in the skin equivalent were decreased by treatment with indomethacin, and the decrease recovered when exogenous PGE₂ was added. These results indicate that keratinocytes stimulate their own proliferation through a double paracrine mechanism mediated by IL-1 and PGE₂. © 2014 by the Wound Healing Society.

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

PubMed Central

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

2017-01-01

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

Integrative analysis of multi-omics data for identifying multi-markers for diagnosing pancreatic cancer

PubMed Central

2015-01-01

Background microRNA (miRNA) expression plays an influential role in cancer classification and malignancy, and miRNAs are feasible as alternative diagnostic markers for pancreatic cancer, a highly aggressive neoplasm with silent early symptoms, high metastatic potential, and resistance to conventional therapies. Methods In this study, we evaluated the benefits of multi-omics data analysis by integrating miRNA and mRNA expression data in pancreatic cancer. Using support vector machine (SVM) modelling and leave-one-out cross validation (LOOCV), we evaluated the diagnostic performance of single- or multi-markers based on miRNA and mRNA expression profiles from 104 PDAC tissues and 17 benign pancreatic tissues. For selecting even more reliable and robust markers, we performed validation by independent datasets from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) data depositories. For validation, miRNA activity was estimated by miRNA-target gene interaction and mRNA expression datasets in pancreatic cancer. Results Using a comprehensive identification approach, we successfully identified 705 multi-markers having powerful diagnostic performance for PDAC. In addition, these marker candidates annotated with cancer pathways using gene ontology analysis. Conclusions Our prediction models have strong potential for the diagnosis of pancreatic cancer. PMID:26328610

mRNA expression of dopamine receptors in peripheral blood lymphocytes of computer game addicts.

PubMed

Vousooghi, Nasim; Zarei, Seyed Zeinolabedin; Sadat-Shirazi, Mitra-Sadat; Eghbali, Fatemeh; Zarrindast, Mohammad Reza

2015-10-01

Excessive playing of computer games like some other behaviors could lead to addiction. Addictive behaviors may induce their reinforcing effects through stimulation of the brain dopaminergic mesolimbic pathway. The status of dopamine receptors in the brain may be parallel to their homologous receptors in peripheral blood lymphocytes (PBLs). Here, we have investigated the mRNA expression of dopamine D3, D4 and D5 receptors in PBLs of computer game addicts (n = 20) in comparison to normal subjects (n = 20), using a real-time PCR method. The results showed that the expression level of D3 and D4 dopamine receptors in computer game addicts were not statistically different from the control group. However, the expression of the mRNA of D5 dopamine receptor was significantly down-regulated in PBLs of computer game addicts and reached 0.42 the amount of the control group. It is concluded that unlike with drug addiction, the expression levels of the D3 and D4 dopamine receptors in computer game addicts are not altered compared to the control group. However, reduced level of the D5 dopamine receptor in computer game addicts may serve as a peripheral marker in studies where the confounding effects of abused drugs are unwanted.

Multiple lipopolysaccharide (LPS) injections alter interleukin 6 (IL-6), IL-7, IL-10 and IL-6 and IL-7 receptor mRNA in CNS and spleen.

PubMed

Szot, Patricia; Franklin, Allyn; Figlewicz, Dianne P; Beuca, Timothy Petru; Bullock, Kristin; Hansen, Kim; Banks, William A; Raskind, Murray A; Peskind, Elaine R

2017-07-04

Neuroinflammation is proposed to be an important component in the development of several central nervous system (CNS) disorders including depression, Alzheimer's disease, Parkinson's disease, and traumatic brain injury. However, exactly how neuroinflammation leads to, or contributes to, these central disorders is unclear. The objective of the study was to examine and compare the expression of mRNAs for interleukin-6 (IL-6), IL-7, IL-10 and the receptors for IL-6 (IL-6R) and IL-7 (IL-7R) using in situ hybridization in discrete brain regions and in the spleen after multiple injections of 3mg/kg lipopolysaccharide (LPS), a model of neuroinflammation. In the spleen, LPS significantly elevated IL-6 mRNA expression, then IL-10 mRNA, with no effect on IL-7 or IL-7R mRNA, while significantly decreasing IL-6R mRNA expression. In the CNS, LPS administration had the greatest effect on IL-6 and IL-6R mRNA. LPS increased IL-6 mRNA expression only in non-neuronal cells throughout the brain, but significantly elevated IL-6R mRNA in neuronal populations, where observed, except the cerebellum. LPS resulted in variable effects on IL-10 mRNA, and had no effect on IL-7 or IL-7R mRNA expression. These studies indicate that LPS-induced neuroinflammation has substantial but variable effects on the regional and cellular patterns of CNS IL-6, IL-7 and IL-10, and for IL-6R and IL-7R mRNA expression. It is apparent that administration of LPS can affect non-neuronal and neuronal cells in the brain. Further research is required to determine how CNS inflammatory changes associated with IL-6, IL-10 and IL-6R could in turn contribute to the development of CNS neurological disorders. Published by Elsevier Ltd.

Digital quantification of gene expression using emulsion PCR.

PubMed

Shi, Xiaolong; Tang, Chao; Wang, Wei; Zhou, Dequan; Lu, Zuhong

2010-01-01

Here we describe a single-molecule quantitative assay of mRNA levels based on mRNA mediate-ligation and BEAMing (beads, emulsion, amplification, and magnetics) technique, which allows accurate and parallel measurement of multiple genes from a small amount of cells. In this method, a pair of oligos complementary target mRNA was used to probe transcripts for each gene of interest. The ligated products of oligos pair were clonally amplified on beads in millions of parallel compartmentalized droplets in a water-in-oil emulsion. The levels of each transcript within a sample were measured by counting the number of the correspondingly amplified beads which were immobilized on a glass surface. To demonstrate its utility, this method has been applied to the quantitation of the mRNA levels for two transcription factors, Klf4 and Sox5, and a housekeeping gene, Gapdh, in human leukemia K562 cells before and after induction with phorbol 12-myristate 13-acetate. Interestingly, we found a significant downregulation of the mRNA level of Sox5 after phorbol 12-myristate 13-acetate treatment. The mRNA mediate-ligation and BEAMing technique provides an accurate and sensitive way to quantify the amount of multiple specific mRNA in a very small number of cells, which may be valuable in the studies requiring precise and parallel quantization of multiple mRNA in the defined cell populations.

The Silencing of RECK Gene is Associated with Promoter Hypermethylation and Poor Survival in Hepatocellular Carcinoma

PubMed Central

Zhang, Changsong; Ling, Yang; Zhang, Chenghui; Xu, Yun; Gao, Lu; Li, Rong; Zhu, Jing; Fan, Lieying; Wei, Lixin

2012-01-01

Background: To evaluate the promoter methylation status of RECK gene and mRNA expression in patients with hepatocellular carcinoma (HCC). Methods: We analyzed RECK methylation by MSP, and RECK mRNA by real-time PCR in 74 HCC. The liver cell lines (7721, Chang and Hep-G2) were treated with 5-Aza-CdR and TSA. Results: RECK mRNA were lower in HCC tissues (Mean -∆Ct = -3.29) than that in Non-Hcc tissues (Mean -∆Ct = -2.42). Expression of RECK was elevated in only 24 (32.43%) of the 74 HCC patients but decreased (-∆∆Ct<0) in 50 (67.57%) of the patients. RECK promoter was hypermethylated in 55.4% (41/74) of HCCs, and in only 17.6% (13/74) of Non-Hcc samples. RECK mRNA were lower in HCC patients with hypermethylation (∆MI>=0.5) (Mean -∆∆Ct = -1.75) than those with demethylation (∆MI<0.5) (Mean -∆∆Ct = 0.05), and there is a decreased tendency for RECK mRNA in HCC patients with promoter hypermethylation (p = 0.002). There was a significantly correlation found between RECK mRNA and poor survival after surgery. After treated by 5-Aza-CdR and TSA, we found that RECK mRNA induced different changes in 7721, Chang and Hep-G2 cells. And RECK demethylation also induced by epigenetic inhibitors. Conclusion: The results suggested that the hypermethylation may lead to promoter silencing of RECK mRNA and associated with poor survival in HCC. PMID:22419890

Gene regulation of atrial natriuretic peptide A, B, and C receptors in rat glomeruli.

PubMed

Itoh, K; Nonoguchi, H; Shiraishi, N; Tomita, K

1999-01-01

Atrial natriuretic peptide (ANP) has three types of receptor. We investigated the gene regulation of three types of ANP receptors (ANPR-A, B, and C) in rat glomeruli using reverse transcription coupled with competitive polymerase chain reaction (PCR). Competitive PCR revealed that ANPR-C mRNA expression was most abundant (ANPR-C > A > B) in glomeruli from control rats among mRNA expressions of three receptors, which were 20- to 15,000-fold higher than those in inner medullary collecting ducts. Two days' dehydration caused reversible decreases of ANPR-A, B, and C mRNAs by 50-80%. To determine the mechanisms of down-regulation of mRNA expression, isolated glomeruli were incubated in isotonic or hypertonic solution. Hyperosmolality induced by NaCl, mannitol or raffinose caused significant increases of ANPR-A, B, and C mRNA expression. Hypertonicity by urea showed smaller effects. ANP stimulated the expression of ANPR-A, B, and C mRNA in vitro. These results indicate that dehydration caused reversible decreases of ANPR-A, B, and C mRNA expression in glomeruli, and these decreases were not caused by increased plasma osmolality but probably by lower circulating levels of ANP.

Expression of pituitary adenylate cyclase-activating polypeptide 1 and 2 receptor mRNA in gallbladder tissue of patients with gallstone or gallbladder polyps.

PubMed

Zhang, Zhen-Hai; Wu, Shuo-Dong; Gao, Hong; Shi, Gang; Jin, Jun-Zhe; Kong, Jing; Tian, Zhong; Su, Yang

2006-03-07

To detect the expression of pituitary adenylate cyclase-activating polypeptide receptor 1 (VPCAP1-R)and VPCAP2-R mRNA in gallbladder tissues of patients with gallstone or gallbladder polyps. The expression of VPCAP1-R and VPCAP2-R mRNA in gallbladder tissues was detected in 25 patients with gallstone,8 patients with gallbladder polyps and 7 donors of liver transplantation by reverse transcription polymerase chain reaction (RT-PCR). The VPCAP2-R mRNA expression level in the control group (1.09+/-0.58) was lower than that in the gallbladder polyp group (1.64+/-0.56) and the gallstone group (1.55+/-0.45) (P<0.05) while the VPCAP1-R mRNA expression level in the control group (1.15+/-0.23) was not apparently different from that in the gallbladder polyp group (1.28+/-0.56) and the gallstone group (1.27+/-0.38). The abnormal expression of VPCAP2-R mRNA in gallbladder tissue may play a role in the formation of gallbladder stone and gallbladder polyps.

Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

NASA Astrophysics Data System (ADS)

Cheglakov, Zoya

Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide a beneficial strategy for simultaneous tracking readily accomplished by multicolor imaging with diverse fluorescent tags. The third method in this thesis will demonstrate the advantage of DNAzymes probes amplification, and offers potential strategy to monitor miRNAs in mammalian live cells.

Effects of electroacupuncture on corticotropin-releasing hormone in rats with chronic visceral hypersensitivity

PubMed Central

Liu, Hui-Rong; Fang, Xiao-Yi; Wu, Huan-Gan; Wu, Lu-Yi; Li, Jing; Weng, Zhi-Jun; Guo, Xin-Xin; Li, Yu-Guang

2015-01-01

AIM: To investigate the effect of electroacupuncture on corticotropin-releasing hormone (CRH) in the colon, spinal cord, and hypothalamus of rats with chronic visceral hypersensitivity. METHODS: A rat model of chronic visceral hypersensitivity was generated according to the internationally accepted method of colorectal balloon dilatation. In the 7th week after the procedure, rats were randomly divided into a model group (MG), electroacupuncture group (EA), and sham electroacupuncture group (S-EA). After treatment, the abdominal withdrawal reflex (AWR) score was used to assess the behavioral response of visceral hyperalgesia. Immunohistochemistry (EnVision method), ELISA, and fluorescence quantitative PCR methods were applied to detect the expression of CRH protein and mRNA in the colon, spinal cord, and hypothalamus. RESULTS: The sensitivity of the rats to the colorectal distension stimulus applied at different strengths (20-80 mmHg) increased with increasing stimulus strength, resulting in increasing AWR scores in each group. Compared with NG, the AWR score of MG was significantly increased (P < 0.01). After conducting EA, the AWR scores of the rats were decreased compared with MG rats. The relative expression of CRH mRNA in the colon, spinal cord, and hypothalamus of MG rats was significantly increased compared with NG rats (P < 0.01). CRH mRNA in the colon and spinal cord of EA and S-EA rats was decreased to varying degrees (P > 0.05) compared with normal rats (NG). However, the decrease in EA compared with MG rats was statistically significant (P < 0.01). The average optical density of CRH expression in the colon of the MG rats was significantly enhanced compared with NG (P < 0.05), while the average optical density of CRH expression in the EA and S-EA rats was significantly decreased compared with MG rats (P < 0.01, P < 0.05, respectively). Compared with MG rats, the CRH concentration in the spinal cord of EA rats was significantly reduced (P < 0.01), but there was no significant change in S-EA rats (P > 0.05). CONCLUSION: Electroacupuncture at the Shangjuxu acupoint was able to significantly reduce the visceral hypersensitivity in rats, and regulated the expression of CRH protein and mRNA in the colon, spinal cord and hypothalamus at different levels, playing a therapeutic role in this model of irritable bowel syndrome. PMID:26109804

[Recombinant expression of Schistosoma japonicum fructose-1, 6-bisphos- phate aldolase and its expression in different developmental stages of S. japonicum].

PubMed

Yan, Ke; Zhong, Zheng-rong; Xu, Yun-xia; Ding, Shu-qin; Hu, Jian-guo; Xu, Yuan-hong; Luo, Qing-lie; Shen, Ji-long

2015-06-01

To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum. FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR. SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum. SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.

The role of EMMPRIN expression in ovarian epithelial carcinomas

PubMed Central

Zhao, Yang; Chen, Shuo; Gou, Wen-feng; Niu, Zhe-feng; Zhao, Shuang; Xiao, Li-jun; Takano, Yasuo; Zheng, Hua-chuan

2013-01-01

Purpose: Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of ovarian epithelial carcinomas. Methods: EMMPRIN siRNA were transfected into ovarian carcinoma cells with the phenotypes and their related molecules examined. EMMPRIN expression was determined in ovarian normal tissue, benign and borderline tumors, and epithelial carcinomas by real-time PCR, western blot, and immunohistochemisty. Results: EMMPRIN siRNA treatment resulted in a lower growth, G1 arrest, apoptotic induction, decreased migration, and invasion. The transfectants showed reduced expression of Wnt5a, Akt, p70s6k, Bcl-xL, survivin, VEGF, and MMP-9 than mock and control cells at both mRNA and protein levels. According to real-time PCR and western blot, EMMPRIN mRNA or protein level was higher in ovarian borderline tumor and carcinoma than normal ovary and benign tumors (P < 0.05), and positively correlated with dedifferentiation and FIGO staging (P < 0.05). Immuhistochemically, EMMPRIN expression was positively correlated with FIGO staging, dedifferentiation, Ki-67 expression, the lower cumulative and relapse-free survival rate (P < 0.05). Conclusions: Upregulated expression of EMMPRIN protein and mRNA might be involved in the pathogenesis, differentiation, and progression of ovarian carcinomas, possibly by modulating cellular events, such as proliferation, cell cycle, apoptosis, migration, and invasion. PMID:23966157

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-09-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed Central

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-01-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility. Images PMID:1697032

Simplified Identification of mRNA or DNA in Whole Cells

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

A recently invented method of detecting a selected messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA) sequence offers two important advantages over prior such methods: it is simpler and can be implemented by means of compact equipment. The simplification and miniaturization achieved by this invention are such that this method is suitable for use outside laboratories, in field settings in which space and power supplies may be limited. The present method is based partly on hybridization of nucleic acid, which is a powerful technique for detection of specific complementary nucleic acid sequences and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes.

Effect of Supplemental Trace Minerals on Hsp-70 mRNA Expression in Commercial Broiler Chicken.

PubMed

Rajkumar, U; Vinoth, A; Reddy, E Pradeep Kumar; Shanmugam, M; Rao, S V Rama

2018-01-02

The effects of supplementing the organic forms of selenium (Se), chromium (Cr), and zinc (Zn) on Hsp-70 mRNA expression and body weight in broiler chickens were evaluated. 200 chicks were equally distributed into stainless steel battery brooders at the rate of 5 birds per pen and reared under heat stress condition up to 42 nd day. The chicks were fed with three experimental diets supplemented with organic forms of Se (0.30 mg/kg), Cr (2 mg/kg), and Zn (40 mg/kg) during the starter and finisher phases and a control diet without any supplementation. On the 21st and 42nd day, 20 birds from each period were sacrificed and samples were collected for analysis. Organic Se, Cr, and Zn supplementation significantly (P < 0.05) reduced the expression of Hsp-70 mRNA levels. The Hsp-70 mRNA expression levels were significantly (P < 0.05) different between the tissues studied with spleen having the lowest expression level. Hsp-70 mRNA expression level was not affected by age of the birds. The study concluded that organic trace mineral (oTM) supplementation resulted in low Hsp-70 mRNA expression, indicating reduced heat stress in broilers.

Assessing the Effect of High Performance Inulin Supplementation via KLF5 mRNA Expression in Adults with Type 2 Diabetes: A Randomized Placebo Controlled Clinical Trail.

PubMed

Ghavami, Abed; Roshanravan, Neda; Alipour, Shahriar; Barati, Meisam; Mansoori, Behzad; Ghalichi, Faezeh; Nattagh-Eshtivan, Elyas; Ostadrahimi, Alireza

2018-03-01

Purpose: The worldwide prevalence of metabolic disorders such as diabetes is increasing rapidly. Currently, the complications of diabetes are the major health concern. The aim of this study was to investigate the effect of high performance (HP) inulin supplementation on glucose homeostasis via KLF5 mRNA expression in adults with type 2 diabetes. Methods: In the present clinical trial conducted for a duration of 6 weeks, 46 volunteers diabetic patients referring to diabetes clinic in Tabriz, Iran, were randomly assigned into intervention (n= 23, consuming 10 gr/d HP inulin) and control groups (n= 23, consuming 10 gr/ d starch). We assessed glycemic and anthropometric indices, blood lipids and plasmatic level of miR-375 as well as KLF5 mRNA expression before and after the intervention. Results: Findings indicated that inulin supplementation significantly decreased fasting plasma glucose (FPG) in comparison to the placebo group (P<0.001). Also Intra-group and between group results showed that inulin supplementation resulted in significant decrease in KLF5 mRNA expression in peripheral blood mononuclear cells (PBMCs) (Fold change: 0.61± 0.11; P-value= 0.001) and significant increase in plasmatic level of miR-375 (Fold change: 3.75± 0.70; P-value=0.004). Conclusion: Considering the improvements of FPG level in diabetic patients, it seems that HP inulin supplementation may be beneficial in controlling diabetes via the expression of some genes. However, further studies are needed to achieve concise conclusions.

Assessing the Effect of High Performance Inulin Supplementation via KLF5 mRNA Expression in Adults with Type 2 Diabetes: A Randomized Placebo Controlled Clinical Trail

PubMed Central

Ghavami, Abed; Roshanravan, Neda; Alipour, Shahriar; Barati, Meisam; Mansoori, Behzad; Ghalichi, Faezeh; Nattagh- Eshtivan, Elyas; Ostadrahimi, Alireza

2018-01-01

Purpose: The worldwide prevalence of metabolic disorders such as diabetes is increasing rapidly. Currently, the complications of diabetes are the major health concern. The aim of this study was to investigate the effect of high performance (HP) inulin supplementation on glucose homeostasis via KLF5 mRNA expression in adults with type 2 diabetes. Methods: In the present clinical trial conducted for a duration of 6 weeks, 46 volunteers diabetic patients referring to diabetes clinic in Tabriz, Iran, were randomly assigned into intervention (n= 23, consuming 10 gr/d HP inulin) and control groups (n= 23, consuming 10 gr/ d starch). We assessed glycemic and anthropometric indices, blood lipids and plasmatic level of miR-375 as well as KLF5 mRNA expression before and after the intervention. Results: Findings indicated that inulin supplementation significantly decreased fasting plasma glucose (FPG) in comparison to the placebo group (P<0.001). Also Intra-group and between group results showed that inulin supplementation resulted in significant decrease in KLF5 mRNA expression in peripheral blood mononuclear cells (PBMCs) (Fold change: 0.61± 0.11; P-value= 0.001) and significant increase in plasmatic level of miR-375 (Fold change: 3.75± 0.70; P-value=0.004). Conclusion: Considering the improvements of FPG level in diabetic patients, it seems that HP inulin supplementation may be beneficial in controlling diabetes via the expression of some genes. However, further studies are needed to achieve concise conclusions. PMID:29670837

p300 expression repression by hypermethylation associated with tumour invasion and metastasis in oesophageal squamous cell carcinoma

PubMed Central

Zhang, Changsong; Li, Ke; Wei, Lixin; Li, Zhengyou; Yu, Ping; Teng, Lijuan; Wu, Kusheng; Zhu, Jin

2007-01-01

Background Aberrant promoter methylation is an important mechanism for gene silencing. Aims To evaluate the promoter methylation status of p300 gene in patients with oesophageal squamous cell carcinoma (OSCC). Methods The methylation status of p300 promoter was analysed by methylation‐specific PCR (MSP) in 50 OSCC tissues and the matching non‐cancerous tissues. Oesophageal cancer cell lines (ECa‐109 and TE‐10) were treated with the demethylation agent 5‐aza‐2′‐deoxycytidine (5‐Aza‐CdR), and p300 mRNA expression was detected by RT‐PCR. Results p300 methylation was found in 42% (21/50) of the OSCC tissues, but in only 20% (10/50) of the corresponding non‐cancerous tissues (p = 0.017). In OSCC samples, 65% of those with deep tumour invasion (adventitia) and 63% samples with metastasis revealed p300 promoter methylation (p<0.05). p300 mRNA expression was observed in 19.0% (4/21) of methylated tumours and 58.6% (17/29) of unmethylated tumours (p = 0.005). In addition, p300 mRNA expression was observed in 40% (4/10) of methylated non‐neoplastic tissues and 87.5% (35/40) of unmethylated non‐tumours (p = 0.001). The demethylation caused by 5‐Aza‐CdR increased the p300 mRNA expression levels in oesophageal cancer cell lines. Conclusions p300 transcription silenced by promoter hypermethylation could play a role in the pathogenesis of oesophageal squamous cell carcinoma. PMID:17965222

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

PubMed

Manzardo, A M; Gunewardena, S; Butler, M G

2013-09-10

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling. Copyright © 2013 Elsevier B.V. All rights reserved.

Role of insulin-like growth factor-1 (IGF-1) in regulating cell cycle progression

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

Ma, Qi-lin; Yang, Tian-lun; Yin, Ji-ye

2009-11-06

Aims: Insulin-like growth factor-1 (IGF-1) is a polypeptide protein hormone, similar in molecular structure to insulin, which plays an important role in cell migration, cell cycle progression, cell survival and proliferation. In this study, we investigated the possible mechanisms of IGF-1 mediated cell cycle redistribution and apoptosis of vascular endothelial cells. Method: Human umbilical vein endothelial cells (HUVECs) were pretreated with 0.1, 0.5, or 2.5 {mu}g/mL of IGF-1 for 30 min before the addition of Ang II. Cell cycle redistribution and apoptosis were examined by flow cytometry. Expression of Ang II type 1 (AT{sub 1}) mRNA and cyclin E proteinmore » were determined by RT-PCR and Western blot, respectively. Results: Ang II (1 {mu}mol/L) induced HUVECs arrested at G{sub 0}/G{sub 1}, enhanced the expression level of AT{sub 1} mRNA in a time-dependent manner, reduced the enzymatic activity of nitric oxide synthase (NOS) and nitric oxide (NO) content as well as the expression level of cyclin E protein. However, IGF-1 enhanced NOS activity, NO content, and the expression level of cyclin E protein, and reduced the expression level of AT{sub 1} mRNA. L-NAME significantly counteracted these effects of IGF-1. Conclusions: Our data suggests that IGF-1 can reverse vascular endothelial cells arrested at G{sub 0}/G{sub 1} and apoptosis induced by Ang II, which might be mediated via a NOS-NO signaling pathway and is likely associated with the expression levels of AT1 mRNA and cyclin E proteins.« less

Effect of Intermittent Hypoxia and Rimonabant on Glucose Metabolism in Rats: Involvement of Expression of GLUT4 in Skeletal Muscle

PubMed Central

Wang, Xiaoya; Yu, Qin; Yue, Hongmei; Zeng, Shuang; Cui, Fenfen

2015-01-01

Background Obstructive sleep apnea (OSA) and its main feature, chronic intermittent hypoxia (IH) during sleep, is closely associated with insulin resistance (IR) and diabetes. Rimonabant can regulate glucose metabolism and improve IR. The present study aimed to assess the effect of IH and rimonabant on glucose metabolism and insulin sensitivity, and to explore the possible mechanisms. Material/Methods Thirty-two rats were randomly assigned into 4 groups: Control group, subjected to intermittent air only; IH group, subjected to IH only; IH+NS group, subjected to IH and treated with normal saline; and IH+Rim group, subjected to IH and treated with 10 mg/kg/day of rimonabant. All rats were killed after 28 days of exposure. Then, the blood and skeletal muscle were collected. We measured fasting blood glucose levels, fasting blood insulin levels, and the expression of glucose transporter 4 (GLUT4) in both mRNA and protein levels in skeletal muscle. Results IH can slow weight gain, increase serum insulin level, and reduce insulin sensitivity in rats. The expressions of GLUT4 mRNA, total GLUT4, and plasma membrane protein of GLUT4 (PM GLUT4) in skeletal muscle were decreased. Rimonabant treatment was demonstrated to improve weight gain and insulin sensitivity of the rats induced by IH. Rimonabant significantly upregulated the expression of GLUT4 mRNA, PM GLUT4, and total GLUT4 in skeletal muscle. Conclusions The present study demonstrates that IH can cause IR and reduced expression of GLUT4 in both mRNA and protein levels in skeletal muscle of rats. Rimonabant treatment can improve IH – induced IR, and the upregulation of GLUT4 expression may be involved in this process. PMID:26503060

Hypoxia-Induced Expression of VEGF Splice Variants and Protein in Four Retinal Cell Types

PubMed Central

Watkins, William M.; McCollum, Gary W.; Savage, Sara R.; Capozzi, Megan E.; Penn, John S.; Morrison, David G.

2014-01-01

The purpose of this study was to investigate the hypoxia-induced Vegf120, Vegf164 and Vegf188 mRNA expression profiles in rat Müller cells (MC), astrocytes, retinal pigmented epithelial cells (RPE) and retinal microvascular endothelial cells (RMEC) and correlate these findings to VEGF secreted protein. Cultured cells were exposed to normoxia or hypoxia. Total RNA was isolated from cell lysates and Vegf splice variant mRNA copy numbers were assayed by a validated qRT-PCR external calibration curve method. mRNA copy numbers were normalized to input total RNA. Conditioned medium was collected from cells and assayed for total VEGF protein by ELISA. Hypoxia increased total Vegf mRNA and secreted protein in all the retinal cell types, with the highest levels observed in MC and astrocytes ranking second. Total Vegf mRNA levels in hypoxic RPE and RMEC were comparable; however, the greatest hypoxic induction of each Vegf splice variant mRNA was observed in RMEC. RPE and RMEC ranked 3rd and 4th respectively, in terms of secreted total VEGF protein in hypoxia. The Vegf120, Vegf164 and Vegf188 mRNA splice variants were all increased in hypoxic cells compared to normoxic controls. In normoxia, the relative Vegf splice variant mRNA levels ranked from highest to lowest for each cell type were Vegf164>Vegf120>Vegf188. Hypoxic induction did not alter this ranking, although it did favor an increased stoichiometry of Vegf164 mRNA over the other two splice variants. MC and astrocytes are likely to be the major sources of total Vegf, and Vegf164 splice variant mRNAs, and VEGF protein in retinal hypoxia. PMID:24076411

Effects of betaine on lipopolysaccharide-induced memory impairment in mice and the involvement of GABA transporter 2

PubMed Central

2011-01-01

Background Betaine (glycine betaine or trimethylglycine) plays important roles as an osmolyte and a methyl donor in animals. While betaine is reported to suppress expression of proinflammatory molecules and reduce oxidative stress in aged rat kidney, the effects of betaine on the central nervous system are not well known. In this study, we investigated the effects of betaine on lipopolysaccharide (LPS)-induced memory impairment and on mRNA expression levels of proinflammatory molecules, glial markers, and GABA transporter 2 (GAT2), a betaine/GABA transporter. Methods Mice were continuously treated with betaine for 13 days starting 1 day before they were injected with LPS, or received subacute or acute administration of betaine shortly before or after LPS injection. Then, their memory function was evaluated using Y-maze and novel object recognition tests 7 and 10-12 days after LPS injection (30 μg/mouse, i.c.v.), respectively. In addition, mRNA expression levels in hippocampus were measured by real-time RT-PCR at different time points. Results Repeated administration of betaine (0.163 mmol/kg, s.c.) prevented LPS-induced memory impairment. GAT2 mRNA levels were significantly increased in hippocampus 24 hr after LPS injection, and administration of betaine blocked this increase. However, betaine did not affect LPS-induced increases in levels of mRNA related to inflammatory responses. Both subacute administration (1 hr before, and 1 and 24 hr after LPS injection) and acute administration (1 hr after LPS injection) of betaine also prevented LPS-induced memory impairment in the Y-maze test. Conclusions These data suggest that betaine has protective effects against LPS-induced memory impairment and that prevention of LPS-induced changes in GAT2 mRNA expression is crucial to this ameliorating effect. PMID:22053950

Effects of quercetin on CDK4 mRNA and protein expression in A549 cells infected by H1N1

PubMed Central

WAN, QIAOFENG; WANG, HAO; LIN, YUAN; GU, LIGANG; HAN, MEI; YANG, ZHIWEI; ZHANG, YANLI; MA, RUI; WANG, LI; WANG, ZHISHENG

2013-01-01

This study was conducted to investigate the effects of quercetin on the expression of cyclin-dependent kinase (CDK4) mRNA and protein in A549 lung epithelial tumor cells infected by H1N1. First, the Thiazolyl Blue Tetrazolium Bromide (MTT) method was used to determine H1N1 virulence, quercetin cytotoxicity and inhibition of the cytopathic effect of H1N1 on A549 cells by quercetin. Subsequently, 100 TCID50 H1N1 was used to infect A549 cells for 2 h prior to culture in maintenance media containing 10 mg/l quercetin. After 4, 12, 24 and 48 h of culture, the cells were collected and total RNA and protein were extracted. Fluorescent quantitative polymerase chain reaction and western blot analysis were then performed to assess the expression of CDK4 mRNA and protein. The experiment demonstrated that the TCID50 of H1N1 in A549 cells was 10−4.75, the maximum non-toxic concentration of quercetin in A549 cells was 30–60 mg/l and the minimum effective concentration of quercetin for the inhibition of the H1N1 cytopathic effect on A549 cells was 10 mg/l. The results indicated that quercetin may significantly inhibit CDK4 mRNA and protein overexpression caused by H1N1 within 4–48 h. In conclusion, quercetin may protect against H1N1 infection by effectively reducing the mRNA and protein expression of CDK4 caused by H1N1 infection. PMID:24649026

Finding new pathway-specific regulators by clustering method using threshold standard deviation based on DNA chip data of Streptomyces coelicolor.

