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.

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.

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

Time-Dependent Expression Profiles of microRNAs and mRNAs in Rat Milk Whey

PubMed Central

Izumi, Hirohisa; Kosaka, Nobuyoshi; Shimizu, Takashi; Sekine, Kazunori; Ochiya, Takahiro; Takase, Mitsunori

2014-01-01

Functional RNAs, such as microRNA (miRNA) and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2) was markedly higher than that in mature milk whey (days 9 and 16). Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes) to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats. PMID:24533154

β-Glucuronidase is a suitable internal control gene for mRNA quantitation in pathophysiological and non-pathological livers.

PubMed

Yamaguchi, Hiromi; Matsumoto, Sawako; Ishibashi, Mariko; Hasegawa, Kiyoshi; Sugitani, Masahiko; Takayama, Tadatoshi; Esumi, Mariko

2013-10-01

The level of expression of housekeeping genes is in general considered stable, and a representative gene such as glyceraldehyde-3-phosphate dehydrogenase is commonly used as an internal control for quantitating mRNA. However, expression of housekeeping genes is not always constant under pathological conditions. To determine which genes would be most suitable as internal controls for quantitative gene expression studies in human liver diseases, we quantified 12 representative housekeeping genes in 27 non-cancerous liver tissues (normal, chronic hepatitis C with and without liver cirrhosis). We identified β-glucuronidase as the most suitable gene for studies on liver by rigorous statistical analysis of inter- and intra-group comparisons. We conclude that it is important to determine the most appropriate control gene for the particular condition to be analyzed. © 2013 Elsevier Inc. All rights reserved.

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.

[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.

RT-qPCR Demonstrates Light-Dependent AtRBCS1A and AtRBCS3B mRNA Expressions in "Arabidopsis thaliana" Leaves

ERIC Educational Resources Information Center

Chang, Ming-Mei; Li, Anna; Feissner, Robert; Ahmad, Talal

2016-01-01

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in diagnosis and research to determine specific mRNA expressions in cells. As RT-qPCR applications increase, it is necessary to provide undergraduates hands-on experience of this modern technique. Here, we report a 3-week laboratory exercise using RT-qPCR to…

Expression of the proto-oncogene Pokemon in colorectal cancer--inhibitory effects of an siRNA.

PubMed

Zhao, Gan-Ting; Yang, Li-Juan; Li, Xi-Xia; Cui, Hui-Lin; Guo, Rui

2013-01-01

This study aimed to investigate expression of the proto-oncogene POK erythroid myeloid ontogenic factor (Pokemon) in colorectal cancer (CRC), and assess inhibitory effects of a small interference RNA (siRNA) expression vector in SW480 and SW620 cells. Semi-quantitative reverse transcription-polymerase chain reaction (PCR) and immunohistochemistry were performed to determine mRNA and protein expression levels of Pokemon in CRC tissues. Indirect immunofluorescence staining was applied to investigate the location of Pokemon in SW480 and SW620 cells. The siRNA expression vectors that were constructed to express a short hairpin RNA against Pokemon were transfected to the SW480 and SW620 cells with a liposome. Expression levels of Pokemon mRNA and protein were examined by real-time quantitative-fluorescent PCR and western blot analysis. The effects of Pokemon silencing on proliferation of SW480 and SW620 cells were evaluated with reference to growth curves with MTT assays. The mRNA expression level of Pokemon in tumor tissues (0.845 ± 0.344) was significantly higher than that in adjacent tumor specimens (0.321 ± 0.197). The positive expression ratio of Pokemon protein in CRC (87.0%) was significantly higher than that in the adjacent tissues (19.6%). Strong fluorescence staining of Pokemon protein was observed in the cytoplasm of the SW480 and SW620 cells. The inhibition ratios of Pokemon mRNA and protein in the SW480 cells were 83.1% and 73.5% at 48 and 72 h, respectively, compared with those of the negative control cells with the siRNA. In the SW620 cells, the inhibition ratios of Pokemon mRNA and protein were 76.3% and 68.7% at 48 and 72 h, respectively. MTT showed that Pokemon gene silencing inhibited the proliferation of SW480 and SW620 cells. Overexpression of Pokemon in CRC may have a function in carcinogenesis and progression. siRNA expression vectors could effectively inhibit mRNA and protein expression of Pokemon in SW480 and SW620 cells, thereby reducing malignant cell proliferation.

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.

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.

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.

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.

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.

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.

Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells.

PubMed

Shi, Jia; Yoshino, Osamu; Osuga, Yutaka; Akiyama, Ikumi; Harada, Miyuki; Koga, Kaori; Fujimoto, Akihisa; Yano, Tetsu; Taketani, Yuji

2012-04-01

To examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary. Molecular studies. Research laboratory. Eight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for in vitro fertilization. Localizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction. The localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR. In the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An in vitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC. GDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

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

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.

Nitric oxide synthase mRNA expression in human fetal membranes: a possible role in parturition.

PubMed

Dennes, W J; Slater, D M; Bennett, P R

1997-04-07

Nitric oxide (NO) is a potent endogenous smooth-muscle relaxant. It is synthesised from 1-arginine by isoforms of nitric oxide synthase (NOS). Whilst it is clear that the uterus responds to NO by relaxation, NOS expression has not been investigated in fetal membranes or myometrium in human pregnancy. This study has shown, using semi-quantitative RT-PCR, expression of cNOS mRNA in human amnion, chorion-decidua, and placenta. iNOS mRNA expression was demonstrated in human amnion, chorion-decidua, and placenta. It is possible that NO synthesised in fetal membranes may act either directly to inhibit myometrial contractility or indirectly to interact with other labour-associated genes, such as cyclo-oxygenase, to coordinate the onset of labour.

Short communication: expression of peptide YY, proglucagon, neuropeptide Y receptor Y2, and glucagon-like peptide-1 receptor in bovine peripheral tissues.

PubMed

Pezeshki, A; Muench, G P; Chelikani, P K

2012-09-01

The role of distal gut signals in control of feed intake and metabolism in cattle has received scant attention. Peptide YY (PYY) and glucagon-like peptide-1, which are secreted from enteroendocrine cells of the distal gut in monogastrics have several functions, including regulation of energy balance. However, little is known of the tissue expression of these peptides and their receptors in cattle. The aim of the current study was to characterize the tissue distribution of PYY, neuropeptide Y receptor Y2 (Y2), proglucagon (GCG), and glucagon-like peptide-1 receptor (GLP1R) in various peripheral tissues of cattle. Four male 7-wk-old dairy calves were euthanized and 16 peripheral tissues were collected. Conventional PCR and quantitative real-time PCR were performed to confirm tissue expression and quantify the transcript abundance in various tissues. The results of conventional PCR revealed that mRNA for both PYY and Y2 was detectable in the rumen, abomasum, duodenum, jejunum, ileum, and colon but not in other tissues. Quantitative real-time PCR data demonstrated that PYY mRNA was 2- to 3-fold greater in the pancreas, kidney, and heart relative to the liver. By conventional PCR, GCG mRNA was detected in the abomasum, duodenum, jejunum, ileum, and colon and GLP1R mRNA was expressed in all gut segments, pancreas, spleen, and kidney. Quantitative real-time PCR data demonstrated that, relative to transcript abundance in the liver, GCG mRNA was 4- to 40-fold higher from abomasum to colon, and GLP1R mRNA was 50- to 300-fold higher from the rumen to colon, 14-fold greater in the pancreas, 18-fold higher in the spleen, and 166-fold greater in the kidney. The tissue distribution of PYY, GCG, and their receptors observed in the current study is, in general, consistent with expression patterns in monogastrics. The predominant expression of PYY, Y2, and GCG in the gut, and the presence of GLP1R in multiple peripheral tissues suggest a role for PYY in controlling gut functions and for GLP-1 in regulating multiple physiological functions in cattle. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. 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

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.

Differences in mitochondrial gene expression profiles, enzyme activities and myosin heavy chain types in yak versus bovine skeletal muscles.

PubMed

Lin, Y Q; Xu, Y O; Yue, Y; Jin, S Y; Qu, Y; Dong, F; Li, Y P; Zheng, Y C

2012-08-29

Hypoxia can affect energy metabolism. We examined gene expression and enzyme activity related to mitochondrial energy metabolism, as well as myosin heavy chain (MyHC) types in yaks (Bos grunniens) living at high altitudes. Real-time quantitative PCR assays indicated that the yak has significantly lower levels of carnitine palmitoyltransferase (CPT) mRNA in the biceps femoris and lower levels of uncoupling protein 3 (UCP3) mRNA in both biceps femoris and longissimus dorsi than in Yellow cattle. No significant differences between yak and Yellow cattle were observed in the activities of mitochondrial β-hydroxyacyl-CoA dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase in the same muscles. Semi-quantitative RT-PCR analysis showed that the MyHC 1 mRNA levels in yak biceps femoris was lower than in Yellow cattle. We conclude that the yak has significantly lower mRNA levels of CPT, UCP3, and MyHC 1 in biceps femoris than in Yellow cattle, suggesting that the yak biceps femoris has lower fatty acid oxidation capacity and greater glycolytic metabolic potential.

The Role of CYP3A4 mRNA Transcript with Shortened 3′-Untranslated Region in Hepatocyte Differentiation, Liver Development, and Response to Drug InductionS⃞

PubMed Central

Li, Dan; Gaedigk, Roger; Hart, Steven N.; Leeder, J. Steven

2012-01-01

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3′-untranslated region (UTR), one had a shorter 3′-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3′-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3′-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3′-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3′-UTR. We conclude that the 3′-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction. PMID:21998292

Technical variations in low-input RNA-seq methodologies.

PubMed

Bhargava, Vipul; Head, Steven R; Ordoukhanian, Phillip; Mercola, Mark; Subramaniam, Shankar

2014-01-14

Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.

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.

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.

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.

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.

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.

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.

The enteric serotonergic system is altered in patients with diverticular disease.

PubMed

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

2013-12-01

Disturbances of the enteric serotonergic system have been implicated in several intestinal motility disorders. Patients with diverticular disease (DD) have been reported to exhibit abnormal intestinal motility and innervation patterns. Gene expression profiles of the serotonergic system and distribution of the serotonin type 4 receptor (5HT-4R) were thus studied in patients with DD. Colonic specimens from patients with DD and controls were subjected to quantitative PCR for serotonin receptors 2B, 3A, 4, serotonin transporter and synthesising enzyme tryptophan hydroxylase. Localisation of 5HT-4R was determined by dual-label immunocytochemistry using smooth muscle actin (α-SMA) and pan-neuronal markers (PGP 9.5) and quantitative analysis was carried out. Site-specific gene expression analysis of 5HT-4R was assessed within myenteric ganglia and muscle layers. Correlation of 5HT-4R with muscarinic receptors 2 and 3 (M2R, M3R) messenger RNA expression was determined. 5HT-4R mRNA expression was downregulated in the tunica muscularis and upregulated in the mucosa of patients with DD, whereas the other components of the serotonergic system remained unchanged. 5HT-4R was detected in ganglia and muscle layers, but was decreased in the circular muscle layer and myenteric ganglia of patients with DD. 5HT-4R mRNA expression correlated with M2R/M3R mRNA expression in controls, but not in patients with DD. The serotonergic system is compromised in DD. Altered expression of 5HT-4R at mRNA and protein levels may contribute to intestinal motor disturbances reported in patients with DD. The findings support the hypothesis that DD is associated and possibly promoted by an enteric neuromuscular pathology.

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.

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.

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.

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.

Alternative-splicing-mediated gene expression

NASA Astrophysics Data System (ADS)

Wang, Qianliang; Zhou, Tianshou

2014-01-01

Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

[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.

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.

The ontogenic expressions of multiple vesicular glutamate transporters during postnatal development of rat pineal gland.

PubMed

Yoshida, S; Ina, A; Konno, J; Wu, T; Shutoh, F; Nogami, H; Hisano, S

2008-03-18

The pineal gland expresses vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2), which are thought to transport glutamate into synaptic-like microvesicles in the pinealocytes. Recently, we reported that the rat pineal gland also expresses VGLUT1v which is a novel variant of VGLUT1 during the perinatal period. To explore the biological significance of these VGLUT expressions in pineal development, we studied the ontogeny of VGLUT in this gland by in situ hybridization, immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rats. Histological analysis revealed that intensities of VGLUT1 hybridization signal and immunostaining drastically increase by postnatal day (P) 7, whereas VGLUT2 expression exhibits high levels of mRNA and protein at birth and decreases gradually from P7 onward. Quantitative RT-PCR analysis supported these histological observations, showing that expressions of VGLUT1 and VGLUT2 exhibit opposite patterns to each other. Coinciding with VGLUT1-upregulation, RT-PCR data showed that expressions of dynamin 1 and endophilin 1, which are factors predictably involved in the endocytotic recovery of VGLUT1-associated vesicle, are also increased by P7. Quantitative RT-PCR analysis of VGLUT1v demonstrated that its mRNA expression is upregulated by P7, kept at the same level until P14, and apparently decreased at P21, suggesting its functional property required for a certain developmental event. Moreover, a comparison of mRNA expressions at daytime and nighttime revealed that neither VGLUT1 nor VGLUT1v shows any difference in both P7 and P21 glands, whereas VGLUT2 is significantly lower at daytime than at nighttime at P21 but not P7, the time point at which the melatonin rhythm is not yet generated. The present study shows that expressions of these VGLUT types are differentially regulated during postnatal pineal development, each presumably participating in physiologically distinct glutamatergic functions.

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.

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

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.

Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies.

PubMed

Tricarico, Carmela; Pinzani, Pamela; Bianchi, Simonetta; Paglierani, Milena; Distante, Vito; Pazzagli, Mario; Bustin, Stephen A; Orlando, Claudio

2002-10-15

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

The peripheral messenger RNA expression of glycogen synthase kinase-3β genes in Alzheimer's disease patients: a preliminary study.

PubMed

Sheng, Jian-Hua; Ng, Tze-Pin; Li, Chun-Bo; Lu, Guang-Hua; He, Wei; Qian, Yi-Ping; Wang, Jing-Hua; Yu, Shun-Ying

2012-12-01

To explore the peripheral leucocytic messenger RNA (mRNA) expression of glycogen synthase kinase-3β (GSK-3β) gene in Alzheimer's disease (AD) patients. Using TaqMan relative quantitative real-time polymerase chain reaction, we analyzed leucocytic gene expression of GSK-3β in 48 AD patients and 49 healthy controls. Clinical data of AD patients were also collected. The mRNA expression level of the GSK-3β gene was significantly higher in the AD group (3.13±0.62) than in the normal group (2.77±0.77). Correlational analyses showed that the mRNA expression level of GSK-3β gene in AD patients was associated with the age of onset (P=0.047), age (P=0.055), and Behavioral Pathology in Alzheimer's Disease Rating Scale total score (P=0.062) and subscores: aggressiveness score (P=0.073) and anxieties and phobias score (P=0.067). Through multivariate regression model, older age, higher anxieties and phobias score and aggressiveness score were associated with higher mRNA expression level of GSK-3β gene. In AD patients, the mRNA expression level of the GSK-3β gene is increased and may be related to age and behavioural pathology in AD. © 2012 The Authors. Psychogeriatrics © 2012 Japanese Psychogeriatric Society.

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.

Repeated use of mifepristone and levonorgestrel and their effect on the ovarian function in mice.

PubMed

Chen, Yuanyuan; Shi, Xiaobo

2016-11-01

To investigate the effects of repeated mifepristone and levonorgestrel use on estrous cycle and expression of ovarian follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in mice. Ovarian FSHR and LHR mRNA expression was measured using real-time quantitative reverse transcription-polymerase chain reaction, while the protein levels were measured using immunohistochemistry. Repeated use of mifepristone and levonorgestrel significantly lengthened the estrous cycle and decreased FSHR and LHR mRNA and protein expression in the ovaries of mice at 4, 24, and 48 days after discontinuing drug use. Repeated use of mifepristone and levonorgestrel had significant main effects on estrous cycle length and the mRNA expression and protein level of ovarian FSHR and LHR. Repeated mifepristone and levonorgestrel use and withdrawal time had a significant interaction with mouse estrous cycle (F = 16.65, P < 0.05), ovarian LHR and FSHR mRNA expression (F = 563.072, P < 0.05), and protein level (F = 6.536, P < 0.05). Repeated use of mifepristone and levonorgestrel can lead to sustained damage to ovarian function through inhibition of ovarian FSHR and LHR expression in mice. © 2016 Japan Society of Obstetrics and Gynecology.

Molecular characterization of melanin-concentrating hormone (MCH) in Schizothorax prenanti: cloning, tissue distribution and role in food intake regulation.

PubMed

Wang, Tao; Yuan, Dengyue; Zhou, Chaowei; Lin, Fangjun; Wei, Rongbin; Chen, Hu; Wu, Hongwei; Xin, Zhiming; Liu, Ju; Gao, Yundi; Chen, Defang; Yang, Shiyong; Wang, Yan; Pu, Yundan; Li, Zhiqiong

2016-06-01

Melanin-concentrating hormone (MCH) is a crucial neuropeptide involved in various biological functions in both mammals and fish. In this study, the full-length MCH cDNA was obtained from Schizothorax prenanti by rapid amplification of cDNA ends polymerase chain reaction. The full-length MCH cDNA contained 589 nucleotides including an open reading frame of 375 nucleotides encoding 256 amino acids. MCH mRNA was highly expressed in the brain by real-time quantitative PCR analysis. Within the brain, expression of MCH mRNA was preponderantly detected in the hypothalamus. In addition, the MCH mRNA expression in the S. prenanti hypothalamus of fed group was significantly decreased compared with the fasted group at 1 and 3 h post-feeding, respectively. Furthermore, the MCH gene expression presented significant increase in the hypothalamus of fasted group compared with the fed group during long-term fasting. After re-feeding, there was a dramatic decrease in MCH mRNA expression in the hypothalamus of S. prenanti. The results indicate that the expression of MCH is affected by feeding status. Taken together, our results suggest that MCH may be involved in food intake regulation in S. prenanti.

Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

NASA Technical Reports Server (NTRS)

Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

2002-01-01

A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

Altered Th1/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed transcribed polymerase chain reaction (RT-PCR).

PubMed Central

Niessner, M; Volk, B A

1995-01-01

Cytokines serve a central function as key factors in the regulation of the intestinal immune response and mediation of tissue damage in inflammatory bowel disease (IBD). Abnormalities in the expression of immunoregulatory cytokines such as IL-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) may indicate a dysregulation of intestinal immunity probably associated with pathogenic events. Therefore, cytokine mRNA concentrations were determined in the mucosa of patients with IBD at sites of active (n = 13) and inactive (n = 12) ulcerative colitis (UC), active (n = 11) and inactive (n = 11) Crohn's disease (CD) and in control patients (n = 14) using quantitative RT-PCR. IL-10 mRNA concentrations were significantly increased in patients with both active UC (P < 0.001) and active CD (P < 0.005) compared with control patients. IFN-gamma mRNA concentrations were also significantly increased both in patients with active UC (P < 0.02) and active CD (P < 0.05) compared with control patients, whereas IL-2 mRNA levels were significantly (P < 0.02) increased only in active CD. IL-4 mRNA expression in the intestinal mucosa was frequently below the detection limit. Our results demonstrate that chronic intestinal inflammation in patients with CD is characterized by an increase of Th1-like cytokines. Furthermore, the increased IL-10 mRNA expression at sites of active IBD suggests that IL-10 is an important regulatory component involved in the control of the inflammatory response in inflammatory bowel disease. Images Fig. 1 Fig. 4 PMID:7664489

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse.

PubMed

Heimeier, Rachel A; Donald, John A

2003-11-01

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.