PubMed

Yang, Yung-Hun; Kim, Ji-Nu; Song, Eunjung; Kim, Eunjung; Oh, Min-Kyu; Kim, Byung-Gee

2008-09-01

In order to identify the regulators involved in antibiotic production or time-specific cellular events, the messenger ribonucleic acid (mRNA) expression data of the two gene clusters, actinorhodin (ACT) and undecylprodigiosin (RED) biosynthetic genes, were clustered with known mRNA expression data of regulators from S. coelicolor using a filtering method based on standard deviation and clustering analysis. The result identified five regulators including two well-known regulators namely, SCO3579 (WlbA) and SCO6722 (SsgD). Using overexpression and deletion of the regulator genes, we were able to identify two regulators, i.e., SCO0608 and SCO6808, playing roles as repressors in antibiotics production and sporulation. This approach can be easily applied to mapping out new regulators related to any interesting target gene clusters showing characteristic expression patterns. The result can also be used to provide insightful information on the selection rules among a large number of regulators.

Expression of NMDA receptor subunits in human blood lymphocytes: A peripheral biomarker in online computer game addiction.

PubMed

Sadat-Shirazi, Mitra-Sadat; Vousooghi, Nasim; Alizadeh, Bentolhoda; Makki, Seyed Mohammad; Zarei, Seyed Zeinolabedin; Nazari, Shahrzad; Zarrindast, Mohammad Reza

2018-05-23

Background and aims Repeated performance of some behaviors such as playing computer games could result in addiction. The NMDA receptor is critically involved in the development of behavioral and drug addictions. It has been claimed that the expression level of neurotransmitter receptors in the brain may be reflected in peripheral blood lymphocytes (PBLs). Methods Here, using a real-time PCR method, we have investigated the mRNA expression of GluN2A, GluN2D, GluN3A, and GluN3B subunits of the NMDA receptor in PBLs of male online computer game addicts (n = 25) in comparison with normal subjects (n = 26). Results Expression levels of GluN2A, GluN2D, and GluN3B subunits were not statistically different between game addicts and the control group. However, the mRNA expression of the GluN3A subunit was downregulated in PBLs of game addicts. Discussion and conclusions Transcriptional levels of GluN2A and GluN2D subunits in online computer game addicts are similar to our previously reported data of opioid addiction and are not different from the control group. However, unlike our earlier finding of drug addiction, the mRNA expression levels of GluN3A and GluN3B subunits in PBLs of game addicts are reduced and unchanged, respectively, compared with control subjects. It seems that the downregulated state of the GluN3A subunit of NMDA receptor in online computer game addicts is a finding that deserves more studies in the future to see whether it can serve as a peripheral biomarker in addiction studies, where the researcher wants to rule out the confusing effects of abused drugs.

Live-cell imaging of endogenous mRNAs with a small molecule.

PubMed

Sato, Shin-ichi; Watanabe, Mizuki; Katsuda, Yousuke; Murata, Asako; Wang, Dan Ohtan; Uesugi, Motonari

2015-02-02

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein 2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

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

Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122; Zhao, Zhe

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels highermore » in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.« less

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis.

PubMed

Spangler, Jacob B; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

PubMed Central

Spangler, Jacob B.; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

Prefrontal mRNA expression of long and short isoforms of D2 dopamine receptor: Possible role in delayed learning deficit caused by early life interleukin-1β treatment.

PubMed

Schwarz, Alexander P; Trofimov, Alexander N; Zubareva, Olga E; Lioudyno, Victoria I; Kosheverova, Vera V; Ischenko, Alexander M; Klimenko, Victor M

2017-08-30

Long (D2L) and short (D2S) isoform of the D2 dopamine receptor are believed to play different roles in behavioral regulation. However, little is known about differential regulation of these isoforms mRNA expression during the process of learning in physiological and pathological states. In this study, we have investigated the combined effect of training in active avoidance (AA) paradigm and chronic early life treatment with pro-inflammatory cytokine interleukin (IL)-1β (1μg/kg i.p., P15-21) on D2S and D2L dopamine receptor mRNA expression in the medial prefrontal cortex (mPFC) of adult rats. We have shown differential regulation of D2 short and long mRNA isoform expression in the mPFC. There was no effect of AA-training on D2S mRNA expression, while D2L mRNA was downregulated in AA-trained control (intact and saline-treated) animals, and this effect was not observed in rats treated with IL-1β. D2S mRNA expression level negatively correlated with learning ability within control (saline-treated and intact) groups but not in IL-1β-treated animals. Thus, prefrontal expression of distinct D2 dopamine receptor splice variants is supposed to be implicated in cognitive decline caused by early life immune challenge. Copyright © 2017 Elsevier B.V. All rights reserved.

Inactivation of MSH3 by promoter methylation correlates with primary tumor stage in nasopharyngeal carcinoma

PubMed Central

Ni, Haifeng; Jiang, Bo; Zhou, Zhen; Yuan, Xiaoyang; Cao, Xiaolin; Huang, Guangwu; Li, Yong

2017-01-01

The aim of this study was to investigate the inactivation of the MutS homolog human 3 (MSH3) gene by promoter methylation in nasopharyngeal carcinoma (NPC). Methylation-specific PCR, semi-quantitative reverse transcription PCR and immunohistochemical analysis were used to detect methylation and the mRNA and protein expression levels of MSH3 in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues. The association between promoter methylation and mRNA expression, and the mRNA and protein expression of the gene and clinical factors was analyzed. The promoter methylation of MSH3 was detected in 50% (27/54) of the primary tumors, but not in the 16 NNE tissues. The mRNA and protein expression levels were significantly decreased in the 54 cases of human NPC as compared to the 16 NNE tissues (P<0.05). The MSH3-methylated cases exhibited significantly lower mRNA and protein expression levels than the unmethylated cases (P<0.05). The MSH3 mRNA and protein expression levels were significantly associated with the variable T stage (P<0.05); however, they did not correlate with the age and sex of the patients, or with the N stage, TNM classification or histopathological subtype (P>0.05). On the whole, MSH3 was frequently inactivated by promoter methylation and its mRNA and protein expression correlated with the primary tumor stage in NPC. PMID:28656302

Overexpression of peptide deformylase in breast, colon, and lung cancers

PubMed Central

2013-01-01

Background Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. Methods The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. Results PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. Conclusions This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation. PMID:23815882

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

PubMed Central

CRUZ, Ariadne Cristiane Cabral; SILVA, Mariana Lúcia; CAON, Thiago; SIMÕES, Cláudia Maria Oliveira

2012-01-01

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. Objectives This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. Material and Methods Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. Conclusions We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs. PMID:23329244

Chorioamnionitis Occurring in Women With Preterm Rupture of the Fetal Membranes Is Associated With a Dynamic Increase in mRNAs Coding Cytokines in the Maternal Circulation

PubMed Central

Gordon, Lavinia; Kapoor, Jada; Walker, Susan P.; Whitehead, Clare; Kaitu’u-Lino, Tu’uhevaha J.; Pell, Gabrielle; Hannan, Natalie J.; Tong, Stephen

2015-01-01

Background: Preterm prelabor rupture of the fetal membranes (PPROM) is a significant contributor to the morbidity and mortality of preterm birth, particularly in the setting of chorioamnionitis. No sensitive or specific diagnostic or predictive test currently exists for the accurate diagnosis of chorioamnionitis. Our aim was to measure messenger RNA (mRNA) coding cytokines in the maternal blood and examine whether they were increased in association with chorioamnionitis at delivery. Methods/Results: We performed a prospective cohort study of women recruited with PPROM at a mean gestational age of 28.9 weeks at risk of developing chorioamnionitis. Blood was sampled from participants, and the expression of mRNA coding for proinflammatory genes was measured in women with and without chorioamnionitis at the time of delivery as well as gestation-matched healthy controls. Expression was measured using quantitative polymerase chain reaction (PCR) and also digital PCR. Interleukin 1β (IL1B) mRNA expression in maternal blood was elevated in women with chorioamnionitis compared to gestation-matched controls. Importantly, among women admitted with PPROM, digital PCR confirmed a significant increase in IL1B expression in maternal blood in women with chorioamnionitis compared to women without chorioamnionitis. Polymerase chain reaction array revealed that CD14, nuclear factor of κ light polypeptide gene enhancer in B-cells 1 (NFKB1), and tumor necrosis factor receptor super family-interacting serine–threonine kinase 1 mRNA were significantly increased in women with chorioamnionitis compared to controls. Digital PCR confirmed that NFKB1 mRNA was significantly increased in patients with chorioamnionitis compared to controls and that CD14 levels increased over time in patients with PPROM having chorioamnionitis. Conclusion: Measuring circulating proinflammatory mRNA in women with PPROM may distinguish those with chorioamnionitis from those without, in turn providing better targeted therapies and appropriate timing of delivery. PMID:25616398

Alteration of Cyclic-AMP Response Element Binding Protein in the Postmortem Brain of Subjects with Bipolar Disorder and Schizophrenia

PubMed Central

Ren, Xinguo; Rizavi, Hooriyah S.; Khan, Mansoor A.; Bhaumik, Runa; Dwivedi, Yogesh; Pandey, Ghanshyam N.

2013-01-01

Background Abnormalities of cyclic-AMP (cAMP) response element binding protein (CREB) function has been suggested in bipolar (BP) illness and schizophrenia (SZ), based on both indirect and direct evidence. To further elucidate the role of CREB in these disorders, we studied CREB expression and function in two brain areas implicated in these disorders, i.e., dorsolateral prefrontal cortex (DLPFC) and cingulate gyrus (CG). Methods We determined CREB protein expression using Western blot technique, CRE-DNA binding using gel shift assay, and mRNA expression using real-time RT-polymerase chain reaction (qPCR) in DLPFC and CG of the postmortem brain of BP (n = 19), SZ (n = 20), and normal control (NC, n = 20) subjects. Results We observed that CREB protein and mRNA expression and CRE-DNA binding activity were significantly decreased in the nuclear fraction of DLPFC and CG obtained from BP subjects compared with NC subjects. However, the protein and mRNA expression and CRE-DNA binding in SZ subjects was significantly decreased in CG, but not in DLPFC, compared with NC. Conclusion These studies thus indicate region-specific abnormalities of CREB expression and function in both BP and SZ. They suggest that abnormalities of CREB in CG may be associated with both BP and SZ, but its abnormality in DLPFC is specific to BP illness. PMID:24148789

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

PubMed

Azimi, Sayyed M; Sheridan, Steven D; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2018-05-01

Identification of optimal transcription-factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription-factor copy-numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH -expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition. © 2018, Azimi et al.

Gill structural integrity changes in fish deficient or excessive in dietary isoleucine: Towards the modulation of tight junction protein, inflammation, apoptosis and antioxidant defense via NF-κB, TOR and Nrf2 signaling pathways.

PubMed

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

2017-04-01

This study firstly aimed to test the impact of dietary isoleucine (Ile) on tight junction protein, inflammation, apoptosis, antioxidant defense and related signaling molecule gene expression in the gill of fish. Young grass carp (Ctenopharyngodon idella) (weighing 256.8 ± 3.5 g) were fed six diets containing graded levels of Ile, namely, 3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg diet for 8 weeks. The results firstly revealed that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and zonula occludens-1 (ZO-1) and up-regulated the mRNA expression of claudin-12, which led to the intercellular structure damage of fish gill. These effects were partially ascribed to the up-regulation of pro-inflammatory cytokines [interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α)] mRNA expressions that referring to up-regulated nuclear factor κB P65 (NF-κB P65) mRNA expression and down-regulated inhibitor factor κBα (IκBα) mRNA expression, and the down-regulation of anti-inflammatory cytokines [interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1)] mRNA expressions that referring to the down-regulated TOR and S6K1 mRNA expression. Interestingly, no change in claudin 15 mRNA level was observed among every treatment. At the same time, the results firstly indicated that Ile deficiency also resulted in the cellular structure damage of fish gill: (1) DNA fragmentation partially due to the up-regulation of caspase-3, caspase-8 and caspase-9 mRNA expression; (2) increase in protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the impaired antioxidant defense [indicated by decreased glutathione (GSH) level and depressed anti-superoxide anion (ASA), anti-hydroxyl radical (a-HR), copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities] that referring to the down-regulation of corresponding antioxidant enzyme mRNA expressions and the related signaling molecules Nrf2 mRNA expression. Ile excess caused similar negative effects that observed in Ile-deficient group, whereas these negative effects were reversed with appropriate Ile supplementation. In conclusion, our results indicated that Ile deficiency or excess disrupted the structural integrity of fish gill, partially due to the trigger of apoptosis, the impairment of antioxidant defense, and the regulation of tight junction protein, inflammatory cytokines, apoptosis-related, antioxidant enzymes and related signaling molecules mRNA expressions in the fish gill. Copyright © 2017 Elsevier Ltd. All rights reserved.

Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

NASA Technical Reports Server (NTRS)

Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

1997-01-01

A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

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.

[Effect of TUBB3, TS and ERCC1 mRNA expression on chemoresponse and clinical outcome of advanced gastric cancer by multiplex branched-DNA liquid chip technology].

PubMed

Huang, Jin; Hu, Huabin; Xie, Yangchun; Tang, Youhong; Liu, Wei; Zhong, Meizuo

2013-06-01

To analyze the impact of β-tubulin-III (TUBB3), thymidylate synthase (TS) and excision repair cross complementation group 1 (ERCC1) mRNA expression on chemoresponse and clinical outcome of patients with advanced gastric cancer treated with TXT/CDDP/FU (DCF) regimen chemotherapy. The study population consisted of 48 patients with advanced gastric cancer. All patients were treated with DCF regimen palliative chemotherapy. The mRNA expressions of TUBB3, TS and ERCC1 of primary tumors were examined by multiplex branched-DNA liquid chip technology. The patients with low TUBB3 mRNA expression had higher response rate to chemotherapy than patients with high TUBB3 expression (P=0.011). There were no significant differences between response rate and TS or ERCC1 expression pattern. Median overall survival (OS) and median time to progression (TTP) were significantly longer in patients with low TUBB3 mRNA expression (P=0.002, P<0.001). TS or ERCC1 expression was not correlated with TTP and OS. In the combined analysis including TUBB3, TS and ERCC1, the patients with 0 or 1 high expression gene had better response rate, TTP and OS than the remaining patients (all P<0.001). Multivariate analysis revealed that ECOG (Eastern Cooperative Oncology Group)≥2 (HR=2.42, P=0.009) and TUBB3 (HR=2.34, P=0.036) mRNA expression significantly impacted on OS. High TUBB3 mRNA expression is correlated with resistance to DCF regimen chemotherapy. TUBB3 might be a predictive and prognostic factor in patients with advanced gastric cancer treated with TXT-based chemotherapy. The combined evaluation of TUBB3, TS and ERCC1 expression can promote the individual treatment in advanced gastric cancer.

Effect of supplementing a fibrous diet with a xylanase and β-glucanase blend on growth performance, intestinal glucose uptake, and transport-associated gene expression in growing pigs.

PubMed

Agyekum, A K; Sands, J S; Regassa, A; Kiarie, E; Weihrauch, D; Kim, W K; Nyachoti, C M

2015-07-01

The present study evaluated supplemental carbohydrase effect on performance, intestinal nutrient uptake, and transporter mRNA expressions in growing pigs offered a high-fiber diet manufactured with distillers dried grains with solubles (DDGS). Twenty-four pigs (22.4 ± 0.7 kg BW) were randomly assigned to 1of 3 nutritionally adequate diets (8 pigs per diet) based on corn and soybean meal (SBM) with either 0 (control) or 30% DDGS (high fiber [HF]). The third diet was supplemented with a xylanase and β-glucanase blend (XB) in addition to the 30% DDGS (HF+XB). Parameters determined were ADFI, ADG, G:F, plasma glucose and plasma urea nitrogen (PUN) concentrations, jejunal tissue electrophysiological properties, and mRNA expressions of the sodium-dependent glucose transport 1 (SGLT1) and cationic AA transporter, bo,+AT, in the jejunal and ileal tissues. In addition, mRNA expressions of the short-chain fatty acid transporters, monocarboxylate transporter 1 (MCT1) and sodium-coupled monocarboxylate transporter, and mucin genes were quantified in the ileum. Feed intake, plasma glucose, and jejunal tissue electrophysiological properties were not affected (P > 0.05) by diet. However, control-fed pigs had superior growth rate and feed efficiency and higher PUN (P < 0.05) than HF- and HF+XB-fed pigs. The HF diet increased (P < 0.05) SGLT1 mRNA expression in the jejunum and decreased (P < 0.05) bo,+ mRNA expression in the ileum. The XB supplementation also increased bo,+ mRNA expression in the ileum relative to HF-fed pigs. Additionally, MCT1 mRNA expression was greater (P < 0.05) in the ileum of the HF- and HF+XB-fed pigs. In the present study, XB supplementation influenced nutrient transporter mRNA expression, although it was not accompanied by improved pig performance.

[Effect of qihuang decoction on mRNA expressions of Bcl-2, Bax, and Caspase-3, 9 in intestinal mucosa epithelium of ischemia/reperfusion injured rats].

PubMed

Yu, Qing-Sheng; Yu, Hong-Liang; Pan, Jin-Fang

2011-02-01

To observe the effect of Qihuang Decoction (QHD) on mRNA expression of apoptosis genes Bcl-2, Bax, and signal transduction molecules Caspase-3, 9 in intestinal mucosa epithelium of ischemia/ reperfusion (I/R) injured rats. Forty Wistar rats were randomized equally into 4 groups, the control group, the model group, the glutamine group, and the QHD group. Rats in the latter two groups were gastric infused with glutamine and QHD respectively for 3 days, but saline was infused instead to rats in the control group and model group. After then, except those in the control group intervened only by sham operation, rats were made into I/R injured model by 45 min occlusion of superior mesenteric artery followed by 1 h reperfusion. Immediately after modeling, mRNA expressions of Bcl-2, Bax, Caspase-3, and Caspase-9 in intestinal mucosa epithelium of rats were detected by reverse transcription-polymerase chain reaction (RT-PCR). Compared with the control group, mRNA expressions of Bcl-2, Bax, Caspase-3 and Caspase-9 were higher in the other three groups (P < 0.05). Compared with the model group, Bcl-2 mRNA expression was higher, while the expressions of the other three indices were lower in both the glutamine group and the QHD group (P < 0.05); and comparisons between the glutamine group and the QHD group showed a more depressed Bax mRNA expression (0.281 +/- 0.087 vs 0.350 +/- 0.053) and higher Bcl-2/Bax ratio (1.648 vs 1. 374) in the QHD group. QHD can reduce the I/R injury in the intestinal mucosa epithelium by inhibiting the cell apoptosis. The mechanism may be correlated with increased Bcl-2 mRNA expressions and decreased mRNA expressions of Bax, Caspase-3 and Caspase-9.

Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics

PubMed Central

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

2000-01-01

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

Biphasic Modulation of NOS Expression, Protein and Nitrite Products by Hydroxocobalamin Underlies Its Protective Effect in Endotoxemic Shock: Downstream Regulation of COX-2, IL-1β, TNF-α, IL-6, and HMGB1 Expression

PubMed Central

Sampaio, André L. F.; Dalli, Jesmond; Brancaleone, Vincenzo; D'Acquisto, Fulvio; Perretti, Mauro; Wheatley, Carmen

2013-01-01

Background. NOS/•NO inhibitors are potential therapeutics for sepsis, yet they increase clinical mortality. However, there has been no in vivo investigation of the (in vitro) •NO scavenger, cobalamin's (Cbl) endogenous effects on NOS/•NO/inflammatory mediators during the immune response to sepsis. Methods. We used quantitative polymerase chain reaction (qPCR), ELISA, Western blot, and NOS Griess assays, in a C57BL/6 mouse, acute endotoxaemia model. Results. During the immune response, pro-inflammatory phase, parenteral hydroxocobalamin (HOCbl) treatment partially inhibits hepatic, but not lung, iNOS mRNA and promotes lung eNOS mRNA, but attenuates the LPS hepatic rise in eNOS mRNA, whilst paradoxically promoting high iNOS/eNOS protein translation, but relatively moderate •NO production. HOCbl/NOS/•NO regulation is reciprocally associated with lower 4 h expression of TNF-α, IL-1β, COX-2, and lower circulating TNF-α, but not IL-6. In resolution, 24 h after LPS, HOCbl completely abrogates a major late mediator of sepsis mortality, high mobility group box 1 (HMGB1) mRNA, inhibits iNOS mRNA, and attenuates LPS-induced hepatic inhibition of eNOS mRNA, whilst showing increased, but still moderate, NOS activity, relative to LPS only. experiments (LPS+D-Galactosamine) HOCbl afforded significant, dose-dependent protection in mice Conclusions. HOCbl produces a complex, time- and organ-dependent, selective regulation of NOS/•NO during endotoxaemia, corollary regulation of downstream inflammatory mediators, and increased survival. This merits clinical evaluation. PMID:23781123

Immunomodulatory Effects of Deokgu Thermomineral Water Balneotherapy on Oxazolone-Induced Atopic Dermatitis Murine Model

PubMed Central

Lee, Young Bok; Kim, Su Jin; Park, Sae Mi; Lee, Kyung Ho; Han, Hyung Jin; Yu, Dong Soo; Woo, So Youn; Yun, Seong Taek; Hamm, Se-Yeong; Kim, Hong Jig

2016-01-01

Background Although the therapeutic mechanism of balneotherapy for atopic dermatitis has not been clarified, many atopic patients who visit thermomineral springs have shown clinical improvements. Objective This study was aimed to evaluate the immunomodulatory effect of thermomineral water balneotherapy on the atopic dermatitis murine model. Methods The oxazolone-induced atopic dermatitis murine model was used to evaluate the therapeutic effect of balneotherapy with Deokgu thermomineral water compared with distilled water. Histologic evaluation and confocal microscopic imaging were performed to analyze the lesional expression of cluster-of-differentiation (CD)4 and forkhead box p3 (Foxp3). Lesional mRNA expression of interleukin (IL) 33, thymic stromal lymphopoietin (TSLP), and Foxp3 was evaluated by real-time reverse transcription polymerase chain reaction. Results Compared with the distilled water bath group, confocal microscopic evaluation of CD4 and Foxp3 merged images showed increased expression of regulatory T cells in the thermomineral balneotherapy group. The lesional mRNA level of IL-33 showed a reduced trend in the thermomineral balneotherapy group, whereas the level of mRNA of Foxp3 was increased. TSLP showed a decreased trend in both distilled water and thermomineral water bath groups. There was a trend of reduced expression in lesional IL-33 mRNA but increased cell count of CD4+ Foxp3+ regulatory T cells in thermomineral balneotherapy compared with distilled water bath. Conclusion Therefore, thermomineral balneotherapy can be an effective and safe adjuvant therapeutic option for atopic dermatitis. PMID:27081266

Oxytocin Intranasal Administration Affects Neural Networks Upstream of GNRH Neurons.

PubMed

Salehi, Mohammad Saied; Khazali, Homayoun; Mahmoudi, Fariba; Janahmadi, Mahyar

2017-08-01

The last decade has witnessed a surge in studies on the clinical applications of intranasal oxytocin as a method of enhancing social interaction. However, the molecular and cellular mechanisms underlying its function are not completely understood. Since oxytocin is involved in the regulation of hypothalamic-pituitary-gonadal axis by affecting the gonadotropin-releasing hormone (GNRH) system, the present study addressed whether intranasal application of oxytocin has a role in affecting GNRH expression in the male rat hypothalamus. In addition, we assessed expression of two excitatory (kisspeptin and neurokinin B) and two inhibitory (dynorphin and RFamide-related peptide-3) neuropeptides upstream of GNRH neurons as a possible route to relay oxytocin information. Here, adult male rats received 20, 40, or 80 μg oxytocin intranasally once a day for 10 consecutive days, and then, the posterior (PH) and anterior hypothalamus (AH) dissected for evaluation of target genes. Using qRT-PCR, we found that oxytocin treatment increased Gnrh mRNA levels in both the PH and AH. In addition, oxytocin at its highest dose increased kisspeptin expression in the AH by around 400%, whereas treatments, dose dependently decreased kisspeptin mRNA in the PH. The expression of neurokinin B was increased from the basal levels following the intervention. Furthermore, although intranasal-applied oxytocin decreased hypothalamic RFamide-related peptide-3 mRNA level, the dynorphin mRNA was not affected. These observations are consistent with the hypothesis that applications of intranasal oxytocin can affect the GNRH system.

Prognostic impact of mRNA levels of osteopontin splice variants in soft tissue sarcoma patients.

PubMed

Hahnel, Antje; Wichmann, Henri; Greither, Thomas; Kappler, Matthias; Würl, Peter; Kotzsch, Matthias; Taubert, Helge; Vordermark, Dirk; Bache, Matthias

2012-04-02

It is well known that osteopontin (OPN) plays an important role in tumor progression and that a high OPN expression level in several tumor entities correlates with poor prognosis in cancer patients. However, little is known about the prognostic relevance of the OPN mRNA splice variants. We analyzed the mRNA expression levels of different OPN splice variants in tumor tissue of 124 soft tissue sarcoma (STS) patients. Quantitative real-time PCR (qRT-PCR) was used to analyze the mRNA expression level of three OPN splice variants (OPN-a, -b and -c). The multivariate Cox's proportional hazard regression model revealed that high mRNA expression levels of OPN splice variants are significantly associated with poor prognosis in STS patients (n = 124). Women (n = 68) with high mRNA expression levels of OPN-a and OPN-b have an especially elevated risk of tumor-related death (OPN-a: RR = 3.0, P = 0.01, CI = 1.3-6.8; OPN-b: RR = 3.4, P = 0.01, CI = 1.4-8.2). In particular, we found that high mRNA expression levels of OPN-b and OPN-c correlated with a high risk of tumor-related death in STS patients that received radiotherapy (n = 52; OPN-b: RR = 10.3, P < 0.01, CI = 2.0-53.7; OPN-c: RR = 11.4, P < 0.01, CI = 2.2-59.3). Our study shows that elevated mRNA expression levels of OPN splice variants are negative prognostic and predictive markers for STS patients. Further studies are needed to clarify the impact of the OPN splice variants on prognosis.

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

PubMed

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

2008-07-01

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

Comparative effects of low-level laser therapy pre- and post-injury on mRNA expression of MyoD, myogenin, and IL-6 during the skeletal muscle repair.

PubMed

Alves, Agnelo Neves; Ribeiro, Beatriz Guimarães; Fernandes, Kristianne Porta Santos; Souza, Nadhia Helena Costa; Rocha, Lília Alves; Nunes, Fabio Daumas; Bussadori, Sandra Kalil; Mesquita-Ferrari, Raquel Agnelli

2016-05-01

This study analyzed the effect of pre-injury and post-injury irradiation with low-level laser therapy (LLLT) on the mRNA expression of myogenic regulatory factors and interleukin 6 (IL-6) during the skeletal muscle repair. Male rats were divided into six groups: control group, sham group, LLLT group, injury group; pre-injury LLLT group, and post-injury LLLT group. LLLT was performed with a diode laser (wavelength 780 nm; output power 40 mW' and total energy 3.2 J). Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior (TA) muscle. After euthanasia, the TA muscle was removed for the isolation of total RNA and analysis of MyoD, myogenin, and IL-6 using real-time quantitative PCR. Significant increases were found in the expression of MyoD mRNA at 3 and 7 days as well as the expression of myogenin mRNA at 14 days in the post-injury LLLT group in comparison to injury group. A significant reduction was found in the expression of IL-6 mRNA at 3 and 7 days in the pre-injury LLLT and post-injury LLLT groups. A significant increase in IL-6 mRNA was found at 14 days in the post-injury LLLT group in comparison to the injury group. LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin. Moreover, the both forms of LLLT administration were able to modulate the mRNA expression of IL-6 during the muscle repair process.

TGF-beta1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k.

PubMed

Lechuga, Carmen G; Hernández-Nazara, Zamira H; Domínguez Rosales, José-Alfredo; Morris, Elena R; Rincón, Ana Rosa; Rivas-Estilla, Ana María; Esteban-Gamboa, Andrés; Rojkind, Marcos

2004-11-01

Transforming growth factor-beta1 (TGF-beta1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-beta1 induces a transient elevation of alpha1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the alpha1(I) collagen mRNA. In the present study, we report that TGF-beta1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of alpha1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-beta1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70(S6k). Altogether, our data suggest that regulation of MMP-13 by TGF-beta1 is a complex process involving transcriptional and posttranscriptional mechanisms.

Intervention of rosiglitazone on myocardium Glut-4 mRNA expression during ischemia-reperfusion injury in cardio-pulmonary bypass in dogs.

PubMed

Liu, Bin; Liang, Guiyou; Xu, Gang; Liu, Daxin; Cai, Qingyong; Gao, Zhenyu

2013-01-01

During cardiac pulmonary bypass (CPB), myocardial ischemia-reperfusion (I/R) induces heart glucose metabolism impairment. Our previous research showed that the decreased glucose utilization is due to decreased glucose transporter-4 (Glut-4) expression and translocation to myocyte surface membranes. This study further examined whether rosiglitazone, a synthetic agonist of peroxisome proliferator-activated receptor γ, could intervene glucose metabolism by regulating Glut-4 mRNA during I/R in dogs. Cardiac ischemia was induced by cardiopulmonary bypass for 30 or 120 min. Plasma insulin and glucose concentrations were measured at pre-bypass (control), aortic cross-clamp off (I/R) at 15, 45, and 75 min. The left ventricle biopsies were taken for the expression of Glut-4 mRNA by real-time RT-PCR. In dogs receiving 120 min ischemia, coronary arterial, venous glucose concentrations, plasma insulin levels, and insulin resistant index (IRI) were increased, but the expression of Glut-4 mRNA was decreased obviously at 15 min of reperfusion, and recovered gradually. On the other hand, these changes were relatively mild in dogs treated with rosiglitazone in cardioplegic solution and expression of Glut-4 mRNA was increased remarkably. It is concluded that the decrease in total amount of Glut-4 mRNA expression could be one of the important molecular mechanisms, which causes the myocardium insulin resistance. The longer the ischemia period, the decrease in amount of Glut-4 mRNA was more dramatic. Adding rosiglitazone into the cardioplegic solution during I/R can increase the amount of Glut-4 mRNA expression, mitigate the myocardium insulin resistance and improve the myocardium I/R injury during CPB.

[Effect of endoplasmic reticulum stress on the expression and osteogenic differentiation of periodontal ligament stem cells].

PubMed

Xue, Peng; Li, Bei; Tan, Jun; An, Ying; Jin, Yan; Wang, Qintao

2015-09-01

To determine the activity of endoplasmic reticulum stress (ERS) and its effect on osteogenic differentiation of periodontal ligament stem cells (PDLSC) in inflammatory microenvironment. PDLSC were obtained from the primary culture of the human tooth and cloned with limited diluted method. Real-time reverse transcription (RT)-PCR was used to examine the different expression of thapsigargin (TG) treated PDLSC and lipopolysaccharide (LPS) treated PDLSC. Real-time RT-PCR, alizarin red staining and cetyl pyridine chloride quantitative analyze were used to examine the osteogenic differentiation of PDLSC, TG + PDLSC, LPS + PDLSC and LPS + PDLSC + 4-PBA. Protein kinase receptor like endoplasmic reticulum kinase (PERK), glucose regulated protein 78 (GRP78), transcription activation factor 4(ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP) mRNA expression in group PDLSC + TG in 6 h were respectively 1.49 ± 0.24, 2.77 ± 0.60, 1.75 ± 0.16, 2.16 ± 0.32, which were all greater than that in group PDLSC (P < 0.05). PERK, CHOP mRNA expression reached the peak at 6 h (1.76 ± 0.08, 2.31 ± 0.17) and were greater than group PDLSC (P < 0.05). ERS could suppress osteogenic differentiation of TG + PDLSC and LPS + PDLSC. The runt-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN) mRNA expression of group TG + PDLSC was respectively 0.73 ± 0.06, 0.01 ± 0.00, 0.20 ± 0.06 (P < 0.05). The RUNX2, ALP, OCN mRNA expression of group LPS + PDLSC was respectively 0.80 ± 0.06, 0.48 ± 0.05, 0.29 ± 0.04 (P < 0.05). The RUNX2, ALP, OCN mRNA expression of group PDLSC + TG + 4-PBA was respectively 1.10 ± 0.09, 0.74 ± 0.05, 0.67 ± 0.13, which were greater higher than that of group LPS + PDLSC (P < 0.05). ERS was activated in PDLSC and suppressed osteogenic differentiation of PDLSC, which can simulate inflammatory microenvironment in vitro. This effect can be recovered by using ERS inhibitor 4-PBA.

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

PubMed

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

2015-01-01

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

Lipoic Acid Exerts Antioxidant and Anti-inflammatory Effects in Response to Heat Shock in C2C12 Myotubes.

PubMed

Lee, Cheng-Tse; Chang, Li-Ching; Wu, Pei-Fung

2016-06-01

This study explored that lipoic acid treatment for 24 h significantly upregulated and promoted heat shock-induced catalase expression and downregulated GPx1 messenger RNA (mRNA) expression, indicating that lipoic acid exhibits antioxidant activity in the decomposition of hydrogen peroxide by upregulating catalase expression. Moreover, lipoic acid treatment for 3 h increased and promoted heat shock-induced interleukin (IL)-6 mRNA and protein levels and that for 24 h downregulated IL-6 mRNA expression, suggesting a dual effect of lipoic acid on IL-6 regulation. Lipoic acid alone failed to increase or reduce tumor necrosis factor (TNF)-α mRNA and protein levels, whereas heat shock alone downregulated TNF-α mRNA and protein expression. These data suggest that lipoic acid does not have a proinflammatory role and that heat shock acts as an anti-inflammatory agent by downregulating TNF-α expression in C2C12 myotubes. Moreover, lipoic acid or heat shock alone upregulated the IL-6 receptor (IL-6R-α) and glycoprotein 130 (gp130) mRNA expression followed by IL-6 expression; these data indicate that the regulation of lipoic acid or heat shock is mediated by IL-6R signaling, thus suggesting that C2C12 myotubes possesses a mechanism for regulating IL-6R and gp130 expression following lipoic acid treatment or heat shock.

[Effect of qinghuobaiduyin on the expression of high mobility group box chromosomal protein 1 in macrophage].

PubMed

Li, Ping; Xu, Dan; Luo, Chengqun

2010-07-01

To observe the expression of high mobility group box chromosomal protein 1(HMGB1) in RAW264.7 macrophages after interfering with burning serum and qinghuobaidu-yin (QHBDY), and to find out the endogenous protection mechanism of QHBDY resisting inflammation reaction. RT-PCR was used to detect the expression of HMGB1 in RAW264.7 macrophages after interfering RAW264.7 macrophages with normal SD rat serum, burning SD rat serum, and QHBDY feeding SD rat serum. Small quantity of HMGB1 mRNA was expressed in RAW264.7. The expression of HMGB1 mRNA fluctuated around the standard level after interfering with normal serum of SD rats. The expression of HMGB1 mRNA rose at 3 h, and then decreased to the standard level; at 18 h, it rose rapidly; at 36 h, it reached the peak; and at 48 h, it remained at the high level after interfering with burning serum. The expression of HMGB1 mRNA increased at 3 h, and then decreased to the standard level. At 24 h, it started to rise after interfering with herb serum, and was lower than that of; the burning serum group (P<0.05). Burning serum can increase the expression of HMGB1 mRNA in RAW264.7. QHBDY can decrease the high expression of HMGB1 mRNA in RAW264.7 caused by burning serum.

Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

DOE PAGES

Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; ...

2016-07-28

Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

Study on expression of CDH4 in lung cancer.

PubMed

Li, Zhupeng; Su, Dan; Ying, Lisha; Yu, Guangmao; Mao, Weimin

2017-01-17

The human CDH4 gene, which encodes the R-cadherin protein, has an important role in cell migration and cell adhesion, sorting, tissue morphogenesis, and tumor genesis. This study analyzed the relationship of CDH4 mRNA expression with lung cancer. Real time PCR was applied to detect CDH4 mRNA transcription in 142 paired cases of lung cancer and noncancerous regions. No correlation was identified between CDH4 mRNA expression and gender, age, lymphnode metastasis, TNM stage, family history, smoking state, drinking state (P > 0.05), but grade and histotype (P < 0.05). The relative CDH4 mRNA value was remarkably decreased in lung cancer tissues compared with noncancerous tissues (P = 0.001). We found that CDH4 mRNA expression was associated with grade and histotype. What is more, the relative CDH4 mRNA value was decreased in the lung cancer tissues. Our results suggested that CDH4 might be a putative tumor suppressor gene (TSG) in lung cancer.

Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland.

PubMed

Inoue, Makiko; Shiina, Tomoya; Aizawa, Sayaka; Sakata, Ichiro; Takagi, Hiroyasu; Sakai, Takafumi

2012-06-01

Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.

Gene expression of apoptosis-related genes, stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress.

PubMed

Guo, Hui; Xian, Jian-An; Li, Bin; Ye, Chao-Xia; Wang, An-Li; Miao, Yu-Tao; Liao, Shao-An

2013-05-01

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress. Copyright © 2013 Elsevier Inc. All rights reserved.

Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: implications for schizophrenia.

PubMed

Curley, Allison A; Eggan, Stephen M; Lazarus, Matt S; Huang, Z Josh; Volk, David W; Lewis, David A

2013-02-01

Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PV(GAD67+/-)) or in all interneurons (GABA(GAD67+/-)) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PV(GAD67+/-) mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABA(GAD67+/-) mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell type-specificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor. Copyright © 2012 Elsevier Inc. All rights reserved.

Paclitaxel-resistant HeLa cells have up-regulated levels of reactive oxygen species and increased expression of taxol resistance gene 1.

PubMed

Bi, Wenxiang; Wang, Yuxia; Sun, Gaoying; Zhang, Xiaojin; Wei, Yongqing; Li, Lu; Wang, Xiaoyuan

2014-07-01

This study is to establish a paclitaxel (PTX)-resistant human cervical carcinoma HeLa cell line (HeLa/PTX) and to investigate its redox characteristics and the expression of taxol resistance gene 1 (Txr1). HeLa cells were treated with PTX and effects of PTX on cell proliferation were detected through cell counting and the MTT assay. Levels of cellular reactive oxygen species (ROS), reduced glutathione (GSH), and oxidized glutathione (GSSG) as well as the ratio of GSH to GSSG were measured by the 2,7-difluorescein diacetate (DCFH-DA) method and the 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) method. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined by the nitrite formation method, the molybdate colorimetric method, and the DTNB colorimetric method, respectively. The level of Txr1 mRNA was determined by real-time PCR. Compared with the regular HeLa cells, HeLa/PTX cells were larger in size and had more cytoplasmic granules. The population doubling time for HeLa/PTX cells was 1.32 times of that of HeLa cells (P<0.01). HeLa/PTX cells showed stronger resistance to PTX than HeLa cells with a resistance index of 122.69. HeLa/PTX cells had higher levels of ROS (P<0.01) and Txr1 mRNA (P<0.01), lower level of GSH (P < 0.05), and lower activities of SOD (P<0.01) and GPx (P < 0.05) than HeLa cells. HeLa/PTX cells, with higher levels of ROS and Txr1 mRNA expression, are more resistant to PTX than HeLa cells.

Rapid, transient, and dose-dependent expression of Hsp70 messenger RNA in the rat brain after morphine treatment

PubMed Central

Ammon-Treiber, Susanne; Grecksch, Gisela; Stumm, Ralf; Riechert, Uta; Tischmeyer, Helga; Reichenauer, Anke; Höllt, Volker

2004-01-01

Induction of Hsp70 in the brain has been reported after intake of drugs of abuse like amphetamine and lysergic acid diethylamide. In this investigation, gene expression of Hsp70 and other heat shock genes in the rat brain was studied in response to morphine. Twenty milligrams per kilogram morphine intraperitoneally resulted in a marked induction of Hsp70 messenger RNA (mRNA) expression in the frontal cortex with a maximum increase of 13.2-fold after 2 hours. A moderate increase of Hsp27 mRNA expression (6.7-fold) could be observed after 4 hours, whereas mRNA expression of Hsp90 and of the constitutive Hsc70 did not exceed a mean factor of 1.8-fold during the 24 hours interval. The increase in Hsp70 mRNA was dose dependent, showing a significant elevation after doses ranging from 10 to 50 mg/kg morphine. In situ hybridization revealed enhanced Hsp70 mRNA expression mainly in cortical areas, in the hippocampus, in the paraventricular and supraoptic nuclei of the hypothalamus, in the locus coeruleus, as well in the pineal body. The double in situ hybridization technique revealed increased Hsp70 mRNA expression mainly in VGLUT1-positive neurons and to a lesser extent in olig1-positive oligodendroglia. Immunohistochemistry revealed a marked increase of Hsp70 protein in neuronal cells and blood vessels after 12 hours. In contrast to animal experiments, morphine did not increase Hsp70 mRNA expression in vitro in μ-opioid receptor (MOR1)–expressing human embryonic kidney 293 cells, suggesting no direct MOR1-mediated cellular effect. To exclude a body temperature–related morphine effect on Hsp70 mRNA expression, the temperature was recorded. Five to 20 mg/kg resulted in hyperthermia (maximum 40.6°), whereas a high dose (50 mg/kg) that produced the highest mRNA induction, showed a clear hypothermia (minimum 37.2°C). These findings argue against the possibility that Hsp70 induction by morphine is caused by its effect on body temperature. It may be speculated that increased expression of Hsp70 after morphine application protects brain structures against potentially hazardous effects of opiates. PMID:15497504

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

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy.

PubMed

Mobley, Christopher Brooks; Fox, Carlton D; Ferguson, Brian S; Amin, Rajesh H; Dalbo, Vincent J; Baier, Shawn; Rathmacher, John A; Wilson, Jacob M; Roberts, Michael D

2014-01-01

The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles.

PubMed

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.

Complexities and sequence similarities of mRNA populations of cholinergic (NS20-Y) and adrenergic (N1E-115) murine neuroblastoma cell lines.

PubMed

Strauss, W L

1990-07-01

The clonal murine neuroblastoma cell lines NS20-Y and N1E-115 have been proposed as models for examining the commitment of neural crest cells to either the cholinergic or adrenergic phenotype, respectively. The validity of this model depends in part on the extent to which these two cell lines have diverged as a result of their transformed, rather than neuronal properties. In order to quantitate differences in gene expression between NS20-Y and N1E-115 cells, the mRNA complexity of each cell type was determined. An analysis of the kinetics of hybridization of NS20-Y cell mRNA with cDNA prepared from NS20-Y cell mRNA demonstrated the presence of approximately 11,700 mRNA species assuming an average length of 1900 nucleotides. A similar analysis using mRNA isolated from N1E-115 cells and cDNA prepared from N1E-115 cell mRNA demonstrated that the adrenergic cell line expressed approximately 11,600 mRNA species. The species of mRNA expressed by each cell line were resolved into high, intermediate, and low abundance populations. In order to determine whether mRNAs were expressed by the cholinergic, but not by the adrenergic cell line, NS20-Y cDNA was hybridized to an excess of N1E-115 cell mRNA. An analysis of the solution hybridization kinetics from this procedure demonstrated that the two cell lines do not differ significantly in the nucleotide complexity of their mRNA populations. The extensive similarity between the two mRNA populations suggests that only a small number of genes are expressed differentially between the two cell lines and supports their use as models for the differentiation of cholinergic and adrenergic neurons.

[miR-503-5p inhibits the proliferation of T24 and EJ bladder cancer cells by interfering with the Rb/E2F signaling pathway].

PubMed

Li, Xiaohui; Han, Xingtao; Yang, Jinhui; Sun, Jiantao; Wei, Pengtao

2017-10-01

Objective To observe the effect of microRNA-503-5p (miR-503-5p) on the growth of T24 and EJ bladder cancer cells, and explore the possible molecular mechanism. Methods The miR-504-5p mimics or miR-NC was transfected into T24 and EJ cells. The target gene of miR-503-5p was predicted by bioinformatics. The expressions of E2F transcription factor 3 (E2F3) mRNA and Rb/E2F signaling pathway mRNA were detected by the real-time quantitative PCR (qPCR). The expressions of Rb/E2F signal pathway proteins E2F3, cyclin E, CDK2, Rb and p-Rb were detected by Western blotting. The cell cycle of bladder cancer cell lines was determined by flow cytometry. MTT assay and plate cloning assay were performed to observe the proliferation ability of bladder cancer cells. Results After miR-503-5p mimics transfection, the expression of miR-503-5p in bladder cancer cells significantly increased. The increased expression of miR-503-5p significantly reduced the expressions of E2F3 mRNA and Rb/E2F signaling pathway mRNA in bladder cancer cells. What's more, the expressions of Rb/E2F signal pathway proteins were down-regulated. The bladder cancer cells were arrested in G0/G1 phase, and their growth was significantly inhibited by miR-503-5p. Conclusion The miR-503-5p over-expression can inhibit the growth of bladder cancer cell lines T24 and EJ by down-regulating the expression of the Rb/E2F signaling pathway.

Differential changes in mGlu2 and mGlu3 gene expression following pilocarpine-induced status epilepticus: A comparative real-time PCR analysis

PubMed Central

Ermolinsky, Boris; Pacheco Otalora, Luis F.; Arshadmansab, Massoud F.; Zarei, Masoud; Garrido-Sanabria, Emilio R.

2008-01-01

Group II metabotropic glutamate (mGlu II) receptors subtype 2 and 3 (mGlu2 and mGlu3) are subtle regulators of neuronal excitability and synaptic plasticity in the hippocampus. In recent years, researchers have investigated the potential neuroprotective and anticonvulsant effects of compounds acting on mGlu II receptors. However, abnormal expression and function of mGlu2 and mGlu3 have been reported in temporal lobe epilepsy, a phenomena that may limit the therapeutic effectiveness of these potentially new antiepileptic drugs. Here, we investigated seizure-induced changes in mGlu2 and mGlu3 mRNA following pilocarpine-inducted status epilepticus (SE) and subsequent epileptogenesis. Relative changes in gene expression were assessed by comparative analysis of quantitative real-time PCR (qrtPCR) by the delta-delta CT method. Pilocarpine-treated and control rats were sacrificed at different periods (24h, 10 days, one month and more than two months) following SE. Total RNA was isolated from microdissected dentate gyrus and processed for RT-PCR and qrtPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control gene. Analysis of relative quantification (RQ) ratios of mGlu2 and mGlu3 mRNA expression revealed a significant down-regulation of both targets at 24h after SE. Gene expression partially recovered at 10 days following SE reaching control levels at one month after SE. Two month after SE, mGlu2 mRNA expression was significantly down-regulated to ~41% of control expression whereas mGlu3 mRNA was comparable to control levels. Our data indicate that mGlu2 and mGlu3 expression is dynamically down-regulated or selectively enhanced during critical periods of epileptogenesis. Seizure-induced differential dysregulation of mGlu2 and mGlu3 receptors may affect the availability of these molecular targets for therapeutic compounds in epilepsy. PMID:18585369

Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes.

PubMed

Gufford, Brandon T; Robarge, Jason D; Eadon, Michael T; Gao, Hongyu; Lin, Hai; Liu, Yunlong; Desta, Zeruesenay; Skaar, Todd C

2018-04-01

Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes ( P  <   .05). Rifampin-induced miRNA expression changes correlated with mRNA changes and miRNAs were identified that may modulate conjugating enzyme expression. NAT2 gene expression was most strongly repressed (1.3-fold) by rifampin while UGT1A4 and UGT1A1 genes were most strongly induced (7.9- and 4.8-fold, respectively). Physiologically based pharmacokinetic modeling (PBPK) was used to simulate the clinical consequences of rifampin induction of CYP3A4- and UGT1A4-mediated midazolam metabolism. Simulations evaluating isolated UGT1A4 induction predicted increased midazolam N-glucuronide exposure (~4-fold) with minimal reductions in parent midazolam exposure (~10%). Simulations accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements.

Increased expression of transforming growth factor beta s after acute oedematous pancreatitis in rats suggests a role in pancreatic repair.

PubMed Central

Riesle, E; Friess, H; Zhao, L; Wagner, M; Uhl, W; Baczako, K; Gold, L I; Korc, M; Büchler, M W

1997-01-01

BACKGROUND: Transforming growth factor beta isoforms (TGF beta s) belong to a family of multifunctional regulators of cellular growth and differentiation. They are mitogenic and chemotactic for fibroblasts and are potent stimulators of extracellular matrix production (collagen) and deposition. Upregulation of TGF beta transcription has been reported for several in vivo systems during repair after injury. AIMS: To study the expression of the three mammalian isoforms of TGF beta (TGF beta 1-3) and their relation to collagen expression as a marker for fibroblast response in acute oedematous pancreatitis in rats. METHODS: Using northern blot analysis and immunohistochemistry, the expression and localisation of TGF beta isoforms, collagen, and amylase were analysed during the course of acute oedematous pancreatitis in rats, experimentally induced by intravenous caerulein infusion. RESULTS: Induction of acute pancreatitis resulted in a biphasic peak pattern of expression of TGF beta 1, beta 2, and beta 3 mRNA, with a pronounced increase from day 1 to day 3 (sixfold, 2.5-fold, fivefold, respectively) and again from day 5 to day 7 (three-fold, 2.3-fold, 3.5-fold, respectively). The temporal changes in TGF beta mRNA identically paralleled the expression in collagen mRNA. In contrast, amylase mRNA expression, used as a general indicator of acinar cell integrity, was slightly decreased after induction of acute pancreatitis. Immunohistochemical analysis of pancreatitis tissue showed that increased expression of TGF beta s was mainly present in the pancreatic acinar and ductal cells; this was evident within one day after pancreatitis induction. CONCLUSION: Overexpression of TGF beta s after induction of acute pancreatitis suggests a role for these proteins in pancreatic repair and remodelling. The increased levels of TGF beta s may help suppress immune activation, and may contribute to the increase in the extracellular matrix including collagen and to the repair of the pancreatic parenchyma. Images PMID:9155579

Differential regulation of preprotachykinin-A mRNA expression in striatum by excitation of hippocampal neurons.

PubMed

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

1993-07-01

In this report we have studied the influence of hippocampal neurons on neuropeptide mRNA expression in both dorsal and ventral striatum in the rat. Intrahippocampal unilateral kainic acid injections were performed in control animals and in animals with a unilateral 6-hydroxydopamine-induced dopamine deafferentation of the striatum. In situ hybridization combined with quantitative image analysis was used to study the expression of preprotachykinin A mRNA encoding the neuropeptides substance P and neurokinin A. The 6-hydroxydopamine-induced lesion caused a decrease of preprotachykinin A mRNA levels in the ipsilateral dorsal striatum and in both sides of the ventral striatum. In normal rats, the intrahippocampal kainic acid injection caused a twofold increase in preprotachykinin A mRNA in the limbic parts of the striatum, which are innervated by the hippocampus. No effect of the kainic acid injection was seen in the lateral parts of the dorsal striatum, a region which does not appear to be innervated by the hippocampus. Animals with a 6-hydroxydopamine lesion showed a similar kainic acid-mediated increase in preprotachykinin A mRNA in parts of the ventral striatum. In the dopamine-lesioned dorsal striatum and ventral striatum the decreased preprotachykinin A mRNA levels were normalized by the intrahippocampal kainic acid injection. These results show that kainic acid-mediated excitation of hippocampal neurons causes a dopamine-independent induction of preprotachykinin A mRNA expression in parts of the ventral striatum, and reverses the dopamine deafferentation-induced decrease of preprotachykinin A mRNA in both dorsal and ventral striatum. Combined, our results suggest that hippocampal neurons can regulate preprotachykinin A mRNA expression in both the ventral and the dorsal striatum.

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

PubMed

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

2002-05-01

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

Proportions of myosin heavy chain mRNAs, protein isoforms and fiber types in the slow and fast skeletal muscles are maintained after alterations of thyroid status in rats.

PubMed

Soukup, T; Diallo, M

2015-01-01

Recently, we have established that slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of euthyroid (EU) Lewis rats posses the same proportions between their four myosin heavy chain (MyHC) mRNAs, protein isoforms and fiber types as determined by real time RT-PCR, SDS-PAGE and 2-D stereological fiber type analysis, respectively. In the present paper we investigated if these proportions are maintained in adult Lewis rats with hyperthyroid (HT) and hypothyroid (HY) status. Although HT and HY states change MyHC isoform expression, results from all three methods showed that proportion between MyHC mRNA-1, 2a, -2x/d, -2b, protein isoforms MyHC-1, -2a, -2x/d, -2b and to lesser extent also fiber types 1, 2A, 2X/D, 2B were preserved in both SOL and EDL muscles. Furthermore, in the SOL muscle mRNA expression of slow MyHC-1 remained up to three orders higher compared to fast MyHC transcripts, which explains the predominance of MyHC-1 isoform and fiber type 1 even in HT rats. Although HT status led in the SOL to increased expression of MyHC-2a mRNA, MyHC-2a isoform and 2A fibers, it preserved extremely low expression of MyHC-2x and -2b mRNA and protein isoforms, which explains the absence of pure 2X/D and 2B fibers. HY status, on the other hand, almost completely abolished expression of all three fast MyHC mRNAs, MyHC protein isoforms and fast fiber types in the SOL muscle. Our data present evidence that a correlation between mRNA, protein content and fiber type composition found in EU status is also preserved in HT and HY rats.

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

Relative IGF-1 and IGF-2 gene expression in maternal and fetal tissues from diabetic swine

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

Wolverton, C.K.; Leaman, D.W.; White, M.E.

1990-02-26

Fourteen pregnant, crossbred gilts were utilized in this study. Seven gilts were injected with alloxan (50 mg/kg) at day 75 of gestation to induce diabetes. Gilts underwent caesarean section on day 105 of gestation. Samples were collected from maternal skeletal muscle, adipose tissue, uterus and endometrium; and from fetal skeletal muscle, adipose tissue, placenta, liver, lung, kidney, heart, brain and spleen. Tissues were frozen in liquid nitrogen for later analysis of IGF-1 and IGF-2 gene expression. Samples were pooled and total RNA was isolated using the guanidine isothiocynate method. Total mRNA was analyzed by dot blot hybridization. Blots were probedmore » with {sup 32}P-cDNA for porcine IGF-1 and rat IGF-2. IGF-1 gene expression in maternal tissues was unaffected by diabetes. Maternal diabetes increased IGF-2 mRNA in maternal adipose tissue but exhibited no effect in muscle or uterus. Expression of IGF-2 by maternal endometrium was decreased by diabetes. Maternal diabetes induced an increase in IGF-1 gene expression in muscle and placenta while causing an increase in IGF-2 expression in fetal liver and placenta. IGF-2 mRNA was lower in lung from fetuses of diabetic mothers than in controls. These results suggest that maternal diabetes alters IGF-1 and IGF-2 gene expression in specific tissues and differential regulation of these genes appears to exist in the mother and developing fetus.« less

Effect of Antisense Oligodeoxynucleotides Glucose Transporter-1 on Enhancement of Radiosensitivity of Laryngeal Carcinoma

PubMed Central

Yan, Sen-Xiang; Luo, Xing-Mei; Zhou, Shui-Hong; Bao, Yang-Yang; Fan, Jun; Lu, Zhong-Jie; Liao, Xin-Biao; Huang, Ya-Ping; Wu, Ting-Ting; Wang, Qin-Ying

2013-01-01

Purpose: Laryngeal carcinomas always resist to radiotherapy. Hypoxia is an important factor in radioresistance of laryngeal carcinoma. Glucose transporter-1 (GLUT-1) is considered to be a possible intrinsic marker of hypoxia in malignant tumors. We speculated that the inhibition of GLUT-1 expression might improve the radiosensitivity of laryngeal carcinoma. Methods: We assessed the effect of GLUT-1 expression on radioresistance of laryngeal carcinoma and the effect of GLUT-1 expressions by antisense oligodeoxynucleotides (AS-ODNs) on the radiosensitivity of laryngeal carcinoma in vitro and in vivo. Results: After transfection of GLUT-1 AS-ODNs: MTS assay showed the survival rates of radiation groups were reduced with the prolongation of culture time (p<0.05); Cell survival rates were significantly reduced along with the increasing of radiation dose (p<0.05). There was significant difference in the expression of GLUT-1mRNA and protein in the same X-ray dose between before and after X-ray radiation (p<0.05). In vivo, the expressions of GLUT-1 mRNA and protein after 8Gy radiation plus transfection of GLUT-1 AS-ODNs were significant decreased compared to 8Gy radiation alone (p<0.001). Conclusion: Radioresistance of laryngeal carcinoma may be associated with increased expression of GLUT-1 mRNA and protein. GLUT-1 AS-ODNs may enhance the radiosensitivity of laryngeal carcinoma mainly by inhibiting the expression of GLUT-1. PMID:23983599

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

PubMed Central

Ohno, Misa; Togashi, Yuto; Tsuda, Kyoko; Okawa, Kazuaki; Kamaya, Minori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

2013-01-01

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific. PMID:23826286

Placental Expressions of CDKN1C and KCNQ1OT1 in Monozygotic Twins with Selective Intrauterine Growth Restriction.

PubMed

Gou, Chenyu; Liu, Xiangzhen; Shi, Xiaomei; Chai, Hanjing; He, Zhi-Ming; Huang, Xuan; Fang, Qun

2017-10-01

CDKN1C and KCNQ1OT1 are imprinted genes that might be potential regulators of placental development. This study investigated placental expressions of CDKN1C and KCNQ1OT1 in monozygotic twins with and without selective intrauterine growth restriction (sIUGR). Seventeen sIUGR and fifteen normal monozygotic(MZ) twin pairs were examined. Placental mRNA expressions of CDKN1C and KCNQ1OT1 were detected by real-time fluorescent quantitative PCR. CDKN1C protein expression was detected by immunohistochemical assay and Western-blotting. In the sIUGR group, smaller fetuses had a smaller share of the placenta, and CDKN1C protein expression was significantly increased while KCNQ1OT1 mRNA expression was significantly decreased. The CDKN1C/KCNQ1OT1 mRNA ratio was lower in the larger fetus than in the smaller fetus (p < .05). In the control group, CDKN1C protein expression showed no difference between larger and smaller fetuses, while KCNQ1OT1 mRNA expression was significantly lower in the larger fetus, and the CDKN1C/KCNQ1OT1 mRNA ratio was higher in the larger fetus than in the smaller fetus (p < .05). Our findings showed that pathogenesis of sIUGR may be related to the co-effect of the up-regulated protein expression of CDKN1C and down-regulated mRNA expression of KCNQ1OT1 in the placenta.