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.

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.

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

PubMed

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

2016-06-01

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

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction.

PubMed

Nishiura, T; Abe, K

1999-01-01

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

PubMed

Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

2014-04-01

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

Temporal and spatial regulation of mRNA export: Single particle RNA-imaging provides new tools and insights

PubMed Central

Heinrich, Stephanie; Derrer, Carina Patrizia; Lari, Azra; Weis, Karsten; Montpetit, Ben

2017-01-01

The transport of messenger RNAs (mRNAs) from the nucleus to cytoplasm is an essential step in the gene expression program of all eukaryotes. Recent technological advances in the areas of RNA-labeling, microscopy, and sequencing are leading to novel insights about mRNA biogenesis and export. This includes quantitative single molecule imaging (SMI) of RNA molecules in live cells, which is providing knowledge of the spatial and temporal dynamics of the export process. As this information becomes available, it leads to new questions, the reinterpretation of previous findings, and revised models of mRNA export. In this review, we will briefly highlight some of these recent findings and discuss how live cell SMI approaches may be used to further our current understanding of mRNA export and gene expression. PMID:28052353

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

Associations of ACE Gene Insertion/Deletion Polymorphism, ACE Activity, and ACE mRNA Expression with Hypertension in a Chinese Population

PubMed Central

He, Qingfang; Fan, Chunhong; Yu, Min; Wallar, Gina; Zhang, Zuo-Feng; Wang, Lixin; Zhang, Xinwei; Hu, Ruying

2013-01-01

Background The present study was designed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion (I/D, rs4646994) polymorphism, plasma ACE activity, and circulating ACE mRNA expression with essential hypertension (EH) in a Chinese population. In addition, a new detection method for circulating ACE mRNA expression was explored. Methods The research was approved by the ethics committee of Zhejiang Provincial Center for Disease Prevention and Control. Written informed consent was obtained prior to the investigation. 221 hypertensives (cases) and 221 normotensives (controls) were interviewed, subjected to a physical examination, and provided blood for biochemical and genetic tests. The ACE mRNA expression was analyzed by real time fluorescent quantitative Reverse Transcription PCR (FQ-RT-PCR). We performed logistic regression to assess associations of ACE I/D genotypes, ACE activity, and ACE mRNA expression levels with hypertension. Results The results of the multivariate logistic regression analysis showed that the additive model (ID, DD versus II) of the ACE genotype revealed an association with hypertension with adjusted OR of 1.43(95% CI: 1.04-1.97), and ACE ID genotype with adjusted OR of 1.72(95% CI: 1.01-2.92), DD genotype with adjusted OR of 1.94(95% CI: 1.01-3.73), respectively. In addition, our data also indicate that plasma ACE activity (adjusted OR was 1.13(95% CI: 1.08-1.18)) was significantly related to hypertension. However, the plasma ACE mRNA expressions were not different between the cases and controls. Conclusion ACE I/D polymorphism and ACE activity revealed significant influence on hypertension, while circulating ACE mRNA expression was not important factors associated with hypertension in this Chinese population. The detection of circulating ACE mRNA expression by FQ-RT-PCR might be a useful method for early screening and monitoring of EH. PMID:24098401

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.

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.

Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

PubMed

Sergio, Luiz Philippe S; Campos, Vera Maria A; Vicentini, Solange C; Mencalha, Andre Luiz; de Paoli, Flavia; Fonseca, Adenilson S

2016-04-01

Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

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.

[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.

Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells.

PubMed

Gao, Rui; Hao, Changlong; Xu, Liguang; Xu, Chuanlai; Kuang, Hua

2018-04-17

Quantitation and in situ monitoring of target mRNA (mRNA) in living cells remains a significant challenge for the chemical and biomedical communities. To quantitatively detect mRNA expression levels in living cells, we have developed DNA-driven gold nanorod coated platinum-upconversion nanoparticle satellite assemblies (termed Au NR@Pt-UCNP satellites) for intracellular thymidine kinase 1 (TK1) mRNA analysis. The nanostructures were capable of recognizing target mRNA in a sequence-specific manner as luminescence of UCNPs was effectively quenched by Au NR@Pt within the assemblies. Following recognition, UCNPs detached from Au NR@Pt, resulting in luminescence restoration to achieve effective in situ imaging and quantifiable detection of target mRNA. The upconversional luminescence intensity of confocal images showed a good linear relationship with intracellular TK1 mRNA ranging from 1.17 to 65.21 fmol/10 μg RNA and a limit of detection (LOD) of 0.67 fmol/10 μg RNA. We believe that our present assay can be broadly applied for detection of endogenous biomolecules at the cellular and tissue levels and restoration of tissue homeostasis in vivo.

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

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.

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.

Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants

PubMed Central

Lattimore, Vanessa L.; Pearson, John F.; Currie, Margaret J.; Spurdle, Amanda B.; Robinson, Bridget A.; Walker, Logan C.

2018-01-01

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance. PMID:29774201

Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.

PubMed

Lattimore, Vanessa L; Pearson, John F; Currie, Margaret J; Spurdle, Amanda B; Robinson, Bridget A; Walker, Logan C

2018-01-01

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2 . The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates ( n  > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

PubMed

Makino, K; Takeichi, O; Imai, K; Inoue, H; Hatori, K; Himi, K; Saito, I; Ochiai, K; Ogiso, B

2018-06-01

To determine whether Porphyromonas endodontalis can reactivate latent Epstein-Barr virus (EBV). The concentrations of short-chain fatty acids (SCFAs) in P. endodontalis culture supernatants were determined using high-performance liquid chromatography. A promoter region of BamHI fragment Z leftward open reading frame 1 (BZLF-1), which is a transcription factor that controls the EBV lytic cycle, was cloned into luciferase expression vectors. Then, the luciferase assay was performed using P. endodontalis culture supernatants. Histone acetylation using Daudi cells treated with P. endodontalis culture supernatants was examined using Western blotting. BZLF-1 mRNA and BamHI fragment Z EB replication activator (ZEBRA) protein were also detected quantitatively using real-time polymerase chain reaction (PCR) and Western blotting. Surgically removed periapical granulomas were examined to detect P. endodontalis, EBV DNA, and BZLF-1 mRNA expression using quantitative real-time PCR. Statistical analysis using Steel tests was performed. The concentrations of n-butyric acid in P. endodontalis culture supernatants were significantly higher than those of other SCFAs (P=0.0173). Using B-95-8-221 Luc cells treated with P. endodontalis culture supernatants, the luciferase assay demonstrated that P. endodontalis induced BZLF-1 expression. Hyperacetylation of histones was also observed with the culture supernatants. BZLF-1 mRNA and ZEBRA protein were expressed by Daudi cells in a dose-dependent manner after the treatment with P. endodontalis culture supernatants. P. endodontalis and BZLF-1 in periapical granulomas were also detected. The expression levels of BZLF-1 mRNA were similar to the numbers of P. endodontalis cells in each specimen. n-butyric acid produced by P. endodontalis reactivated latent EBV. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

PubMed Central

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

2012-01-01

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

A PCR-aided transcript titration assay (PATTY) to measure topoisomerase I gene expression in human tumor specimens.

PubMed

Meersma, G J; Bakker, M; Groen, H J; Van der Zee, A G; Jensen, P B; Giaccone, G; De Vries, E G; Smit, E F

1999-01-01

Topoisomerase I (topo I) inhibitors are promising anticancer agents with demonstrated activity against a wide range of solid tumors. Quantitative information on topol mRNA levels in tumor biopsies may predict response to topo I inhibitors. A polymerase chain reaction aided transcript titration assay (PATTY) was developed to allow quantitation of topol mRNA in small samples. Concentrations of topol mRNA in total RNA samples were estimated by RT-PCR analysis in a human small cell lung cancer (SCLC) cell line (GLC,) and its topotecan (GL2C/SK and F) and camptothecin (GL2C/Campt) resistant sublines, human non-small cell lung cancer (NSCLC) and ovarian carcinoma samples. Topol PATTY showed a decreased topo I mRNA level in GLC2/SK and F (4.5 pg/100 ng total RNA) and GLC,/Campt (2.2 pg/100 ng total RNA), respectively, compared to the parent cell line GLC2 (5.4 pg/100 ng total RNA). Topol protein levels as measured by Western blotting were compatible with topol mRNA levels. Median (range) topol mRNA levels were 3.23 (2.33-5.10) pg/100 ng total RNA in resected NSCLC specimen (n = 6), and 2.03 (0.54-0.95) pg/100 ng total RNA in resected ovarian cancer specimen (n = 6). We conclude that topol PATTY is a new assay that quantitates topol mRNA levels in cell lines and small tumor samples.

A novel mechanism of protamine expression deregulation highlighted by abnormal protamine transcript retention in infertile human males with sperm protamine deficiency.

PubMed

Aoki, V W; Liu, L; Carrell, D T

2006-01-01

Sperm protamine deficiency has been associated with human male infertility. However, the aetiology of deregulated protamine expression remains elusive. The objective of this study was to evaluate the underlying aetiology of protamine deficiency in male infertility patients with deregulated protamine expression. Protamine-1 (P1) and protamine-2 (P2) protein concentrations were compared against P1 and P2 mRNA levels in the sperm of 166 male infertility patients and 27 men of known fertility. Protamine protein concentrations were quantified by nuclear protein extraction, gel electrophoresis and densitometry analysis. Semi-quantitative real-time RT-PCR was used to quantify P1 and P2 mRNA levels. P1 mRNA concentrations were significantly increased in patients underexpressing P1 protein versus those with normal and increased P1 levels. In patients with an abnormally low ratio of P1 to P2 (P1/P2 <0.8), there was a significant increase in P1 mRNA retention. Patients underexpressing P2 also had significantly increased mean P2 mRNA levels, although the majority of these P2-deficient patients showed an increased frequency of significantly reduced P2 mRNA levels. This is the first study to concomitantly evaluate P1 and P2 protein and mRNA levels in mature human sperm. Abnormally elevated protamine mRNA retention appears to be associated with aberrant protamine expression in infertile human males. These data suggest that defects in protamine translation regulation may contribute to protamine deficiency in infertile males.

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

PubMed

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

2017-10-01

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

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.

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.

Quantifying Extrinsic Noise in Gene Expression Using the Maximum Entropy Framework

PubMed Central

Dixit, Purushottam D.

2013-01-01

We present a maximum entropy framework to separate intrinsic and extrinsic contributions to noisy gene expression solely from the profile of expression. We express the experimentally accessible probability distribution of the copy number of the gene product (mRNA or protein) by accounting for possible variations in extrinsic factors. The distribution of extrinsic factors is estimated using the maximum entropy principle. Our results show that extrinsic factors qualitatively and quantitatively affect the probability distribution of the gene product. We work out, in detail, the transcription of mRNA from a constitutively expressed promoter in Escherichia coli. We suggest that the variation in extrinsic factors may account for the observed wider-than-Poisson distribution of mRNA copy numbers. We successfully test our framework on a numerical simulation of a simple gene expression scheme that accounts for the variation in extrinsic factors. We also make falsifiable predictions, some of which are tested on previous experiments in E. coli whereas others need verification. Application of the presented framework to more complex situations is also discussed. PMID:23790383

Quantifying extrinsic noise in gene expression using the maximum entropy framework.

PubMed

Dixit, Purushottam D

2013-06-18

We present a maximum entropy framework to separate intrinsic and extrinsic contributions to noisy gene expression solely from the profile of expression. We express the experimentally accessible probability distribution of the copy number of the gene product (mRNA or protein) by accounting for possible variations in extrinsic factors. The distribution of extrinsic factors is estimated using the maximum entropy principle. Our results show that extrinsic factors qualitatively and quantitatively affect the probability distribution of the gene product. We work out, in detail, the transcription of mRNA from a constitutively expressed promoter in Escherichia coli. We suggest that the variation in extrinsic factors may account for the observed wider-than-Poisson distribution of mRNA copy numbers. We successfully test our framework on a numerical simulation of a simple gene expression scheme that accounts for the variation in extrinsic factors. We also make falsifiable predictions, some of which are tested on previous experiments in E. coli whereas others need verification. Application of the presented framework to more complex situations is also discussed. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Expression of DNA repair genes in burned skin exposed to low-level red laser.

PubMed

Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

2014-11-01

Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

Effect of developmental exposure to chlorpyrifos on the expression of neurotrophin growth factors and cell-specific markers in neonatal rat brain.

PubMed

Betancourt, Angela M; Burgess, Shane C; Carr, Russell L

2006-08-01

Chlorpyrifos (CPS), a known neurotoxicant, is a widely used agricultural organophosphorus insecticide. The effects of postnatal exposure to CPS on the expression of mRNA for two factors critical to brain development, nerve growth factor (NGF) and reelin, were investigated in the forebrain of rats. In addition, the expression of mRNA for the muscarinic acetylcholine receptor (mAChR) M(1) subtype and cell-specific markers for developing neurons (beta-III tubulin), astrocytes (glial fibrillary acidic protein, GFAP), and oligodendrocytes (myelin-associated glycoprotein, MAG) was also investigated. Oral administration of CPS (1.5 or 3.0 mg/kg) or the corn oil vehicle was performed daily from postnatal days (PNDs) 1 through 6. No signs of overt toxicity or of cholinergic hyperstimulation were observed after CPS administration. Body weight was significantly different from controls on PND7 in both males and females exposed to 3.0 mg/kg CPS. Quantitative PCR was performed on the forebrain. The expression of NGF, reelin, and M(1) mAChR mRNA was significantly reduced with both dosages of CPS in both sexes. beta-III Tubulin mRNA expression remained unchanged after exposure, whereas MAG mRNA expression was significantly decreased with both dosages of CPS in both sexes, suggesting effects on the developing oligodendrocytes. In contrast, GFAP mRNA levels were significantly increased with both dosages of CPS in both sexes, suggesting increased astrocyte reactivity. Our findings indicate that dosages of CPS which cause significant cholinesterase inhibition but do not exert overt toxicity can adversely affect the expression levels of critical genes involved in brain development during the early postnatal period in the rat.

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.

Effect of electroacupuncture on brain-derived neurotrophic factor mRNA expression in mouse hippocampus following cerebral ischemia-reperfusion injury.

PubMed

Zhao, Jianxin; Xu, Huazhou; Tian, Yuanxiang; Hu, Manxiang; Xiao, Hongling

2013-04-01

This work aims to observe the effects of electroacupuncture on brain-derived neurotrophic factor (BDNF) mRNA expression in mouse hippocampus following cerebral ischemia-reperfusion injury. The models of mouse cerebral ischemia-reperfusion injury were established. A total of 96 healthy mice were randomly assigned into 4 groups, namely, the sham surgery, model, model + electroacupuncture, and mode + hydergine groups. Mice in the model + electroacupuncture group were treated through electroacupuncture at the Shenshu (BL 23), Geshu (BL 17), and Baihui (GV 20) acupoints. Mice in the model+hydergine group were intragastrically administered with hydergine (0.77 mg/kg(-1) x day(-1)). The levels of BDNF mRNA expressions in the hippocampus were ana lyzed through a semi-quantitative reverse transcription-polymerase chain reaction assay on days 1 and 7 after the surgeries. BDNF mRNA expressions in the mouse hippocampus of the model group on days 1 and 7 after the surgery were higher than those of the sham surgery group (both P < 0.01). On days 1 and 7 of the electroacupuncture treatment, BDNF mRNA expression in the mouse hippocampus of the model + electroacupuncture group was significantly elevated compared with the model group (both P < 0.01) or the model + hydergine group (both P < 0.01). On days 1 and 7 of the hydergine treatment, BDNF mRNA expression in the mouse hippocampus of the model + hydergine group tended to increase compared with the model group; however, statistical significance was not achieved (both P > 0.05). Electroacupuncture treatment enhances endogenous BDNF expression, which may improve the survival environment for intracerebral neurons and inhibit the apoptosis of hippocampal cells.

Validation of reference genes for gene expression studies in soybean aphid, Aphis glycines Matsumura

USDA-ARS?s Scientific Manuscript database

Quantitative real-time PCR (qRT-PCR) is a common tool for quantifying mRNA transcripts. To normalize results, a reference gene is mandatory. Aphis glycines is a significant soybean pest, yet gene expression and functional genomics studies are hindered by a lack of stable reference genes. We evalu...

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

Postnatal changes and sexual dimorphism in collagen expression in mouse skin

PubMed Central

Arai, Koji Y.; Hara, Takuya; Nagatsuka, Toyofumi; Kudo, Chikako; Tsuchiya, Sho; Nomura, Yoshihiro; Nishiyama, Toshio

2017-01-01

To investigate sexual dimorphism and postnatal changes in skin collagen expression, mRNA levels of collagens and their regulatory factors in male and female skin were examined during the first 120 days of age by quantitative realtime PCR. Levels of mRNAs encoding extracellular matrices did not show any differences between male and female mice until day 15. Col1a1 and Col1a2 mRNAs noticeably increased at day 30 and remained at high levels until day 120 in male mice, while those in female mice remained at low levels during the period. Consistent with the mRNA expression, pepsin-soluble type I collagen contents in skin was very high in mature male as compared to female. Col3a1 mRNA in male mice also showed significantly high level at day 120 as compared to female. On the other hand, expression of mRNAs encoding TGF-ßs and their receptors did not show apparent sexual dimorphism although small significant differences were observed at some points. Castration at 60 days of age resulted in a significant decrease in type I collagen mRNA expression within 3 days, and noticeably decreased expression of all fibril collagen mRNAs examined within 14 days, while administration of testosterone tube maintained the mRNA expression at high levels. Despite the in vivo effect of testosterone, administration of physiological concentrations of testosterone did not affect fibril collagen mRNA expression in either human or mouse skin fibroblasts in vitro, suggesting that testosterone does not directly affect collagen expression in fibroblasts. In summary, present study demonstrated dynamic postnatal changes in expression of collagens and their regulatory factors, and suggest that testosterone and its effects on collagen expression are responsible for the skin sexual dimorphism but the effects of testosterone is not due to direct action on dermal fibroblasts. PMID:28494009

Expression of myosin heavy chain isoforms mRNA transcripts in the temporalis muscle of common chimpanzees (Pan troglodytes).

PubMed

Ciurana, Neus; Artells, Rosa; Muñoz, Carmen; Arias-Martorell, Júlia; Bello-Hellegouarch, Gaëlle; Casado, Aroa; Cuesta, Elisabeth; Pérez-Pérez, Alejandro; Pastor, Juan Francisco; Potau, Josep Maria

2017-11-01

The common chimpanzee (Pan troglodytes) is the primate that is phylogenetically most closely related to humans (Homo sapiens). In order to shed light on the anatomy and function of the temporalis muscle in the chimpanzee, we have analyzed the expression patterns of the mRNA transcripts of the myosin heavy chain (MyHC) isoforms in different parts of the muscle. We dissected the superficial, deep and sphenomandibularis portions of the temporalis muscle in five adult P. troglodytes and quantified the expression of the mRNA transcripts of the MyHC isoforms in each portion using real-time quantitative polymerase chain reaction. We observed significant differences in the patterns of expression of the mRNA transcripts of the MyHC-IIM isoform between the sphenomandibularis portion and the anterior superficial temporalis (33.6% vs 47.0%; P=0.032) and between the sphenomandibularis portion and the anterior deep temporalis (33.6% vs 43.0; P=0.016). We also observed non-significant differences between the patterns of expression in the anterior and posterior superficial temporalis. The differential expression patterns of the mRNA transcripts of the MyHC isoforms in the temporalis muscle in P. troglodytes may be related to the functional differences that have been observed in electromyographic studies in other species of primates. Our findings can be applicable to the fields of comparative anatomy, evolutionary anatomy, and anthropology. Copyright © 2017 Elsevier GmbH. All rights reserved.

Efficacy of Omega Fatty Acid Supplementation on mRNA Expression Level of Tumor Necrosis Factor Alpha in Patients with Gastric Adenocarcinoma.

PubMed

Hosseinzadeh, Asghar; Ardebili, Seyed Mojtaba Mohaddes

2016-09-01

Tumor necrosis factor alpha (TNF-α), a multifunctional cytokine, is involved in apoptosis, cell proliferation, cell survival, and inflammation. It plays a dual role in cancer development and progression. It has been revealed that polyunsaturated fatty acids (PUFAs) modulate the production and activity of TNF family cytokines. The objective of the present study was to evaluate the effect of PUFAs on messenger RNA expression levels of TNF-α in patients with gastric adenocarcinoma. Thirty-four chemotherapy-naive patients diagnosed with gastric adenocarcinoma were randomly divided into two groups. The first group (17 individuals) received cisplatin without supplements and the second group (17 individuals) received cisplatin plus orally administered PUFA supplements for 3 weeks, based on treatment strategies. The gastric biopsy samples were obtained from all participants before and after treatment, and TNF-α mRNA expression levels were evaluated by quantitative real-time PCR procedure. Our findings revealed that TNF-α mRNA expression is downregulated in group II, after receiving cisplatin and omega fatty acid supplement for 3 weeks. However, this difference is not statistically significant (p > 0.05). TNF-α mRNA expression did not show significant alteration in group I, after receiving cisplatin alone. Taken together, we concluded that omega fatty acids reduce TNF-α expression at the mRNA level in patients with gastric adenocarcinoma. These data suggest that TNF-α may act as a potential target for the therapy of human gastric adenocarcinoma.

Inorganic arsenic exposure increased expression of Fas and Bax gene in vivo and vitro.

PubMed

He, Yuefeng; Zhang, Ruobing; Xiaoxiao, Song; Li, Shang; Xinan, Wu; Huang, Dahai

2018-06-01

Accumulating evidences have shown that apoptosis plays an important role in mediating the therapeutic effects and toxicity of arsenic. Fas and Bax genes are critical regulatory genes for apoptosis. In this study, we investigated the association between levels of Fas and Bax expression and the three arsenic species (inorganic arsenic (iAs), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) in vivo and vitro. Three arsenic species in urine were measured and levels of Fas and Bax expression were examined by the quantitative real-time PCR (qPCR) for all subjects. We found that Fas and Bax mRNA expression in the exposed group were significantly higher than that in the control group. The levels of gene expression were positively correlated with the concentrations of urinary iAs, MMA and DMA in all subjects. Sodium arsenite induced Fas and Bax mRNA expression, then MMA and DMA did not induce mRNA expression in MDA-MB-231 and XWLC-05 cells. The findings of the present study indicated that iAs, MMA, and DMA had different effects on expression of Bax and Fas gene. Copyright © 2017. Published by Elsevier B.V.

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

Effects of forced swimming stress on thyroid function, pituitary thyroid-stimulating hormone and hypothalamus thyrotropin releasing hormone expression in adrenalectomy Wistar rats.

PubMed

Sun, Qiuyan; Liu, Aihua; Ma, Yanan; Wang, Anyi; Guo, Xinhong; Teng, Weiping; Jiang, Yaqiu

2016-11-01

In order to study the impact that is imposed on the hypothalamic-pituitary-thyroid (HPT) axis of adrenalectomy male Wistar rats by stress caused by swimming, the blood level of triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH), the expression of TSHβ mRNA at the pituitary and thyrotropin releasing hormone (TRH) expression at the paraventricular nucleus (PVN) were measured. A total of 50 male Wistar rats of 6-8 weeks of age and with an average weight of 190-210 grams were randomly divided into the following two groups: The surgical (without adrenal glands) and non-surgical (adrenalectomy) group. These two groups were then divided into the following five groups, according to the time delay of sacrifice following forced swim (10 min, 2 h, 12 h and 24 h) and control (not subjected to swimming) groups. A bilateral adrenalectomy animal model was established. Serum TSH in the blood was measurement by chemiluminescent immunoassay, and cerebrum tissue were excised for the measurement of TRH expression using an immunohistochemistry assay. In addition, pituitaries were excised for the extraction of total RNA. Finally, reverse transcription-quantitative polymerase chain reaction was performed for quantitation of TSHβ. Following swimming, the serum T3, T4 and TSH, the TSHβ mRNA expression levels in the pituitary and the TRH expression in the PVN of the surgical group were gradually increased. In the non-surgical group, no significant differences were observed in the serum T3, T4 and TSH levels compared with the control group. The TSHβ mRNA expression at the pituitary showed a similar result. Furthermore, the TRH expression at PVN was gradually increased and stress from swimming could increase the blood T4, T3 and TSH levels, TSHβ mRNA expression at the pituitary and TRH expression at the PVN in adrenalectomy Wistar rats. Moreover, the index in the surgical group changed significantly compared with the non-surgical group. In conclusion, the results suggest that there is a positive correlation between stress from forced swimming and the variation of the HPT axis.

Different expression profiles of the lysyl oxidases and matrix metalloproteinases in human ACL fibroblasts after co-culture with synovial cells.

PubMed

Wang, Chunli; Xu, Chunming; Chen, Rongfu; Yang, Li; Sung, Kl Paul

2018-02-12

Purposes The anterior cruciate ligament (ACL) has poor functional healing response. The synovial tissue surrounding ACL ligament might be a major regulator of the microenvironment in the joint cavity after ACL injury, thus affecting the repair process. Using transwell co-culture, this study explored the direct influence of human synovial cells (HSCs) on ACL fibroblasts (ACLfs) by characterizing the differential expression of the lysyl oxidase family (LOXs) and matrix metalloproteinases (MMP-1, -2, -3), which facilitate extracellular matrix (ECM) repair and degradation, respectively. Methods The mRNA expression levels of LOXs and MMP-1, -2, -3 were analyzed by semi-quantitative PCR and quantitative real-time PCR. The protein expression levels of LOXs and MMP-1, -2, -3 were detected by western blot. Results We found that co-culture resulted in an increase in the mRNAs of LOXs in normal ACLfs and differentially regulated the expression of MMPs. Then we applied 12% mechanical stretch on ACLfs to induce injury and found the mRNA expression levels of LOXs in injured ACLfs were decreased in the co-culture group relative to the mono-culture group. Conversely, the mRNA expression levels of MMPs in injured ACLfs were promoted in the co-culture group compared with the mono-culture group. At translational level, we found that LOXs were lower while MMPs were highly expressed in the co-culture group compared to the mono-culture group. Conclusions The co-culture of ACLfs and HSCs, which mimicked the cell-to-cell contact in a micro-environment, could contribute to protein modulators for wound healing, inferring the potential reason for the poor self-healing of injured ACL.

Eotaxin/CCL11 expression by infiltrating macrophages in rat heart transplants during ongoing acute rejection.

PubMed

Zweifel, Martin; Mueller, Christoph; Schaffner, Thomas; Dahinden, Clemens; Matozan, Katja; Driscoll, Robert; Mohacsi, Paul

2009-10-01

Eotaxin/CCL11 chemokine is expressed in different organs, including the heart, but its precise cellular origin in the heart is unknown. Eotaxin is associated with Th2-like responses and exerts its chemotactic effect through the chemokine receptor-3 (CCR3), which is also expressed on mast cells (MC). The aim of our study was to find the cellular origin of eotaxin in the heart, and to assess whether expression is changing during ongoing acute heart transplant rejection, indicating a correlation with mast cell infiltration which we observed in a previous study. In a model of ongoing acute heart transplant rejection in the rat, we found eotaxin mRNA expression within infiltrating macrophages, but not in mast cells, by in situ-hybridization. A five-fold increase in eotaxin protein in rat heart transplants during ongoing acute rejection was measured on day 28 after transplantation, compared to native and isogeneic control hearts. Eotaxin concentrations in donor hearts on day 28 after transplantation were significantly higher compared to recipient hearts, corroborating an origin of eotaxin from cells within the heart, and not from the blood. The quantitative comparison of eotaxin mRNA expression between native hearts, isografts, and allografts, respectively, revealed no statistically significant difference after transplantation, probably due to an overall increase in the housekeeping gene's 18S rRNA during rejection. Quantitative RT-PCR showed an increase in mRNA expression of CCR3, the receptor for eotaxin, during ongoing acute rejection of rat heart allografts. Although a correlation between increasing eotaxin expression by macrophages and mast cell infiltration is suggestive, functional studies will elucidate the role of eotaxin in the process of ongoing acute heart transplant rejection.

Effects of ACTH, dexamethasone, and adrenalectomy on 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) gene expression in the rat central nervous system

PubMed Central

Ye, Ping; Kenyon, Christopher J; MacKenzie, Scott M; Nichol, Katherine; Seckl, Jonathan R; Fraser, Robert; Connell, John M C; Davies, Eleanor

2008-01-01

Using a highly sensitive quantitative RT-PCR method for the measurement of CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) mRNAs, we previously demonstrated that CYP11B2 expression in the central nervous system (CNS) is subject to regulation by dietary sodium. We have now quantified the expression of these genes in the CNS of male Wistar Kyoto (WKY) rats in response to systemic ACTH infusion, dexamethasone infusion, and to adrenalectomy. CYP11B1 and CYP11B2 mRNA levels were measured in total RNA isolated from the adrenal gland and discrete brain regions using real-time quantitative RT-PCR. ACTH infusion (40 ng/day for 7 days, N=8) significantly increased CYP11B1 mRNA in the adrenal gland, hypothalamus, and cerebral cortex compared with animals infused with vehicle only. ACTH infusion decreased adrenal CYP11B2 expression but increased expression in all of the CNS regions except the cortex. Dexamethasone (10 μg/day for 7 days, N=8) reduced adrenal CYP11B1 mRNA compared with control animals but had no significant effect on either gene's expression in the CNS. Adrenalectomy (N=6 per group) significantly increased CYP11B1 expression in the hippocampus and hypothalamus and raised CYP11B2 expression in the cerebellum relative to sham-operated animals. This study confirms the transcription of CYP11B1 and CYP11B2 throughout the CNS and demonstrates that gene transcription is subject to differential regulation by ACTH and circulating corticosteroid levels. PMID:18252953

The mRNA level of MLH1 in peripheral blood is a biomarker for the diagnosis of hereditary nonpolyposis colorectal cancer.

PubMed

Yu, Hong; Li, Hui; Cui, Yongan; Xiao, Wei; Dai, Guihong; Huang, Junxing; Wang, Chaofu

2016-01-01

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by functional defects in mismatch repair (MMR) genes, including mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2). This study aimed to assess whether the mRNA expression of MLH1 in peripheral blood could be used as a biomarkers for the diagnosis of HNPCC. The mRNA level of MLH1 was determined in 19 HNPCC families (46 members) using real-time quantitative polymerase chain reaction (qPCR). The mRNA levels of MLH1 in HNPCC were significantly lower than controls (P < 0.001). Receiver operating characteristic (ROC) curve showed a high diagnostic value of the mRNA level of MLH1 for the diagnosis of HNPCC with the area under curve of 0.858. At an optimal cut-off value (0.511), the mRNA level of MLH1 had a sensitivity of 81.3% and a specificity of 86.7% for distinguishing HNPCC from controls. In conclusion, the mRNA expression of MLH1 in peripheral blood may serve as a biomarker for the diagnosis of HNPCC.

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

PubMed

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

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.

Leptin receptor mRNA in rat brain astrocytes

PubMed Central

Hsuchou, Hung; Pan, Weihong; Barnes, Maria J.; Kastin, Abba J.

2009-01-01

We recently reported that mouse astrocytes express leptin receptors (ObR), and that obesity induces upregulation of astrocytic ObR. To provide further evidence of the importance of astrocytic ObR expression, we performed double-labeling fluorescent in-situ hybridization (FISH) and immunohistochemistry in the rat hypothalamus. Laser confocal microscopic image analysis showed that ObR mRNA was present in glial fibrillary acidic protein (+) cells that show distinctive astrocytic morphology as well as in neurons. In addition to the presence of ObR mRNA, ObR protein was shown in both astrocytes and neurons in the rat hypothalamus by double-labeling immunohistochemistry. In cultured rat C6 astrocytoma cells treated with different doses of lipopolysaccharide for 6 h, the mRNA for ObRa or ObRb did not show significant changes, as measured by quantitative RT-PCR. However, the protein expression of both ObRa and ObRb, determined by western blotting, was increased after the C6 cells were treated with either lipopolysaccharide or tumor necrosis factor-α. The results indicate that astrocytic ObR expression is present in rats as well as mice, and that it probably plays a role in the neuroinflammatory response. PMID:19747514

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.

Decreased expression of fibulin-4 in aortic wall of aortic dissection.

PubMed

Huawei, P; Qian, C; Chuan, T; Lei, L; Laing, W; Wenlong, X; Wenzhi, L

2014-02-01

In this research, we will examine the expression of Fibulin-4 in aortic wall to find out its role in aortic dissection development. The samples of aortic wall were obtained from 10 patients operated for acute ascending aortic dissection and five patients for chronic ascending aortic dissection. Another 15 pieces of samples from patients who had coronary artery bypass were as controls. The aortic samples were stained with aldehyde magenta dyeing to evaluate the arrangement of elastic fibers. The Fibulin-4 protein and mRNA expression were both determined by Western blot and realtime quantitative polymerase chain reaction. Compared with the control group, both in acute and chronic ascending aortic dissection, elastic fiber fragments increased and the expression of fibulin-4 protein significantly decreased (P= 0.045 < 0.05). The level of fibulin-4 mRNA decreased in acute ascending aortic dissection (P= 0.034 < 0.05), while it increased in chronic ascending aortic dissection (P=0.004 < 0.05). The increased amounts of elastic fiber fragments were negatively correlated with the expression of fibulin-4 mRNA in acute ascending aortic dissection. In conclusion, in aortic wall of ascending aortic dissection, the expression of fibulin-4 protein decreased and the expression of fibulin-4 mRNA was abnormal. Fibulin-4 may play an important role in the pathogenesis of aortic dissection.

Subchronic exposure to arsenic through drinking water alters expression of cancer-related genes in rat liver.

PubMed

Cui, Xing; Li, Song; Shraim, Amjad; Kobayashi, Yayoi; Hayakawa, Toru; Kanno, Sanae; Yamamoto, Megumi; Hirano, Seishiro

2004-01-01

Although arsenic exposure causes liver disease and/or hepatoma, little is known about molecular mechanisms of arsenic-induced liver toxicity or carcinogenesis. We investigated the effects of arsenic on expression of cancer-related genes in a rat liver following subchronic exposure to sodium arsenate (1, 10, 100 ppm in drinking water), by using real-time quantitative RT-PCR and immunohistochemical analyses. Arsenic accumulated in the rat liver dose-dependently and caused hepatic histopathological changes, such as disruption of hepatic cords, sinusoidal dilation, and fatty infiltration. A 1-month exposure to arsenic significantly increased hepatic mRNA levels of cyclin D1 (10 ppm), ILK (1 ppm), and p27(Kip1) (10 ppm), whereas it reduced mRNA levels of PTEN (1 ppm) and beta-catenin (100 ppm). In contrast, a 4-month arsenic exposure showed increased mRNA expression of cyclin D1 (100 ppm), ILK (1 ppm), and p27(Kip1) (1 and 10 ppm), and decreased expression of both PTEN and beta-catenin at all 3 doses. An immunohistochemical study revealed that each protein expression accords closely with each gene expression of mRNA level. In conclusion, subchronic exposure to inorganic arsenate caused pathological changes and altered expression of cyclin D1, p27(Kip1), ILK, PTEN, and beta-catenin in the liver. This implies that arsenic liver toxicity involves disturbances of some cancer-related molecules.

Sarcomeric Myosin Expression in the Tongue Body of Humans, Macaques and Rats

PubMed Central

Rahnert, Jill A.; Sokoloff, Alan J.; Burkholder, Thomas J.

2010-01-01

Expression of developmental and unconventional myosin heavy chain (MHC) isoforms in some adult head and neck muscles is thought to reflect specific contractile demands of muscle fibers active during kinematically complex movements. Mammalian tongue muscles are active during oromotor behaviors that encompass a wide range of tongue movement speeds and tongue shape changes (e.g. respiration, oral transport, swallowing, rejection), but the extent to which tongue muscles express developmental and unconventional MHC is not known. Quantitative PCR was used to determine the mRNA content of conventional MHC-beta, MHC-2a, MHC-2b and MHC-2x, the developmental isoforms embryonic MHC and neonatal MHC and the unconventional isoforms atrial/cardiac-α MHC (MHC-alpha), extraocular MHC, masseter MHC and slow tonic MHC in tongue body muscles of the rat, macaque and human. In all species, conventional MHC isoforms predominate. MHC-2b and MHC-2x account for 98% of total MHC mRNA in the rat. MHC-2a, MHC-2x and MHC-beta account for 94% of total MHC mRNA in humans and 96% of total MHC mRNA in macaque. With the exception of MHC-alpha in humans (5%), developmental and unconventional MHC mRNA represents less than 0.3% of total MHC mRNA. We conclude that in these species, there is limited expression of developmental and unconventional MHC and that diversity of tongue body muscle fiber contractile properties is achieved primarily by MHC-beta, MHC-2a, MHC-2x and MHC-2b. Whether expression of MHC-alpha mRNA in tongue is unique to humans or present in other hominoids awaits further investigation. PMID:19907142

Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia

PubMed Central

Scarr, Elizabeth; Udawela, Madhara; Greenough, Mark A; Neo, Jaclyn; Suk Seo, Myoung; Money, Tammie T; Upadhyay, Aradhana; Bush, Ashley I; Everall, Ian P; Thomas, Elizabeth A; Dean, Brian

2016-01-01

Our expression microarray studies showed messenger RNA (mRNA) for solute carrier family 39 (zinc transporter), member 12 (SLC39A12) was higher in dorsolateral prefrontal cortex from subjects with schizophrenia (Sz) in comparison with controls. To better understand the significance of these data we ascertained whether SLC39A12 mRNA was altered in a number of cortical regions (Brodmann’s area (BA) 8, 9, 44) from subjects with Sz, in BA 9 from subjects with mood disorders and in rats treated with antipsychotic drugs. In addition, we determined whether inducing the expression of SLC39A12 resulted in an increased cellular zinc uptake. SLC39A12 variant 1 and 2 mRNA was measured using quantitative PCR. Zinc uptake was measured in CHO cells transfected with human SLC39A12 variant 1 and 2. In Sz, compared with controls, SLC39A12 variant 1 and 2 mRNA was higher in all cortical regions studied. The were no differences in levels of mRNA for either variant of SLC39A12 in BA 9 from subjects with mood disorders and levels of mRNA for Slc39a12 was not different in the cortex of rats treated with antipsychotic drugs. Finally, expressing both variants in CHO-K1 cells was associated with an increase in radioactive zinc uptake. As increased levels of murine Slc39a12 mRNA has been shown to correlate with increasing cellular zinc uptake, our data would be consistent with the possibility of a dysregulated zinc homeostasis in the cortex of subjects with schizophrenia due to altered expression of SLC39A12. PMID:27336053

Increased Expression of Interleukin-18 mRNA is Associated with Carotid Artery Stenosis

PubMed

Arapi, Berk; Bayoğlu, Burcu; Cengiz, Müjgan; Dirican, Ahmet; Deser, Serkan Burç; Junusbekov, Yerik; Arslan, Caner

2018-05-29

Carotid artery stenosis is the atherosclerotic narrowing of the proximal internal carotid artery and one of the primary causes of stroke. Elevated expression of the pleiotropic proinflammatory cytokine interleukin-18 has been demonstrated in human atherosclerotic plaques. To investigate whether the mRNA expression levels of interleukin-18 and interleukin-18-binding protein and interleukin-18 −137 G/C (rs187238) variants are associated with carotid artery stenosis development. Case-control study. The mRNA expression levels of interleukin-18 and interleukin-18-binding protein and interleukin-18 rs187238 variants were evaluated by quantitative real-time polymerase chain reaction and real-time polymerase chain reaction, respectively, in the peripheral blood mononuclear cells of 70 patients with carotid artery stenosis (36 symptomatic, 34 asymptomatic) and 75 healthy controls. Interleukin-18 mRNA expression was significantly increased in carotid artery stenosis patients compared to that in healthy controls (p=0.01). However, no significant difference was observed between interleukin-18-binding protein mRNA expression levels in patients with carotid artery stenosis and those in controls (p=0.101). Internal carotid artery stenosis severity was significantly higher in symptomatic patients than that in asymptomatic patients (p<0.001). A significant relationship was identified between interleukin-18 expression and internal carotid artery stenosis severity in patients with carotid artery stenosis (p=0.051). Interleukin-18 rs187238 polymorphism genotype frequencies did not significantly differ between patients with carotid artery stenosis and controls (p=0.246). A significant difference was identified between interleukin-18-binding protein gene expression and symptomatic and asymptomatic patients (p=0.026), but there was no difference in interleukin-18 expression between the symptomatic and asymptomatic subgroups (p=0.397). Interleukin-18 mRNA expression may affect carotid artery stenosis etiopathogenesis and internal carotid artery stenosis severity and also may play a mechanistic role in the pathogenesis of carotid artery stenosis, influencing the appearance of symptoms.