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

PubMed

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

2008-03-01

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

Immunological and pathological changes in the placenta during infection with Listeria monocytogenes in pregnant guinea pigs.

PubMed

Irvin, Elizabeth Ann; Williams, Denita; Hamler, Sarah E; Smith, Mary Alice

2008-10-01

Exposure to Listeria monocytogenes during pregnancy can result in spontaneous abortion and stillbirths; however, the mechanisms are unknown. Our objective was to determine the effects of infection on specific inflammatory and anti-inflammatory cytokine mRNA expression and apoptosis in the placenta after infection with L. monocytogenes. Pregnant guinea pigs were treated on gestation day (gd) 35 with 10(8) colony forming units L. monocytogenes and sacrificed on gd 37, 41, 44, or 55. At gd 41, IFN-gamma and IL-2 mRNA expression was significantly decreased in placentas from treated dams (0.0012-fold and 0.131-fold, respectively). At gd 55, TNF-alpha mRNA expression was significantly decreased (0.19-fold), while IFN-gamma mRNA expression was significantly increased (32-fold), and apoptosis was detected in 100% of placentas from treated dams. In conclusion, inflammatory cytokine mRNA expression is altered and apoptosis is increased in the placenta after treatment with L. monocytogenes, and these changes may contribute to fetal death.

[Effects of the escharectomy during burn shock stage on expression of glucose translator-4 mRNA in skeletal muscle and adipose tissue].

PubMed

Shuai, Xiu-rong; Liu, Tong-fa; Guo, Zhen-rong; Yu, Shun-xian; He, Peng-fei; Yuan, Wen-zhou; Li, Feng; He, Li-xin

2004-04-07

To investigate the effect of the escharectomy during burn shock stage on expression of glucose translator-4 (GLUT4) mRNA in skeletal muscle and adipose tissue. 30% TBSA scalded rats were employed. Escharectomy were conducted at 8 h, 24 h, 168 h after burns respectively. Insulin, glucagon, cortisol and glucose levels in serum were analyzed. RT-PCR were employed to analyze GLUT4 mRNA expression in skeletal muscle and adipose tissue. Glucagon, cortisol and glucose levels in serum were declined in groups which escharectomy were conducted during burn shock stage. GLUT4 mRNA expression in both skeletal muscle and adipose tissue were downregulated after burns and escharectomy conducted during burn shock stage made it restored to near normal. GLUT4 mRNA expression will declined after major burns in skeletal muscle and adipose tissue. Escharectomy during shock stage could make it upregulated, which will be helpful to improve glucose metabolism and hypermetabolism after major burns.

The expression of KRT2 and its effect on melanogenesis in alpaca skins.

PubMed

Cui, Yucong; Song, Yajun; Geng, Qingling; Ding, Zengfeng; Qin, Yilong; Fan, Ruiwen; Dong, Changsheng; Geng, Jianjun

2016-06-01

In order to investigate the effects of the keratin 2 (KRT2) on alpaca melanocyte in vivo and vitro, the immunohistochemistry (IHC), quantitative real-time PCR (qPCR), Western blot, and alpaca melanocytes transfection methods were used. The results showed that mRNA and protein expression of KRT2 was highly expressed in brown skin in comparison with that in white skin. Moreover, we found that KRT2 was expressed in alpaca melanocytes in vitro by immunocytochemistry. After transfection with KRT2 in alpaca melanocytes, the relative mRNA and protein expression of KRT2, microphthalmia-associtated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) in alpaca skin melanocytes was increased with significant differences; a further result was the increase of melanin production. The results suggested that KRT2 functions in alpaca hair color formation, which offered an essential theoretical basis for further exploration of the role of melanogenesis. Copyright © 2016 Elsevier GmbH. All rights reserved.

Regulation of p21/CIP1/WAF-1 mediated cell-cycle arrest by RNase L and tristetraprolin, and involvement of AU-rich elements

PubMed Central

Al-Haj, Latifa; Blackshear, Perry J.; Khabar, Khalid S.A.

2012-01-01

The p21Cip1/WAF1 plays an important role in cell-cycle arrest. Here, we find that RNase L regulates p21-mediated G1 growth arrest in AU-rich elements-dependent manner. We found a significant loss of p21 mRNA expression in RNASEL−/− MEFs and that the overexpression of RNase L in HeLa cells induces p21 mRNA expression. The p21 mRNA half-life significantly changes as a result of RNase L modulation, indicating a post-transcriptional effect. Indeed, we found that RNase L promotes tristetraprolin (TTP/ZFP36) mRNA decay. This activity was not seen with dimerization- and nuclease-deficient RNase L mutants. Deficiency in TTP led to increases in p21 mRNA and protein. With induced ablation of RNase L, TTP mRNA and protein expressions were higher, while p21 expression became reduced. We further establish that TTP, but not C124R TTP mutant, binds to, and accelerates the decay of p21 mRNA. The p21 mRNA half-life was prolonged in TTP−/− MEFs. The TTP regulation of p21 mRNA decay required functional AU-rich elements. Thus, we demonstrate a novel mechanism of regulating G1 growth arrest by an RNase L-TTP-p21 axis. PMID:22718976

Expression of Flk-1 and Cyclin D2 mRNA in the Myocardium of Rats with Doxorubicin-Induced Cardiomyopathy and after Treatment with Betulonic Acid Amide.

PubMed

Mzhelskaya, M M; Klinnikova, M G; Koldysheva, E V; Lushnikova, E L

2017-10-01

The expression of VEGFR2 (Flk-1, according to immunohistochemistry) and of cyclin D2 mRNA (according to real-time PCR) in the myocardium of rats is studied in doxorubicin-induced cardiomyopathy and in response to betulonic acid amide. Doxorubicin alone and in combination with betulonic acid amide causes after 3 days a manifest reduction of cyclin D2 mRNA expression (by 38 and 63%, respectively), while injection of betulonic acid amide alone causes a 23-fold increase of cyclin D2 mRNA expression. An increase of cyclin D2 mRNA expression has been detected in all experimental groups after 14 days of experiment, the most pronounced in response to betulonic acid amide (63 times). The expression of Flk-1 in cardiomyocytes increases significantly in response to both chemical agents starting from day 3 of experiment. These results indicate that doxorubicin and betulonic acid amide induce cytoprotective reactions in the myocardium, first at the intracellular, then at the cellular levels.

[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.

Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow

NASA Technical Reports Server (NTRS)

Hillsley, M. V.; Frangos, J. A.

1997-01-01

It is our hypothesis that interstitial fluid flow plays a role in the bone remodeling response to mechanical loading. The fluid flow-induced expression of three proteins (collagen, osteopontin, and alkaline phosphatase) involved in bone remodeling was investigated. Rat calvarial osteoblasts subjected to pulsatile fluid flow at an average shear stress of 5 dyne/cm2 showed decreased alkaline phosphatase (AP) mRNA expression after only 1 hour of flow. After 3 hours of flow, AP mRNA levels had decreased to 30% of stationary control levels and remained at this level for an additional 5 hours of flow. Steady flow (4 dyne/cm2 fluid shear stress), in contrast, resulted in a delayed and less dramatic decrease in AP mRNA expression to 63% of control levels after 8 hours of flow. The reduced AP mRNA expression under pulsatile flow conditions was followed by reduced AP enzyme activity after 24 hours. No changes in collagen or osteopontin mRNA expression were detected over 8 hours of pulsatile flow. This is the first time fluid flow has been shown to affect gene expression in osteoblasts.

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.

Placental expression of D6 decoy receptor in preeclampsia

PubMed Central

Cho, Geum Joon; Lee, Eun Sung; Jin, Hye Mi; Lee, Ji Hye; Kim, Yeun Sun; Seol, Hyun-Joo; Hong, Soon-Cheol; Kim, Hai-Joong

2015-01-01

Objective The purpose of this study was to investigate the expression of the D6 decoy receptor that can bind chemokines and target them for degradation, resulting in inhibition of inflammation in placentas from preeclamptic and normal pregnancies. Methods The current study was carried out in 35 pregnant women (23 patients with preeclampsia and 12 healthy, normotensive pregnant women) during the third trimester of pregnancy. The expressions of D6 decoy receptor in the placenta were determined with real time reverse transcriptase polymerase chain reaction and western blotting. Results The mRNA and protein of D6 decoy receptor were detected in all of placentas from preeclamptic and normal pregnancies. Placental D6 decoy receptor mRNA expression was significantly lower in patients with preeclampsia than in patients with normal pregnancies. Western blot analyses revealed decreased protein expression in cases of preeclampsia. Conclusion The expression of the D6 decoy receptor in preeclamptic placentas was significantly lower than in normal placentas. Further studies are needed to clarify the underlying mechanisms that link decreased expression of placental D6 decoy receptor and preeclampsia. PMID:26430656

mRNA expression of CDH3, IGF2BP3, and BIRC5 in biliary brush cytology specimens is a useful adjunctive tool of cytology for the diagnosis of malignant biliary stricture

PubMed Central

Kim, Tae Ho; Chang, Jae Hyuck; Lee, Hee Jin; Kim, Jean A; Lim, Yeon Soo; Kim, Chang Whan; Han, Sok Won

2016-01-01

Abstract Although advances have been made in diagnostic tools, the distinction between malignant and benign biliary strictures still remains challenging. Intraductal brush cytology is a convenient and safe method that is used for the diagnosis of biliary stricture, but, low sensitivity limits its usefulness. This study aimed to demonstrate the usefulness of mRNA expression levels of target genes in brush cytology specimens combined with cytology for the diagnosis of malignant biliary stricture. Immunohistochemistry for cadherin 3 (CDH3), p53, insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), and baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) was performed in 4 benign and 4 malignant bile duct tissues. Through endoscopic or interventional radiologic procedures, brush cytology specimens were prospectively obtained in 21 and 35 paitents with biliary strictures. In the brush cytology specimens, the mRNA expressions levels of 5 genes were determined by real-time polymerase chain reaction. Immunohistochemistry for CDH3, p53, IGF2BP3, HOXB7, and BIRC5 all showed positive staining in malignant tissues in contrast to benign tissues, which were negative. In the brush cytology specimens, the mRNA expression levels of CDH3, IGF2BP3, HOXB7, and BIRC5 were significantly higher in cases of malignant biliary stricture compared with cases of benign stricture (P = 0.006, P < 0.001, P < 0.001, and P = 0.001). The receiver-operating characteristic curves of these 4 mRNAs demonstrated that mRNA expression levels are useful for the prediction of malignant biliary stricture (P = 0.006, P < 0.001, P < 0.001, and P = 0.002). The sensitivity and specificity, respectively, for malignant biliary stricture were 57.1% and 100% for cytology, 57.1% and 64.3% for CDH3, 76.2% and 100% for IGF2BP3, 71.4% and 57.1% for HOXB7, and 76.2% and 64.3% for BIRC5. When cytology was combined with the mRNA levels of CDH3, IGF2BP3, or BIRC5, the sensitivity for malignant biliary stricture improved to 90.5%. The measurement of the mRNA expression levels of CDH3, IGF2BP3, and BIRC5 by real-time polymerase chain reaction combined with cytology was useful for the differentiation of malignant and benign biliary strictures in brush cytology specimens. PMID:27399126

CMG2 Expression Is an Independent Prognostic Factor for Soft Tissue Sarcoma Patients

PubMed Central

Wedler, Alice; Rot, Swetlana; Keßler, Jacqueline; Kehlen, Astrid; Holzhausen, Hans-Jürgen; Bache, Matthias; Würl, Peter; Kappler, Matthias

2017-01-01

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (rs = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients’ disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression. PMID:29215551

CMG2 Expression Is an Independent Prognostic Factor for Soft Tissue Sarcoma Patients.

PubMed

Greither, Thomas; Wedler, Alice; Rot, Swetlana; Keßler, Jacqueline; Kehlen, Astrid; Holzhausen, Hans-Jürgen; Bache, Matthias; Würl, Peter; Taubert, Helge; Kappler, Matthias

2017-12-07

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (r s = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients' disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression.

Prostaglandin E1 reduces the glomerular mRNA expression of monocyte-chemoattractant protein 1 in anti-thymocyte antibody-induced glomerular injury.

PubMed

Jocks, T; Zahner, G; Freudenberg, J; Wolf, G; Thaiss, F; Helmchen, U; Stahl, R A

1996-06-01

To study whether prostaglandins (PG) can regulate the mRNA expression of monocyte-chemoattractant protein 1 (MCP-1) in glomerular immune injury, MCP-1 mRNA levels were evaluated in anti-thymocyte antibody (ATS) -induced glomerular injury by Northern blotting and reverse transcription-polymerase chain reaction. Immune injury was induced in vivo by the intravenous application of ATS to male Wistar rats and in vitro by the perfusion of isolated rat kidneys with ATS and rat serum. In vivo 3 h and 5 days after antibody application, glomerular mRNA expression of MCP-1 was markedly enhanced compared with controls. In the isolated perfused kidney, antibody and complement also induced an increase in MCP-1 expression at 10 min and 60 min after antibody perfusion. When the rats were treated with PGE (250 micrograms, twice daily), the increase in MCP-1 expression was reduced. This was associated with a reduction of intraglomerular recruitment of monocytes/macrophages. In the isolated perfused kidneys, PGE1 (1 mg/L) prevented the antibody- and rat serum-stimulated increase in glomerular MCP-1 mRNA expression. These data demonstrate that PGE1 reduces glomerular MCP-1 mRNA expression in glomerulonephritis and in the isolated perfused rat kidney after induction of immune injury with antibody and complement. The data suggest that prostaglandins might mediate MCP-1 effects in glomerular immune injuries.

Inactivation of MSH3 by promoter methylation correlates with primary tumor stage in nasopharyngeal carcinoma.

PubMed

Ni, Haifeng; Jiang, Bo; Zhou, Zhen; Yuan, Xiaoyang; Cao, Xiaolin; Huang, Guangwu; Li, Yong

2017-09-01

The aim of this study was to investigate the inactivation of the MutS homolog human 3 (MSH3) gene by promoter methylation in nasopharyngeal carcinoma (NPC). Methylation‑specific PCR, semi‑quantitative reverse transcription PCR and immunohistochemical analysis were used to detect methylation and the mRNA and protein expression levels of MSH3 in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues. The association between promoter methylation and mRNA expression, and the mRNA and protein expression of the gene and clinical factors was analyzed. The promoter methylation of MSH3 was detected in 50% (27/54) of the primary tumors, but not in the 16 NNE tissues. The mRNA and protein expression levels were significantly decreased in the 54 cases of human NPC as compared to the 16 NNE tissues (P<0.05). The MSH3‑methylated cases exhibited significantly lower mRNA and protein expression levels than the unmethylated cases (P<0.05). The MSH3 mRNA and protein expression levels were significantly associated with the variable T stage (P<0.05); however, they did not correlate with the age and sex of the patients, or with the N stage, TNM classification or histopathological subtype (P>0.05). On the whole, MSH3 was frequently inactivated by promoter methylation and its mRNA and protein expression correlated with the primary tumor stage in NPC.

Gnrh mRNA expression in the brain of cooperatively breeding female Damaraland mole-rats.

PubMed

Voigt, Cornelia; Bennett, Nigel C

2017-04-01

The Damaraland mole-rat ( Fukomys damarensis ) is a eusocial, subterranean rodent, in which breeding is limited to a single reproductive pair within each colony. Non-reproductive females, while in the confines of the colony, exhibit socially induced infertility. Anovulation is thought to be caused by a disruption in the normal gonadotropin-releasing hormone (GNRH) secretion from the hypothalamus. To assess whether social suppression is associated with altered Gnrh mRNA expression in the brain, we investigated the distribution and gene expression levels by means of in situ hybridization in female breeders and non-breeders from field captured colonies of the Damaraland mole-rat. We found expression of Gnrh mRNA as a loose network in several forebrain areas of female Damaraland mole-rats with the majority of labelling in the preoptic and anterior hypothalamus. The distribution matched previous findings using immunocytochemistry in this and other social mole-rat species. Quantification of the hybridisation signal revealed no difference between breeding and non-breeding females in the average optical density of the hybridization signal and the size of the total area covered by Gnrh mRNA. However, analysis along the rostro-caudal axis revealed significantly elevated Gnrh mRNA expression in the rostral preoptic region of breeders compared to non-breeders, whereas the latter had increased Gnrh mRNA expression at the caudal level of the anterior hypothalamus. This study indicates that social suppression affects the expression of Gnrh mRNA in female Damaraland mole-rats. Furthermore, differential regulation occurs within different neuron subpopulations. © 2017 Society for Reproduction and Fertility.

Effects of different dietary intake on mRNA levels of MSTN, IGF-I, and IGF-II in the skeletal muscle of Dorper and Hu sheep hybrid F1 rams.

PubMed

Xing, H J; Wang, Z Y; Zhong, B S; Ying, S J; Nie, H T; Zhou, Z R; Fan, Y X; Wang, F

2014-07-24

MSTN, IGF-І(insulin-like growth factor-І) and IGF-II (insulin-like growth factor-II) regulate skeletal muscle growth. This study investigated the effects of different dietary intake levels on skeletal muscles. Sheep was randomly assigned to 3 feeding groups: 1) the maintenance diet (M), 2) 1.4 x the maintenance diet (1.4M), and 3) 2.15 x the maintenance diet (2.15M). Before slaughtering the animals, blood samples were collected to measure plasma urea, growth hormone, and insulin concentrations. After slaughtering, the longissimus dorsi, semitendinosus, semimembranosus, gastrocnemius, soleus, and chest muscle were removed to record various parameters, including the mRNA expression levels of MSTN and IGFs, in addition to skeletal muscle fiber diameter and cross-sectional area. The result showed that as dietary intake improved, the mRNA expression levels of MSTN and IGF-II decreased, whereas IGF-Іexpression increased. The mRNA expression levels of MSTN and IGFs were significantly different in the same skeletal muscle under different dietary intake. The skeletal muscle fiber diameter and cross-sectional area increased with greater dietary intake, as observed for the mRNA expression of IGF-І; however, it contrasted to that observed for the mRNA expression of MSTN and IGF-II. In conclusion, dietary intake levels have a certain influence on MSTN and IGFs mRNA expression levels, in addition to skeletal muscle fiber diameter and cross-sectional area. This study contributes valuable information for enhancing the molecular-based breeding of sheep.

mRNA expression of corticotropin-releasing factor and urocortin 1 after restraint and foot shock together with alprazolam administration.

PubMed

Cespedes, Isabel C; de Oliveira, Amanda R; da Silva, Joelcimar M; da Silva, André V; Sita, Luciane V; Bittencourt, Jackson C

2010-12-01

Corticotropin-releasing factor (CRF) is expressed in the paraventricular nucleus of the hypothalamus (PVN), and act centrally to provoke stress-like autonomic and behavioral responses. Urocortins 1-3 are additional ligands to the CRF receptors 1 and 2. Ucn 1 neurons are primarily concentrated in the Edinger-Westphal (EW) nucleus and also have been associated with stress responses. It is also known that UCN 1 respond in different ways depending on the stressor presented. Benzodiazepines can act via the CRF peptidergic system and chronic administration of alprazolam does not interfere with CRF mRNA expression in the PVN, but significantly increase Ucn 1 mRNA expression in the EW. The aim of our study was to investigate the relationship between different stressor stimuli, foot shock (FS) and restraint (R), and the mRNA expression of CRF and Ucn 1 in the PVN and EW using alprazolam (A). We employed fos activation and in situ hybridization. Restraint group presented increased fos-ir and CRF mRNA expression in the PVN compared to FS group. The stress responses of R group were prevented by A. In the EW, fos-ir was higher in the FS group than in the R group, whereas Ucn 1 mRNA expression was higher in the R group than in the FS group. Alprazolam significantly increased fos-ir and Ucn 1 mRNA expression in both groups. Our results show that PVN and EW respond in different ways to the same stressors. Furthermore, EW of stressed animals replies in a complementary way comparing to PVN with the use of Alprazolam. Copyright © 2010 Elsevier Inc. All rights reserved.

Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats.

PubMed

Komar, Carolyn M; Curry, Thomas E

2002-05-01

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.

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

PubMed

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

2004-05-01

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

Promoter hypermethylation of the RECK gene is associated with its low expression and poor survival of esophageal squamous cell carcinoma

PubMed Central

Zhu, Jing; Ling, Yang; Xu, Yun; Lu, Mingzhu; Liu, Yongping; Zhang, Changsong

2017-01-01

The present study aimed to investigate the association between the methylation status of the reversion-inducing cysteine-rich protein with kazal motifs (RECK) gene and its mRNA expression levels in patients with esophageal squamous cell carcinoma (ESCC). The methylation status of RECK was analyzed by methylation-specific polymerase chain reaction (PCR), and RECK mRNA expression levels were analyzed by quantitative PCR, in 310 paired ESCC tissues. The mean RECK methylation index (MI) was 0.65 in ESCCs and 0.49 in non-tumor samples. There was a significant association between RECK methylation and the American Joint Committee on Cancer stage and lymph node metastasis in ESCC (P<0.0001; P=0.001). The mRNA expression level of RECK was lower in ESCC tissues (mean-∆Cq=−4.66) compared with non-tumor tissues (mean-∆Cq=−2.79), and decreased RECK mRNA expression levels were associated with lymph node metastasis in ESCC. In addition, RECK mRNA levels were decreased in ESCC patients with hypermethylation of the RECK gene (∆MI >0.16; mean-∆∆Cq=−2.85) compared with those with hypomethylation of the RECK gene (∆MI ≤0.16; mean-∆∆Ct=−0.83), and there was a significant difference in the mRNA expression levels of RECK between those with N0–1 and N2–3 lymph node metastasis (P<0.0001). A significant correlation was observed between RECK mRNA expression levels, the MI of RECK and poor postoperative survival (P=0.0003; P<0.0001). The results of the present study suggested that promoter hypermethylation may be an important factor for loss of RECK mRNA expression and may be an indicator of poor survival in ESCC. PMID:28454343

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

Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai

Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

TNF-α potentiates uric acid-induced interleukin-1β (IL-1β) secretion in human neutrophils.

PubMed

Yokose, Kohei; Sato, Shuzo; Asano, Tomoyuki; Yashiro, Makiko; Kobayashi, Hiroko; Watanabe, Hiroshi; Suzuki, Eiji; Sato, Chikako; Kozuru, Hideko; Yatsuhashi, Hiroshi; Migita, Kiyoshi

2018-05-01

Monosodium urate (MSU) has been shown to promote interleukin-1β (IL-1β) secretion in human monocytes, but the priming signals for NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway remains elusive. In this study, we investigated the role of Tumor necrosis factor-alpha (TNF-α) on MSU-mediated IL-1β induction in human neutrophils. Human neutrophils were stimulated with MSU, in the presence or absence of TNF-α priming. The cellular supernatants were analyzed for IL-1β, IL-18, and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1β mRNA expressions in human neutrophils were analyzed by real-time PCR method. TNF-α stimulation induced pro-IL-1β mRNA expression; however, MSU stimulation did not induce pro-IL-1β mRNA expression in human neutrophils. TNF-α alone or MSU stimulation did not result in efficient IL-1β secretion in human neutrophils, whereas in TNF-α-primed neutrophils, MSU stimulation resulted in a marked IL-1β and IL-18 secretion. TNF-α-primed neutrophils secreted cleaved caspase-1 (p20), in response to MSU stimulation. Our data demonstrate that priming of human neutrophils with TNF-α promotes uric acid-mediated IL-1β secretion in the absence of microbial stimulation. These findings provide insights into the neutrophils-mediated inflammatory processes in gouty arthritis.

Association between EML4-ALK fusion gene and thymidylate synthase mRNA expression in non-small cell lung cancer tissues

PubMed Central

XU, CHUN-WEI; WANG, GANG; WANG, WU-LONG; GAO, WEN-BIN; HAN, CHUAN-JUN; GAO, JING-SHAN; ZHANG, LI-YING; LI, YANG; WANG, LIN; ZHANG, YU-PING; TIAN, YU-WANG; QI, DONG-DONG

2015-01-01

This study aimed to investigate the association of the mRNA expression of the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene with that of thymidylate synthase (TYMS) in non-small cell lung cancer (NSCLC) tissues. Quantitative polymerase chain reaction was used to detect the expression of EML4-ALK fusion gene and TYMS mRNA in 257 cases of NSCLC. The positive rate of EML4-ALK fusion gene was 4.28% in the NSCLC tissues (11/257), and was higher in nonsmokers than in smokers (P<0.05); TYMS mRNA expression was detected in 63.42% (163/257) of cases. An association of the EML4-ALK fusion gene with TYMS expression was detected; a low expression level of TYMS mRNA was observed more frequently when the EML4-ALK fusion gene was present than when it was not detected (P<0.05). In conclusion, patients positive for the EML4-ALK fusion gene in NSCLC tissues are likely to have a low expression level of TYMS, and may benefit from the first-line chemotherapy drug pemetrexed. PMID:26136951

Association between EML4-ALK fusion gene and thymidylate synthase mRNA expression in non-small cell lung cancer tissues.

PubMed

Xu, Chun-Wei; Wang, Gang; Wang, Wu-Long; Gao, Wen-Bin; Han, Chuan-Jun; Gao, Jing-Shan; Zhang, Li-Ying; Li, Yang; Wang, Lin; Zhang, Yu-Ping; Tian, Yu-Wang; Qi, Dong-Dong

2015-06-01

This study aimed to investigate the association of the mRNA expression of the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene with that of thymidylate synthase (TYMS) in non-small cell lung cancer (NSCLC) tissues. Quantitative polymerase chain reaction was used to detect the expression of EML4-ALK fusion gene and TYMS mRNA in 257 cases of NSCLC. The positive rate of EML4-ALK fusion gene was 4.28% in the NSCLC tissues (11/257), and was higher in nonsmokers than in smokers (P<0.05); TYMS mRNA expression was detected in 63.42% (163/257) of cases. An association of the EML4-ALK fusion gene with TYMS expression was detected; a low expression level of TYMS mRNA was observed more frequently when the EML4-ALK fusion gene was present than when it was not detected (P<0.05). In conclusion, patients positive for the EML4-ALK fusion gene in NSCLC tissues are likely to have a low expression level of TYMS, and may benefit from the first-line chemotherapy drug pemetrexed.

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.

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

Upregulation of estrogen receptor subtypes and vitellogenin mRNA in cinnamon clownfish Amphiprion melanopus during the sex change process: profiles on effects of 17beta-estradiol.

PubMed

Kim, Na Na; Jin, Deuk-Hee; Lee, Jehee; Kil, Gyung-Suk; Choi, Cheol Young

2010-10-01

In the present study, we investigated the expression pattern of estrogen receptors (esr) and vitellogenin (vtg) mRNA in the gonads and liver during sex change in cinnamon clownfish by using quantitative polymerase chain reaction. We divided gonadal development during the sex change from male to female into 3 stages (mature male, male at 90days after removing female, and mature female) and investigated esr and vtg mRNA expressions during the sex change. With female, the esr and vtg mRNA expressions increased. In western blot analysis, Esr1 protein was detected only in the ovaries of female cinnamon clownfish. Also, to understand the effect of 17beta-estradiol (E(2)), we investigated the esr and vtg mRNA expression patterns in the gonads and liver, and the changes in plasma E(2) level after E(2) injection. E(2) treatment increased both mRNA expression levels of esr and vtg and plasma E(2) levels. The present study describes the molecular characterization of esr subtypes and the interactions between esr and vtg after E(2) treatment in cinnamon clownfish. 2010 Elsevier Inc. All rights reserved.

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

PubMed

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

2014-10-09

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

Expression of phosphatidylserine-specific phospholipase A(1) mRNA in human THP-1-derived macrophages.

PubMed

Hosono, Hiroyuki; Homma, Masato; Ogasawara, Yoko; Makide, Kumiko; Aoki, Junken; Niwata, Hideaki; Watanabe, Machiko; Inoue, Keizo; Ohkohchi, Nobuhiro; Kohda, Yukinao

2010-01-01

The expression of phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) is most upregulated in the genes of peripheral blood cells from chronic rejection model rats bearing long-term surviving cardiac allografts. The expression profile of PS-PLA(1) in peripheral blood cells responsible for the immune response may indicate a possible biological marker for rejection episodes. In this study, PS-PLA(1) mRNA expression was examined in human THP-1-derived macrophages. The effects of several immunosuppressive agents on this expression were also examined in in vitro experiments. A real-time RT-PCR analysis revealed that PS-PLA(1) mRNA expression was found in human THP-1-derived macrophages. This expression was enhanced in the cells stimulated with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. Other TLR ligands (TLR2, 3, 5, 7, and 9) did not show a significant induction of PS-PLA(1) mRNA. The time course of the mRNA expression profiles was different between PS-PLA(1) and tumor necrosis factor-α (TNF-α), which showed a maximal expression at 12 and 1 h after LPS stimulation, respectively. Among the observed immunosuppressive agents, corticosteroids, prednisolone, 6α-methylprednisolone, dexamethasone, and beclomethasone inhibited PS-PLA(1) expression with half-maximal inhibitory concentrations less than 3.0 nM, while methotrexate, cyclosporine A, tacrolimus, 6-mercaptopurine, and mycophenoic acid showed either a weak or moderate inhibition. These results suggest that the expression of PS-PLA(1) mRNA in THP-1-derived macrophages is activated via TLR4 and it is inhibited by corticosteroids, which are used at high dosages to suppress chronic allograft rejection.

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression

PubMed Central

Tavares, Catarina; Eloy, Catarina; Melo, Miguel; Gaspar da Rocha, Adriana; Pestana, Ana; Batista, Rui; Rios, Elisabete; Sobrinho Simões, Manuel

2018-01-01

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression. PMID:29757257

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression.

PubMed

Tavares, Catarina; Eloy, Catarina; Melo, Miguel; Gaspar da Rocha, Adriana; Pestana, Ana; Batista, Rui; Bueno Ferreira, Luciana; Rios, Elisabete; Sobrinho Simões, Manuel; Soares, Paula

2018-05-13

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression.

Amphiphysin I but not dynamin I nor synaptojanin mRNA expression increased after repeated methamphetamine administration in the rat cerebrum and cerebellum.

PubMed

Hamamura, Mitsuko; Okouchi, Jiro; Ozawa, Hidetoshi; Kimuro, Yoshihiko; Iwaki, Akiko; Fukumaki, Yasuyuki

2013-07-01

Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3' region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3' UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca²⁺ and Mg²⁺ devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.

The effect of dietary carbohydrate on genes for fatty acid synthase and inflammatory cytokines in adipose tissues from lean and obese subjects.

PubMed

Hudgins, Lisa C; Baday, Aline; Hellerstein, Marc K; Parker, Thomas S; Levine, Daniel M; Seidman, Cynthia E; Neese, Richard A; Tremaroli, Jolanta D; Hirsch, Jules

2008-04-01

Hepatic de novo lipogenesis (DNL) is markedly stimulated in humans by low-fat diets enriched in simple sugars. However, the dietary responsiveness of the key enzyme controlling DNL in human adipose tissue, fatty acid synthase (FAS), is uncertain. Adipose tissue mRNA for FAS is increased in lean and obese subjects when hepatic DNL is elevated by a eucaloric, low-fat, high-sugar diet. Twelve lean and seven obese volunteers were given two eucaloric diets (10% vs. 30% fat; 75% vs. 55% carbohydrate; sugar/starch 60/40) each for 2 weeks by a random-order cross-over design. FAS mRNA in abdominal and gluteal adipose tissues was compared to hepatic DNL measured in serum by isotopic and nonisotopic methods. Adipose tissue mRNA for tumor necrosis factor-alpha and IL-6, which are inflammatory cytokines that modulate DNL, was also assayed. The low-fat high-sugar diet induced a 4-fold increase in maximum hepatic DNL (P<.001) but only a 1.3-fold increase in adipose tissue FAS mRNA (P=.029) and no change in cytokine mRNA. There was a borderline significant positive correlation between changes in FAS mRNA and hepatic DNL (P=.039). Compared to lean subjects, obese subjects had lower levels of FAS mRNA and higher levels of cytokine mRNA (P<.001). The results suggest that key elements of human adipose tissue DNL are less responsive to dietary carbohydrate than is hepatic DNL and may be regulated by diet-independent factors. Irrespective of diet, there is reduced expression of the FAS gene and increased expression of cytokine genes in adipose tissues of obese subjects.

Skeletal muscle deiodinase type 2 regulation during illness in mice.

PubMed

Kwakkel, J; van Beeren, H C; Ackermans, M T; Platvoet-Ter Schiphorst, M C; Fliers, E; Wiersinga, W M; Boelen, A

2009-11-01

We have previously shown that skeletal muscle deiodinase type 2 (D2) mRNA (listed as Dio2 in MGI Database) is upregulated in an animal model of acute illness. However, human studies on the expression of muscle D2 during illness report conflicting data. Therefore, we evaluated the expression of skeletal muscle D2 and D2-regulating factors in two mouse models of illness that differ in timing and severity of illness: 1) turpentine-induced inflammation, and 2) Streptococcus pneumoniae infection. During turpentine-induced inflammation, D2 mRNA and activity increased compared to pair-fed controls, most prominently at day 1 and 2, whereas after S. pneumoniae infection D2 mRNA decreased. We evaluated the association of D2 expression with serum thyroid hormones, (de-)ubiquitinating enzymes ubiquitin-specific peptidase 33 and WD repeat and SOCS box-containing 1 (Wsb1), cytokine expression and activation of inflammatory pathways and cAMP pathway. During chronic inflammation the increased muscle D2 expression is associated with the activation of the cAMP pathway. The normalization of D2 5 days after turpentine injection coincides with increased Wsb1 and tumor necrosis factor alpha expression. Muscle interleukin-1beta (Il1b) expression correlated with decreased D2 mRNA expression after S. pneumoniae infection. In conclusion, muscle D2 expression is differentially regulated during illness, probably related to differences in the inflammatory response and type of pathology. D2 mRNA and activity increases in skeletal muscle during the acute phase of chronic inflammation compared to pair-fed controls probably due to activation of the cAMP pathway. In contrast, muscle D2 mRNA decreases 48 h after a severe bacterial infection, which is associated with local Il1b mRNA expression and might also be due to diminished food-intake.

Effect of peritoneal dialysis on expression of vascular endothelial growth factor, basic fibroblast growth factor and endostatin of the peritoneum in peritoneal dialysis patients.

PubMed

Gao, Dan; Zhao, Zhan-Zheng; Liang, Xian-Hui; Li, Yan; Cao, Ying; Liu, Zhang-Suo

2011-11-01

The aim of this study is to investigate the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endostatin (ES) in human peritoneum and investigate the relationship between them and peritoneum neoangiogensis in the patients with uraemia and peritoneal dialysis (PD). Peritoneal biopsies were obtained from normal subjects (n = 8), uraemic predialysis patients (n = 12) and PD patients (n = 10). The mRNA expression of VEGF, bFGF and ES in peritoneal tissues were measured through real-time polymerase chain reaction. The protein expression of VEGF, bFGF and ES in peritoneal tissues were determined through western blot. Microvessel density (MVD) of peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody. The mRNA and protein of VEGF, bFGF and ES were expressed in all peritoneal samples. Compared with the normal control group, the mRNA and protein expression of VEGF and bFGF in peritoneal tissues were all significantly upregulated in the uraemic predialysis and PD group (all P < 0.05). Compared with the normal control group, the protein expression of ES were significantly upregulated in the uraemic predialysis and PD group (all (P < 0.05), but the mRNA expression of ES did not have obvious differences in the uraemic predialysis and PD group as compared to the normal control group (P > 0.05). MVD of peritoneal tissue were increased in the uraemic predialysis and PD group compared with the normal group (all P < 0.05). A significant positive correlation was found between VEGF mRNA expression and MVD, bFGF mRNA expression and MVD. The mRNA expression of VEGF and bFGF, the protein expression of VEGF, bFGF, and ES and microvessel density (MVD) are increased both in the uraemic predialysis and PD patients. These results show that uraemia circumstances and non-physiological compatibility of peritoneal dialysis solution might increase VEGF, bFGF and ES expression and MVD, which might participate in the increment of the peritoneum neoangiogensis and ultrafiltration failure in PD patients. © 2011 The Authors. Nephrology © 2011 Asian Pacific Society of Nephrology.

Human organotypic retinal flat-mount culture (HORFC) as a model for retinitis pigmentosa11.

PubMed

Azizzadeh Pormehr, Leila; Daftarian, Narsis; Ahmadian, Shahin; Rezaei Kanavi, Mozhgan; Ahmadieh, Hamid; Shafiezadeh, Mahshid

2018-05-10

The splicing factor PRPF31 is the most commonly mutated general splicing factor in the retinitis pigmentosa. We used a rapid, convenient and cost effective transfection method with an efficient PRPF31 knockdown in HORFC in order to study the effect of PRPF31 downregulation on retinal gene expressions in an ex vivo model. Modified calcium phosphate method was used to transfect HORFC by PRPF31 siRNA. Different times and doses of siRNA for transfection were assayed and optimum condition was obtained. PRPF31 mRNA and protein downregulation were assessed by qRTPCR and Western blot. The tissue viability of HORFC was measured using the MTT. ImageJ analysis on stained retinal sections by immunohistochemistry was used for thickness measurement of outer nuclear photoreceptor layer. The PRPF31 gene downregulation effects on retinal specific gene expression were analyzed by qRTPCR. A total of 50 nM of PRPF31 siRNA transfection after 63 h in HORFC, showed the optimum reduction in the level of PRPF31 mRNA and protein as shown by qRTPCR and Western blot (over 90% and 50% respectively). The PRPF31 mRNA silencing with calcium phosphate had no effect on cell viability in the period of the experiment. Thickness measurement of outer nuclear photoreceptor layer with IHC showed the significant reduction after 63 h of study (P value = 0.02). siRNA induced PRPF31 knockdown, led to reduction of retinal specific mRNA gene expression involved in phototransduction (RHO, GNAT1, RP1), photoreceptor structure (ROM1, FSCN2, CA4, SEMA4) and transcription factor (CRX) (fold change >5), after 63 h. © 2018 Wiley Periodicals, Inc.

An Assessment of Database-Validated microRNA Target Genes in Normal Colonic Mucosa: Implications for Pathway Analysis.

PubMed

Slattery, Martha L; Herrick, Jennifer S; Stevens, John R; Wolff, Roger K; Mullany, Lila E

2017-01-01

Determination of functional pathways regulated by microRNAs (miRNAs), while an essential step in developing therapeutics, is challenging. Some miRNAs have been studied extensively; others have limited information. In this study, we focus on 254 miRNAs previously identified as being associated with colorectal cancer and their database-identified validated target genes. We use RNA-Seq data to evaluate messenger RNA (mRNA) expression for 157 subjects who also had miRNA expression data. In the replication phase of the study, we replicated associations between 254 miRNAs associated with colorectal cancer and mRNA expression of database-identified target genes in normal colonic mucosa. In the discovery phase of the study, we evaluated expression of 18 miR-NAs (those with 20 or fewer database-identified target genes along with miR-21-5p, miR-215-5p, and miR-124-3p which have more than 500 database-identified target genes) with expression of 17 434 mRNAs to identify new targets in colon tissue. Seed region matches between miRNA and newly identified targeted mRNA were used to help determine direct miRNA-mRNA associations. From the replication of the 121 miRNAs that had at least 1 database-identified target gene using mRNA expression methods, 97.9% were expressed in normal colonic mucosa. Of the 8622 target miRNA-mRNA associations identified in the database, 2658 (30.2%) were associated with gene expression in normal colonic mucosa after adjusting for multiple comparisons. Of the 133 miRNAs with database-identified target genes by non-mRNA expression methods, 97.2% were expressed in normal colonic mucosa. After adjustment for multiple comparisons, 2416 miRNA-mRNA associations remained significant (19.8%). Results from the discovery phase based on detailed examination of 18 miRNAs identified more than 80 000 miRNA-mRNA associations that had not previously linked to the miRNA. Of these miRNA-mRNA associations, 15.6% and 14.8% had seed matches for CRCh38 and CRCh37, respectively. Our data suggest that miRNA target gene databases are incomplete; pathways derived from these databases have similar deficiencies. Although we know a lot about several miRNAs, little is known about other miRNAs in terms of their targeted genes. We encourage others to use their data to continue to further identify and validate miRNA-targeted genes.

Molecular signaling in intervertebral disk development.

PubMed

DiPaola, Christian P; Farmer, James C; Manova, Katia; Niswander, Lee A

2005-09-01

The purpose of this investigation is to identify and study the expression pattern of pertinent molecular factors involved in the differentiation of the intervertebral disk (IVD). It is likely that hedgehog genes and the BMP inhibitors are key factors involved in spinal joint formation. Radioactive in situ hybridization with mRNA probes for pax-1, SHH, IHH and Noggin gene was performed on mouse embryo and adult tissue. Immunohistochemistry was performed to localize hedgehog receptor, "patched" (ptc). From 14.5 dpc until birth pax-1 mRNA was expressed in the developing anulus fibrosus (AF). During the same developmental period Noggin mRNA is highly expressed throughout the spine, in the developing AF, while ptc protein and SHH mRNA were expressed in the developing nucleus pulposus (NP). IHH mRNA was expressed by condensing chondrocytes of the vertebral bodies and later becomes confined to the vertebral endplate. We show for the first time that pax-1 is expressed in the adult intervertebral disk. Ptc expression in the NP is an indicator of hedgehog protein signaling in the developing IVD. The expression pattern of the BMP inhibitor Noggin appears to be important for the normal formation of the IVD and may prove to play a role in its segmental pattern formation.

Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood

PubMed Central

Clinton, Sarah M.; Glover, Matthew E.; Maltare, Astha; Laszczyk, Ann M.; Mehi, Stephen J.; Simmons, Rebecca K.; King, Gwendalyn D.

2013-01-01

Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development. PMID:23838326

Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities

PubMed Central

Baril, Patrick; Ezzine, Safia; Pichon, Chantal

2015-01-01

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy. PMID:25749473

Monitoring the spatiotemporal activities of miRNAs in small animal models using molecular imaging modalities.

PubMed

Baril, Patrick; Ezzine, Safia; Pichon, Chantal

2015-03-04

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

PubMed

Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

2015-12-01

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

Localization of complement factor H gene expression and protein distribution in the mouse outer retina

PubMed Central

Smit-McBride, Zeljka; Oltjen, Sharon L.; Radu, Roxana A.; Estep, Jason; Nguyen, Anthony T.; Gong, Qizhi

2015-01-01

Purpose To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC. PMID:25684976

Prediagnostic Smoking Is Associated with Binary and Quantitative Measures of ER Protein and ESR1 mRNA Expression in Breast Tumors.

PubMed

Butler, Eboneé N; Bensen, Jeannette T; Chen, Mengjie; Conway, Kathleen; Richardson, David B; Sun, Xuezheng; Geradts, Joseph; Olshan, Andrew F; Troester, Melissa A

2018-01-01

Background: Smoking is a possible risk factor for breast cancer and has been linked to increased risk of estrogen receptor-positive (ER + ) disease in some epidemiologic studies. It is unknown whether smoking has quantitative effects on ER expression. Methods: We examined relationships between smoking and ER expression from tumors of 1,888 women diagnosed with invasive breast cancer from a population-based study in North Carolina. ER expression was characterized using binary (±) and continuous measures for ER protein, ESR1 mRNA, and a multigene luminal score (LS) that serves as a measure of estrogen signaling in breast tumors. We used logistic and linear regression models to estimate temporal and dose-dependent associations between smoking and ER measures. Results: The odds of ER + , ESR1 + , and LS + tumors among current smokers (at the time of diagnosis), those who smoked 20 or more years, and those who smoked within 5 years of diagnosis were nearly double those of nonsmokers. Quantitative levels of ESR1 were highest among current smokers compared with never smokers overall [mean (log 2 ) = 9.2 vs. 8.7, P < 0.05] and among ER + cases; however, we did not observe associations between smoking measures and continuous ER protein expression. Conclusions: In relationship to breast cancer diagnosis, recent smoking was associated with higher odds of the ER + , ESR1 + , and LS + subtype. Current smoking was associated with elevated ESR1 mRNA levels and an elevated LS, but not with altered ER protein. Impact: A multigene LS and single-gene ESR1 mRNA may capture tumor changes associated with smoking. Cancer Epidemiol Biomarkers Prev; 27(1); 67-74. ©2017 AACR . ©2017 American Association for Cancer Research.

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

PubMed Central

2011-01-01

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

Molecular cloning and functional analysis of MRLC2 in Tianfu, Boer, and Chengdu Ma goats.

PubMed

Xu, H G; Xu, G Y; Wan, L; Ma, J

2013-03-15

To determine the molecular basis of heterosis in goats, fluorescence quantitative polymerase chain reaction (PCR) was performed to investigate myosin-regulatory light chain 2 (MRLC2) gene expression in the longissimus dorsi muscle tissues of the Tianfu goat and its parents, the Boer and Chengdu Ma goats. The goat MRLC2 gene was differentially expressed in the crossbreed, and the purebred mRNA were isolated and identified using fluorescence quantitative reverse transcription-PCR (RT-PCR). The complete coding sequence of MRLC2 was obtained using the cDNA method, and the full-length coding sequence consisted of 513 bp encoding 172 amino acids. The EF-hand superfamily domain of the MRLC2 protein is well conserved in caprine and other animals. The deduced amino acid sequence of MRLC2 shared significant identity with MRLC2 from other mammals. Phylogenetic tree analysis revealed that the MRLC2 protein was closely related to MRLC2 in other mammals. Several predicted miRNA target sites were found in the coding sequence of caprine MRLC2 mRNA. Analysis by RT-PCR showed that MRLC2 mRNA was present in the heart, stomach, liver, spleen, lung, small intestine, kidney, leg muscle, abdominal muscle, and longissimus dorsi muscles. In particular, the high expression of MRLC2 mRNA was detected in the longissimus dorsi, leg muscle, abdominal muscle, stomach, and heart, but low levels of expression were also observed in the liver, spleen, lung, small intestine, and kidney. The expression of the MRLC2 gene was upregulated in the longissimus dorsi muscle of Boer and Tianfu goats, and it was moderately upregulated in Chengdu Ma goats.

Depot-specific Regulation of the Conversion of Cortisone to Cortisol in Human Adipose Tissue

PubMed Central

Lee, Mi-Jeong; Fried, Susan K.; Mundt, Steven S.; Wang, Yanxin; Sullivan, Sean; Stefanni, Alice; Daugherty, Bruce L.; Hermanowski-Vosatka, Anne

2015-01-01

Objective Our main objective was to compare the regulation of cortisol production within omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue. Methods and Procedures Om and Abd sc adipose tissue were obtained at surgery from subjects with a wide range of BMI. Hydroxysteroid dehydrogenase (HSD) activity (3H-cortisone and 3H-cortisol interconversion) and expression were measured before and after organ culture with insulin and/or dexamethasone. Results Type 1 HSD (HSD1) mRNA and reductase activity were mainly expressed within adipocytes and tightly correlated with adipocyte size within both depots. There was no depot difference in HSD1 expression or reductase activity, while cortisol inactivation and HSD2 mRNA expression (expressed in stromal cells) were higher in Om suggesting higher cortisol turnover in this depot. Culture with insulin decreased HSD reductase activity in both depots. Culture with dexamethasone plus insulin compared to insulin alone increased HSD reductase activity only in the Om depot. This depot-specific increase in reductase activity could not be explained by an alteration in HSD1 mRNA or protein, which was paradoxically decreased. However, in Om only, hexose-6-phosphate dehydrogenase (H6PDH) mRNA levels were increased by culture with dexamethasone plus insulin compared to insulin alone, suggesting that higher nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) production within the endoplasmic reticulum (ER) contributed to the higher HSD reductase activity. Discussion We conclude that in the presence of insulin, glucocorticoids cause a depot-specific increase in the activation of cortisone within Om adipose tissue, and that this mechanism may contribute to adipocyte hypertrophy and visceral obesity. PMID:18388900

Influence of performance on gene expression in skeletal muscle: effects of forced inactivity

NASA Technical Reports Server (NTRS)

Thomason, D. B.; Booth, F. W.

1989-01-01

Joint immobilization and hindlimb suspension are used to examine muscle protein expression and mRNA quantities in rats. A decrease in protein synthesis was not associated with alteration in alpha-actin mRNA, cytochrome c mRNA, or beta-myosin heavy chain mRNA early in treatment. Percentage declines after seven days are compared with early treatment quantities to determine acute and chronic response to muscular atrophy.

Expression of chemokines CXCL4 and CXCL7 by synovial macrophages defines an early stage of rheumatoid arthritis

PubMed Central

Yeo, L; Adlard, N; Juarez, M; Smallie, T; Snow, M; Buckley, C D; Raza, K; Filer, A; Scheel-Toellner, D

2016-01-01

Background and objectives For our understanding of the pathogenesis of rheumatoid arthritis (RA), it is important to elucidate the mechanisms underlying early stages of synovitis. Here, synovial cytokine production was investigated in patients with very early arthritis. Methods Synovial biopsies were obtained from patients with at least one clinically swollen joint within 12 weeks of symptom onset. At an 18-month follow-up visit, patients who went on to develop RA, or whose arthritis spontaneously resolved, were identified. Biopsies were also obtained from patients with RA with longer symptom duration (>12 weeks) and individuals with no clinically apparent inflammation. Synovial mRNA expression of 117 cytokines was quantified using PCR techniques and analysed using standard and novel methods of data analysis. Synovial tissue sections were stained for CXCL4, CXCL7, CD41, CD68 and von Willebrand factor. Results A machine learning approach identified expression of mRNA for CXCL4 and CXCL7 as potentially important in the classification of early RA versus resolving arthritis. mRNA levels for these chemokines were significantly elevated in patients with early RA compared with uninflamed controls. Significantly increased CXCL4 and CXCL7 protein expression was observed in patients with early RA compared with those with resolving arthritis or longer established disease. CXCL4 and CXCL7 co-localised with blood vessels, platelets and CD68+ macrophages. Extravascular CXCL7 expression was significantly higher in patients with very early RA compared with longer duration RA or resolving arthritis Conclusions Taken together, these observations suggest a transient increase in synovial CXCL4 and CXCL7 levels in early RA. PMID:25858640

Tissue-specific mRNA expression profiling in grape berry tissues

PubMed Central

Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

2007-01-01

Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented. Conclusion These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality. PMID:17584945

Anti-nociceptive mechanism of baicalin involved in intervention of TRPV1 in DRG neurons in vitro.

PubMed

Sui, Feng; Zhang, Chang-Bin; Yang, Na; Li, Lan-Fang; Guo, Shu-Ying; Huo, Hai-Ru; Jiang, Ting-Liang

2010-06-16

Scutellaria baicalensis Georgi (Lamiaceae) is often included as an ingredient in traditional Chinese compound prescriptions for the treatment of fever-related or inflammatory conditions. The present work was to further uncover the analgesic mechanisms of baicalin (a known principal constituent of Scutellaria baicalensis) by investigating its effects on the expression of TRPV1 mRNA as well as on its functions as mediators of calcium entrance into the cytoplasm of dorsal root ganglion (DRG) neurons in vitro. By using CPT as an agent to eliminate the non-neuronal cells and using serum-free neurobasal as culture medium, primary cultures of rat DRG neurons with high purity and viability were established. On this basis, effects of baicalin on both the expression of TRPV1 mRNA and on the function of TRPV1 in vitro under two various temperature conditions were studied. The TRPV1 mRNA expression levels were examined by using qRT-PCR and analyzed by the method of 2(-DeltaDeltaCT). The elevation amplitudes of intracellular [Ca(2+)]i evoked by TRPV1 agonist capsaicin in DRG neurons were examined by the calcium fluorescence imaging method under confocal microscopy. Baicalin was shown to down-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and under the latter temperature, the intracellular fluorescent intensity evoked by capsaicin was significantly decreased following incubation with baicalin in vitro. We also demonstrated that the actions of baicalin to TRPV1 were not achieved through pathways of TRPA1 or TRPV subfamily members. Collectively, these results provide compelling evidence that the down-regulated actions of baicalin to TRPV1 in DRG neurons might account for part of the anti-nociceptive mechanism of baicalin. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Apoptosis and Inflammation Associated Gene Expressions in Monocrotaline-Induced Pulmonary Hypertensive Rats after Bosentan Treatment

PubMed Central

Hong, Young Mi; Kwon, Jung Hyun; Choi, Shinkyu

2014-01-01

Background and Objectives Vascular wall remodeling in pulmonary hypertension can be caused by an aberration in the normal balance between proliferation and apoptosis of endothelial cell in the pulmonary artery. The objective of this study was to evaluate the effect of bosentan on apoptosis in monocrotaline (MCT)-induced pulmonary hypertension. Materials and Methods Sprague-Dawley rats were divided into three groups: control (C) group, M group (MCT 60 mg/kg) and B group (MCT 60 mg/kg plus bosentan 20 mg/day orally). Gene expressions of Bcl (B cell leukemia/lymphoma)-2, caspase-3, complement component (C)-6, vascular endothelial growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were analyzed by real time polymerase chain reaction and western blot analysis. Results The messenger ribonucleic acid (mRNA) expressions of caspase-3 and VEGF were significantly increased in the M group compared with the C group, and significantly decreased in the B group compared with the M group in week 4. mRNA expression of IL-6 was significantly decreased in weeks 1, 2, and 4 in the B group compared with the M group. mRNA expression of TNF-α was significantly decreased on day 5 and in weeks 1 and 2 in the B group compared with the M group. Conclusion Bosentan may have potential for preventing apoptosis and inflammation. PMID:24653739

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

PubMed

Zheng, Jun; Takagi, Hiroyasu; Tsutsui, Chihiro; Adachi, Akihito; Sakai, Takafumi

2008-03-01

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua.

PubMed

Kolkova, Z; Noskova, V; Ehinger, A; Hansson, S; Casslén, B

2010-10-01

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERβ mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

Expression Changes of Apoptotic Genes in Tissues from Mice Exposed to Nicotine

PubMed Central

Jalili, Cyrus; Salahshoor, Mohammad Reza; Moradi, Mohammad Taher; Ahookhash, Maryam; Taghadosi, Mehdi; Sohrabi, Maryam

2017-01-01

Objective: Smoking is the leading preventable cause of various diseases such as lung cancer, chronic obstructive pulmonary disease and cardiovascular disease. Nicotine, one of the major toxic components of tobacco, contributes to the pathogenesis of different diseases. Methods: Given the controversy about nicotine toxicity, the present study was conducted to determine apoptotic effects of nicotine on the heart, kidney, lung and liver of male mice. Real-time PCR was performed to identify mRNA expression changes in apoptotic-related genes between nicotine treated and control mice. Result: In the heart and lung, nicotine caused significant decrease in P53, Bax and Caspase-3 mRNA expression levels compared to the control group. However, in the kidney and liver, the result was significant increase in Bax, Caspase-2, Caspase-3 and a significant decrease in P53 mRNA expression (p<0.01). DNA fragmentation assays indicated no fragmentation in the heart and lung, but in the kidney and liver of nicotine treated mice, isolated DNA was fragmented. Conclusion: Our study provided insight into the molecular mechanisms of nicotine anti-apoptotic effects on the heart and lung as well as pro-apoptotic effects on kidney and liver via a P53-independent pathway. PMID:28240526

GROWTH OF HUMAN PANCREATIC CANCER IS INHIBITED BY DOWN-REGULATION OF GASTRIN GENE EXPRESSION

PubMed Central

Matters, Gail L.; Harms, John F.; McGovern, Christopher O.; Jayakumar, Calpurnia; Crepin, Keisha; Smith, Zachary P.; Nelson, Melissa C.; Stock, Heather; Fenn, Craig W.; Kaiser, James; Kester, Mark; Smith, Jill P.

2009-01-01

Objectives This study evaluated the effects of gastrin mRNA down-regulation on growth of human pancreatic cancer. Methods Gastrin expression was examined in human pancreatic cancer cell lines by RT-PCR and peptide expression was assessed by immunocytochemistry. Gastrin was down-regulated using either stable transfection of an antisense gastrin cDNA or one of three shRNA (short hairpin RNA) constructs. Tumor formation was evaluated following either subcutaneous or orthotopic injections into nude mice. The effect of nanoliposomes loaded with gastrin siRNA was tested in mice bearing pancreatic tumors. Results Stable transfection of gastrin antisense or shRNAs into BxPC-3 cells resulted in clones with >90% reduction in gastrin mRNA. Tumor growth rate and incidence of metastases in both wild type and transfected pancreatic cancer cells was directly proportional to the degrees of gastrin mRNA expression. Immunofluoresence analysis confirmed that gastrin peptide levels were decreased in antisense and shRNA tumors. Gastrin knockdown clones had lower Ki-67 and increased cleaved caspase-3 staining, consistent with known effects of gastrin on proliferation and apoptosis. Tumors in mice treated with gastrin siRNA were smaller than controls. Conclusions These results suggest that RNAi targeting of gastrin could serve as an effective treatment for pancreatic cancer. PMID:19465883

A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

PubMed Central

Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

2014-01-01

Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

Synthetic mRNA is a more reliable tool for the delivery of DNA-targeting proteins into the cell nucleus than fusion with a protein transduction domain.

PubMed

Leontovyc, Ivan; Habart, David; Loukotova, Sarka; Kosinova, Lucie; Kriz, Jan; Saudek, Frantisek; Koblas, Tomas

2017-01-01

Cell reprogramming requires efficient delivery of reprogramming transcription factors into the cell nucleus. Here, we compared the robustness and workload of two protein delivery methods that avoid the risk of genomic integration. The first method is based on fusion of the protein of interest to a protein transduction domain (PTD) for delivery across the membranes of target cells. The second method relies on de novo synthesis of the protein of interest inside the target cells utilizing synthetic mRNA (syn-mRNA) as a template. We established a Cre/lox reporter system in three different cell types derived from human (PANC-1, HEK293) and rat (BRIN-BD11) tissues and used Cre recombinase to model a protein of interest. The system allowed constitutive expression of red fluorescence protein (RFP), while green fluorescence protein (GFP) was expressed only after the genomic action of Cre recombinase. The efficiency of protein delivery into cell nuclei was quantified as the frequency of GFP+ cells in the total cell number. The PTD method showed good efficiency only in BRIN-BD11 cells (68%), whereas it failed in PANC-1 and HEK293 cells. By contrast, the syn-mRNA method was highly effective in all three cell types (29-71%). We conclude that using synthetic mRNA is a more robust and less labor-intensive approach than using the PTD-fusion alternative.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

PubMed

Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

2018-01-01

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P < 0.05 and more than 5.96% genes presented very strong correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

PubMed

Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

2017-01-01

Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 10 6 to approximately 10 8 cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.