Diagnostic performance of HPV E6/E7 mRNA assay for detection of cervical high-grade intraepithelial neoplasia and cancer among women with ASCUS Papanicolaou smears.

PubMed

Ren, Chenchen; Zhu, Yuanhang; Yang, Li; Zhang, Xiaoan; Liu, Ling; Ren, Chunying

2018-02-01

The aim of this study was to investigate the clinical performance of high risk (HR) HPV E6/E7 mRNA assay in detecting cervical high-grade intraepithelial neoplasia and cancer among women with atypical squamous cells of undetermined significance (ASCUS) Papanicolaou (Pap) smears. A total of 160 patients with ASCUS who underwent HR-HPV DNA assay, HR-HPV E6/E7 mRNA assay and colposcopy biopsy at Third Affiliated Hospital of Zhengzhou University, China, from December 2015 to March 2017, were enrolled. Logistic regression analysis was used to evaluate the relationship between pathological results with clinical biologic factors. Univariate analysis showed that the qualitative results of HR-HPV DNA, qualitative results of HR-HPV E6/E7 mRNA and expression levels of HR-HPV E6/E7 mRNA were risk factors of high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer (all P < 0.05). Multivariable analysis found that only the expression levels of HR-HPV E6/E7 mRNA was associated with high-grade CIN and cervical cancer (OR = 8.971, 95% CI = 2.572-31.289, P = 0.001). An optimal cut-off value of ≥ 558.26 copies/ml was determined using receiver operating characteristic curve, and specificity of cut-off value were higher than E6/E7 mRNA qualitative assay and DNA qualitative assay. HPV E6/E7 mRNA quantitative assay may be a valuable tool in triage of ASCUS pap smears. A high specificity of E6/E7 mRNA quantitative assay as a triage test in women with ASCUS can be translated into a low referral for colposcopy.

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

Regulation of LPS-induced mRNA expression of pro-inflammatory cytokines via alteration of NF-κB activity in mouse peritoneal macrophages exposed to fluoride.

PubMed

Tian, Yuhu; Huo, Meijun; Li, Guangsheng; Li, Yanyan; Wang, Jundong

2016-10-01

F toxicity to immune system, especially to macrophage, has been studied a lot recently. Nuclear factor-kappa B (NF-κB), as a transcription factor, plays a central role in immune and inflammatory responses via the regulation of downstream gene expression. Recent studies indicated that fluoride effect on inflammatory cytokine secretion, however, the molecular mechanism was less understood. In our study, peritoneal macrophages (PMs) were divided several groups and were administrated sodium fluoride (NaF, 50, 100, 200, 400, 800 μM) and/or lipopolysaccharide (LPS, 30 ng/mg). The mRNA expression of p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in macrophages exposed to fluoride was determined by quantitative real-time RT-PCR respectively. The translocation of NF-κB from cytoplasm to nucleus, which in a way reflects NF-κB activity, was demonstrated by Immunofluorescence and ELISA. Our results showed that fluoride had a dose-dependent effect on NF-κB activity, which coincided with LPS-induced mRNA expression of its downstream genes, iNOS and IL-1β. Fluoride alone causes no effect on gene expression. However, the mRNA expression of TNF-α showed non-NF-κB-dependent manner. Therefore, we come to the conclusion that fluoride can regulate LPS-induced mRNA expression of iNOS and IL-1β via NF-κB pathway in mouse peritoneal macrophages. Copyright © 2016 Elsevier Ltd. All rights reserved.

Influence of A-21T and C-262T genetic polymorphisms at the promoter region of the catalase (CAT) on gene expression.

PubMed

Saify, Khyber; Saadat, Iraj; Saadat, Mostafa

2016-09-01

Catalase (CAT, OMIM: 115500) is one of the major antioxidant enzymes, which plays an important role in the clearance of reactive oxygen species. Three genetic polymorphisms of A-21T (rs7943316), C-262T (rs1001179), and C-844T (rs769214) in the promoter region of the CAT have been reported. It has been suggested that these polymorphisms may alter the recognition sites of transcriptional factors, therefore it might be concluded that these polymorphisms may alter the expression levels of the gene. The aim of the present study is to evaluate the associations between these genetic variations and the CAT mRNA levels in human peripheral blood cells. The present study consisted of 47 healthy students of Shiraz University (south-west Iran). Genotypes of the CAT polymorphisms were determined by PCR based method. The quantitative CAT mRNA expression levels were investigated using quantitative real-time PCR. Analysis of variance revealed significant differences between the study genotypes (For A-21T polymorphism: F = 7.45; df = 2, 44; P = 0.002; For C-262T polymorphism: F = 15.17; df = 2, 44; P < 0.001). The studied polymorphisms showed linkage disequilibrium (D' = 1.0, r 2  = 0.1813, χ 2  = 17.03, P < 0.0001). The mRNA levels of CAT in the AC/TT, TC/TC, TC/TT, and TC/TC diplotypes significantly were higher than the mRNA levels in AC/AC diplotype. There was a significant difference between the study genotypes (F = 9.24; df = 5, 41; P < 0.001). The TC/TC and TT/TT diplotypes showed about 2 and 4 folds CAT mRNA levels compared with the AC/AC diplotype. The present findings indicated that these polymorphisms were significantly associated with the gene expression.

Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes.

PubMed

Mogal, Ashish; Abdulkadir, Sarki A

2006-04-01

In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

Aging alters mRNA expression of amyloid transporter genes at the blood-brain barrier.

PubMed

Osgood, Doreen; Miller, Miles C; Messier, Arthur A; Gonzalez, Liliana; Silverberg, Gerald D

2017-09-01

Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aβ) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aβ transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aβ receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aβ accumulation. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Trefoil factors: Tumor progression markers and mitogens via EGFR/MAPK activation in cholangiocarcinoma

PubMed Central

Kosriwong, Kanuengnuch; Menheniott, Trevelyan R; Giraud, Andrew S; Jearanaikoon, Patcharee; Sripa, Banchob; Limpaiboon, Temduang

2011-01-01

AIM: To investigate trefoil factor (TFF) gene copy number, mRNA and protein expression as potential biomarkers in cholangiocarcinoma (CCA). METHODS: TFF mRNA levels, gene copy number and protein expression were determined respectively by quantitative reverse transcription polymerase chain reaction (PCR), quantitative PCR and immunohistochemistry in bile duct epithelium biopsies collected from individuals with CCA, precancerous bile duct dysplasia and from disease-free controls. The functional impact of recombinant human (rh)TFF2 peptide treatment on proliferation and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling was assessed in the CCA cell line, KMBC, by viable cell counting and immunoblotting, respectively. RESULTS: TFF1, TFF2 and TFF3 mRNA expression was significantly increased in CCA tissue compared to disease-free controls, and was unrelated to gene copy number. TFF1 immunoreactivity was strongly increased in both dysplasia and CCA, whereas TFF2 immunoreactivity was increased only in CCA compared to disease-free controls. By contrast, TFF3 immunoreactivity was moderately decreased in dysplasia and further decreased in CCA. Kaplan-Meier analysis found no association of TFF mRNA, protein and copy number with age, gender, histological subtype, and patient survival time. Treatment of KMBC cells with rhTFF2 stimulated proliferation, triggered phosphorylation of EGFR and downstream extracellular signal related kinase (ERK), whereas co-incubation with the EGFR tyrosine kinase inhibitor, PD153035, blocked rhTFF2-dependent proliferation and EGFR/ERK responses. CONCLUSION: TFF mRNA/protein expression is indicative of CCA tumor progression, but not predictive for histological sub-type or survival time. TFF2 is mitogenic in CCA via EGFR/MAPK activation. PMID:21472131

Molecular cloning, characterization, tissue distribution and mRNA expression changes during the hibernation and reproductive periods of estrogen receptor alpha (ESR1) in Chinese alligator, Alligator sinensis.

PubMed

Zhang, Ruidong; Hu, Yuehong; Wang, Huan; Yan, Peng; Zhou, Yongkang; Wu, Rong; Wu, Xiaobing

2016-10-01

Chinese alligator, Alligator sinensis, is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha (ERα/ESR1) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period (P<0.05) in cerebellum, pituitary gland, liver, spleen, lung, kidney and ovary, while no significant change in other examined tissues (P>0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles. Copyright © 2016 Elsevier Inc. All rights reserved.

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 cyclooxygenase-2 in the canine lower urinary tract with regard to the effects of gonadal status and gender.

PubMed

Ponglowhapan, S; Church, D B; Khalid, M

2009-05-01

As pituitary gonadotrophins can induce prostaglandin (PG) synthesis and receptors for LH and FSH are present in the canine lower urinary tract (LUT), the objectives of this study were to (i) investigate the expression of COX-2, a key rate-limiting enzyme in PG production, in the canine LUT and (ii) determine if COX-2 expression differs between gender, gonadal status (intact and gonadectomised) and LUT regions. Four regions (body and neck of the bladder as well as proximal and distal urethra) of the LUT were obtained from 20 clinically healthy dogs (5 intact males, 5 intact anoestrous females, 4 castrated males, 6 spayed females). In situ hybridization and immunohistochemistry were performed to determine the presence of COX-2 mRNA and protein, respectively. The mRNA and protein expression was semi-quantitatively assessed. The scoring system combined both the distribution and intensity of positive staining and was carried out separately on the three tissue layers (epithelium, sub-epithelial stroma and muscle) for each of four regions of the LUT. In comparison to intact dogs, lower expression (P<0.001) of COX-2 and its mRNA in gonadectomised males and females was observed in all tissue layers of each region of the LUT except in the distal urethra where there was no difference in mRNA expression between gonadal statuses. Regardless of region and tissue layer, intact females expressed more (P<0.05) COX-2 and its mRNA than intact males. However, in gonadectomised dogs, mRNA expression of COX-2 did not differ between genders; males had higher (P<0.001) protein level of COX-2 compared to females. In conclusion, both COX-2 and its mRNA were expressed in the canine LUT and COX-2-regulated PG synthesis in the canine LUT may differ between gonadal statuses and genders. The lower expression of COX-2 in gonadectomised dogs may impair normal function of the LUT and probably implicated in the development of neutering-induced urinary incontinence in the dog.

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.

Identification of Suitable Reference Genes for mRNA Studies in Bone Marrow in a Mouse Model of Hematopoietic Stem Cell Transplantation.

PubMed

Li, H; Chen, C; Yao, H; Li, X; Yang, N; Qiao, J; Xu, K; Zeng, L

2016-10-01

Bone marrow micro-environment changes during hematopoietic stem cell transplantation (HSCT) with subsequent alteration of genes expression. Quantitative polymerase chain reaction (q-PCR) is a reliable and reproducible technique for the analysis of gene expression. To obtain more accurate results, it is essential to find a reference during HSCT. However, which gene is suitable during HSCT remains unclear. This study aimed to identify suitable reference genes for mRNA studies in bone marrow after HSCT. C57BL/6 mice were treated with either total body irradiation (group T) or busulfan/cyclophosphamide (BU/CY) (group B) followed by infusion of bone marrow cells. Normal mice without treatments were served as a control. All samples (group T + group B + control) were defined as group G. On days 7, 14, and 21 after transplantation, transcription levels of 7 candidate genes, ACTB, B2M, GAPDH, HMBS, HPRT, SDHA, and YWHAZ, in bone marrow cells were measured by use of real-time quantitative PCR. The expression stability of these 7 candidate reference genes were analyzed by 2 statistical software programs, GeNorm and NormFinder. Our results showed that ACTB displayed the highest expression in group G, with lowest expression of PSDHA in group T and HPRT in groups B and G. Analysis of expression stability by use of GeNorm or NormFinder demonstrated that expression of B2M in bone marrow were much more stable during HSCT, compared with other candidate genes including commonly used reference genes GAPDH and ACTB. ACTB could be used as a suitable reference gene for mRNA studies in bone marrow after HSCT. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

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

2005-01-01

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

Pleiotrophin Expression during Odontogenesis

PubMed Central

Ames, Jennifer E.; Tamkenath, Amena; Mamaeva, Olga; Stidham, Katherine; Wilson, Mary E.; Perez-Pinera, Pablo; Deuel, Thomas F.; MacDougall, Mary

2012-01-01

Pleiotrophin (PTN) is an extracellular matrix–associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) and odontoblast-like (M06-G3) and ameloblast-like (EOE-3M) cell lines were grown and samples prepared for immunocytochemistry, Western blot, and conventional and quantitative PCR analysis. Effects of BMP2, BMP4, and BMP7 treatment on PTN expression in odontoblast-like M06-G3 cells were tested by quantitative PCR. Finally, immunohistochemistry of sectioned mice mandibles and maxillaries at developmental stages E16, E18, P1, P6, P10, and P28 was performed. The experiments showed that PTN, at both the mRNA and protein level, was expressed in all tested epithelial and mesenchymal dental cell lines and that the level of PTN mRNA was influenced differentially by the bone morphogenetic proteins. The authors observed initial expression of PTN in the inner enamel epithelium with prolonged expression in the ameloblasts and odontoblasts throughout their stages of maturation and strong expression in the terminally differentiated and enamel matrix–secreting ameloblasts and odontoblasts of the adult mouse incisors and molars. PMID:22382872

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.

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

PubMed

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

2018-05-09

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

[Gene promoter methylation in glucose-6-phosphate dehydrogenase deficiency].

PubMed

Xu, Dan-Dan; Wen, Fei-Qiu; Lv, Rong-Yu; Zhang, Min; Chen, Yun-Sheng; Chen, Xiao-Wen

2016-05-01

To investigate the features of methylation in the promoter region of glucose-6-phosphate dehydrogenase (G6PD) gene and the association between gene promoter methylation and G6PD deficiency. Fluorescent quantitative PCR was used to measure the mRNA expression of G6PD in 130 children with G6PD deficiency. Sixty-five children without G6PD deficiency served as the control group. The methylation-sensitive high-resolution melting curve analysis and bisulfite PCR sequencing were used to analyze gene promoter methylation in 22 children with G6PD deficiency and low G6PD mRNA expression. The G6PD gene promoter methylation was analyzed in 44 girls with normal G6PD mRNA expression (7 from G6PD deficiency group and 37 from control group). Twenty-two (16.9%) children with G6PD deficiency had relatively low mRNA expression of G6PD; among whom, 16 boys showed no methylation, and 6 girls showed partial methylation. Among the 44 girls with normal G6PD mRNA expression, 40 showed partial methylation, and 4 showed no methylation (1 case in the G6PD group and 3 cases in the control group). Gene promoter methylation is not associated with G6PD deficiency in boys. Girls have partial methylation or no methylation in the G6PD gene, suggesting that the methylation may be related to G6PD deficiency in girls.

Effects of mycophenolate mofetil on proliferation and mucin-5AC expression in human conjunctival goblet cells in vitro.

PubMed

He, Hong; Ding, Hui; Liao, Aiping; Liu, Qiong; Yang, Jun; Zhong, Xingwu

2010-10-01

To investigate the effects of mycophenolate mofetil (MMF) on proliferation and mucin-5AC (MUC5AC) mRNA expression of normal human conjunctival goblet cells (CGCs) in vitro and to understand mechanisms of MMF in treatment of dry eye syndrome at molecular level. Purified human CGCs were treated with a series of graded concentrations of MMF after being confirmed by immunocytochemistry and flow cytometry. Proliferation and MUC5AC mRNA expression of CGCs were measured by Cell Count Kit-8 (CCK-8) and quantitative nested real-time reverse transcription polymerase chain reaction (QNRT-PCR at 24 h after treatment. The cell proliferation and MUC5AC mRNA expressiion were compared among different doses of MMF. MMF induced a dose-dependent upregulation of MUC5AC mRNA expression (F=238.851, p<0.01) but a biphase effect on proliferation of the CGCs over 24 h of co-incubation. This biphase effect manifested as a dose-dependent increase in cell numbers with MMF from 0.25 to 2.5 ng/ml, an unchanged population of the cells from 2.5 to 10 ng/ml and a reduced population of the cells from 25 to 100 ng/ml. MMF exerts biphase effects on cell regeneration and upregulates MUC5AC mRNA expression in CGCs in vitro. It appears that the use of MMF at low concentrations is attractive in dry eye (DE) treatment.

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.

Platelet-derived growth factor A mRNA in platelets is associated with the degree of hepatic fibrosis in chronic hepatitis C.

PubMed

Tanikawa, Aline Aki; Grotto, Rejane Maria Tommasini; Silva, Giovanni Faria; Ferrasi, Adriana Camargo; Sarnighausen, Valéria Cristina Rodrigues; Pardini, Maria Inês de Moura Campos

2017-01-01

Transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor (PDGF) are the main cytokines related to hepatic fibrogenesis. RNA isolated from the platelets and hepatic tissue of 43 HCV carriers was used for quantitative polymerase chain reaction to determine TGFB1, PDGFA, and PDGFB RNA expression. The mRNA expression of PDGFA in platelets was significantly lower in the group with advanced fibrosis than in the group with early-stage fibrosis. TGFB1 was more frequently expressed in platelets than in hepatic tissue, which was different from PDGFB. A pathway mediated by overexpression of TGFB1 via PDGFA in megakaryocytes could be involved in the development of fibrosis.

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.

Housekeeping gene expression during fetal brain development in the rat-validation by semi-quantitative RT-PCR.

PubMed

Al-Bader, Maie Dawoud; Al-Sarraf, Hameed Ali

2005-04-21

Mammalian gene expression is usually carried out at the level of mRNA where the amount of mRNA of interest is measured under different conditions such as growth and development. It is therefore important to use a "housekeeping gene", that does not change in relative abundance during the experimental conditions, as a standard or internal control. However, recent data suggest that expression of some housekeeping genes may vary with the extent of cell proliferation, differentiation and under various experimental conditions. In this study, the expression of various housekeeping genes (18S rRNA [18S], glyceraldehydes-3-phosphate dehydrogenase [G3PDH], beta-glucuronidase [BGLU], histone H4 [HH4], ribosomal protein L19 [RPL19] and cyclophilin [CY]) was investigated during fetal rat brain development using semi-quantitative RT-PCR at 16, 19 and 21 days gestation. It was found that all genes studied, with exception to G3PDH, did not show any change in their expression levels during development. G3PDH, on the other hand, showed increased expression with development. These results suggest that the choice of a housekeeping gene is critical to the interpretation of experimental results and should be modified according to the nature of the study.