Long Noncoding RNAs and mRNA Regulation in Peripheral Blood Mononuclear Cells of Patients with Chronic Obstructive Pulmonary Disease

PubMed Central

Wang, Weijia; Xu, Dan

2018-01-01

Background Inflammation plays a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD). We evaluated the lncRNA and mRNA expression profile of peripheral blood mononuclear cells (PBMCs) from healthy nonsmokers, smokers without airflow limitation, and COPD patients. Methods lncRNA and mRNA profiling of PBMCs from 17 smokers and 14 COPD subjects was detected by high-throughput microarray. The expression of dysregulated lncRNAs was validated by qPCR. The lncRNA targets in dysregulated mRNAs were predicted and the GO enrichment was analyzed. The regulatory role of lncRNA ENST00000502883.1 on CXCL16 expression and consequently the effect on PBMC recruitment were investigated by siRNA knockdown and chemotaxis analysis. Results We identified 158 differentially expressed lncRNAs in PBMCs from COPD subjects compared with smokers. The dysregulated expression of 5 selected lncRNAs NR_026891.1 (FLJ10038), ENST00000502883.1 (RP11-499E18.1), HIT000648516, XR_429541.1, and ENST00000597550.1 (CTD-2245F17.3), was validated. The GO enrichment showed that leukocyte migration, immune response, and apoptosis are the main enriched processes that previously reported to be involved in the pathogenesis of COPD. The regulatory role of ENST00000502883.1 on CXCL16 expression and consequently the effect on PBMC recruitment was confirmed. Conclusion This study may provide clues for further studies targeting lncRNAs to control inflammation in COPD. PMID:29725270

[Study on the inhibition effect of siRNA on herpes simplex virus type 2 ICP4 gene].

PubMed

Liu, Ji-feng; Guan, Cui-ping; Tang, Xu; Xu, Ai-e

2010-06-01

To explore the inhibition effect of RNA interference on the ICP4 expression and DNA replication of herpes simplex virus type 2 (HSV2). Four pairs of siRNA targeted to HSV2 ICP4 gene and negative control siRNA were synthetized by chemical method, named as siRNA-1, siRNA-2, siRNA-3, siRNA-4 and siRNA-N respecticely. HSV2 HG52 was used to attack Vero cell after transfection overnight. Vero cell and supernatant were collected at 1d, 2d, 3d, 4d and 5d after virus attacking. Flurogenic quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR)was used to detect the expression of HSV2 ICP4 mRNA, flurogenic quantitative polymerase chain reaction(FG-PCR) was used to detect the expression of HSV2 DNA and Western-Blot was used to detect the expression of HSV2 ICP4 protein. All the four pairs of siRNA could significantly inhibit the expression of HSV2 ICP4 mRNA and protein, especially siRNA-2. The above siRNAs could significantly decrease HSV2 DNA copy number,too. siRNAs targeted to HSV2 ICP4 gene could significantly inhibit expression of HSV2 ICP4 mRNA and protein, and decrease HSV2 DNA copy number, suggesting that siRNA can inhibit HSV2 DNA replication through silencing ICP4 gene.

Comparison of the in vitro effects of TCDD, PCB 126 and PCB 153 on thyroid-restricted gene expression and thyroid hormone secretion by the chicken thyroid gland.

PubMed

Katarzyńska, Dorota; Hrabia, Anna; Kowalik, Kinga; Sechman, Andrzej

2015-03-01

The aim of this study was to compare the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB 126; a coplanar PCB congener) and 2,2'4,4',5,5'-hexachlorobiphenyl (PCB153; non-coplanar PCB) on mRNA expression of thyroid-restricted genes, i.e. sodium iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), and thyroid hormone secretion from the thyroid gland of the laying chicken. Relative expression levels of NIS, TG and TPO genes and thyroxine (T4) and triiodothyronine (T3) secretion from the thyroidal explants were quantified by the real-time qPCR and RIA methods, respectively. In comparison with the control group, TCDD and PCB 126 significantly increased mRNA expression of TPO and TG genes. TCDD did not affect NIS mRNA levels, but PCB 126 decreased its expression. No effect of PCB 153 on the expression of these genes was observed. TCDD and PCB 126 significantly decreased T4 and T3 secretion. There was no significant effect of PCB 153 on these hormone secretions. In conclusion, the results obtained show that in comparison with non-coplanar PCB 153, TCDD and coplanar PCB 126 can directly affect thyroid hormone synthesis and secretion, and in consequence, they may disrupt the endocrine function of the thyroid gland of the laying chicken. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of electroacupuncture on corticotropin-releasing hormone in rats with chronic visceral hypersensitivity.

PubMed

Liu, Hui-Rong; Fang, Xiao-Yi; Wu, Huan-Gan; Wu, Lu-Yi; Li, Jing; Weng, Zhi-Jun; Guo, Xin-Xin; Li, Yu-Guang

2015-06-21

To investigate the effect of electroacupuncture on corticotropin-releasing hormone (CRH) in the colon, spinal cord, and hypothalamus of rats with chronic visceral hypersensitivity. A rat model of chronic visceral hypersensitivity was generated according to the internationally accepted method of colorectal balloon dilatation. In the 7(th) week after the procedure, rats were randomly divided into a model group (MG), electroacupuncture group (EA), and sham electroacupuncture group (S-EA). After treatment, the abdominal withdrawal reflex (AWR) score was used to assess the behavioral response of visceral hyperalgesia. Immunohistochemistry (EnVision method), ELISA, and fluorescence quantitative PCR methods were applied to detect the expression of CRH protein and mRNA in the colon, spinal cord, and hypothalamus. The sensitivity of the rats to the colorectal distension stimulus applied at different strengths (20-80 mmHg) increased with increasing stimulus strength, resulting in increasing AWR scores in each group. Compared with NG, the AWR score of MG was significantly increased (P < 0.01). After conducting EA, the AWR scores of the rats were decreased compared with MG rats. The relative expression of CRH mRNA in the colon, spinal cord, and hypothalamus of MG rats was significantly increased compared with NG rats (P < 0.01). CRH mRNA in the colon and spinal cord of EA and S-EA rats was decreased to varying degrees (P > 0.05) compared with normal rats (NG). However, the decrease in EA compared with MG rats was statistically significant (P < 0.01). The average optical density of CRH expression in the colon of the MG rats was significantly enhanced compared with NG (P < 0.05), while the average optical density of CRH expression in the EA and S-EA rats was significantly decreased compared with MG rats (P < 0.01, P < 0.05, respectively). Compared with MG rats, the CRH concentration in the spinal cord of EA rats was significantly reduced (P < 0.01), but there was no significant change in S-EA rats (P > 0.05). Electroacupuncture at the Shangjuxu acupoint was able to significantly reduce the visceral hypersensitivity in rats, and regulated the expression of CRH protein and mRNA in the colon, spinal cord and hypothalamus at different levels, playing a therapeutic role in this model of irritable bowel syndrome.

Preparation of a nano emodin transfersome and study on its anti-obesity mechanism in adipose tissue of diet-induced obese rats

PubMed Central

2014-01-01

Objective To describe the preparation of nano emodin transfersome (NET) and investigate its effect on mRNA expression of adipose triglyceride lipase (ATGL) and G0/G1 switch gene 2 (G0S2) in adipose tissue of diet-induced obese rats. Methods NET was prepared by film-ultrasonic dispersion method. The effects of emodin components at different ratios on encapsulation efficiency were investigated.The NET envelopment rate was determined by ultraviolet spectrophotometry. The particle size and Zeta potential of NET were evaluated by Zetasizer analyzer. Sixty male SD rats were assigned to groups randomly. After 8-week treatment, body weight, wet weight of visceral fat and the percentage of body fat (PBF) were measured. Fasting blood glucose and serum lipid levels were determined. The adipose tissue section was HE stained, and the cellular diameter and quantity of adipocytes were evaluated by light microscopy. The mRNA expression of ATGL and G0S2 from the peri-renal fat tissue was assayed by RT-PCR. Results The appropriate formulation was deoxycholic acid sodium salt vs. phospholipids 1:8, cholesterol vs. phospholipids 1:3, vitamin Evs. phospholipids 1:20, and emodin vs. phospholipid 1:6. Zeta potential was −15.11 mV, and the particle size was 292.2 nm. The mean encapsulation efficiency was (69.35 ± 0.25)%. Compared with the obese model group, body weight, wet weight of visceral fat, PBF and mRNA expression of G0S2 from peri-renal fat tissue were decreased significantly after NET treatment (all P < 0.05), while high-density lipoprotein cholesterol (HDL-C), the diameter of adipocytes and mRNA expression of ATGL from peri-renal fat tissue were increased significantly (all P < 0.05). Conclusion The preparation method is simple and reasonable. NET with negative electricity was small and uniform in particle size, with high encapsulation efficiency and stability. NET could reduce body weight and adipocyte size, and this effect was associated with the up-regulation of ATGL, down-regulation of G0S2 expression in the adipose tissue, and improved insulin sensitivity. PMID:24641917

Expression of beta 3-adrenoceptor mRNA in rat tissues.

PubMed

Evans, B A; Papaioannou, M; Bonazzi, V R; Summers, R J

1996-01-01

1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical beta-adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by beta 3-AR.

VR-10 Thrombospondin-1 Synthetic Polypeptide's Impact on Rhesus Choroid-Retinal Endothelial Cells.

PubMed

Tian, Run; Han, Fang; Yang, Jun; Zhao, Hai-Yan; Mei, Yan; Deng, Ai-Ping; Fang, Lin; Zhang, Xi-Rui

2018-01-01

This study aimed to investigate the effects of the VR-10 TSP-1 synthetic polypeptide on cytokines and the proliferation and migration of endothelial cells, as well as exploring a new method for anti-ocular neoangiogenesis. We measured the proliferation of RF/6A cells by an MTT assay and investigated the migration of RF/6A cells by a Transwell chamber assay. We examined the mRNA transcript levels of TGF-β2, VEGF, PEDF, Bcl-2 and FasL in RF/6A cells by RT-PCR and evaluated the expression of Fas and caspase-3 proteins in RF/6A cells by western blot analysis. 1. TSP-1 (1 µg/ml) and synthetic peptide VR-10 (0.1 µg/ml, 1 µg/ml and 10 µg/ml) inhibited the proliferation of RF/6A cells in a time and dose-dependent way. 2. TSP-1 and synthetic peptide VR-10 could inhibit the migration of RF/6A cells in a Transwell chamber (P < 0.001). It was demonstrated that 10 µg/ml synthetic peptide VR-10 had the strongest effect. 3. The expression of TGF-β2 mRNA in RF/6A cells increased after treatment with 1 µg/ml TSP-1 (P < 0.0001). However, there was no significant difference between the synthetic peptide VR-10 and the control group (P > 0.05). Expression of PEDF mRNA in RF/6A cells was increased after treatment with 1 µg/ml TSP-1 and synthetic peptide VR-10. We demonstrated that 10 µg/ml synthetic peptide VR-10 had the strongest effect (P < 0.001). There were significant differences between groups (P < 0.001). Expression of TGF-β2 mRNA in RF/6A cells increased after treatment with 1 µg/ml TSP-1 (P = 0.000). There was no significant difference between the synthetic peptide VR-10 and the control group (P > 0.05). PEDF mRNA expression in RF/6A cells decreased after 1 µg/ml TSP-1 and synthetic peptide VR-10 therapy, among which 10 µg/ml synthetic peptide VR-10 demonstrated the strongest effect (P < 0.001). There were significant differences between groups (P < 0.001), except for the 1 µg/ml synthetic peptide VR-10 and 1 µg/ml synthetic peptide VR-10 groups (P = 0.615). 4. Compared with the control group, FasL mRNA expression was significantly increased in the 10 µg/ml synthetic peptide VR-10 treatment group; however, Bcl-2 mRNA expression was decreased. 5. Western blotting showed that RF/6A cells in the control group mainly expressed the 32 kD procaspase-3 forms. For the 10 µg/ml synthetic peptide, VR-10 treatment group, it showed decreased expression of procaspase-3 (32 kD) and concomitant increased expression of its shorter pro apoptotic forms (20 kD). Compared with the control group, Fas protein expression significantly increased in the 10 µg/ml synthetic peptide VR-10 treatment group. Synthetic peptide VR-10 had an inhibitory action on the proliferation and migration of RF/6A cells. VR-10 inhibited angiogenesis by its combined actions, which included up-regulating the expression of an anti-angiogenesis gene, namely, pigment epithelium-derived factor (PEDF), down-regulating the expression of the pro-angiogenic vascular endothelial growth factor (VEGF), and mediated endothelial cell apoptosis. © 2018 The Author(s). Published by S. Karger AG, Basel.

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.

INDUCTION OF GENE EXPRESSION IN SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) TREATED WITH 17B-ESTRADIOL, DIETHYLSTILBESTROL, OR ETHINYLESTRADIOL: THE USE OF MRNA FINGERPRINTS AS AN INDICATOR OF GENE REGULATION

EPA Science Inventory

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variega...

Increase of CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells.

PubMed

Kunzmann, Steffen; Krempl, Christine; Seidenspinner, Silvia; Glaser, Kirsten; Speer, Christian P; Fehrholz, Markus

2018-04-16

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase of CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

PubMed

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

1992-01-01

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

Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement

PubMed Central

Qin, Bolin; Arvy, Nathalie; Poulet, Laurent; Batandier, Cécile; Roussel, Anne-Marie; Anderson, Richard A.

2018-01-01

In occidental societies, high fat and high sugar diets often coincide with episodes of stress. The association is likely to modify brain energy control. Brain insulin signalling is rarely studied in stressed individuals consuming high fat diets. Furthermore the effects of cinnamon supplement are not known in these conditions. Therefore, we exposed rats, over a 12-week period, to a control (C) or a high fat/high fructose (HF/HFr) diet that induces peripheral insulin resistance. A cinnamon supplement (C+CN and HF/HFr +CN) was added or not. After diet exposure, one group of rats was exposed to a 30-min restraint followed by a 10-min open-field test, their combination featuring a moderate stressor, the other rats staying unstressed in their home cages. The insulin signalling in hippocampus and frontal cortex was studied through the mRNA expression of the following genes: insulin receptor (Ir), insulin receptor substrate (Irs1), glucose transporters (Glut1 and Glut3), glycogen synthase (Gys1) and their modulators, Akt1 and Pten. In C rats, stress enhanced the expression of Ir, Irs1, Glut1, Gys1 and Akt1 mRNA. In C+CN rats, stress induced an increase in Pten but a decrease in Gys1 mRNA expression. In HF/HFr rats, stress was associated with an increase in Pten mRNA expression. In HF/HFr+CN rats, stress increased Pten mRNA expression but also decreased Gys1 mRNA expression. This suggests that a single moderate stress favours energy refilling mechanisms, an effect blunted by a previous HF/HFr diet and cinnamon supplement. PMID:29813096

Hypothalamic neuropeptides, not leptin sensitivity, contributes to the hyperphagia in lactating Brandt's voles, Lasiopodomys brandtii.

PubMed

Cui, Jian-Guo; Tang, Gang-Bing; Wang, De-Hua

2011-07-01

Both pregnancy and lactation are associated with hyperphagia, and circulating leptin levels are elevated during pregnancy but decreased during lactation in Brandt's voles, Lasiopodomys brandtii. Previous findings suggest that impaired leptin sensitivity contributes to hyperphagia during pregnancy. The present study aimed to examine whether the decreased circulating leptin level and/or hypothalamic leptin sensitivity contributed to the hyperphagia during lactation in Brandt's voles. The serum leptin level and mRNA expression of the long form of the leptin receptor (Ob-Rb), suppressor-of-cytokine-signalling-3 (SOCS-3), neuropeptide Y (NPY), agouti-related protein (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus were examined on dioestrous, day 5, day 17 of lactation and day 27 (1 week after weaning) in Brandt's voles. Compared with controls, hypothalamic Ob-Rb and SOCS-3 mRNA expression was not significantly changed during lactation. The serum leptin level was significantly lower in lactating females than in the non-reproductive group. Hypothalamic NPY and AgRP mRNA expression significantly increased whereas POMC mRNA expression was significantly decreased during lactation compared with controls. However, there were no significant changes in hypothalamic CART mRNA expression. Food intake was positively correlated with NPY and AgRP mRNA expression but negatively correlated with POMC mRNA expression during lactation. These data suggest that hyperphagia during lactation was associated with low leptin levels, but not impaired leptin sensitivity, and that the hypothalamic neuropeptides NPY, AgRP and POMC are involved in mediating the role of leptin in food intake regulation in lactating Brandt's voles.

Molecular effects of leptin on peroxisome proliferator activated receptor gamma (PPAR-γ) mRNA expression in rat's adipose and liver tissue.

PubMed

Abbasi, A; Moghadam, A A; Kahrarian, Z; Abbsavaran, R; Yari, K; Alizadeh, E

2017-08-15

Leptin is a 16-kDa peptide hormone secreted by adipose tissue that participates in the regulation of energy homeostasis. The aim of this study was to determine the effect of leptin injection on mRNA expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) and comparison of PPAR-γ mRNA expression in rat's adipose and liver tissue. Twenty adult male rats were divided into the following groups: Group 1asa control (n=10) that did not receive any treatment. Group 2as a treatment (n=10) that received leptin (30 µg ⁄ kg BW) intraperitoneally (ip) for two successive days. Blood samples were taken before and one day after second leptin injection for triglyceride (TG), Free Fatty Acid (FFA), HLD-cholesterol, and LDL-cholesterol measurement. Total RNA was extractedfrom the adipose tissue and liver tissues of rats.  Adipose and liver tissue cells' cDNA was synthesized to characterize the expression of PPAR-γ. Gene expression of PPAR-γ mRNA was tested by RT- PCR technique. Results show leptin decreases expression of PPAR-γ on rat. Low levels of PPAR-γ mRNA were detected in adipose and liver tissues of treatment rats in comparison to control group. In treatment group, the level of PPAR-γ mRNA in liver tissue was very lower than the adipose tissue. The levels of HDL and FFA in treatment rats were increased whereas serum levels TG, VLDL and LDL were not changed. It is concluded that leptin signal with suppressing of PPAR-γ mRNA expression in rat's adipose and liver tissues can result in lipolysis instead of lipogenesis.

Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement.

PubMed

Canini, Frédéric; Qin, Bolin; Arvy, Nathalie; Poulet, Laurent; Batandier, Cécile; Roussel, Anne-Marie; Anderson, Richard A

2018-01-01

In occidental societies, high fat and high sugar diets often coincide with episodes of stress. The association is likely to modify brain energy control. Brain insulin signalling is rarely studied in stressed individuals consuming high fat diets. Furthermore the effects of cinnamon supplement are not known in these conditions. Therefore, we exposed rats, over a 12-week period, to a control (C) or a high fat/high fructose (HF/HFr) diet that induces peripheral insulin resistance. A cinnamon supplement (C+CN and HF/HFr +CN) was added or not. After diet exposure, one group of rats was exposed to a 30-min restraint followed by a 10-min open-field test, their combination featuring a moderate stressor, the other rats staying unstressed in their home cages. The insulin signalling in hippocampus and frontal cortex was studied through the mRNA expression of the following genes: insulin receptor (Ir), insulin receptor substrate (Irs1), glucose transporters (Glut1 and Glut3), glycogen synthase (Gys1) and their modulators, Akt1 and Pten. In C rats, stress enhanced the expression of Ir, Irs1, Glut1, Gys1 and Akt1 mRNA. In C+CN rats, stress induced an increase in Pten but a decrease in Gys1 mRNA expression. In HF/HFr rats, stress was associated with an increase in Pten mRNA expression. In HF/HFr+CN rats, stress increased Pten mRNA expression but also decreased Gys1 mRNA expression. This suggests that a single moderate stress favours energy refilling mechanisms, an effect blunted by a previous HF/HFr diet and cinnamon supplement.

Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

PubMed Central

Guyon, T; Levasseur, P; Truffault, F; Cottin, C; Gaud, C; Berrih-Aknin, S

1994-01-01

Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss. Images PMID:8040257

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

PubMed

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

2018-04-24

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

Colonic mucosal gene expression and genotype in irritable bowel syndrome patients with normal or elevated fecal bile acid excretion

PubMed Central

Carlson, Paula; Acosta, Andres; Busciglio, Irene

2015-01-01

The mucosal gene expression in rectosigmoid mucosa (RSM) in irritable bowel syndrome with diarrhea (IBS-D) is unknown. Our objectives were, first, to study mRNA expression [by RT2 PCR of 19 genes pertaining to tight junctions, immune activation, intestinal ion transport and bile acid (BA) homeostasis] in RSM in IBS-D patients (n = 47) and healthy controls (n = 17) and study expression of a selected protein (PDZD3) in 10 IBS-D patients and 4 healthy controls; second, to assess RSM mRNA expression according to genotype and fecal BA excretion (high ≥2,337 μmol/48 h); and third, to determine whether genotype or mucosal mRNA expression is associated with colonic transit or BA parameters. Fold changes were corrected for false detection rate for 19 genes studied (P < 0.00263). In RSM in IBS-D patients compared with controls, mRNA expression of GUC2AB, PDZD3, and PR2Y4 was increased, whereas CLDN1 and FN1 were decreased. One immune-related gene was upregulated (C4BP4) and one downregulated (CCL20). There was increased expression of a selected ion transport protein (PDZD3) on immunohistochemistry and Western blot in IBS-D compared with controls (P = 0.02). There were no significant differences in mucosal mRNA in 20 IBS-D patients with high compared with 27 IBS-D patients with normal BA excretion. GPBAR1 (P < 0.05) was associated with colonic transit. We concluded that mucosal ion transport mRNA (for several genes and PDZD3 protein) is upregulated and barrier protein mRNA downregulated in IBS-D compared with healthy controls, independent of genotype. There are no differences in gene expression in IBS-D with high compared with normal fecal BA excretion. PMID:25930081

Developmental expression of the neuroligins and neurexins in fragile X mice.

PubMed

Lai, Jonathan K Y; Doering, Laurie C; Foster, Jane A

2016-03-01

Neuroligins and neurexins are transsynaptic proteins involved in the maturation of glutamatergic and GABAergic synapses. Research has identified synaptic proteins and function as primary contributors to the development of fragile X syndrome. Fragile X mental retardation protein (FMRP), the protein that is lacking in fragile X syndrome, binds neuroligin-1 and -3 mRNA. Using in situ hybridization, we examined temporal and spatial expression patterns of neuroligin (NLGN) and neurexin (NRXN) mRNAs in the somatosensory (S1) cortex and hippocampus in wild-type (WT) and fragile X knockout (FMR1-KO) mice during the first 5 weeks of postnatal life. Genotype-based differences in expression included increased NLGN1 mRNA in CA1 and S1 cortex, decreased NLGN2 mRNA in CA1 and dentate gyrus (DG) regions of the hippocampus, and increased NRXN3 mRNA in CA1, DG, and S1 cortex between female WT and FMR1-KO mice. In male mice, decreased expression of NRXN3 mRNA was observed in CA1 and DG regions of FMR1-KO mice. Sex differences in hippocampal expression of NLGN2, NRXN1, NRXN2, and NRXN3 mRNAs and in S1 cortex expression of NRXN3 mRNAs were observed WT mice, whereas sex differences in NLGN3, NRXN1, NRXN2, and NRXN3 mRNA expression in the hippocampus and in NLGN1, NRXN2 and NRXN3 mRNA expression in S1 cortex were detected in FMR1-KO mice. These results provide a neuroanatomical map of NLGN and NRXN expression patterns over postnatal development in WT and FMR1-KO mice. The differences in developmental trajectory of these synaptic proteins could contribute to long-term differences in CNS wiring and synaptic function. © 2015 Wiley Periodicals, Inc.

Colonic mucosal gene expression and genotype in irritable bowel syndrome patients with normal or elevated fecal bile acid excretion.

PubMed

Camilleri, Michael; Carlson, Paula; Acosta, Andres; Busciglio, Irene

2015-07-01

The mucosal gene expression in rectosigmoid mucosa (RSM) in irritable bowel syndrome with diarrhea (IBS-D) is unknown. Our objectives were, first, to study mRNA expression [by RT(2) PCR of 19 genes pertaining to tight junctions, immune activation, intestinal ion transport and bile acid (BA) homeostasis] in RSM in IBS-D patients (n = 47) and healthy controls (n = 17) and study expression of a selected protein (PDZD3) in 10 IBS-D patients and 4 healthy controls; second, to assess RSM mRNA expression according to genotype and fecal BA excretion (high ≥ 2,337 μmol/48 h); and third, to determine whether genotype or mucosal mRNA expression is associated with colonic transit or BA parameters. Fold changes were corrected for false detection rate for 19 genes studied (P < 0.00263). In RSM in IBS-D patients compared with controls, mRNA expression of GUC2AB, PDZD3, and PR2Y4 was increased, whereas CLDN1 and FN1 were decreased. One immune-related gene was upregulated (C4BP4) and one downregulated (CCL20). There was increased expression of a selected ion transport protein (PDZD3) on immunohistochemistry and Western blot in IBS-D compared with controls (P = 0.02). There were no significant differences in mucosal mRNA in 20 IBS-D patients with high compared with 27 IBS-D patients with normal BA excretion. GPBAR1 (P < 0.05) was associated with colonic transit. We concluded that mucosal ion transport mRNA (for several genes and PDZD3 protein) is upregulated and barrier protein mRNA downregulated in IBS-D compared with healthy controls, independent of genotype. There are no differences in gene expression in IBS-D with high compared with normal fecal BA excretion. Copyright © 2015 the American Physiological Society.

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.

Effects of interleukin-8 on estradiol and progesterone production by bovine granulosa cells from large follicles and progesterone production by luteinizing granulosa cells in culture.

PubMed

Shimizu, Takashi; Kaji, Ayami; Murayama, Chiaki; Magata, Fumie; Shirasuna, Koumei; Wakamiya, Kaori; Okuda, Kiyoshi; Miyamoto, Akio

2012-01-01

Interleukin 8 (IL-8) is a chemoattractant involved in the recruitment and activation of neutrophils and is associated with the ovulate process. We examined the possible role of IL-8 in steroid production by bovine granulosa cells before and after ovulation. The concentration of IL-8 in the follicular fluid of estrogen-active dominant (EAD) and pre-ovulatory follicles (POF) was higher than that of small follicles (SF). CXCR1 mRNA expression was higher in the granulosa cells of EAD and POF than that of SF. In contrast, CXCR2 mRNA expression was lower in granulosa cells of EAD and POF than in SF. IL-8 inhibited estradiol (E2) production in follicle-stimulating hormone (FSH)-treated granulosa cells at 48 h of culture. IL-8 also suppressed CYP19A1 mRNA expression in FSH-treated granulosa cells. IL-8 stimulated progesterone (P4) production in luteinizing hormone (LH)-treated granulosa cells at 48 h of culture. Although IL-8 did not alter the expression of genes associated with P4 production, it induced StAR protein expression in LH-treated granulosa cells. The expression of CXCR1 mRNA in corpus luteum (CL) did not change during the luteal phase. In contrast, the expression of CXCR2 mRNA in middle CL was significantly higher than in early and regression CL during the luteal phase. In luteinizing granulosa cells, an in vitro model of granulosa cell luteinization, CXCR2 mRNA expression was downregulated, whereas CXCR1 mRNA expression was unchanged. IL-8 also stimulated P4 production in luteinizing granulosa cells. These data provide evidence that IL-8 functions not only as a chemokine, but also act as a regulator of steroid synthesis in granulosa cells to promote luteinization after ovulation. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2009-01-01

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

[Effect of Panax notoginseng saponins on liver drug metablic enzyme activity, mRNA and protein expressions in rats].

PubMed

Chen, Yan-Jin; Wang, Yu-Guang; Ma, Zeng-Chun; Xiao, Cheng-Rong; Tan, Hong-Ling; Liang, Qian-De; Tang, Xiang-Lin; Zhao, Yong-Hong; Wang, Dong-Gen; Gao, Yue

2014-10-01

To study the effect of Panax notoginseng saponins (PNS) on liver drug metabolic enzyme activity, mRNA and protein expressions in rats. Male Wistar rats were randomly divided into nine groups. After administration of the test drugs, their liver microsomes, liver total RNA and total protein were extracted to detect the regulating effect of PNS on liver drug metabolic enzyme activity-related subtype enzymatic activity, mRNA and protein expression by substrate probe, quantitative PCR and Western Blot technology. The result of this experiment was that PNS could significantly induce CYP1A2 and CYP2E1 enzyme activity, mRNA expression, CYP2E1 protein expression level. PNS significantly induced CYP3A mRNA expression, but with no significant effect in CYP3A enzyme activity level. PNS had no significant effect CYP1A1 and CYP2B mRNA expressions and enzyme activity levels. PNS had selective regulations on different P450 subtypes, and the major subtypes were CYP1A2 and CYP2E1. In clinical practice, particularly in the combination with CYP1A2 and CYP2E1 metabolism-related drugs, full consideration shall be given to the possible drug interactions in order to avoid potential toxic and side effects. Meanwhile, whether the induction effect of CYP2E1 gets involved in ginsenoside's effect incavenging free radicals deserves further studies.

Increased expression of glutamic acid decarboxylase mRNA in rat substantia nigra after an ibotenic acid lesion in the caudate-putamen.

PubMed

Lindefors, N; Brené, S; Persson, H

1990-04-01

In situ hybridization histochemistry and RNA blots were used to study expression of glutamic acid decarboxylase (GAD) mRNA in rat caudate-nucleus and substantia nigra. In situ hybridization combined with computerized image analysis revealed that in the intact substantia nigra reticulata the cross-section area of GAD mRNA positive neurons were 25% larger in the dorsolateral part as compared with the ventromedial part. A unilateral ibotenic acid injection in caudate-putamen lesioned neurons, some of which project to the ipsilateral substantia nigra. An increased level of GAD mRNA was observed in substantia nigra ipsilateral to the lesion. Computerized image analysis of sections from in situ hybridization revealed an increase in the number of silver grains over GAD mRNA positive neurons in the dorsolateral substantia nigra reticulata ipsilateral to the lesion. However, no change was observed in the ventromedial part suggesting that GAD mRNA expression in this part of the nigra is less sensitive to inhibition by caudate-putamen afferents. In agreement with in situ experiments, RNA blots showed a 2-fold increased level of GAD mRNA in substantia nigra ipsilateral to the lesion. The increased GAD mRNA expression in the deafferented substantia nigra suggests a disinhibition of nigral GABA neurons, resulting in an increased utilization of GABA in these substantia nigra neurons.