Aquaporin-1 and aquaporin-3 expressions in the temporo-mandibular joint condylar cartilage after an experimentally induced osteoarthritis.

PubMed

Meng, Juan-hong; Ma, Xu-chen; Li, Zhi-min; Wu, Deng-cheng

2007-12-20

Over 70% of the total tissue weight in the cartilage matrix consists of water, and the early-stage osteoarthritic cartilage is characterized by swelling. Water transport in the cartilage matrix and across the membranes of chondrocytes may be important in normal and pathological conditions of cartilage. The purpose of this study was to identify aquaporin-1 (AQP1) and aquaporin-3 (AQP3) expressions in the mandibular condylar cartilage after experimentally induced osteoarthritis (OA) in rats. An experimental temporomandibular joint OA was induced by partial discectomy in rats. The pathological characteristics of the normal, early-stage, and late-stage osteoarthritic TMJ cartilages were verified by histological techniques. The AQP1 and AQP3 gene expressions in the normal and osteoarthritic cartilages were measured using quantitative real-time reverse-transcription PCR analysis. The cartilage sections were incubated in primary polyclonal antibodies to AQP3; immunofluorescent microscopy was used to examine the AQP3 expression shown by its protein level. The mRNA expression levels of AQP1 and AQP3, analyzed using quantitative PCR, revealed that AQP3 mRNA was highly up-regulated in the OA cartilage, which was considered significant. There was no notable difference in the expression of AQP1 mRNA between OA and normal controls. With the progressing of the OA, the localization of the AQP3 protein was quite different from that of the normal cartilage. Compared to the normal cartilage, the expressions of AQP3 protein were observed mainly in the proliferative zone and the upper mid-zone chondrocytes at the early-stage of OA, and were observed to appear frequently throughout the mid- and deep zone during the late-stage of OA. The high expression of AQP3 mRNA in the OA cartilage and the different localization of the AQP3 protein suggest that it may play a particular role in OA pathogenesis. Further study of AQP3 function may provide new insight into the understanding of the molecular mechanisms underlying OA.

Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

PubMed

Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2013-10-10

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots. Copyright © 2013 Elsevier B.V. All rights reserved.

Mitochondrial MnSOD mRNA expression in human chorioamniotic membranes and its association with labor, inflammation and infection

PubMed Central

Than, Nandor Gabor; Romero, Roberto; Tarca, Adi L.; Draghici, Sorin; Erez, Offer; Chaiworapongsa, Tinnakorn; Kim, Yeon Mee; Kim, Sun Kwon; Vaisbuch, Edi; Tromp, Gerard

2010-01-01

Objective Human parturition is characterized by the activation of genes involved in acute inflammatory in the fetal membranes. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that scavenges reactive oxygen species (ROS). MnSOD is up-regulated in sites of inflammation and has an important role in the down-regulation of acute inflammatory processes. Therefore, the aim of this study was to determine the differences in MnSOD mRNA expression in the fetal membranes in patients with term and preterm labor as well as in acute chorioamnionitis. Study design Fetal membranes were obtained from patients in the following groups: 1) term not in labor (n=29); 2) term in labor (n=29); 3) spontaneous preterm labor with intact mebranes (n=16); 4) PTL with histological chorioamnionitis (n=12); 5) preterm prelabor rupture of membranes (PPROM; n=17); and 6) PPROM with histological chorioamnionitis (n=21). MnSOD mRNA expression in the membranes was determined by quantitative real-time RT-PCR. Results 1) MnSOD mRNA expression was higher in the fetal membranes of patients at term in labor than those not in labor (2.4-fold; p=0.02); 2) the amount of MnSOD mRNA in the fetal membranes was higher in PTL than in term labor or in PPROM (7.2-fold, p=0.03; 3.2-fold, p=0.03, respectively); 3) MnSOD mRNA expression was higher when histological chorioamnionitis was present both among patients with PPROM (3.8-fold, p=0.02) and with PTL (5.4-fold, p=0.02) than in patients with these conditions without histological chorioamnionitis; 4) expression of MnSOD mRNA was higher in PTL with chorioamnionitis than in PPROM with chorioamnionitis (4.3-fold, p=0.03); Conclusion The increase in MnSOD mRNA expression by fetal membranes in term labor and in histological chorioamnionitis in PTL and PPROM suggests that the fetus deploys anti-oxidant mechanisms to constrain the inflammatory processes in the chorioamniotic membranes. PMID:19900038

Shikonin, an ingredient of Lithospermum erythrorhizon, down-regulates the expression of steroid sulfatase genes in breast cancer cells.

PubMed

Zhang, Yi; Qian, Rui-Qin; Li, Ping-Ping

2009-10-18

Steroid sulfatase (STS) has an important role in regulating the biosynthesis of estrogen within breast tumors. We aimed to investigate whether shikonin, an ingredient of Lithospermum erythrorhizon, could modulate STS expression in breast cancer cells. By MTT assay, shikonin inhibited the cell proliferation of breast cancer cells MCF-7 and SK-BR-3. Moreover, by semi-quantitative/quantitative reverse transcription polymerase chain reaction and dual-luciferase reporter based bioluminescent measurements, the mRNA and enzymatic activity levels of STS were decreased after shikonin treatment. Concluding, shikonin could act as a selective estrogen enzyme modulator by down-regulating the STS expression.

[Expression of connective tissue growth factor in colorectal cancer and its association with prognosis].

PubMed

Sun, Zheng; Yang, Ping; Liang, Li-yuan; Zhang, Tong; Zhang, Wei-jian; Cao, Jie

2012-11-01

To investigate the expression of connective tissue growth factor (CTGF) in colorectal cancer(CRC) and its association with clinicopathologic parameters and overall survival rate. Fresh tumor tissues and matched distal normal colon tissues were collected from 92 patients diagnosed as CRC by surgical operation. The expression level of CTGF mRNA was quantified by quantitative reverse transcription PCR. Thirty out of 92 pairs of tissue specimens were selected randomly to detect CTGF protein by immunohistochemistry. All the cases were followed up to identify prognostic factors for survival. CTGF mRNA expression was up-regulated in CRC. The positive rate of CTGF protein expression tissues (73.3%) was significantly higher than that in the corresponding normal tissues (23.3%, P<0.01). CTGF expression was lower in patients with lymphatic metastasis or stage III/IIII disease (all P<0.05). A negative association was also observed between the CTGF protein positive rate and tumor infiltration depth (P<0.05). The relative expression of CTGF mRNA in tumor tissues was classified into high and low expression groups. The 5-year cumulative survival rate was lower in patients with low CTGF expression (29.3%) as compared to those with high CTGF expressions (68.3%) (P<0.01). Cox regression analysis revealed that the relative expression level of CTGF was independent factor of overall survival (RR=2.960, 95%CI:1.491-1.587, P<0.01). ROC curve analysis showed that sensitivity and specificity of CTGF mRNA expression for prediction of 5-year survival were 64.9% and 74.5%, respectively. The aberrant expression of CTGF is associated with the malignant biological behaviors of CRC. Low expression of CTGF is associated with worse prognosis of CRC.

Regulation of. beta. -cell glucose transporter gene expression

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

Chen, Ling; Alam, Tausif; Johnson, J.H.

1990-06-01

It has been postulated that a glucose transporter of {beta} cells (GLUT-2) may be important in glucose-stimulated insulin secretion. To determine whether this transporter is constitutively expressed or regulated, the authors subjected conscious unrestrained Wistar rats to perturbations in glucose homeostasis and quantitated {beta}-cell GLUT-2 mRNA by in situ hybridization. After 3 hr of hypoglycemia, GLUT-2 and proinsulin mRNA signal densities were reduced by 25% of the level in control rats. After 4 days, GLUT-2 and proinsulin mRNA densities were reduced by 85% and 65%, respectively. After 12 days of hypoglycemia, the K{sub m} for 3-O-methyl-D-glucose transport in isolated ratmore » islets, normally 18-20 mM, was 2.5 mM. This provides functional evidence of a profound reduction of high K{sub m} glucose transporter in {beta} cells. In contrast, GLUT-2 was only slightly reduced by hypoglycemia in liver. To determine the effect of prolonged hyperglycemia, they also infused animals with 50% (wt/vol) glucose for 5 days. Hyperglycemic clamping increased GLUT-2 mRNA by 46% whereas proinsulin mRNA doubled. They conclude that GLUT-2 expression in {beta} cells, but not liver, is subject to regulation by certain perturbations in blood glucose homeostasis.« less

[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.

Genome Wide Meta-analysis Highlights the Role of Genetic Variation in RARRES2 in the Regulation of Circulating Serum Chemerin

PubMed Central

Breitfeld, Jana; Marzi, Carola; Grallert, Harald; Gross, Arnd; Ladenvall, Claes; Schleinitz, Dorit; Krause, Kerstin; Kirsten, Holger; Laurila, Esa; Kriebel, Jennifer; Thorand, Barbara; Rathmann, Wolfgang; Groop, Leif; Prokopenko, Inga; Isomaa, Bo; Beutner, Frank; Kratzsch, Jürgen; Thiery, Joachim; Fasshauer, Mathias; Klöting, Nora; Gieger, Christian; Blüher, Matthias; Stumvoll, Michael; Kovacs, Peter

2014-01-01

Chemerin is an adipokine proposed to link obesity and chronic inflammation of adipose tissue. Genetic factors determining chemerin release from adipose tissue are yet unknown. We conducted a meta-analysis of genome-wide association studies (GWAS) for serum chemerin in three independent cohorts from Europe: Sorbs and KORA from Germany and PPP-Botnia from Finland (total N = 2,791). In addition, we measured mRNA expression of genes within the associated loci in peripheral mononuclear cells by micro-arrays, and within adipose tissue by quantitative RT-PCR and performed mRNA expression quantitative trait and expression-chemerin association studies to functionally substantiate our loci. Heritability estimate of circulating chemerin levels was 16.2% in the Sorbs cohort. Thirty single nucleotide polymorphisms (SNPs) at chromosome 7 within the retinoic acid receptor responder 2 (RARRES2)/Leucine Rich Repeat Containing (LRRC61) locus reached genome-wide significance (p<5.0×10−8) in the meta-analysis (the strongest evidence for association at rs7806429 with p = 7.8×10−14, beta = −0.067, explained variance 2.0%). All other SNPs within the cluster were in linkage disequilibrium with rs7806429 (minimum r2 = 0.43 in the Sorbs cohort). The results of the subgroup analyses of males and females were consistent with the results found in the total cohort. No significant SNP-sex interaction was observed. rs7806429 was associated with mRNA expression of RARRES2 in visceral adipose tissue in women (p<0.05 after adjusting for age and body mass index). In conclusion, the present meta-GWAS combined with mRNA expression studies highlights the role of genetic variation in the RARRES2 locus in the regulation of circulating chemerin concentrations. PMID:25521368

Long-term treatment with haloperidol affects neuropeptide S and NPSR mRNA levels in the rat brain.

PubMed

Palasz, Artur; Rojczyk, Ewa; Golyszny, Milosz; Filipczyk, Lukasz; Worthington, John J; Wiaderkiewicz, Ryszard

2016-04-01

The brainstem-derived neuropeptide S (NPS) has a multidirectional regulatory activity, especially as a potent anxiolytic factor. Accumulating data suggests that neuroleptics affect peptidergic signalling in various brain structures. However, there is no information regarding the influence of haloperidol on NPS and NPS receptor (NPSR) expression. We assessed NPS and NPSR mRNA levels in brains of rats treated with haloperidol using quantitative real-time polymerase chain reaction. Chronic haloperidol treatment (4 weeks) led to a striking upregulation of NPS and NPSR expression in the rat brainstem. Conversely, the NPSR mRNA expression was decreased in the hippocampus and striatum. This stark increase of NPS in response to haloperidol treatment supports the hypothesis that this neuropeptide is involved in the dopamine-dependent anxiolytic actions of neuroleptics and possibly also in the pathophysiology of mental disorders. Furthermore, our findings underline the complex nature of potential interactions between dopamine receptors and brain peptidergic pathways, which has potential clinical applications.

Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stål).

PubMed

Vontas, J G; Small, G J; Hemingway, J

2000-12-01

Organophosphorus and carbamate insecticide resistance in Nilaparvata lugens is based on amplification of a carboxylesterase gene, Nl-EST1. An identical gene occurs in susceptible insects. Quantitative real-time PCR was used to demonstrate that Nl-EST1 is amplified 3-7-fold in the genome of resistant compared to susceptible planthoppers. Expression levels were similar to amplification levels, with 1-15-fold more Nl-EST1 mRNA in individual insects and 5-11-fold more Nl-EST1 mRNA in mass whole body homogenates of resistant females compared to susceptibles. These values corresponded to an 8-10-fold increase in esterase activity in the head and thorax of individual resistant insects. Although amplification, expression and activity levels of Nl-EST1 in resistant N. lugens were similar, the correlation between esterase activity and Nl-EST1 mRNA levels in resistant individuals was not linear.

The effects of splicing variant of PXR PAR-2 on CYP3A4 and MDR1 mRNA expressions.

PubMed

Liu, Yan; Ji, Wei; Yin, You; Fan, Lan; Zhang, Jian; Yun, Huang; Wang, Nianci; Li, Qing; Wei, Zhang; Ouyang, Dongshen; Zhou, Hong-Hao

2009-05-01

PAR-2(SV1), a splicing variant of PXR, has similar activity as PXR wild type. Currently, a 6bp-deletion variant ((-133)GAGAAG(-128)) in promoter region of PAR-2(SV1) was reported, which could diminish the hPAR-2 promote activity in HepG2 cells. The distribution and functions of 6bp-deletion in Chinese were investigated. The PXR genotype was analyzed from 56 liver samples and 177 blood samples. Then the mRNA expression of PAR-2(SV1), total PXR, CYP3A4 and MDR1 were quantitatively analyzed by real-time PCR. The allelic frequencies of 6bp-deletion were 22.4%, 38.4% and 23.7%, in blood of Chinese healthy (n=177), hepatic carcinoma samples (n=33) and calculus of bile duct ones (n=23) respectively. PAR-2(SV1) transcript represented approximately 15.3% of the total PXR transcripts in all liver samples. The 6bp-deletion cut down PAR-2(SV1) mRNA and total PXR mRNA transcriptional expression, and then led to down regulations of MDR1 and CYP3A4. PAR-2(SV1) plays an important role in total PXR mRNA expression. The 6bp-deletion affects the PAR-2(SV1) expression greatly, and then contributes to the adjustment of expression and function of total PXR. Thus it leads to the changed target gene expressions, which may partly explain interindividual variations in CYP3A4 and MDR1. And these phenomena suggest that individuals with 6bp-deletion are prone to carcinoma when exposed to toxicity.

[Expression of SLP-2 mRNA in endometrial cancer and its significance].

PubMed

Feng, Wang-qin; Cui, Zhu-mei; Feng, Feng-zhi; Qi, Xiu-juan; Ding, Fang; Li, Wen-dong; Liu, Zhi-hua

2005-08-01

To characterize the differential expression of SLP-2 in endometrial cancer, and to study the effect of human SLP-2 gene on human endometrial cancer cell line. The expression of SLP-2 gene in 32 cases of endometrial cancer and 28 cases of normal endometrial tissues was examined by semi-quantitative RT-PCR. Eukaryotic expression vectors of sense and antisense SLP-2 were constructed and transfected into HEC-1B cell line using lipofectamine 2000 respectively. The morphological changes of the cell were observed by phase contrast microscopy. The cell growth was detected by methyl thiazolyl tetrazolium (MTT) assay, and the cell cycles were analyzed by flow cytometry. The expression of SLP-2 mRNA in endometrial cancer tissues was higher than that in normal endometrial tissues (1.6 +/- 0.7 vs 0.7 +/- 0.3, P < 0.05). Sense and antisense human SLP-2 constructs were transfected into HEC-1B cell line respectively. After being transfected with sense SLP-2, the expression of SLP-2 mRNA in HEC-1B cell line was increased by about 2.4 times that of the control group, the cell growth was accelerated, and the number of cells in G(1) phase was decreased by 12.5%, S phase was increased by 8.0%. After being transfected with antisense SLP-2, the expression of SLP-2 mRNA was declined 50%. The transfected cells showed slower growth, and the number of cells in G(1) phase was significantly increased by 10.5%, and S phase was declined by 9.8%. SLP-2 mRNA shows up-regulation in endometrial cancer tissues, and it may have some relationship with carcinogenesis of endometrial cancer.

The relationship between deiodinase activity and inflammatory responses under the stimulation of uremic toxins.

PubMed

Xu, Gaosi; Tu, Weiping; Qin, Shulan

2014-08-31

It is unclear to what extent uremic toxins participate in inflammatory responses and the activities of deiodinases, as well as the effects of deiodinases on inflammatory cytokines. Hepatocellular carcinoma cell lines (HepG2) were transfected with small interfering ribonucleic acid (siRNA) specific for deiodinase type 1 (DIO1) and cultured with or without uremic toxins. The mRNA expression of DIO1, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α was detected by quantitative real-time PCR. The presence of selenoprotein M (SelM) and DIO1 was assessed by western blotting. Sonicate deiodinase activities in HepG2 cells were measured by a dithiothreitol-stimulated assay. The NF-κB, AP-1 and CREB-1 inflammatory signal pathways were confirmed by EMSA. After culturing for 24 h, the mRNA expression of DIO1 was significantly decreased by the specific siRNA (reduced by 76%, P = 0.0002). Uremic toxins significantly increased the mRNA expression (P < 0.01) of IL-1β, IL-6 and TNF-α and inhibited DIO1 mRNA expression (P < 0.01) compared with controls. Suppression of DIO1 by siRNA significantly decreased the mRNA expression of IL-1β and IL-6 (P < 0.05) but not TNF-α (P = 0.093). Uremic toxins and specific siRNA synchronously reduced the protein expression of SelM and DIO1. Uremic toxins activate the expression of inflammatory cytokines. The major findings of this study indicate that the uremic toxins, more than inflammatory cytokines, play direct inhibitory roles in DIO1 enzyme activity, which then provides a negative feedback to the growing accumulation of inflammatory cytokines.

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

PubMed

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

2001-11-01

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

Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops

PubMed Central

Meurens, François; Berri, Mustapha; Auray, Gael; Melo, Sandrine; Levast, Benoît; Virlogeux-Payant, Isabelle; Chevaleyre, Claire; Gerdts, Volker; Salmon, Henri

2009-01-01

Salmonella enterica subspecies enterica serovar Typhimurium, commonly called S. Typhimurium, can cause intestinal infections in humans and various animal species such as swine. To analyze the host response to Salmonella infection in the pig we used an in vivo gut loop model, which allows the analysis of multiple immune responses within the same animal. Four jejunal gut-loops were each inoculated with 3×108 cfu of S. Typhimurium in 3 one-month-old piglets and mRNA expressions of various cytokines, chemokines, transcription factors, antimicrobial peptides, toll like and chemokine receptors were assessed by quantitative real-time PCR in the Peyer’s patch and the gut wall after 24 h. Several genes such as the newly cloned CCRL1/CCX-CKR were assessed for the first time in the pig at the mRNA level. Pro-inflammatory and T-helper type-1 (Th1) cytokine mRNA were expressed at higher levels in infected compared to non-infected control loops. Similarly, some B cell activation genes, NOD2 and toll like receptor 2 and 4 transcripts were more expressed in both tissues while TLR5 mRNA was down-regulated. Interestingly, CCL25 mRNA expression as well as the mRNA expressions of its receptors CCR9 and CCRL1 were decreased both in the Peyer’s patch and gut wall suggesting a potential Salmonella strategy to reduce lymphocyte homing to the intestine. In conclusion, these results provide insight into the porcine innate mucosal immune response to infection with entero-invasive microorganisms such as S. Typhimurium. In the future, this knowledge should help in the development of improved prophylactic and therapeutic approaches against porcine intestinal S. Typhimurium infections. PMID:18922229

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

PubMed

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

2016-11-01

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

[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.