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

PubMed Central

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

2001-01-01

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

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

PubMed

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

2007-06-01

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

Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation.

PubMed

Rubenstein, R C; Lyons, B M

2001-07-01

Intracellular trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is repaired by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA downregulates protein and mRNA expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family) by approximately 40-50% and decreases formation of a HSC70-DeltaF508 CFTR complex that may be important in the intracellular degradation of DeltaF508 CFTR. We examined the potential mechanisms by which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells, 1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70 promoter or of a human HSC70 promoter fragment in luciferase reporter assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays. In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA degradation by approximately 40%. The initial rate of 35S-HSC70 protein synthesis in 4PBA-treated IB3-1 cells was reduced by approximately 40%, consistent with the steady-state mRNA level, whereas its rate of degradation was unaltered by 4PBA. 4PBA also reduced the steady-state accumulation of (35)S-HSC70 by approximately 40%. These data suggest that 4PBA decreases the expression of HSC70 mRNA and protein by inducing cellular adaptations that result in the decreased stability of HSC70 mRNA.

Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia.

PubMed

Hisaoka-Nakashima, Kazue; Kajitani, Naoto; Kaneko, Masahiro; Shigetou, Takahiro; Kasai, Miho; Matsumoto, Chie; Yokoe, Toshiki; Azuma, Honami; Takebayashi, Minoru; Morioka, Norimitsu; Nakata, Yoshihiro

2016-03-01

A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action. Copyright © 2016 Elsevier B.V. All rights reserved.

Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver.

PubMed

Odbayar, Tseye-Oidov; Kimura, Toshinori; Tsushida, Tojiro; Ide, Takashi

2009-05-01

The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague-Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.

Effects of Coriolus versicolor polysaccharide B on monocyte chemoattractant protein 1 gene expression in rat.

PubMed

Song, Lie-Chang; Chen, Hai-Sheng; Lou, Ning; Song, Chang; Zeng, Jun; Fu, Ting-Huan

2002-06-01

To investigate the effect of Coriolus versicolor polysaccharide B (CVPS-B), a new water-soluble component of polysaccharides from the fungus Coriolus versicolor (Fr) L on monocyte chemoattractant protein-1 (MCP-1) gene expression in rat splenocytes. Expression of MCP-1 mRNA in rat splenocytes was examined by reverse transcription-polymerase chain reaction (RT-PCR) with beta- actin as an internal standard. Sequencing of RT-PCR products was performed to confirm their specificity in MCP-1 gene composition. (1) Without pre-treatment of lipopolysaccharide (LPS), the relative MCP-1 mRNA expression ratios (MCP-1/beta-actin) for the saline control group and for CVPS-B groups in 3 different doses (10, 20, and 30 mg . kg-1 . d-1, ip, for 4 d) were 1.4 +/- 0.3, 1.6 +/- 0.4, 1.7 +/- 0.5, and 1.5 +/- 0.4, respectively (P > 0.05); (2) LPS (10 microg . kg-1, ip) enhanced the expression of MPC-1 mRNA by the ratio of 114 %; (3) pre-treatment with CVPS-B of 4 different doses (5, 10, 30, and 50 mg . kg-1 . d-1, ip, for 4 d) decreased the LPS induced expression of MPC-1 mRNA by the ratios of 51 %, 70 %, 84 %, and 99 %, respectively (n = 6). In a dose-related fashion, CVPS-B inhibited the expression of MCP-1 mRNA induced by LPS in the rat splenocytes, but did not significantly affect the expression of MPC-1 mRNA in the normal rat.

Evaluation of folate receptor 1 (FOLR1) mRNA expression, its specific promoter methylation and global DNA hypomethylation in type I and type II ovarian cancers.

PubMed

Notaro, Sara; Reimer, Daniel; Fiegl, Heidi; Schmid, Gabriel; Wiedemair, Annamarie; Rössler, Julia; Marth, Christian; Zeimet, Alain Gustave

2016-08-02

In this retrospective study we evaluated the respective correlations and clinical relevance of FOLR1 mRNA expression, FOLR1 promoter specific methylation and global DNA hypomethylation in type I and type II ovarian cancer. Two hundred fifty four ovarian cancers, 13 borderline tumours and 60 samples of healthy fallopian epithelium and normal ovarian epithelium were retrospectively analysed for FOLR1 expression with RT-PCR. FOLR1 DNA promoter methylation and global DNA hypomethylation (measured by means of LINE1 DNA hypomethylation) were evaluated with MethyLight technique. No correlation between FOLR1 mRNA expression and its specific promoter DNA methylation was found neither in type I nor in type II cancers, however, high FOLR1 mRNA expression was found to be correlated with global DNA hypomethylation in type II cancers (p = 0.033). Strong FOLR1 mRNA expression was revealed for Grades 2-3, FIGO stages III-IV, residual disease > 0, and serous histotype. High FOLR1 expression was found to predict increased platinum sensitivity in type I cancers (odds ratio = 3.288; 1.256-10.75; p = 0.020). One-year survival analysis showed in type I cancers an independent better outcome for strong expression of FOLR1 in FIGO stage III and IV. For the entire follow up period no significant independent outcome for FOLR1 expression was revealed. In type I cancers LINE 1 DNA hypomethylation was found to exhibit a worse PFS and OS which were confirmed to be independent in multivariate COX regression model for both PFS (p = 0.026) and OS (p = 0.012). No correlations were found between FOLR1 expression and its specific promoter methylation, however, high FOLR1 mRNA expression was associated with DNA hypomethylation in type II cancers. FOLR1 mRNA expression did not prove to predict clinical outcome in type II cancers, although strong FOLR1 expression generally denotes ovarian cancers with highly aggressive phenotype. In type I cancers, however, strong FOLR1 expression has been found to be a reliable indicator of improved platinum responsiveness reflecting a transient better one-year follow up outcome in highly FOLR1 expressing type I cancers. An independent prognostic role of global DNA hypomethylation was demonstrated in type I tumours.

[Effects of sika pilose antler type collagen on ROS1728 cell and its molecular mechanism].

PubMed

Wang, Yan-Shuang; Luo, Su; Zhang, Da-Fang; Qu, Xiao-Bo; Li, Feng

2016-09-01

In this paper, effect and molecular mechanism of sika pilose antler type I collagen(SPC-I) of ROS1728 cell were explored. For the SPC-I provides the theory basis for the treatment of osteoporosis. The adherent method was used to cultivate rat osteosarcoma osteogenesis sample cell line ROS1728. The effect of SPC-I on ROS1728 cells proliferation was tested by CCK-8 method. Runx2, osernix, ALP, Coll-I, OC osteogenesis related genes expression was tested by RT-PCR, and Runx2 protein expression was tested by Western-bolt. Results showed that 5 g•L ⁻¹ SPC-I could inhibit ROS1728 cell proliferation, and significantly promote the expression of ROS1728 cell specific transcription factor Runx2 and osterix mRNA, Runx2 protein and marker gene ALP, Coll-I, OC mRNA expression(P<0.01). 2.5 g•L ⁻¹ and 10 g•L ⁻¹ SPC-I could significantly inhibit the ROS1728 cell proliferation(P<0.01), and inhibit the expression of related genes. In conclusion, 5 g•L ⁻¹ SPC-I could inhibit ROS1728 cell proliferation, obviously enhance ROS1728 cell function, promote ROS1728 cell differentiation, maturation. Copyright© by the Chinese Pharmaceutical Association.

The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

PubMed Central

2011-01-01

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

Human leucocyte antigen (HLA-DR) gene expression is reduced in sepsis and correlates with impaired TNFα response: A diagnostic tool for immunosuppression?

PubMed Central

Winkler, Martin Sebastian; Rissiek, Anne; Priefler, Marion; Schwedhelm, Edzard; Robbe, Linda; Bauer, Antonia; Zahrte, Corinne; Zoellner, Christian; Kluge, Stefan; Nierhaus, Axel

2017-01-01

Background Sepsis is defined as a dysregulated immune response to infection. Impaired immune response in sepsis, often described as endotoxin tolerance, is characterized by unresponsiveness of monocytes on lipopolysaccharide (LPS) stimulation to release tumor necrosis factor α (TNFα). Furthermore, decreased monocyte surface protein expression of human leucocyte antigen DR (HLA-DR) is a marker for changes of the innate immune response during sepsis. Quantitative polymerase chain reaction (qPCR) and flow-cytometry (FACS) have been used to measure protein or gene expression of HLA-DR. We aimed to determine whether changes in mRNA expression of HLA-DR are associated with impaired TNFα response in human sepsis. Methods Surface protein together with mRNA expression of HLA-DR were measured by FACS and qPCR in a cohort of 9 sepsis patients and compared to 10 pre-operative control patients in a prospective study. In addition, 20 patients with post-surgical inflammation, 20 patients with sepsis or septic shock were included and TNFα was determined following ex vivo stimulation of whole blood with 500 pg/mL LPS. Total RNA was prepared from whole blood and subjected to qPCR analysis for expression analysis of HLA-DR alpha (HLA-DRA) to correlate TNFα response with HLA-DRA expression. Results Patients with sepsis presented higher numbers of monocytes in peripheral blood (P<0.001) but decreased surface protein and mRNA HLA-DR levels when compared to controls. In all patients mRNA expression of HLA-DRA was decreased by approximately 70% compared to controls (P<0.01) and was lowest in patients with sepsis or septic shock (P<0.01). TNFα response to LPS was decreased in all patients (median 319 pg/mL versus controls 1256 pg/mL; P<0.01) and lowest in patients with sepsis or septic shock (median 128 pg/mL; P<0.01). HLA-DRA correlated positively with TNFα response in all study participants (r +0.60, P<0.001) and within patients (r +0.67, P<0.001). The TNFα:HLA-DRA ratio correlated negatively with severity and the Sequential Organ Failure Assessment (SOFA) score (Spearman’s rho -0.59, P<0.001) Conclusion In this study, HLA-DRA expression was associated with a functional assay of the innate immune response. Future interventional studies aimed at the immune response during sepsis could make use of these methods for optimizing target groups based on biological plausibility and intervention effectiveness. PMID:28771573

MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms

PubMed Central

2011-01-01

Background Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge. Therefore, a reverse strategy, which identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the SNP-related mRNAs will be useful. Those mRNA transcripts that are expressed at two distinct abundances in proportion to SNP allele frequency may warrant further study. Matrix metalloproteinase 1 (MMP1) is important in both normal development and in numerous pathologies. Although much research has been conducted to investigate the expression of MMP1 in many different cell types and conditions, the regulation of its expression is still not fully understood. Results In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals. We found that MMP1 mRNA abundance was bimodally distributed in un-treated HUVECs and showed a bimodal response to inflammatory mediator treatment. RT-PCR and MMP1 activity assays confirmed the bimodal regulation and DNA sequencing of 69 individuals identified an MMP1 gene promoter polymorphism that segregated precisely with the MMP1 bimodal expression. Chromatin immunoprecipation (ChIP) experiments indicated that the transcription factors (TFs) ETS1, ETS2 and GATA3, bind to the MMP1 promoter in the region of this polymorphism and may contribute to the bimodal expression. Conclusions We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticans to understand and use. This method identified bimodal endothelial cell expression of MMP1, which appears to be biologically significant with implications for inflammatory disease. (271 Words) PMID:21244711

[Expression profiles of miRNA-182 and Clock mRNA in the pineal gland of neonatal rats with hypoxic-ischemic brain damage].

PubMed

Han, Xing; Ding, Xin; Xu, Li-Xiao; Liu, Ming-Hua; Feng, Xing

2016-03-01

To study the changes of miRNA expression in the pineal gland of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the possible roles of miRNA in the pathogenesis of circadian rhythm disturbance after HIBD. Seven-day-old Sprague-Dawley (SD) rats were randomly divided into 2 groups: HIBD and sham-operated. HIBD was induced according to the Rice-Vannucci method. The pineal glands were obtained 24 hours after the HIBD event. The expression profiles of miRNAs were determined using GeneChip technigue and quantitative real-time PCR (RT-PCR). Then the miRNA which was highly expressed was selected. The expression levels of the chosen miRNA were detected in different tissues (lungs, intestines, stomach, kidneys, cerebral cortex, pineal gland). RT-PCR analysis was performed to measure the expression profiles of the chosen miRNA and the targeted gene Clock mRNA in the pineal gland at 0, 24, 48 and 72 hours after HIBD. miRNA-182 that met the criteria was selected by GeneChip and RT-PCR. miRNA-182 was highly expressed in the pineal gland. Compared with the sham-operated group, the expression of miRNA-182 was significantly up-regulated in the pineal gland at 24 and 48 hours after HIBD (P<0.05). Compared with the sham-operated group, Clock mRNA expression in the HIBD group increased at 0 hour after HIBD, decreased at 48 hours after HIBD and increased at 72 hours after HIBD (P<0.05). miRNA-182 may be involved in the pathogenesis of circadian rhythm disturbance after HIBD.

Three-Dimensional mRNA Measurements Reveal Minimal Regional Heterogeneity in Esophageal Squamous Cell Carcinoma

PubMed Central

Yan, Wusheng; Shih, Joanna; Rodriguez-Canales, Jaime; Tangrea, Michael A.; Player, Audrey; Diao, Lixia; Hu, Nan; Goldstein, Alisa M.; Wang, Jing; Taylor, Philip R.; Lippman, Scott M.; Wistuba, Ignacio I.; Emmert-Buck, Michael R.; Erickson, Heidi S.

2014-01-01

The classic tumor clonal evolution theory postulates that cancers change over time to produce unique molecular subclones within a parent neoplasm, presumably including regional differences in gene expression. More recently, however, this notion has been challenged by studies showing that tumors maintain a relatively stable transcript profile. To examine these competing hypotheses, we microdissected discrete subregions containing approximately 3000 to 8000 cells (500 to 1500 μm in diameter) from ex vivo esophageal squamous cell carcinoma (ESCC) specimens and analyzed transcriptomes throughout three-dimensional tumor space. Overall mRNA profiles were highly similar in all 59 intratumor comparisons, in distinct contrast to the markedly different global expression patterns observed in other dissected cell populations. For example, normal esophageal basal cells contained 1918 and 624 differentially expressed genes at a greater than twofold level (95% confidence level of <5% false positives), compared with normal differentiated esophageal cells and ESCC, respectively. In contrast, intratumor regions had only zero to four gene changes at a greater than twofold level, with most tumor comparisons showing none. The present data indicate that, when analyzed using a standard array-based method at this level of histological resolution, ESCC contains little regional mRNA heterogeneity. PMID:23219752

[Effect of soy isoflavone on gene expression of leptin and insulin sensibility in insulin-resistant rats].

PubMed

Chen, Shi-wei; Zhang, Li-shi; Zhang, Hong-min; Feng, Xiao-fan; Peng, Xiao-li

2006-04-18

To explore the effects of soy isoflavone (SIF) on gene expression of leptin and insulin sensibility in insulin-resistant (IR) rats induced by high-fat, and to reveal the mechanisms of SIF in ameliorating insulin sensibility. IR rats were randomly divided into four groups based on their insulin-resistant indexes (IRI): one model control group and three SIF groups that were gavaged with water solutions with SIF at doses of 0 mg/kg, 50 mg/kg, 150 mg/kg, and 450 mg/kg, respectively. After one month, fasting glucose, fasting insulin, leptin in serum, and leptin mRNA in the perirenal adipocyte were detected by enzymic method, radioimmunoassay, enzyme linked immunosorbent assay, and real time quantitative RT-PCR, respectively. The model control group was used to compare against the other groups: (1) Insulin and IRI were lower in the 150 mg/kg and 450 mg/kg groups; (2) In the 450 mg/kg group, body weight and leptin mRNA expression were lower, serum leptin content was higher. These results indicate that soy isoflavone might decrease body weight of rats and leptin mRNA, increase serum leptin level, and ameliorate leptin and insulin sensitivities.

Differential expression of decorin and biglycan genes during mouse tooth development

NASA Technical Reports Server (NTRS)

Matsuura, T.; Duarte, W. R.; Cheng, H.; Uzawa, K.; Yamauchi, M.

2001-01-01

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.

Selective probing of mRNA expression levels within a living cell.

PubMed

Nawarathna, D; Turan, T; Wickramasinghe, H Kumar

2009-08-24

We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high ( approximately 2500) and extremely low (11 0) copy number mRNA from a living cell mRNA in less than 10 s.

Changes in proliferating and apoptotic markers of leiomyoma following treatment with a selective progesterone receptor modulator or gonadotropin-releasing hormone agonist.

PubMed

Yun, Bo Seong; Seong, Seok Ju; Cha, Dong Hyun; Kim, Ji Yeon; Kim, Mi-La; Shim, Jeong Yun; Park, Ji Eun

2015-08-01

To evaluate changes in proliferating and apoptotic markers of myoma tissue from patients treated with a selective progesterone receptor modulator (SPRM) or GnRH agonist by measuring expression of PDGF-A mRNA, IGF-1 mRNA, bcl-2 mRNA, and PCNA and caspase-3 protein. Between December 2013 and July 2014, women with symptomatic leiomyoma were divided into control (no treatment before surgery), SPRM (treatment with ulipristal acetate [SPRM] for 3 months before surgery), and GnRHa (treatment with leuprolide acetate [GnRH agonist] for 3 months before surgery) groups. Tissue specimens were collected from the myoma core and normal myometrium of all patients. The expression of mRNA and protein was assessed by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot. A total of 38 patients were enrolled (control group, n=14; SPRM group, n=13; GnRHa group, n=11). PDGF-A mRNA expression was lower in both the myoma core and normal myometrium tissues of the SPRM compared with the control group, but there was no difference between the control and GnRHa group. There were also no group differences in bcl-2 mRNA or IGF-1 mRNA expression. Both PCNA and caspase-3 protein expression were higher in the leiomyoma tissue of the SPRM compared with the control group, but there was no difference between the control and GnRHa groups in the expression of either protein. Both proliferation and apoptosis were increased in the leiomyoma of patients after SPRM treatment, but there was no change following GnRH agonist treatment, in vivo. However, PDGF-A mRNA was decreased after SPRM treatment, indicating a dual effect of progesterone on the regulation of growth factors. Furthermore, there was an increase in caspase-3 protein, but not bcl-2 mRNA, expression in the SPRM group suggesting that SPRM may exert its effects in pathways other than the bcl-2 apoptotic pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mushroom β-Glucan May Immunomodulate the Tumor-Associated Macrophages in the Lewis Lung Carcinoma

PubMed Central

Wang, Wan-Jhen; Wu, Yu-Sheng; Chen, Sherwin; Liu, Chi-Feng

2015-01-01

The present study showed that oral mushroom beta-glucan treatment significantly increased IFN-γ mRNA expression but significantly reduced COX-2 mRNA expression within the lung. For LLC tumor model, oral Ganoderma lucidum or Antrodia camphorata polysaccharides treatments significantly reduced TGF-β production in serum. In addition, IL-12 and IFN-γ mRNA expression were significantly increased, but IL-6, IL-10, COX-2, and TGF-β mRNA expression were substantially following oral mushroom polysaccharides treatments. The study highlights the efficacious effect of mushroom polysaccharides for ameliorating the immune suppression in the tumor microenvironment. Increased M1 phenotype of tumor-associated macrophages and attenuated M2 phenotype of tumor-associated macrophages could be achieved by ingesting mushroom polysaccharides. PMID:26167490

L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

Gene expression analysis in lymphoblasts derived from patients with autism spectrum disorder.

PubMed

Yasuda, Yuka; Hashimoto, Ryota; Yamamori, Hidenaga; Ohi, Kazutaka; Fukumoto, Motoyuki; Umeda-Yano, Satomi; Mohri, Ikuko; Ito, Akira; Taniike, Masako; Takeda, Masatoshi

2011-05-26

The autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that result in severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare mutations of some genes, such as neuroligin (NLGN) 3/4, neurexin (NRXN) 1, SHANK3, MeCP2 and NHE9, have been reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD. We measured mRNA expression levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction assays. The mRNA expression levels of NLGN3 and SHANK3 normalized by β-actin or TBP were significantly decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not quantitatively measurable in lymphoblastoid cells. Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the possibility that decreased mRNA expression levels of these genes might be involved in the pathophysiology of ASD in a substantial population of ASD patients.

Molecular analysis of nicotinic receptor expression in autism.

PubMed

Martin-Ruiz, C M; Lee, M; Perry, R H; Baumann, M; Court, J A; Perry, E K

2004-04-07

Autism is a developmental disorder of unknown aetiopathology and lacking any specific pharmacological therapeutic intervention. Neurotransmitters such as serotonin, gamma-aminobutyric acid (GABA) and acetylcholine have been implicated. Abnormalities in nicotinic acetylcholine receptors have been identified including cortical loss of binding to the alpha4/beta2 subtype and increase in cerebellar alpha7 binding. Receptor expression (mRNA) has not so far been systematically examined. This study aims to further explore the role of nicotinic receptors in autism by analysing nicotinic receptor subunit mRNA in conjunction with protein levels and receptor binding in different brain areas. Quantitative RT-PCR for alpha4, alpha7 and beta2 subunit mRNA expression levels; alpha3, alpha4, alpha7 and beta2 subunit protein expression immunochemistry and specific radioligand receptor binding were performed in adult autism and control brain samples from cerebral cortex and cerebellum. Alpha4 and beta2 protein expression and receptor binding density as well as alpha4 mRNA levels were lower in parietal cortex in autism, while alpha7 did not change for any of these parameters. In cerebellum, alpha4 mRNA expression was increased, whereas subunit protein and receptor levels were decreased. Alpha7 receptor binding in cerebellum was increased alongside non-significant elevations in mRNA and protein expression levels. No significant changes were found for beta2 in cerebellum. The data obtained, using complementary measures of receptor expression, indicate that reduced gene expression of the alpha4beta2 nicotinic receptor in the cerebral cortex is a major feature of the neurochemical pathology of autism, whilst post-transcriptional abnormalities of both this and the alpha7 subtype are apparent in the cerebellum. The findings point to dendritic and/or synaptic nicotinic receptor abnormalities that may relate to disruptions in cerebral circuitry development.

Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression.

PubMed

Yan, Aifen; Zhang, Lingjuang; Tang, Zhiguo; Zhang, Yanhong; Qin, Chaobin; Li, Bo; Li, Wensheng; Lin, Haoran

2011-07-01

Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro. Copyright © 2011 Elsevier Inc. All rights reserved.

Glutamatergic and Dopaminergic Neurons in the Mouse Ventral Tegmental Area

PubMed Central

Yamaguchi, Tsuyoshi; Qi, Jia; Wang, Hui-Ling; Zhang, Shiliang; Morales, Marisela

2014-01-01

The ventral tegmental area (VTA) comprises dopamine (DA), GABA and glutamate (Glu) neurons. Some rat VTA Glu neurons, expressing vesicular glutamate transporter 2 (VGluT2), co-express tyrosine hydroxylase (TH). While transgenic mice are now being used in attempts to determine the role of VGluT2/TH neurons in reward and neuronal signaling, such neurons have not been characterized in mouse tissue. By cellular detection of VGluT2-mRNA and TH-immunoreactivity (TH-IR), we determined the cellular expression of VGluT2-mRNA within VTA TH-IR neurons in the mouse. We found that some mouse VGluT2 neurons co-expressed TH-IR, but their frequency was lower than in the rat. To determine whether low expression of TH mRNA or TH-IR accounts for this low frequency, we evaluated VTA cellular co-expression of TH-transcripts and TH-protein. Within the medial aspects of the VTA, some neurons expressed TH mRNA but lacked TH-IR; among them a subset co-expressed VGluT2 mRNA. To determine if lack of VTA TH-IR was due to TH trafficking, we tagged VTA TH neurons by cre-inducible expression of mCherry in TH::Cre mice. By dual immunofluorescence, we detected axons containing mCherry, but lacking TH-IR, in the lateral habenula, indicating that mouse low frequency of VGluT2 mRNA (+)/TH-IR (+) neurons is due to lack of synthesis of TH protein, rather than TH-protein trafficking. In conclusion, VGluT2 neurons are present in the rat and mouse VTA, but they differ in the populations of VGluT2/TH and TH neurons. We reveal that under normal conditions, the translation of TH protein is suppressed in the mouse mesohabenular TH neurons. PMID:25572002

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma.

PubMed

Zhu, Yi; Zhang, Jing-jing; Zhu, Rong; Zhu, Yan; Liang, Wen-biao; Gao, Wen-tao; Yu, Jun-bo; Xu, Ze-kuan; Miao, Yi

2011-12-01

The MUC4 gene could have a key role in the progression of pancreatic cancer, but the quantitative measurement of its expression in clinical tissue samples remains a challenge. The correlations between MUC4 promoter methylation status in vivo and either pancreatic cancer progression or MUC4 mRNA expression need to be demonstrated. We used the techniques of quantitative real-time PCR and DNA methylation-specific PCR combined microdissection to precisely detect MUC4 expression and promoter methylation status in 116 microdissected foci from 57 patients with pancreatic ductal adenocarcinoma. Both mRNA expression and hypomethylation frequency increased from normal to precancerous lesions to pancreatic cancer. Multivariate Cox regression analysis showed that high-level MUC4 expression (P = 0.008) and tumor-node-metastasis staging (P = 0.038) were significant independent risk factors for predicting the prognosis of 57 patients. The MUC4 mRNA expression was not significantly correlated with promoter methylation status in 30 foci of pancreatic ductal adenocarcinoma. These results suggest that high mRNA expression and hypomethylation of the MUC4 gene could be involved in carcinogenesis and in the malignant development of pancreatic ductal adenocarcinoma. The MUC4 mRNA expression may become a new prognostic marker for pancreatic cancer. Microdissection-based quantitative real-time PCR and methylation-specific PCR contribute to the quantitative detection of MUC4 expression in clinical samples and reflect the epigenetic regulatory mechanisms of MUC4 in vivo.

[Influence of macrophages on the expression of vascular endothelial growth factor receptor mRNA, homeobox B2 mRNA, and integrin alpha nu beta3 in vascular endothelial strain].

PubMed

Liu, Liang; Liu, Chang; Zhang, Xiao-qi; Ming, Jia; Liu, Xu-sheng; Xu, Hui; Cheng, Tian-min

2005-06-01

To investigate the influence of macrophages on the expression of the vascular endothelial growth factor (VEGF) receptor (KDR) mRNA, homeobox B2 (HOXB2) mRNA, and integrin alpha nu beta3 in vitro in vascular endothelial strain. Human umbilical vein cells (ECV304) were cultured in vitro and divided into 4 groups, i.e. (1) ECV304 group, (2) ECV304 + conA group [with conA (25 microg/ml in culture) added to ECV304], (3) ECV304 + U937 group (with 1 x 10(5)/ml of U937 cells added to 1 x 10(5)/ml ECV 304), (4) ECV304 + U937 + conA group [with 1 x 10(5)/ml of U937 cells and conA (25 microg/ml in culture)] groups. Forty-eight hours after culturing, the expression of integrin receptor alpha nu beta3 and the changes in the expression of KDR mRNA and HOXB2 mRNA in each group were determined by immunofluorescent technique and RT-PCR, respectively. The expression of integrin receptor alpha nu beta3, KDR mRNA, and HOXB2 mRNA in ECV304 group were 6.7 +/- 1.5, 0.633 +/- 0.012, and 0.674 +/- 0.004, respectively, while those in ECV304 + U937 + conA group (10.2 +/- 1.7, 0.879 +/- 0.003, 0.947 +/- 0.003) were obviously more upregulated when compared with those in ECV304 group (P < 0.01). No difference in the above indices was found between ECV304 and ECV304 + conA, ECV304 + U937 groups (P > 0.05). Macrophages activated by ConA can accelerate the proliferation, migration and adhesion to the basement membrane matrix of vascular endothelial cells through the influence on the expression of KDR mRNA, HOXB2 mRNA and integrin alpha nu beta3, and through this pathway the angiogenesis is modulated.

Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence

PubMed Central

Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; D. van der Vaart, Andrew; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S.; Miles, Michael F.; Dick, Danielle; Riley, Brien P.; Dumur, Catherine; Vladimirov, Vladimir I.

2015-01-01

Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

Aberrant expression of erythropoietin in uterine leiomyoma: implications in tumor growth.

PubMed

Asano, Ryoko; Asai-Sato, Mikiko; Miyagi, Yohei; Mizushima, Taichi; Koyama-Sato, Makiko; Nagashima, Yoji; Taguri, Masataka; Sakakibara, Hideya; Hirahara, Fumiki; Miyagi, Etsuko

2015-08-01

Myomatous erythrocytosis syndrome is a rare complication of uterine leiomyoma caused by erythropoietin (EPO) that is produced by tumor cells. We assessed the EPO expression in leiomyomas and investigated the effects of EPO on the tumor growth. Tissue samples were collected from 114 patients with uterine leiomyomas who underwent myomectomy or hysterectomy in Yokohama City University Hospital. From 17 patients, the corresponding normal myometrium was also collected. All samples were analyzed for EPO messenger RNA (mRNA) expression by real-time reverse transcription-polymerase chain reaction. EPO protein expression was determined by an enzyme-linked immunosorbent assay. The relationships between EPO expression and clinicopathological features were retrospectively analyzed using the patients' charts. Blood vessel density and maturity were assessed using hematoxylin-eosin staining and CD34 immunohistochemistry. EPO mRNA expression was detected in 108 of 114, or 95%, of the leiomyomas. The mean EPO mRNA expression in the leiomyoma was higher than the corresponding normal myometrium (3836 ± 4122 vs 1455 ± 2141; P = .025 by Wilcoxon rank test). The EPO mRNA expression in the leiomyomas varied extensively among samples, ranging from undetectable levels to 18-fold above the mean EPO mRNA of normal myometrium. EPO protein production was observed concomitant with mRNA expression. A positive correlation of leiomyoma size and EPO mRNA expression was shown by Spearman rank correlation coefficient (ρ = 0.294; P = .001), suggesting the involvement of EPO in leiomyoma growth. The blood vessel maturity was also significantly increased in EPO-producing leiomyomas (high vessel maturity in high vs low EPO group: 67% vs 20%; P = .013 by Fisher exact test). This report demonstrates that EPO is produced in most of conventional leiomyomas and supports a model in which EPO accelerates tumor growth, possibly by inducing vessel maturity. Our study suggests one possible mechanism by which some uterine leiomyomas reach a large size, and the understanding of EPO expression patterns in these tumors may be useful for management of the patients with leiomyomas. Copyright © 2015 Elsevier Inc. All rights reserved.