High Intensity High Volume Interval Training Improves Endurance Performance and Induces a Nearly Complete Slow-to-Fast Fiber Transformation on the mRNA Level.

PubMed

Eigendorf, Julian; May, Marcus; Friedrich, Jan; Engeli, Stefan; Maassen, Norbert; Gros, Gerolf; Meissner, Joachim D

2018-01-01

We present here a longitudinal study determining the effects of two 3 week-periods of high intensity high volume interval training (HIHVT) (90 intervals of 6 s cycling at 250% maximum power, P max /24 s) on a cycle ergometer. HIHVT was evaluated by comparing performance tests before and after the entire training (baseline, BSL, and endpoint, END) and between the two training sets (intermediate, INT). The mRNA expression levels of myosin heavy chain (MHC) isoforms and markers of energy metabolism were analyzed in M. vastus lateralis biopsies by quantitative real-time PCR. In incremental tests peak power (P peak ) was increased, whereas V ˙ O 2peak was unaltered. Prolonged time-to-exhaustion was found in endurance tests with 65 and 80% P max at INT and END. No changes in blood levels of lipid metabolites were detected. Training-induced decreases of hematocrit indicate hypervolemia. A shift from slow MHCI/β to fast MHCIIa mRNA expression occurred after the first and second training set. The mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a master regulator of oxidative energy metabolism, decreased after the second training set. In agreement, a significant decrease was also found for citrate synthase mRNA after the second training set, indicating reduced oxidative capacity. However, mRNA expression levels of glycolytic marker enzyme glyceraldehyde-3-phosphate dehydrogenase did not change after the first and second training set. HIHVT induced a nearly complete slow-to-fast fiber type transformation on the mRNA level, which, however, cannot account for the improvements of performance parameters. The latter might be explained by the well-known effects of hypervolemia on exercise performance.

[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.

[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.

Effect of the heat shock protein HSP27 on androgen receptor expression and function in prostate cancer cells.

PubMed

Stope, Matthias B; Schubert, Tina; Staar, Doreen; Rönnau, Cindy; Streitbörger, Andreas; Kroeger, Nils; Kubisch, Constanze; Zimmermann, Uwe; Walther, Reinhard; Burchardt, Martin

2012-06-01

Heat shock proteins (HSP) are involved in processes of folding, activation, trafficking and transcriptional activity of most steroid receptors including the androgen receptor (AR). Accumulating evidence links rising heat shock protein 27 (HSP27) levels with the development of castration-resistant prostate cancer. In order to study the functional relationship between HSP27 and the AR, we modulated the expression of the small heat shock protein HSP27 in human prostate cancer (PC) cell lines. HSP27 protein concentrations in LNCaP and PC-3 cells were modulated by over-expression or silencing of HSP27. The effects of HSP27 on AR protein and mRNA levels were monitored by Western blotting and quantitative RT-PCR. Treatment for the AR-positive LNCaP with HSP27-specific siRNA resulted in a down-regulation of AR levels. This down-regulation of protein was paralleled by a decrease in AR mRNA. Most interestingly, over-expression of HSP27 in PC-3 cells led to a significant increase in AR mRNA although the cells were unable to produce functional AR protein. The observation that HSP27 is involved in the regulation of AR mRNA by a yet unknown mechanism highlights the complexity of HSP27-AR signaling network.

A disintegrin and metalloproteinase 17 mRNA and protein expression in esophageal squamous cell carcinoma, as well as its clinicopathological factors and prognosis

PubMed Central

LIU, HONG-BIN; YANG, QI-CHANG; SHEN, YI; ZHU, YAN; ZHANG, XIAO-JUAN; CHEN, HAO

2015-01-01

The aim of the present study was to explore a disintegrin and metalloproteinase 17 (ADAM17) mRNA and protein expression in esophageal squamous cell carcinoma and its association with clinicopathological factors and prognosis. Through semi-quantitative reverse transcription polymerase chain reaction, the ADAM17 mRNA expression in 50 cases of esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were detected. Using streptavidin peroxidase conjugated immunohistochemistry, ADAM17 protein levels were detected in 80 cases of esophageal squamous cell carcinoma and corresponding normal esophageal mucosa. A log rank test and the Cox proportional hazards model were used for the esophageal cancer survival analysis. ADAM17 mRNA expression levels in esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were 0.937±0.241 and 0.225±0.077, respectively (P<0.01). ADAM17 mRNA expression in esophageal squamous cell carcinoma was correlated with lymph node metastasis (P<0.01) and tumor, node and metastasis (TNM) staging (P<0.05), however, it was not correlated with gender, age or histological grade (P>0.05). ADAM17 protein expression rates in esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were 66.25 and 6.25% respectively, a difference that was statistically significant (P<0.01). In addition, ADAM17 protein expression in esophageal squamous cells was correlated with lymph node metastasis and TNM stage (P<0.05), while it was not correlated with gender, age or histological grade (P>0.05). ADAM17 protein expression and epidermal growth factor receptor (EGFR) protein expression were positively correlated (P<0.01). Lymph node metastasis, TNM stage, ADAM17 and EGFR protein expression may be used as independent prognostic indicators of esophageal squamous cell carcinoma (all P<0.05). ADAM17 mRNA and protein were highly expressed in esophageal squamous cell carcinoma; they have important roles in invasion and metastasis and a certain value in judging the prognosis of patients with esophageal squamous cell carcinoma. PMID:25351873

Diphlorethohydroxycarmalol of Ishige okamurae and Caffeine Modified the Expression of Extracellular Fibrillars during Adipogenesis of Mouse Subcutaneous Adipose Derived Stem Cell

PubMed Central

Jeon, Younmi; Song, Siyoung; Kim, Hagju; Cheon, Yong-Pil

2013-01-01

Although, one of the etiologies of localized lipodystrophy of the subcutaneous connective tissue (cellulite) is the histological alternation of adipose tissue, the characteristics of expression of the components of extracellular matrix (ECM) components during adipogenesis are not uncovered. In this study, the effects of caffeine and Ishige okamurae originated diphlorethohydroxycarmalol (DPHC) on the expression of extracellualr fibers was analyzed with quantitative RT-PCR during differentiation induction of mouse subcutaneous adipose derived stem cells (msADSC) into adipocyte. The expression levels of Col1a, Col3a1, and Col61a were decreased by the adipogenci induction in a time-dependent manners. However, Col2a mRNA and Col4a1 mRNA expressions were oposit to them. Caffeine and DPHC stimulated the changes of the expression of these collagens. Eln mRNA expression was increased by induction. DPHC stimulated the expression of it. Mfap5 mRNA expression was deceased in both adipogenic cell and matured adipocytes. Caffeine suppressed the expression of Mfap5 but the effect of DPHC was different by the concentration. The expression of bioglycan, decorin, and lumican were also modified by caffeine and DPHC in a concentration-dependent manner. Based on this study, we revealed firstly the effects of caffeine and DPHC on the expression of collagens, elastin, and glycoproteins during adipogenesis of msADSCs. Those results suggest that DPHC may have antiadipogenic effect and has more positive effets on normal adipose tissue generation and work as suppressor the abnormality of ECM structure. Such results indicate that DPHC can be applied in keeping the stability of the ECM of adipogenic tissues. PMID:25949143

mRNA expression OF IFN-λs in the gingival tissue of chronic and aggressive periodontitis patients: A polymerase chain reaction study.

PubMed

Bilichodmath, Shivaprasad; Nair, Sruthi K; Bilichodmath, Rekha; Mangalekar, Sachin B

2018-05-01

Several studies have proven the existence of herpesviruses in periodontal pockets of chronic and aggressive periodontitis patients. Recently discovered interferon lambda (IFN-λ) has antiviral properties and is induced by herpesviruses. The present study was aimed at quantitative analysis of mRNA expression of IFN-λs (IFN-λ1, IFN-λ2, IFN-λ3) in the gingival tissues of chronic and aggressive periodontitis patients. A total of 90 participants (50 males and 40 females; age ranging from 19 to 50 years, mean age 31.50±7.8) were categorized into three groups as healthy participants, chronic periodontitis patients and aggressive periodontitis patients. mRNA expression of IFN-λs in gingival tissues was estimated using reverse transcriptase polymerase chain reaction and was correlated with clinical parameters such as gingival index (GI), probing pocket depth (PPD), and clinical attachment level (CAL). mRNA of IFN-λ1, IFN-λ2 and IFN-λ3 was expressed in gingival tissues of healthy participants, chronic and aggressive periodontitis patients. Highest level of IFN-λ1 was observed in aggressive periodontitis patients (3.049±9.793), whereas IFN-λ2 (4.322±11.310) and IFN-λ3 (11.932±27.479) were maximum in chronic periodontitis patients. The difference in the mRNA expression of IFN-λ1 (p = 0.008) and IFN-λ3 (p = 0.043) among three groups was statistically significant CONCLUSION: Increased quantity of IFN-λs in chronic and aggressive periodontitis patients suggests a role in periodontitis. Variation in the expression of IFN-λ1 and IFN-λ3 in periodontitis patients needs to be further evaluated. The mRNA expression of antiviral IFN-λs in gingival tissues might enhance our understanding related to viral pathogenesis of periodontal diseases. This article is protected by copyright. All rights reserved. © 2018 American Academy of Periodontology.

A non-inflammatory form of immune competence prevails in acute pre-pubescent malnutrition: new evidence based on critical mRNA transcripts in the mouse.

PubMed

Monk, Jennifer M; Richard, Cynthia L; Woodward, Bill

2012-05-01

The declining inflammatory immune competence of acute (i.e. wasting) pre-pubescent protein-energy malnutrition has been regarded as reflecting an unregulated immunological disintegration. Recent evidence, however, suggests that malnutrition stimulates a regulated immunological reconfiguration to achieve a non-inflammatory form of competence, perhaps offering protection against autoimmune reactions - the 'Tolerance Model'. Our objective was to determine the influence of acute pre-pubescent malnutrition on the expression of genes critical to tolerogenic regulation. Male and female C57BL/6J mice, initially 19 d old, consumed a complete purified diet either ad libitum (age-matched controls) or in restricted daily quantities (mimicking marasmus), or consumed an isoenergetic low-protein diet ad libitum (mimicking incipient kwashiorkor) for 14 d (six animals per dietary group). Gene expression in the spleen, typically an inflammatory organ, and in the small intestine, a site designed for non-inflammatory defence, was assessed by real-time quantitative RT-PCR, and normalised to β-actin. In the spleen of the malnourished groups, both IL-10 and transforming growth factor-β1 mRNA expression increased compared with controls (P < 0.05), whereas mRNA expression of IL-12p40 decreased (P < 0.05). Conversely, malnutrition exerted no influence on the expression of mRNA for these cytokines in the small intestine (P>0.05). Moreover, forkhead box P3 mRNA expression, indicative of cell-based tolerogenic potential, was sustained in both the spleen and intestine of the malnourished groups (P>0.05). Thus, despite limited supplies of energy and substrates, the spleen shifted towards a non-inflammatory character and the intestine was sustained in this mode in advanced pre-pubescent weight loss. These findings provide the first support for the Tolerance Model at the level of mRNA transcript expression.

Down-regulation of PRKCB1 expression in Han Chinese patients with subsyndromal symptomatic depression.

PubMed

Guo, Xiaoyun; Li, Zezhi; Zhang, Chen; Yi, Zhenghui; Li, Haozhe; Cao, Lan; Yuan, Chengmei; Hong, Wu; Wu, Zhiguo; Peng, Daihui; Chen, Jun; Xia, Weiping; Zhao, Guoqing; Wang, Fan; Yu, Shunying; Cui, Donghong; Xu, Yifeng; Golam, Chowdhury M I; Smith, Alicia K; Wang, Tong; Fang, Yiru

2015-10-01

Subsyndromal symptomatic depression (SSD) is a common disease with significant social dysfunction. However, SSD is still not well understood and the pathophysiology of it remains unclear. We classified 48 candidate genes for SSD according to our previous study into clusters and pathways using DAVID Bioinformatics Functional Annotation Tool. We further replicated the result by using real-time Quantitative PCR (qPCR) studies to examine the expression of identified genes (i.e., STAT5b, PKCB1, ABL1 and NRAS) in another group of Han Chinese patients with SSD (n = 50). We further validated the result by examining PRKCB1 expression collected from MDD patients (n = 20). To test whether a deficit in PRKCB1 expression leads to dysregulation in PRKCB1 dependent transcript networks, we tested mRNA expression levels for the remaining 44 genes out of 48 genes in SSD patients. Finally, the power of discovery was improved by incorporating information from Quantitative Trait (eQTL) analysis. The results showed that the PRCKB1 gene expression in peripheral blood mononuclear cells (PBMC) was 33.3% down-regulated in SSD patients (n = 48, t = 3.202, p = 0.002), and a more dramatic (n = 17, 49%) down-regulation in MDD patients than control (n = 49, t = 2.114, p = 0.001). We also identified 37 genes that displayed a strong correlation with PRKCB1 mRNA expression levels in SSD patients. The expression of PRKCB1 was regulated by multiple single nucleotide polymorphisms (SNPs) both at the transcript level and exon level. In conclusion, we first found a significant decrease of PRCKB1 mRNA expression in SSD, suggesting PRKCB1 might be the candidate gene and biomarker for SSD. Copyright © 2015 Elsevier Ltd. All rights reserved.

Unusual Stability of Messenger RNA in Snake Venom Reveals Gene Expression Dynamics of Venom Replenishment

PubMed Central

Currier, Rachel B.; Calvete, Juan J.; Sanz, Libia; Harrison, Robert A.; Rowley, Paul D.; Wagstaff, Simon C.

2012-01-01

Venom is a critical evolutionary innovation enabling venomous snakes to become successful limbless predators; it is therefore vital that venomous snakes possess a highly efficient venom production and delivery system to maintain their predatory arsenal. Here, we exploit the unusual stability of messenger RNA in venom to conduct, for the first time, quantitative PCR to characterise the dynamics of gene expression of newly synthesised venom proteins following venom depletion. Quantitative PCR directly from venom enables real-time dynamic studies of gene expression in the same animals because it circumvents the conventional requirement to sacrifice snakes to extract mRNA from dissected venom glands. Using qPCR and proteomic analysis, we show that gene expression and protein re-synthesis triggered by venom expulsion peaks between days 3–7 of the cycle of venom replenishment, with different protein families expressed in parallel. We demonstrate that venom re-synthesis occurs very rapidly following depletion of venom stores, presumably to ensure venomous snakes retain their ability to efficiently predate and remain defended from predators. The stability of mRNA in venom is biologically fascinating, and could significantly empower venom research by expanding opportunities to produce transcriptomes from historical venom stocks and rare or endangered venomous species, for new therapeutic, diagnostic and evolutionary studies. PMID:22879897

Validation of Reference Genes in mRNA Expression Analysis Applied to the Study of Asthma.

PubMed

Segundo-Val, Ignacio San; Sanz-Lozano, Catalina S

2016-01-01

The quantitative Polymerase Chain Reaction is the most used technique for the study of gene expression. To correct putative experimental errors of this technique is necessary normalizing the expression results of the gene of interest with the obtained for reference genes. Here, we describe an example of the process to select reference genes. In this particular case, we select reference genes for expression studies in the peripheral blood mononuclear cells of asthmatic patients.

Identification of Relationships Between Interleukin 15 mRNA and Brain-Derived Neurotrophic Factor II mRNA Levels With Formal Components of Temperament in Asthmatic Patients.

PubMed

Panek, Michał; Jonakowski, Mateusz; Zioło, Jan; Pietras, Tadeusz; Wieteska, Łukasz; Małachowska, Beata; Mokros, Łukasz; Szemraj, Janusz; Kuna, Piotr

2017-04-01

Asthma is a chronic inflammatory and heterogeneous disease developing mostly through allergic inflammation, which modifies the expression of various cytokines and neurotrophins. Previous studies suggest the involvement of interleukin (IL)-15 in the regulation of immune response in asthma. Brain-derived neurotrophic factor (BDNF) II plays an important role as a regulator of development and survival of neurons as well as maintenance of their physiological activity. Chronic stress associated with asthma and elevated IL-15 mRNA and BDNFII mRNA levels may affect the mood and a subjective sensation of dyspnoea-inducing anxiety. Psychopathological variables and numerous cytokine/neurotrophin interactions influence the formation of temperament and strategies of coping with stress. The aim of the study was to identify the role of IL-15 mRNA and BDNFII mRNA expressions and their effect on components of temperament and strategies of coping with stress in asthmatics. A total of 352 subjects (176 healthy volunteers and 176 asthmatic patients) participated in the study. The Formal Characteristic of Behaviour-Temperament Inventory (FCB-TI), Coping Inventory for Stressful Situations (CISS), Beck Depression Inventory, State-Trait Anxiety Inventory, and Borg Rating of Perceived Exertion (RPE) Scale were applied in all the subjects. The expression of IL-15 and BDNFII gene was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Different levels of IL-15 and BDNFII expressions between healthy volunteers and patients were revealed in the study. IL-15 enhanced the BDNFII mRNA expression among patients with bronchial asthma. The depression level negatively correlated with the BDNFII mRNA expression. This neurotrophin modified the temperament variable. BDNFII significantly affected (proportional relationship) the level of briskness in asthmatic patients. BDNFII might influence the level and style of coping with stress (emotion-oriented style). This hypothesis requires further studies on protein functional models. The obtained data confirms the role of IL-15 and BDNFII in the pathomechanisms of depression and formation of selected traits defining the temperament in asthmatics.

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

NASA Technical Reports Server (NTRS)

Polacek, Denise C.; Passerini, Anthony G.; Shi, Congzhu; Francesco, Nadeene M.; Manduchi, Elisabetta; Grant, Gregory R.; Powell, Steven; Bischof, Helen; Winkler, Hans; Stoeckert, Christian J Jr;

[Expression of PTEN in Myocardial Tissue in Coronary Heart Disease].

PubMed

Li, Xue-rong; He, Yong; Lei, Yu-jia; Qin, Xe-he; Wei, Qing-tao; Pan, Xin-min; Li, Li-juan; Zhang, Lin

2016-04-01

To observe the expression of phosphatase and tensin homology deleted on chromosome ten (PTEN) in myocardial tissue in patients with coronary heart disease, and explore the relevance between the expression of PTEN and the occurrence and development of coronary heart disease. A total of 16 death cases with pathological diagnosis of coronary heart disease were collected as experimental group, and 19 cases without myocardial lesions were selected as control group. The expression of PTEN protein and its mRNA were detected by immunohistochemistry and real-time fluorescence quantitative PCR respectively. The correlation between the expression of PTEN and the pathogenesis of coronary heart disease was analyzed. The expression of PTEN protein in myocardium in cases with coronary heart disease was significantly lower compared with the control group (P < 0.05). There was no statistical difference of the expression of PTEN mRNA between experimental and control group (P > 0.05). PTEN may be involved in the occurrence and development of coronary heart disease.

Expression of ATF4 and VEGF in chorionic villus tissue in early spontaneous abortion.

PubMed

Chai, Luwei; Ling, Kang; He, Xiaoxi; Yang, Rong

2013-10-01

To explore the relationship between early spontaneous abortion (SA) and the expression of activating transcription factor 4 (ATF4) and vascular endothelial growth factor (VEGF). The expression of ATF4 and VEGF protein and mRNA in villi from first trimester spontaneous abortion (SA, n=30) and normal pregnancy (NP, n=30) were detected by immunohistochemistry and fluorescent quantitative polymerase chain reaction (FQ-PCR). Both protein and mRNA expressions of ATF4 and VEGF in the SA group were significantly lower than in the NP group (P<0.01). Their proteins are expressed mainly in syncytiotrophoblast, cytotrophoblast and villous stromal cells. Correlation analysis showed that the expression of ATF4 was positively correlated with that of VEGF in the SA group (r=0.717, P<0.01). Lower expression of ATF4 and VEGF genes in chorionic villus tissue may participate in the pathogenesis of spontaneous abortion. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Patterns of mRNA and protein expression during minus-lens compensation and recovery in tree shrew sclera.

PubMed

Gao, Hong; Frost, Michael R; Siegwart, John T; Norton, Thomas T

2011-04-12

To increase our understanding of the mechanisms that remodel the sclera during the development of lens-induced myopia, when the sclera responds to putative "go" signals of retinal origin, and during recovery from lens-induced myopia, when the sclera responds to retinally-derived "stop" signals. Seven groups of tree shrews were used to examine mRNA levels during minus lens compensation and recovery. Starting 24 days after eye opening (days of visual experience [VE]) lens compensation animals wore a monocular -5D lens for 1, 4, or 11 days. Recovery animals wore the -5D lens for 11 days, which was then removed for 1 or 4 days. Normal animals were examined at 24 and 38 days of VE. All groups contained 8 animals. Scleral mRNA levels were examined in the treated and contralateral control eyes with quantitative real-time polymerase chain reaction (qPCR) for 27 genes divided into four categories: 1) signaling molecules, 2) matricellular proteins, 3) metalloproteinases (MPs) and tissue inhibitors of metalloproteinases (TIMPs), and 4) cell adhesion and other proteins. Four groups (n=5 per group) were used to examine protein levels. One group wore a -5D lens for 4 days. A second group recovered for 4 days after 11 days of -5D lens treatment. Two groups were used to examine age-matched normal protein levels at 28 and 39 days of VE. The levels of six scleral proteins that showed differential mRNA expression were examined with quantitative western blots. Nineteen of the genes showed differential (treated eye versus control eye) expression of mRNA levels in at least one group of animals. Which genes showed differential expression differed after 1 and 4 days of compensation and after 1 or 4 days of recovery. The mRNA level for one gene, a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), was upregulated in the treated eyes after 1 day of compensation. After 4 days, transforming growth factor beta receptor 3 (TGFBR3), transforming growth factor-beta-induced protein ig-h3 (TGFBI), and matrix metalloproteinase 14 (MMP14) mRNA levels were upregulated. Downregulated were mRNA levels for transforming growth factor beta-1 (TGFB1), transforming growth factor beta-2 (TGFB2), thrombospondin 1 (THBS1), tenascin (TNC), osteonectin (SPARC), osteopontin (SPP1), tissue inhibitor of metalloproteinases 3 (TIMP3), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5). After 11 days of lens wear, there was no differential expression. During recovery, after 1 day, treated-eye mRNA downregulation was found for TGFB2, TGFBR1, TGFBR2, TGFBR3, SPARC, ADAMTS1, ADAMTS5, syndecan 4 (SDC4), and collagen type VI, alpha 1 (COL6A1). After 4 days, TGFB1, TGFB2, TGFB3, THBS2, and TIMP3 mRNA levels were upregulated in the recovering eye. Significant downregulation, relative to normal eyes, was found in both the control and treated eyes for most genes after 1 day of compensation; a similar decrease was found, compared to lens-compensated eyes, after one day of recovery. Protein levels for THBS1 showed positive correlation with the differential mRNA levels and TGFBR3 showed a negative correlation. No differential protein expression was found for TGFB2, TGFBI, MMP14, and TIMP3. The different patterns of differential mRNA expression during minus lens compensation (hyperopia) and recovery (myopia) show that scleral fibroblasts distinguish between "go" and "stop" conditions. There is evidence of binocular global downregulation of genes at the start of both lens wear and recovery. As additional information accumulates about changes in gene expression that occur during compensation and recovery the "signature" of differential changes may help us to understand in more detail how the sclera responds in "go" and "stop" conditions.

UV-laser microdissection and mRNA expression analysis of individual neurons from postmortem Parkinson's disease brains.

PubMed

Gründemann, Jan; Schlaudraff, Falk; Liss, Birgit

2011-01-01

Cell specificity of gene expression analysis is essential to avoid tissue sample related artifacts, in particular when the relative number of target cells present in the compared tissues varies dramatically, e.g., when comparing dopamine neurons in midbrain tissues from control subjects with those from Parkinson's disease (PD) cases. Here, we describe a detailed protocol that combines contact-free UV-laser microdissection and quantitative PCR of reverse-transcribed RNA of individual neurons from postmortem human midbrain tissue from PD patients and unaffected controls. Among expression changes in a variety of dopamine neuron marker, maintenance, and cell-metabolism genes, we found that α-synuclein mRNA levels were significantly elevated in individual neuromelanin-positive dopamine midbrain neurons from PD brains when compared to those from matched controls.

Upregulation of Ryk expression in rat dorsal root ganglia after peripheral nerve injury.

PubMed

Li, Xin; Li, Yao-hua; Yu, Shun; Liu, Yaobo

2008-10-22

To study changes of Ryk expression in dorsal root ganglia (DRG) after peripheral nerve injury, we set up an animal model of unilateral sciatic nerve lesioned rats. Changes of Ryk protein expression in DRG neurons after unilateral sciatic nerve injury were investigated by immunostaining. Changes of Ryk mRNA were also tested by semi-quantitative PCR concurrently. We found, both at the level of protein and mRNA, that Ryk could be induced in cells of ipsilateral DRG after unilateral sciatic nerve lesion. Further investigation by co-immunostaining confirmed that the Ryk-immunoreactive (Ryk-IR) cells were NeuN-immunoreactive (NeuN-IR) neurons of DRG. We also showed the pattern of Ryk induction in DRG neurons after sciatic nerve injury: the number of Ryk IR neurons peaked at 2 weeks post-lesion and decreased gradually by 3 weeks post-lesion. The proportions of different sized Ryk IR neurons were also observed and counted at various stages after nerve lesion. Analysis of Ryk mRNA by RT-PCR showed the same induction pattern as by immunostaining. Ryk mRNA was not expressed in normal or contralateral DRG, but was expressed 1, 2 and 3 weeks post-lesion in the ipsilateral DRG. Ryk mRNA levels increased slightly from 1 to 2 weeks, decreased then by 3 weeks post-lesion. These results indicate that Ryk might be involved in peripheral nerve plasticity after injury. This is a novel function apart from its well-known fundamental activity as a receptor mediating axon guidance and outgrowth.

The molecular basis of urgency: regional difference of vanilloid receptor expression in the human urinary bladder.

PubMed

Liu, Lu; Mansfield, Kylie J; Kristiana, Ika; Vaux, Kenneth J; Millard, Richard J; Burcher, Elizabeth

2007-01-01

Treatments targeting vanilloid receptor TRPV1 are effective in some bladder disorders. Our aim was to determine the expression profiles of TRPV1 in regions of human bladder and test the hypothesis that there would be an upregulation of TRPV1 in mucosa of patients with bladder hypersensitivity but not idiopathic detrusor overactivity (IDO). Women with sensory urgency (SU), interstitial cystitis (IC), and IDO were investigated by videourodynamics and cystoscopy. Control biopsies were used for comparison. Biopsies were dissected into mucosa and muscle, and evaluated for TRPV1 mRNA expression using quantitative competitive RT-PCR (QC-RT-PCR). TRPV1 mRNA from SU trigonal mucosa was significantly higher than control trigonal mucosa or SU bladder body mucosa. In contrast, in IDO patients, there was no difference between trigonal mucosa and body mucosa. In IC biopsies, RNA quality was substandard and unable to be used for analysis. The most striking finding was that TRPV1 mRNA expressed in SU trigonal mucosa was significantly inversely correlated with the bladder volume at first sensation of filling during cystometry. No such relationship was seen for IDO trigonal mucosa. No difference was seen in bladder body mucosa from any disease groups compared with age-matched control. The symptoms of SU were associated with the increased expression of TRPV1 mRNA in the trigonal mucosa. No upregulation or regional differences of TRPV1 mRNA were seen in IDO patients. TRPV1 may play a role in SU and premature first bladder sensation on filling.

Analysis of myosin heavy chain mRNA expression by RT-PCR

NASA Technical Reports Server (NTRS)

Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

1997-01-01

An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

[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

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

PubMed

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

2015-06-01

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

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

PubMed Central

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

2015-01-01

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

Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.

PubMed

Wang, Hye-Young; Ahn, Sungwoo; Park, Sunyoung; Kim, SeungIl; Lee, Hyeyoung

2017-01-01

Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression. © 2016 S. Karger AG, Basel.

Synovial chemokine expression and relationship with knee symptoms in patients with meniscal tears

PubMed Central

Nair, Anjali; Gan, Justin; Bush-Joseph, Charles; Verma, Nikhil; Tetreault, Matthew W.; Saha, Kanta; Margulis, Arkady; Fogg, Louis; Scanzello, Carla R.

2015-01-01

Objective In patients with knee OA, synovitis is associated with knee pain and symptoms. We previously identified synovial mRNA expression of a set of chemokines (CCL19, IL-8, CCL5, XCL-1, CCR7) associated with synovitis in patients with meniscal tears but without radiographic OA. CCL19 and CCR7 were also associated with knee symptoms. This study sought to validate expression of these chemokines and association with knee symptoms in more typical patients presenting for meniscal arthroscopy, many who have pre-existing OA. Design Synovial biopsies and fluid (SF) were collected from patients undergoing meniscal arthroscopy. Synovial mRNA expression was measured using quantitative RT-PCR. The Knee Injury and Osteoarthritis Outcome Score (KOOS) was administered preoperatively. Regression analyses determined if associations between chemokine mRNA levels and KOOS scores were independent of other factors including radiographic OA. CCL19 in SF was measured by ELISA, and compared to patients with advanced knee OA and asymptomatic organ donors. Results 90% of patients had intra-operative evidence of early cartilage degeneration. CCL19, IL-8, CCL5, XCL1, CCR7 transcripts were detected in all patients. Synovial CCL19 mRNA levels independently correlated with KOOS Activities of Daily Living scores (95% CI [-8.071, -0.331], p= 0.036), indicating higher expression was associated with more knee-related dysfunction. SF CCL19 was detected in 7 of 10 patients, compared to 4 of 10 asymptomatic donors. Conclusion In typical patients presenting for meniscal arthroscopy, synovial CCL19 mRNA expression was associated with knee-related difficulty with activities of daily living, independent of other factors including presence of radiographic knee OA. PMID:25724256

Conjunctival mucin mRNA expression in contact lens wear.

PubMed

Corrales, Rosa M; Galarreta, David; Herreras, Jose M; Saez, Victoria; Arranz, Isabel; González, Maria J; Mayo, Agustin; Calonge, Margarita; Chaves, Felipe J

2009-09-01

To investigate the influence of the water content in non-ionic hydrogel contact lenses (HCL) on the mRNA levels of human conjunctival mucin genes (MUCs). Sixteen healthy subjects with no history of contact lenses wear were selected and randomized into two equal groups. Group 1 subjects wore low water content (38%, Soflens 38) non-ionic HCLs. Group 2 wore high water content (66%, Soflens 66) non-ionic HCLs. Conjunctival impression cytology was applied to the superior bulbar conjunctiva of both eyes before, 6 months, and 1 year after HCL fitting, and 15 days after discontinuation of wearing. Total RNA was isolated, retrotranscribed, and amplified by conventional polymerase chain reaction (PCR) and by quantitative real time PCR to study the mRNA levels of MUCs and to analyze variations during the study period. Time- and HCL-dependent variations in mRNA expression were analyzed using Student's test. From the known MUCs, transcripts from MUC1, MUC2, MUC4, MUC5AC, MUC7, MUC13, MUC15, MUC16, and MUC17 genes were detected in all subjects before HCL fitting. Except for MUC2, the expression of some MUC genes significantly increased whereas others significantly decreased at either the 6- and 12-month period. Statistically significant differences between both HCL groups (p < 0.001) were found in the MUC4, MUC13, and MUC15 mRNA expression after 1 year of wear and after the 15 days without HCL wear. However, these differences were not clearly related to the water content of the lenses. Low and high water content non-ionic HCLs induced different changes in the mRNA levels of several MUCs, but the water content was not related to the changes. Recovery to basal levels of conjunctival MUC mRNA expression after wearing HCL lenses for a year takes longer than 15 days for some MUCs.

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

Quantification and molecular characterization of the feline leukemia virus A receptor.

PubMed

Katrin Helfer-Hungerbuehler, A; Cattori, Valentino; Bachler, Barbara; Hartnack, Sonja; Riond, Barbara; Ossent, Pete; Lutz, Hans; Hofmann-Lehmann, Regina

2011-12-01

Virus receptors and their expression patterns on the cell surface determine the cell tropism of the virus, host susceptibility and the pathogenesis of the infection. Feline thiamine transport protein 1 (fTHTR1) has been identified as the receptor for feline leukemia virus (FeLV) A. The goal of the present study was to develop a quantitative, TaqMan real-time PCR assay to investigate fTHTR1 mRNA expression in tissues of uninfected and FeLV-infected cats, cats of different ages, in tumor tissues and leukocyte subsets. Moreover, the receptor was molecularly characterized in different feline species. fTHTR1 mRNA expression was detected in all 30 feline tissues investigated, oral mucosa scrapings and blood. Importantly, identification of significant differences in fTHTR1 expression relied on normalization with an appropriate reference gene. The lowest levels were found in the blood, whereas high levels were measured in the oral mucosa, salivary glands and the musculature. In the blood, T lymphocytes showed significantly higher fTHTR1 mRNA expression levels than neutrophil granulocytes. In vitro activation of peripheral blood mononuclear cells with concanavalin A alone or followed by interleukin-2 led to a transient increase of fTHTR1 mRNA expression. In the blood, but not in the examined tissues, FeLV-infected cats tended to have lower fTHTR1 mRNA levels than uninfected cats. The fTHTR1 mRNA levels were not significantly different between tissues with lymphomas and the corresponding non-neoplastic tissues. fTHTR1 was highly conserved among different feline species (Iberian lynx, Asiatic and Indian lion, European wildcat, jaguarundi, domestic cat). In conclusion, while ubiquitous fTHTR1 mRNA expression corresponded to the broad target tissue range of FeLV, particularly high fTHTR1 levels were found at sites of virus entry and shedding. The differential susceptibility of different species to FeLV could not be attributed to variations in the fTHTR1 sequence. Copyright © 2011 Elsevier B.V. All rights reserved.

The Effect of Repeated Electroacupuncture Analgesia on Neurotrophic and Cytokine Factors in Neuropathic Pain Rats

PubMed Central

Wang, Junying; Duanmu, Chenlin; Feng, Xiumei; Yan, Yaxia

2016-01-01

Chronic pain is a common disability influencing quality of life. Results of previous studies showed that acupuncture has a cumulative analgesic effect, but the relationship with spinal cytokines neurotrophic factors released by astrocytes remains unknown. The present study was designed to observe the effect of electroacupuncture (EA) treatment on spinal cytokines neurotrophic factors in chronic neuropathic pain rats. The chronic neuropathic pain was established by chronic constrictive injury (CCI). EA treatment was applied at Zusanli (ST36) and Yanglingquan (GB34) (both bilateral) once a day, for 30 min. IL-1β mRNA, TNF-α mRNA, and IL-1 mRNA were detected by quantitative real-time PCR, and the proteins of BDNF, NGF, and NT3/4 were detected by Western blot. The expression levels of cytokines such as IL-1β mRNA, TNF-α mRNA, IL-6 mRNA, and neurotrophic factors such as BDNF, NGF, and NT3/4 in the spinal cord were increased significantly after CCI. The astrocytes released more IL-1β and BDNF after CCI. Repeated EA treatment could suppress the elevated expression of IL-1β mRNA, TNFα mRNA, and BDNF, NGF, and NT3/4 but had no effect on IL-6 mRNA. It is suggested that cytokines and neurotrophic factors which may be closely associated with astrocytes participated in the process of EA relieving chronic pain. PMID:27800006

Loss of Thrombomodulin in Placental Dysfunction in Preeclampsia.

PubMed

Turner, Rosanne J; Bloemenkamp, Kitty W M; Bruijn, Jan A; Baelde, Hans J

2016-04-01

Preeclampsia is a pregnancy-specific syndrome characterized by placental dysfunction and an angiogenic imbalance. Systemically, levels of thrombomodulin, an endothelium- and syncytiotrophoblast-bound protein that regulates coagulation, inflammation, apoptosis, and tissue remodeling, are increased. We aimed to investigate placental thrombomodulin dysregulation and consequent downstream effects in the pathogenesis of preeclampsia. Placentas from 28 preeclampsia pregnancies, 30 uncomplicated pregnancies, and 21 pregnancies complicated by growth restriction as extra controls were included. Immunohistochemical staining of thrombomodulin, caspase-3, and fibrin was performed. Placental mRNA expression of thrombomodulin, inflammatory markers, matrix metalloproteinases 2 and 9, and soluble Flt-1 were measured with quantitative polymerase chain reaction. Thrombomodulin mRNA expression was determined in vascular endothelial growth factor-transfected trophoblast cell lines. Thrombomodulin protein and mRNA expression were decreased in preeclampsia as compared with both control groups (P=0.001). Thrombomodulin mRNA expression correlated with maternal body mass index (P<0.01) and diastolic blood pressure (P<0.05) in preeclampsia. An increase in placental apoptotic cells was associated with preeclampsia (P<0.001). Thrombomodulin expression correlated positively with matrix metalloproteinase expression (P<0.01) in preeclampsia, but not with fibrin deposits or inflammatory markers. Placental soluble Flt-1 expression correlated with decreased thrombomodulin expression. Vascular endothelial growth factor induced upregulation of thrombomodulin expression in trophoblast cells. Decreased thrombomodulin expression in preeclampsia may play a role in placental dysfunction in preeclampsia and is possibly caused by an angiogenic imbalance. Hypertension and obesity are associated with thrombomodulin downregulation. These results set the stage for further basic and clinical research on thrombomodulin in the pathogenesis of preeclampsia and other syndromes characterized by endothelial dysfunction. © 2016 American Heart Association, Inc.

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

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

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

PubMed

Qiu, Miao-Zhen; Li, Zhuang-Hua; Zhou, Zhi-Wei; Li, Yu-Hong; Wang, Zhi-Qiang; Wang, Feng-Hua; Huang, Peng; Aziz, Fahad; Wang, Dao-Yuan; Xu, Rui-Hua

2010-10-31

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. 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. 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. 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.

Comparison of glutathione peroxidase 1 and iodothyronine deiodinase 1 mRNA expression in murine liver after feeding selenite or selenized yeast.