[The mRNA expression of mitogen-activated protein kinase signal pathway related genes in the blood of arseniasis patients caused by burning coal].

PubMed

Luo, Peng; Zhang, Ai-hua; Xiao, Yun; Pan, Xue-li; Dong, Xue-xin; Huang, Xiao-xin

2013-09-01

To detect the mRNA expression of ERK1, ERK2, JNK1 and P38 gene in mitogen-activated protein kinase(MAPK) path way in the arseniasis patients caused by burning coal. 70 arseniasis patients caused by burning coal at Jiaole village XingRen county in December 2006 were selected as case group, and another 30 villagers with similar living habits, matched gender and age, healthy physical condition without history of burning high arsenic coal were selected as control group from 12 km nearby the same village.Silver diethyl dithiocarbamate method (Ag-DDC) was taken to detect the arsenic contents in the environmental media, food, and arsenic level in the urine and hair of arseniasis patients.On the principle of informed consent, the peripheral blood was collected from the patients. The total RNA was extracted with Trizol method and cDNA was reversed from it. The mRNA expression of ERK1, ERK2, JNK1 and P38 gene in MAPK path way were tested by real-time fluorescent quantitative PCR (QT-PCR). A total of 70 cases of arseniasis patients (31 cases of mild, 25 cases of moderate and 14 cases of severe) and 30 cases of control were chosen. The median (quartile) of arsenic contents in the indoor air, outdoor air, coal, chili and corn were 0.079 (0.053-0.117) mg/m(3) ,0.007 (0.002-0.015) mg/m(3) , 93.010 (39.460-211.740) mg/kg, 3.460(0.550-16.760) mg/kg and 1.500(0.300-4.140) mg/kg respectively. They were above the national health standards. The median (quartile) of arsenic contents in the soil, rice and drinking water were separately 12.130(4.230-24.820) mg/kg, 0.650(0.300-0.980) mg/kg and 0.043(0.012-0.089)mg/kg, which were within the national health standards. Compared with the control group ((26.97 ± 9.71)µg/g Cr), arsenic level in the patients' urine ((71.48 ± 22.74)µg/g Cr) increased significantly, the differences were significant (F = 90.38, P < 0.01). Compared with the control group ((1.58 ± 1.07)µg/g), arsenic level in the patients' hair ((4.45 ± 2.78) µg/g) increased significantly, the differences were significant (F = 48.22, P < 0.01). The relative expression amount of the median(quartile) for ERK2, JNK1 mRNA were 0.0667 (0.0378-0.1371) and 0.0013 (0.0009-0.0025), respectively. Compared with the control group 0.1744 (0.1009-0.1985) and 0.0022 (0.0017-0.0030) , only the decreases of ERK2, JNK1 mRNA expression was significant (χ(2) = 15.10, 14.25, P < 0.01), and no significance in the other index. ERK2 mRNA relative expression for mild, medium and severe groups were separately 0.0818 (0.0408-0.1509) ,0.0582 (0.0154-0.1699) and 0.0588 (0.0399-0.1034) . Compared with the control group (0.1744 (0.1099-0.1985) ), there was significant difference (Z = -2.89, -3.19, -2.67, P < 0.01). JNK1 mRNA relative expression were 0.0012 (0.0007-0.001 57), 0.0019 (0.0011-0.0035), 0.0013 (0.0010-0.0026), respectively. Compared with the control group (0.0022 (0.0017-0.0030) ), significances were found in the mild groups (Z = -3.72, P < 0.01). Arsenic could induce the changes of ERK2 and JNK1mRNA expression in the MAPK path way in arseniasis patients.It suggests that the MAPK signaling pathway take part in the occurrence and development process of arseniasis caused by burning coal.

Possible involvement of AMPK in acute exercise-induced expression of monocarboxylate transporters MCT1 and MCT4 mRNA in fast-twitch skeletal muscle.

PubMed

Takimoto, Masaki; Takeyama, Mirei; Hamada, Taku

2013-11-01

The regulatory mechanisms responsible for acute exercise-induced expression of monocarboxylate transporters MCT1 and MCT4 mRNA in skeletal muscle remain unclear. 5'-adenosine-activated protein kinase (AMPK) is a key signaling molecule that regulates gene expression at the mRNA level. We examined whether AMPK activation is involved in acute exercise-induced expression of MCT1 and MCT4 mRNA in fast-twitch muscle. Male Sprague-Dawley rats were subjected to an acute bout of either 5min high-intensity intermittent swimming (HIS) or 6-h low-intensity prolonged swimming (LIS). The effects of acute exercise on the phosphorylation of AMPK (p-AMPK), calcium/calmodulin pendent kinase II (p-CaMKII), p38 mitogen-activated protein kinase (p-p38MAPK), and MCTs mRNA were analyzed in vivo. To observe the direct effects of AMPK activation on MCTs mRNA, the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), caffeine, and dantrolene were analyzed in vitro using an isolated muscle incubation model. The p-AMPK increased in response to both HIS and LIS, although the p-CaMKII and p-p38MAPK were increased only following HIS. Irrespective of exercise intensity, MCT1 and MCT4 mRNA was also transiently upregulated by both HIS and LIS. Direct exposure of the epitrochlearis muscle to 0.5mmol/L AICAR or 1mmol/L caffeine, which activated p-AMPK increased both MCT1 and MCT4 mRNA levels. When pAMPK was inhibited by dantrolene, neither MCT1 nor MCT4 mRNA was increased. These results suggest that acute exercise-induced increases in MCT1 and MCT4 mRNA expression may be possibly mediated by AMPK activation, at least in part in fast-twitch muscle. © 2013.

[Effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction].

PubMed

Tan, Wenqing; Wang, Wei; Zheng, Xuan; Chen, Jiying; Yuan, Xiangning; Zhang, Fangfang; Wang, Shuting; Tao, Lijian

2018-05-28

To investigate the effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction (UUO) and to observe the effect of fluorofenidone on the expressions of collagen type I (Col I), collagen type III (Col III), α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), platelet derived growth factor (PDGF) in the renal tissues of UUO rats.
 Methods: Male Sprague-Dawley (SD) rats were randomly divided into a sham-operated group, a UUO group, and a flurofenidone group (n=5). UUO model was induced by ligating the left ureter in rats. The rats were treated with 125 mg/(kg.d) fluorofenidone by gastric gavage in the fluorofenidone group at 24 h before the operation, and the rats were treated with the identical dose of 0.5% sodium carboxyl methyl cellulose (CMC-Na) in the other 2 groups. The rats were sacrificed at 14 days after UUO. Pathological changes of the renal tissue were observed by HE and Masson staining, the mRNA expressions of Col I, Col III, α-SMA, PDGF and CTGF were detected by real-time PCR, and the protein expressions of Col I, Col III, PDGF and CTGF were detected by immunohistochemical staining.
 Results: The renal interstitial damage index, relative collagen area and mRNA and protein expressions of Col I and Col III in the renal tissues of the rats in the UUO group significantly increased (P<0.05), and fluorofenidone could reduce these indexes (P<0.05). Compared with the sham-operated group, the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF in the renal tissues of the rats in the UUO group were increased, but fluorofenidone could decrease the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF (P<0.05).
 Conclusion: Fluorofenidone (125 mg/kg.d) could attenuate renal interstitial fibrosis through inhibition of fibroblast proliferation, myofibroblastic activation, PDGF and CTGF expression.

Prostate cancer targeting motifs: expression of αν β3, neurotensin receptor 1, prostate specific membrane antigen, and prostate stem cell antigen in human prostate cancer cell lines and xenografts.

PubMed

Taylor, Robert M; Severns, Virginia; Brown, David C; Bisoffi, Marco; Sillerud, Laurel O

2012-04-01

Membrane receptors are frequent targets of cancer therapeutic and imaging agents. However, promising in vitro results often do not translate to in vivo clinical applications. To better understand this obstacle, we measured the expression differences in receptor signatures among several human prostate cancer cell lines and xenografts as a function of tumorigenicity. Messenger RNA and protein expression levels for integrin α(ν) β(3), neurotensin receptor 1 (NTSR1), prostate specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) were measured in LNCaP, C4-2, and PC-3 human prostate cancer cell lines and in murine xenografts using quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and immunohistochemistry. Stable expression patterns were observed for integrin α(ν) and PSMA in all cells and corresponding xenografts. Integrin β(3) mRNA expression was greatly reduced in C4-2 xenografts and greatly elevated in PC-3 xenografts compared with the corresponding cultured cells. NTSR1 mRNA expression was greatly elevated in LNCaP and PC-3 xenografts. PSCA mRNA expression was elevated in C4-2 xenografts when compared with C4-2 cells cultured in vitro. Furthermore, at the protein level, PSCA was re-expressed in all xenografts compared with cells in culture. The regulation of mRNA and protein expression of the cell-surface target proteins α(ν) β(3), NTSR1, PSMA, and PSCA, in prostate cancer cells with different tumorigenic potential, was influenced by factors of the microenvironment, differing between cell cultures and murine xenotransplants. Integrin α(ν) β(3), NTRS1 and PSCA mRNA expression increased with tumorigenic potential, but mRNA expression levels for these proteins do not translate directly to equivalent expression levels of membrane bound protein. Copyright © 2011 Wiley Periodicals, Inc.

In situ synthesis of protein arrays.

PubMed

He, Mingyue; Stoevesandt, Oda; Taussig, Michael J

2008-02-01

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

[The effects of methionine and choline on the expression levels of CaMKII and CREB mRNA and proteins in rats exposed to lead].

PubMed

Feng, Chang; Fan, Guang-qin; Wu, Feng-yun; Lin, Fen; Li, Yan-shu; Chen, Ying

2012-07-01

To study the effects of methionine and choline on the expression levels of CaMKII and CREB mRNA and proteins in hippocampus of rats exposed to lead. Male SD rats were divided into five groups. (1) control group, (2) group exposed to lead+2 by drinking water with 0.40 g/L lead acetate, (3) group exposed to methionine and choline (1:1, 400 mg/kg), (4) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 100 mg/kg), (5) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 400 mg/kg). In 8 weeks after exposure, all rats were killed. Then CREB mRNA and CaMK II mRNA expression levels in hippocampus were detected by real-time PCR, CREB and CaMK II protein expression levels in hippocampus were measured by western blot assay. The expression levels (0.743 ± 0.185 and 0.729 ± 0.199) of CaMKII mRNA and CREB mRNA in the hippocampus of lead group were significantly lower than those (0.950 ± 0.238 and 0.901 ± 0.232) of control group (P < 0.05), also the expression levels (0.271 ± 0.045 and 0.212 ± 0.058) of CREB protein and pCREB protein in the hippocampus of lead group were significantly lower than those (0.319 ± 0.058 and 0.506 ± 0.125) of control group (P < 0.05). The expression levels (1.014 ± 0.210 and 1.126 ± 0.379) of CaMKII mRNA and the expression levels (1.029 ± 0.335 and 0.932 ± 0.251) of CREB mRNA in the hippocampus of 2 groups exposed to lead acetate plus methionine and choline were significantly higher than those of lead group (P < 0.05). The expression levels (0.407 ± 0.951 and 0.563 ± 0.178) of CREB protein and pCREB protein in the hippocampus of group exposed to lead acetate plus 400 mg/kg methionine and choline were significantly higher than those of lead group (P < 0.05). Methionine and choline could decrease the inhibition effects of lead on the expression of CaMKII and CREB mRNA or CREB and pCREB proteins in the hippocampus of rats.

Sex-lethal promotes nuclear retention of msl2 mRNA via interactions with the STAR protein HOW

PubMed Central

Graindorge, Antoine; Carré, Clément; Gebauer, Fátima

2013-01-01

Female-specific repression of male-specific-lethal-2 (msl2) mRNA in Drosophila melanogaster provides a paradigm for coordinated control of gene expression by RNA-binding complexes. Repression is orchestrated by Sex-lethal (SXL), which binds to the 5′ and 3′ untranslated regions (UTRs) of the mRNA and inhibits splicing in the nucleus and subsequent translation in the cytoplasm. Here we show that SXL ensures msl2 silencing by yet a third mechanism that involves inhibition of nucleocytoplasmic transport of msl2 mRNA. To identify SXL cofactors in msl2 regulation, we devised a two-step purification method termed GRAB (GST pull-down and RNA affinity binding) and identified Held-Out-Wings (HOW) as a component of the msl2 5′ UTR-associated complex. HOW directly interacts with SXL and binds to two sequence elements in the msl2 5′ UTR. Depletion of HOW reduces the capacity of SXL to repress the expression of msl2 reporters without affecting SXL-mediated regulation of splicing or translation. Instead, HOW is required for SXL to retain msl2 transcripts in the nucleus. Cooperation with SXL confers a sex-specific role to HOW. Our results uncover a novel function of SXL in nuclear mRNA retention and identify HOW as a mediator of this function. PMID:23788626

Dysregulated glutamate and dopamine transporters in postmortem frontal cortex from bipolar and schizophrenic patients

PubMed Central

Rao, Jagadeesh Sridhara; Kellom, Matthew; Reese, Edmund Arthur; Rapoport, Stanley Isaac; Kim, Hyung-Wook

2012-01-01

Background Dysregulated glutamate, serotonin and dopamine neurotransmission has been reported in bipolar disorder (BD) and schizophrenia (SZ), but the underlying mechanisms of dysregulation are not clear. We hypothesized that they involve alterations in excitatory amino acid transporters (EAATs), the serotonin reuptake transporter (SERT), and the dopamine reuptake transporter (DAT). Methods To test this hypothesis, we determined protein and mRNA levels of EAAT subtypes 1–4, of the SERT and of the DAT in postmortem frontal cortex from BD (n=10) and SZ (n=10) patients and from healthy control (n=10) subjects. Results Compared to control levels, protein and mRNA levels of EAAT1 were increased significantly in cortex from both BD and SZ patients. EAAT2 protein and mRNA levels were decreased significantly in BD but not in SZ cortices. EAAT3 and EAAT 4 protein and mRNA levels were significantly higher in SZ but not in BD compared with control. DAT protein and mRNA levels were decreased significantly in both BD and SZ cortex. There was no significant change in SERT expression in either BD or SZ. Conclusions The altered EAATs and DAT expression could result in altered glutamatergic and hyperdopaminergic function in BD and SZ. Differently altered EAATs involved in glutamatergic transmission could be therapeutic targets for treating BD and SZ. PMID:21925739

Developmental expression of VGF mRNA in the prenatal and postnatal rat.

PubMed

Snyder, S E; Pintar, J E; Salton, S R

1998-04-27

VGF is a developmentally regulated, secretory peptide precursor that is expressed by neurons and neuroendocrine cells and that has its transcription and secretion induced rapidly by neurotrophins and by depolarization. To gain insight into the possible functions and regulation of VGF in vivo, we have characterized the distribution of VGF mRNA in the developing rat nervous system. VGF expression was first detectable at embryonic day 11.5 in the primordia of cranial, sympathetic, and dorsal root ganglia, and its distribution expanded throughout development to include significant expression throughout the brain, spinal cord, and retina of the adult rat. The earliest expression of VGF, therefore, appeared in the peripheral nervous system as developing neurons settled in their designated ganglia. In many regions of the brain, VGF mRNA levels were found to be highest during periods when axonal outgrowth and synaptogenesis predominate. Areas of the central nervous system that contain predominantly dividing cells never displayed any VGF mRNA expression, nor did the vast majority of nonneural tissues.

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

PubMed

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

[Study of signal transduction pathway in the expression of inflammatory factors stimulated by lipopolysaccharides from Porphyromonas endodontalis in osteoblasts].

PubMed

Yang, Di; Qiu, Li-hong; Li, Ren; Li, Zi-mu; Li, Chen

2010-04-01

To quantify the interleukin (IL)-1beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides ([PS) extracted from Porphyromonoas endodontalis (P. endodontalis) in osteoblasts, and to relate P. endodontalis LPS to the bone resorptive pathogenesis in the lesions of chronic apical periodontitis. MG63 cells was pretreated with PD98059 or SB203580 for 1 h and then treated with P. endodontolis LPS for 6 h. The expression of IL-1beta mRNA and IL-6 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) technique. The production of IL-1beta mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with PD98059. Both of the production of IL-1beta mRNA and JL-6 mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with SB203580. The synthesis of IL-1beta mRNA stimulated by Pendodontalis LPS in MG63 probably occur via extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen activated protein kinase (MAPK) signal transduction system. The synthesis of IL-6 mRNA stimulated by P.endodontalis LPS in MG63 probahly occur via p38MAPK signal transduction system.

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

PubMed

Sato, K; Yamazaki, K; Shizume, K; Kanaji, Y; Obara, T; Ohsumi, K; Demura, H; Yamaguchi, S; Shibuya, M

1995-09-01

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

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

Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M.

2009-10-30

The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, amore » previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.« less

MHC2TA mRNA levels and human herpesvirus 6 in multiple sclerosis patients treated with interferon beta along two-year follow-up

PubMed Central

2012-01-01

Background In previous studies we found that MHC2TA +1614 genotype frequency was very different when MS patients with and without human herpesvirus 6 (HHV-6) in serum samples were compared; a different clinical behavior was also described. The purpose of the study was: 1. To evaluate if MHC2TA expression in MS patients was influenced by interferon beta (IFN-beta) treatment. 2. To study MHC2TA expression in MS patients with and without minor allele C. 3. To analyze the relation between MHC2TA mRNA levels and HHV-6 active infection in MS patients. Methods Blood and serum samples of 154 MS patients were collected in five programmed visits: basal (prior to beginning IFN-beta treatment), six, twelve, eighteen and twenty-four months later. HHV-6 in serum and MHC2TA mRNA levels were evaluated by PCR and RT-PCR, respectively. Neutralizing antibodies (NAbs) against IFN-beta were analyzed by the cytopathic effect assay. Results We found that MHC2TA mRNA levels were significantly lower among MS patients with HHV-6 active infection at the basal visit (without treatment) than in those MS patients without HHV-6 active infection at the basal visit (p = 0.012); in all the positive samples we only found variant A. Furthermore, 58/99 (58.6%) MS patients without HHV-6 along the five programmed visits and an increase of MHC2TA expression after two-years of IFN-beta treatment were clinical responders vs. 5/21 (23.8%) among those MS patients with HHV-6 and a decrease of MHC2TA mRNA levels along the two-years with IFN-beta treatment (p = 0.004); no differences were found between patients with and without NAbs. Conclusions MHC2TA mRNA levels could be decreased by the active replication of HHV-6; the absence of HHV-6 in serum and the increase of MHC2TA expression could be further studied as markers of good clinical response to IFN-beta treatment. PMID:23009575

Polygalacturonase Gene Expression in Rutgers, rin, nor, and Nr Tomato Fruits 1

PubMed Central

DellaPenna, Dean; Kates, David S.; Bennett, Alan B.

1987-01-01

Polygalacturonase (PG) gene expression was studied in normally ripening tomato fruit (Lycopersicon esculentum Mill, cv Rutgers) and in three ripening-impaired mutants, rin, nor, and Nr. Normal and mutant fruit of identical chronological age were analyzed at 41, 49, and 62 days after pollination. These stages corresponded to mature-green, ripe, and overripe, respectively, for Rutgers. The amount of PG mRNA in Rutgers was highest at 49 days and accounted for 2.3% of the total mRNA mass but at 62 days had decreased to 0.004% of the total mRNA mass. In Nr, the amount of PG mRNA steadily increased between 41 and 62 days after pollination, reaching a maximum level of 0.5% of the total mRNA mass. The mutant nor exhibited barely detectable levels of PG mRNA at all stages tested. Surprisingly, PG mRNA, comprising approximately 0.06% of the mRNA mass, was detected in 49 day rin fruit. This mRNA accumulation occurred in the absence of elevated ethylene production by the fruit and resulted in the synthesis of enzymically active PG I. The different patterns of PG mRNA accumulation in the three mutants suggests that distinct molecular mechanisms contribute to reduced PG expression in each ripening-impaired mutant. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:16665727

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

PubMed Central

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-01-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA. PMID:8943327

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

PubMed

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-12-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

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

PubMed

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

2006-04-01

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

[Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of interleukin-34 in mouse osteoblasts].

PubMed

Yu, Ya-Qiong; Guo, Jia-Jie; Qiu, Li-Hong; Li, Xiao-Lin; Yang, Di; Guo, Yan

2017-02-01

To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (P.e) on the expression of interleukin-34 (IL-34) mRNA in MC3T3-E1 cells and the role of p38MAPK, ERK1/2, NF-κB and SIRT1 in the process. MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-24 h). The expression of IL-34 mRNA was detected by real-time reverse transcription-polymerase chain reaction (real time RT-PCR). MC3T3-E1 cells were pretreated with inhibitor of NF-κB(BAY 11-7082),inhibitor of p38MAPK (SB203580), inhibitor of ERK1/2 (PD98059), agonist of sirtuin1 (SIRT1) [resveratrol (RES)] and inhibitor of SIRT1 (EX-527) for 1 h, and then were treated with 20 mg/L P.e-LPS. The expression of IL-34 mRNA was detected by real time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. The level of IL-34 mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L)，which indicated that P.e-LPS induced osteoblasts to express IL-34 mRNA in a dose-dependent manner. Maximal induction of IL-34 mRNA expression was observed in MC3T3-E1 cells treated with 20 mg/L P.e-LPS for 24 h.At 48 h, the expression of IL-34 mRNA decreased gradually. The mRNA of IL-34 decreased significantly after pretreatment with 10 μmol/L BAY-117082, SB203580 and PD98059 for 1 h. P.e-LPS-induced IL-34 upregulation was attenuated by pretreatment with RES, but increased by EX-527. These results suggest that P.e-LPS may mediate IL-34 mRNA expression in MC3T3-E1 cells. This process is dependent, at least in part, on p38MAPK, ERK1/2, NF-κB and SIRT1 signaling pathways.

Periapical cytokine expression in sickle cell disease.

PubMed

Ferreira, Shirlene Barbosa Pimentel; de Brito, Luciana Carla Neves; Oliveira, Michelle Pimenta; Maciel, Kamilla Faria; Martelli Júnior, Hercílio; Vieira, Leda Quercia; Sobrinho, Antônio Paulino Ribeiro

2015-03-01

Sickle cell anemia (SCA) is the most prevalent genetic disease worldwide. Patients with SCA exhibit increased levels of proinflammatory mediators as part of a permanently activated immunoinflammatory status. The aim of this study was to evaluate the mRNA expression levels of the cytokines interferon (IFN-γ), tumor necrosis factor, interleukin (IL-1β, IL-17A, IL-10), receptor activator for nuclear factor kappa B ligand, and the chemokines CCL2/MCP-1 and CCL5 in the periapical interstitial fluid from SCA individuals compared with healthy individuals. Samples were collected from 12 teeth of SCA patients and 12 non-SCA patients with apical periodontitis. In addition, 12 teeth were sampled from the periapical region of healthy patients with vital pulp (control). The expression of cytokine mRNA was detected by using real-time polymerase chain reaction. The expression of mRNA for the Th1-associated cytokines IFN-γ, tumor necrosis factor-α, and IL-1β were significantly higher in SCA individuals than in the control individuals (P < .05). Among Th1-associated cytokines, only IFN-γ was significantly increased in non-SCA compared with control patients (vital pulp). The expression of IL-17A mRNA was significant higher in SCA cases than in control samples (P < .05), whereas the IL-10 mRNA expression was significantly increased in SCA and non-SCA individuals when compared with the control group. Similar levels of receptor activator for nuclear factor kappa B ligand, CCL2, and CCL5 mRNA expression were observed in all samples. However, no significant differences were observed in the expression of cytokine or chemokine mRNA between SCA and non-SCA individuals (P > .05). The results were able to demonstrate that SCA patients presented prone proinflammatory ability, despite the fact that any differences in periapical immune responses between SCA and non-SCA individuals were not observed. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

The expression of 11β-hydroxysteroid dehydrogenase type 1 is increased in experimental periodontitis in rats.

PubMed

Nakata, Takaya; Umeda, Makoto; Masuzaki, Hiroaki; Sawai, Hirofumi

2016-10-03

The involvement of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive glucocorticoids into active glucocorticoids intracellularly, in metabolic diseases and chronic inflammatory diseases has been elucidated. We recently reported that an increase in 11β-HSD1 expression was associated with chronic periodontitis in humans irrespective of obesity. To further clarify the role of 11β-HSD1 in chronic periodontitis, the expression of 11β-HSD1 was investigated in experimental periodontitis model in rats. Experimental periodontitis was induced by silk ligature of left maxillary second molars of 7-week-old male Wistar rats, and periodontal tissues were collected at day 3. The expression of 11β-HSD1, 11β-HSD2, and TNFα mRNA was examined using real time reverse transcription-polymerase chain reaction. The expression of TNFα was used as an indicator of inflammation. Thus, the rats in which the levels of TNFα mRNA were increased in the ligature-induced periodontitis compared with the control were analysed. The findings demonstrated that the expression of 11β-HSD1 mRNA was significantly increased in experimental periodontitis compared with the control. The increase in the levels of 11β-HSD1 mRNA in the ligature-induced periodontitis compared with the control was positively correlated with that of TNFα mRNA. On the other hand, the expression of 11β-HSD2 mRNA, which inactivates glucocorticoids, was slightly decreased in experimental periodontitis. Therefore, the ratio of 11β-HSD1 versus 11β-HSD2 mRNA was significantly higher in experimental periodontitis than in the control. These results suggest that the increased expression of 11β-HSD1, which would result in the increased levels of intracellular glucocorticoids, may play a role in the pathophysiology of experimental periodontitis.

Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

PubMed Central

Catts, Vibeke Sørensen; Shannon Weickert, Cynthia

2012-01-01

An increase in apoptotic events may underlie neuropathology in schizophrenia. By data-mining approaches, we identified significant expression changes in death receptor signaling pathways in the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia, particularly implicating the Tumor Necrosis Factor Superfamily member 6 (FAS) receptor and the Tumor Necrosis Factor [ligand] Superfamily member 13 (TNFSF13) in schizophrenia. We sought to confirm and replicate in an independent tissue collection the noted mRNA changes with quantitative real-time RT-PCR. To test for regional and diagnostic specificity, tissue from orbital frontal cortex (OFC) was examined and a bipolar disorder group included. In schizophrenia, we confirmed and replicated significantly increased expression of TNFSF13 mRNA in the DLPFC. Also, a significantly larger proportion of subjects in the schizophrenia group had elevated FAS receptor expression in the DLPFC relative to unaffected controls. These changes were not observed in the bipolar disorder group. In the OFC, there were no significant differences in TNFSF13 or FAS receptor mRNA expression. Decreases in BH3 interacting domain death agonist (BID) mRNA transcript levels were found in the schizophrenia and bipolar disorder groups affecting both the DLPFC and the OFC. We tested if TNFSF13 mRNA expression correlated with neuronal mRNAs in the DLPFC, and found significant negative correlations with interneuron markers, parvalbumin and somatostatin, and a positive correlation with PPP1R9B (spinophilin), but not DLG4 (PSD-95). The expression of TNFSF13 mRNA in DLPFC correlated negatively with tissue pH, but decreasing pH in cultured cells did not cause increased TNFSF13 mRNA nor did exogenous TNFSF13 decrease pH. We concluded that increased TNFSF13 expression may be one of several cell-death cytokine abnormalities that contribute to the observed brain pathology in schizophrenia, and while increased TNFSF13 may be associated with lower brain pH, the change is not necessarily causally related to brain pH. PMID:22545112

Colonization by non-pathogenic bacteria alters mRNA expression of cytochromes P450 in originally germ-free mice.

PubMed

Jourová, L; Anzenbacher, P; Lišková, B; Matušková, Z; Hermanová, P; Hudcovic, T; Kozáková, H; Hrnčířová, L; Anzenbacherová, E

2017-11-01

Gut microbiota provides a wide range of beneficial function for the host and has an immense effect on the host's health state. It has also been shown that gut microbiome is often involved in the biotransformation of xenobiotics; however, the molecular mechanisms of the interaction between the gut bacteria and the metabolism of drugs by the host are still unclear. To investigate the effect of microbial colonization on messenger RNA (mRNA) expression of liver cytochromes P450 (CYPs), the main drug-metabolizing enzymes, we used germ-free (GF) mice, lacking the intestinal flora and mice monocolonized by non-pathogenic bacteria Lactobacillus plantarum NIZO2877 or probiotic bacteria Escherichia coli Nissle 1917 compared to specific pathogen-free (SPF) mice. Our results show that the mRNA expression of Cyp1a2 and Cyp2e1 was significantly increased, while the expression of Cyp3a11 mRNA was decreased under GF conditions compared to the SPF mice. The both bacteria L. plantarum NIZO2877 and E. coli Nissle 1917 given to the GF mice decreased the level of Cyp1a2 mRNA and normalized it to the control level. On the other hand, the colonization by these bacteria had no effect on the expression of Cyp3a11 mRNA in the liver of the GF mice (which remained decreased). Surprisingly, monocolonization with chosen bacterial strains has shown a different effect on the expression of Cyp2e1 mRNA in GF mice. Increased level of Cyp2e1 expression observed in the GF mice was found also in mice colonized by L. plantarum NIZO2877 ; however, the colonization with probiotic E. coli Nissle 1917 caused a decrease in Cyp2e1 expression and partially restored the SPF mice conditions.