PubMed

Qin, Shunyi; Huang, Kehe; Gao, Jianzhong; Huang, Da; Cai, Tanxi; Pan, Cuiling

2009-01-01

The experiment was conducted to compare the effect of different selenium sources on the expression of glutathione peroxidase 1 (GPx1) and iodothyronine deiodinase 1 (Dio1) mRNA in mice by quantitative real-time PCR. A total of 60 male Kunming mice at average body weight of 20 g were allotted to three groups in a randomized complete block design, namely two treatments and one control. Mice in Group 1 were fed a basal diet as control, while mice in Groups 2 and 3 were fed the basal diet supplemented with 0.1mg/kg selenium as sodium selenite or selenized yeast, respectively. Whole feeding experiment lasted for 30 d. At the end of the feeding trial, liver mRNA levels of GPx1 and Dio1 were determined by quantitative real-time PCR, as well as growth performance, body composition, blood and GPx activity were determined. The results showed that no significant differences in overall growth performance and body composition, including body weight, body length, heart weight, kidney weight and liver weight, were found between the experimental groups (P>0.05). Blood GPx activity increased in all of the selenium supplemented groups compared with control group (P<0.01). However, blood GPx activity in selenized yeast group was higher than that in sodium selenite group (P<0.05). Liver mRNA levels of GPx1 and Dio1 also increased in the two selenium supplemented groups compared with the control group (P<0.05), while there was no significant difference between the sodium selenite and selenized yeast groups (P>0.05). In conclusion, selenium increased the mRNA expression of GPx1 and Dio1 genes in murine liver, and there was no significant difference between the organic or inorganic form of selenium used.

Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas

PubMed Central

Lau, Tze Pheng; Roslani, April Camilla; Lian, Lay Hoong; Chai, Hwa Chia; Lee, Ping Chin; Hilmi, Ida; Goh, Khean Lee; Chua, Kek Heng

2014-01-01

Objectives To characterise the mRNA expression patterns of early and advanced stage colorectal adenocarcinomas of Malaysian patients. Design Comparative expression analysis. Setting and participants We performed a combination of annealing control primer (ACP)-based PCR and reverse transcription-quantitative real-time PCR for the identification of differentially expressed genes (DEGs) associated with early and advanced stage primary colorectal tumours. We recruited four paired samples from patients with colorectal cancer (CRC) of Dukes’ A and B for the preliminary differential expression study, and a total of 27 paired samples, ranging from CRC stages I to IV, for subsequent confirmatory test. The tumouric samples were obtained from the patients with CRC undergoing curative surgical resection without preoperative chemoradiotherapy. The recruited patients with CRC were newly diagnosed with CRC, and were not associated with any hereditary syndromes, previously diagnosed cancer or positive family history of CRC. The paired non-cancerous tissue specimens were excised from macroscopically normal colonic mucosa distally located from the colorectal tumours. Primary and secondary outcome measures The differential mRNA expression patterns of early and advanced stage colorectal adenocarcinomas compared with macroscopically normal colonic mucosa were characterised by ACP-based PCR and reverse transcription-quantitative real-time PCR. Results The RPL35, RPS23 and TIMP1 genes were found to be overexpressed in both early and advanced stage colorectal adenocarcinomas (p<0.05). However, the ARPC2 gene was significantly underexpressed in early colorectal adenocarcinomas, while the advanced stage primary colorectal tumours exhibited an additional overexpression of the C6orf173 gene (p<0.05). Conclusions We characterised two distinctive gene expression patterns to aid in the stratification of primary colorectal neoplasms among Malaysian patients with CRC. Further work can be done to assess and compare the mRNA expression levels of these identified DEGs between each CRC stage group, stages I–IV. PMID:25107436

Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark.

PubMed

Cutler, Christopher P; Maciver, Bryce; Cramb, Gordon; Zeidel, Mark

2011-01-01

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5' and 3' RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill >  intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (P(f)) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.

Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark

PubMed Central

Cutler, Christopher P; MacIver, Bryce; Cramb, Gordon; Zeidel, Mark

2012-01-01

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill >  intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (Pf) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species. PMID:22291652

[Effects of Fluoxetine on Nogo Expression and Collagen Production with Decrease of Pulmonary Artery Pressure in Rats with Right Ventricular Failure.

PubMed

Ran, Xun; Zhao, Jian-Xun; Nie, Hu; Chen, Yu-Cheng

2016-11-01

To investigate the effect of fluoxetine on neurite growth inhibitor (Nogo) expession and collagen production of cardiac tissue in rats with right heart failure and pulmonary hypertension. Thirty one male SD rats were randomly divided into the treatment group,right heart failure group and normal control group.The rats in the treatment group and right heart failure group received intrapertioneal injection of monocrotaline (MCT,60 mg/kg) to induce pulmonary hypertension and right heart failure.After 21 days,the rats in treatment group were given fluoxetine of 10 mg/(kg×d) by gavage per day for 21 days,the rats in the other two groups were given saline.HE staining was used to observe the pulmonary artery and right ventricular myocardial tissue in rats.The collagen formation in right ventricular myocardium was observed by Masson staining.The expressions of Nogo-A, Nogo-B ,type1collagen and type 3 collagen mRNA in myocardium were measured by real-time fluorescence quantitative PCR,while the semi quantitative measurement of Nogo protein level was detected by Western blot. After the intervention of fluoxetine,pulmonary artery stenosis was significantly reduced,myocardial tissue lesion decreased,collagen synthesis decreased in right ventricular myocardium.RT-PCR showed that mRNA of Nogo-A decreased,and mRNA of Nogo-B increased ( P <0.05).Western blot showed that the expression of Nogo-A protein decreased,while Nogo-B1 protein expression increased ( P <0.05),Nogo-B2 expression was not significantly changed ( P >0.05). Nogo may affect the collagen synthesis in right heart failure,and partly involved in myocardial fibrosis.

The CesA Gene Family of Barley. Quantitative Analysis of Transcripts Reveals Two Groups of Co-Expressed Genes1

PubMed Central

Burton, Rachel A.; Shirley, Neil J.; King, Brendon J.; Harvey, Andrew J.; Fincher, Geoffrey B.

2004-01-01

Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis. PMID:14701917

SMN blood levels in a Porcine Model of Spinal Muscular Atrophy

PubMed Central

Iyer, Chitra; Wang, Xueqian; Renusch, Samantha R.; Duque, Sandra I.; Wehr, Allison M.; Mo, Xiaokui-Molly; McGovern, Vicki L.; Arnold, W. David; Burghes, Arthur H.M.; Kolb, Stephen J.

2017-01-01

Spinal Muscular Atrophy (SMA) is an autosomal recessive motor neuron disease that results in loss of spinal motor neurons, muscular weakness and, in severe cases, respiratory failure and death. SMA is caused by a deletion or mutation of the SMN1 gene and retention of the SMN2 gene that leads to low SMN expression levels. The measurement of SMN mRNA levels in peripheral blood samples has been used in SMA clinical studies as a pharmacodynamic biomarker for response to therapies designed to increase SMN levels. We recently developed a postnatal porcine model of SMA by the viral delivery of a short-hairpin RNA (shRNA) targeting porcine pSMN. scAAV9-mediated knockdown of pSMN mRNA at postnatal day 5 reliably resulted in denervation, weakness and motor neuron and ventral root axon loss that began 3–4 weeks after viral delivery, and this phenotype could be ameliorated by subsequent viral delivery of human SMN (hSMN). To determine if the effect of modulating SMN levels using gene therapy can be measured in blood, we measured expression of pSMN mRNA and hSMN mRNA by quantitative droplet digital PCR (ddPCR). We found that the endogenous expression of pSMN mRNA in blood increases in the first month of life. However, there were no significant differences in blood levels of pSMN mRNA after knock-down or of human SMN mRNA after gene therapy. Our results, obtained in a large animal model of SMA that is similar in size and anatomy to human infants, suggest that measurement of SMN mRNA levels in blood may not be informative in SMA clinical trials involving intrathecal delivery of SMN-modulating therapies. PMID:28269795

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

PubMed

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

2012-03-01

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

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.

BRCA1: A Novel Prognostic Factor in Resected Non-Small-Cell Lung Cancer

PubMed Central

Rosell, Rafael; Skrzypski, Marcin; Jassem, Ewa; Taron, Miquel; Bartolucci, Roberta; Sanchez, Jose Javier; Mendez, Pedro; Chaib, Imane; Perez-Roca, Laia; Szymanowska, Amelia; Rzyman, Witold; Puma, Francesco; Kobierska-Gulida, Grazyna; Farabi, Raffaele; Jassem, Jacek

2007-01-01

Background Although early-stage non-small-cell lung cancer (NSCLC) is considered a potentially curable disease following complete resection, patients have a wide spectrum of survival according to stage (IB, II, IIIA). Within each stage, gene expression profiles can identify patients with a higher risk of recurrence. We hypothesized that altered mRNA expression in nine genes could help to predict disease outcome: excision repair cross-complementing 1 (ERCC1), myeloid zinc finger 1 (MZF1) and Twist1 (which regulate N-cadherin expression), ribonucleotide reductase subunit M1 (RRM1), thioredoxin-1 (TRX1), tyrosyl-DNA phosphodiesterase (Tdp1), nuclear factor of activated T cells (NFAT), BRCA1, and the human homolog of yeast budding uninhibited by benzimidazole (BubR1). Methodology and Principal Findings We performed real-time quantitative polymerase chain reaction (RT-QPCR) in frozen lung cancer tissue specimens from 126 chemonaive NSCLC patients who had undergone surgical resection and evaluated the association between gene expression levels and survival. For validation, we used paraffin-embedded specimens from 58 other NSCLC patients. A strong inter-gene correlation was observed between expression levels of all genes except NFAT. A Cox proportional hazards model indicated that along with disease stage, BRCA1 mRNA expression significantly correlated with overall survival (hazard ratio [HR], 1.98 [95% confidence interval (CI), 1.11-6]; P = 0.02). In the independent cohort of 58 patients, BRCA1 mRNA expression also significantly correlated with survival (HR, 2.4 [95%CI, 1.01-5.92]; P = 0.04). Conclusions Overexpression of BRCA1 mRNA was strongly associated with poor survival in NSCLC patients, and the validation of this finding in an independent data set further strengthened this association. Since BRCA1 mRNA expression has previously been linked to differential sensitivity to cisplatin and antimicrotubule drugs, BRCA1 mRNA expression may provide additional information for customizing adjuvant antimicrotubule-based chemotherapy, especially in stage IB, where the role of adjuvant chemotherapy has not been clearly demonstrated. PMID:17987116

Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle

PubMed Central

Nagler, James J.; Cavileer, Timothy D.; Verducci, Joseph S.; Schultz, Irvin R.; Hook, Sharon E.; Hayton, William L.

2012-01-01

Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (β), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erβ1, erβ2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17β, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erβ1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17β levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too. PMID:22732076

Endurance training blocks uncoupling protein 1 up-regulation in brown adipose tissue while increasing uncoupling protein 3 in the muscle tissue of rats fed with a high-sugar diet.

PubMed

de Queiroz, Karina Barbosa; Rodovalho, Gisele Vieira; Guimarães, Juliana Bohnen; de Lima, Daniel Carvalho; Coimbra, Cândido Celso; Evangelista, Elísio Alberto; Guerra-Sá, Renata

2012-09-01

The mitochondrial uncoupling proteins (UCPs) of interscapular brown adipose tissue (iBAT) and of muscles play important roles in energy balance. For instance, the expression of UCP1 and UCP3 are modulated by free fatty acid gradients induced by high-sugar diets and acute exercise that is dependent on sympathetic stimulation. However, the effects of endurance training in animals fed with high-sugar diets are unknown. This study aims to evaluate the long-term effects of diet and exercise on UCP1 and UCP3 levels and energy balance efficiency. Rats fed with standard or high-sugar (HSD) diets were simultaneously subjected to running training over an 8-week period. After the training period, the rats were decapitated, and the iBAT and gastrocnemius muscle tissues were removed for evaluation of the β₃-receptor, Ucp1, and Ucp3 mRNA and protein expression, which were analyzed by quantitative reverse transcriptase polymerase chain reaction and Western blot, respectively. Groups fed with an HSD displayed a higher adiposity index and iBAT weight (P < .05), whereas exhibited an up-regulation of Ucp1 mRNA and protein levels (P < .05). Training increased β₃-receptor mRNA in iBAT and reduced the Ucp3 mRNA in muscle tissues. In association with an HSD, training restored the increasing β₃-receptor mRNA and greatly up-regulated the levels of Ucp3 mRNA. Therefore, training blocked the HSD-induced up-regulation of UCP1 expression in iBAT, whereas it up-regulated the expression of Ucp3 mRNA in muscle. These results suggest that training enhances the relationship between Ucp1/Ucp3 mRNA levels, which could result in higher energy efficiency, but not when HSD-induced elevated sympathetic activity is maintained. Copyright © 2012. Published by Elsevier Inc.

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

Nagy, Edit, E-mail: edit.nagy@karolinska.se; Department of Cardiology, Karolinska University Hospital, Stockholm; Caidahl, Kenneth

Highlights: Black-Right-Pointing-Pointer Oxidative stress has been implicated in the pathomechanism of calcific aortic valve stenosis. Black-Right-Pointing-Pointer We assessed the transcript levels for PARP-1 (poly(ADP-ribose) polymerase), acts as a DNA damage nick sensor in stenotic valves. Black-Right-Pointing-Pointer Early stage of diseased tricuspid valves exhibited higher mRNA levels for PARP-1 compared to bicuspid valves. Black-Right-Pointing-Pointer The mRNA levels for PARP-1 inversely correlated with the clinical stenosis severity in tricuspid valves. Black-Right-Pointing-Pointer Our data demonstrated that DNA damage pathways might be associated with stenosis severity only in tricuspid valves. -- Abstract: Oxidative stress may contribute to the hemodynamic progression of aortic valve stenosis,more » and is associated with activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) 1. The aim of the present study was to assess the transcriptional profile and the topological distribution of PARP-1 in human aortic valves, and its relation to the stenosis severity. Human stenotic aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery and used for mRNA extraction followed by quantitative real-time PCR to correlate the PARP-1 expression levels with the non invasive hemodynamic parameters quantifying the stenosis severity. Primary isolated valvular interstitial cells (VICs) were used to explore the effects of cytokines and leukotriene C{sub 4} (LTC{sub 4}) on valvular PARP-1 expression. The thickened areas of stenotic valves with tricuspid morphology expressed significantly higher levels of PARP-1 mRNA compared with the corresponding part of bicuspid valves (0.501 vs 0.243, P = 0.01). Furthermore, the quantitative gene expression levels of PARP-1 were inversely correlated with the aortic valve area (AVA) (r = -0.46, P = 0.0469) and AVA indexed for body surface area (BSA) (r = -0.498; P = 0.0298) only in tricuspid aortic valves. LTC{sub 4} (1 nM) significantly elevated the mRNA levels of PARP-1 by 2.38-fold in VICs. Taken together, these data suggest that valvular DNA-damage pathways may be associated with inflammation and the stenosis severity in tricuspid aortic valves.« less

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

RNA-based determination of ESR1 and HER2 expression and response to neoadjuvant chemotherapy.

PubMed

Denkert, C; Loibl, S; Kronenwett, R; Budczies, J; von Törne, C; Nekljudova, V; Darb-Esfahani, S; Solbach, C; Sinn, B V; Petry, C; Müller, B M; Hilfrich, J; Altmann, G; Staebler, A; Roth, C; Ataseven, B; Kirchner, T; Dietel, M; Untch, M; von Minckwitz, G

2013-03-01

Hormone and human epidermal growth factor receptor 2 (HER2) receptors are the most important breast cancer biomarkers, and additional objective and quantitative test methods such as messenger RNA (mRNA)-based quantitative analysis are urgently needed. In this study, we investigated the clinical validity of RT-PCR-based evaluation of estrogen receptor (ESR1) and HER2 mRNA expression. A total of 1050 core biopsies from two retrospective (GeparTrio, GeparQuattro) and one prospective (PREDICT) neoadjuvant studies were evaluated by quantitative RT-PCR for ESR1 and HER2. ESR1 mRNA was significantly predictive for reduced response to neoadjuvant chemotherapy in univariate and multivariate analysis in all three cohorts. The complete pathologically documented response (pathological complete response, pCR) rate for ESR1+/HER2- tumors was 7.3%, 8.0% and 8.6%; for ESR1-/HER2- tumors it was 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT, respectively (P < 0.001 in each cohort). In the Kaplan-Meier analysis in GeparTrio patients with ESR1+/HER2- tumors had the best prognosis, compared with ESR1-/HER2- and ESR1-/HER2+ tumors [disease-free survival (DFS): P < 0.0005, overall survival (OS): P < 0.0005]. Our results suggest that mRNA levels of ESR1 and HER2 predict response to neoadjuvant chemotherapy and are significantly associated with long-term outcome. As an additional option to standard immunohistochemistry and gene-array-based analysis, quantitative RT-PCR analysis might be useful for determination of the receptor status in breast cancer.

Expression of key ion channels in the rat cardiac conduction system by laser capture microdissection and quantitative real-time PCR.

PubMed

Ou, Yan; Niu, Xiao-lin; Ren, Fu-xian

2010-09-01

The objective of this study was to investigate the molecular basis of the inferior nodal extension (INE) in the atrioventricular junctional area that accounts for arrhythmias. The INE was separated from the adult rat heart by laser capture microdissection. The mRNA expression of ion channels was detected by quantitative real-time PCR. Hierarchical clustering was used to demonstrate clustering of expression of genes in sections. The mRNA expression of HCN4, Ca(v)3.1 and Ca(v)3.2 was high in the INE, atrioventricular node and sino-atrial node, and that of Ca(v)3.2 high in Purkinje fibres. Although the expression of HCN1 and Ca(v)1.3 was low in the rat heart, it was relatively higher in the INE, atrioventricular node and sino-atrial node than in right atrial and right ventricular (working) myocytes. Both HCN2 and Ca(v)1.2 were expressed at higher levels in working myocytes than in nodal tissues and in the INE. Hierarchical clustering analysis demonstrated that the expression of the HCN and calcium channels in INE was similar to that in the slow-response automatic cells and different from that in working myocytes and Purkinje fibres. The expression of HCN and calcium channels in the INE of the adult rat heart is similar to that of slow-response automatic cells and provides a substrate for automatic phase 4 depolarization in cells.

Quantitation of CYP1A1 and 1B1 mRNA in polycyclic aromatic hydrocarbon-treated human T-47D and HepG2 cells by a modified bDNA assay using fluorescence detection.

PubMed

Wu, Susan J; Spink, David C; Spink, Barbara C; Kaminsky, Laurence S

2003-01-15

The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2'-(2-benzothiazol)-6'-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by benzo[a]pyrene (B[a]P) and dibenzo[a,h]anthracene (DB[a,h]A). The fluorescence response increased linearly for 200 min without photobleaching and increased linearly (r2=0.997) up to at least 0.2 microg total RNA. The data revealed that at 0.5 and 1.0 microM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DB[a,h]A exceeded that by B[a]P by 18- and 6-fold, respectively. In T-47D cells B[a]P induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A B[a]P cocontaminant in the environment, arsenite, did not affect B[a]P-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid-throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.

Fatty acid binding protein 5 promotes tumor angiogenesis and activates the IL6/STAT3/VEGFA pathway in hepatocellular carcinoma.

PubMed

Pan, Long; Xiao, Heng; Liao, Rui; Chen, Qingsong; Peng, Chong; Zhang, Yuchi; Mu, Tong; Wu, Zhongjun

2018-06-25

Tumor angiogenesis is an essential process for facilitating tumor growth and metastasis. Fatty acid binding protein 5(FABP5)is highly expressed in hepatocellular carcinoma (HCC). Thus, we investigated the role of FABP5 in tumor angiogenesis during HCC development. In this study, the protein and mRNA levels of FABP5 in matched HCC and adjacent noncancerous liver tissues from 43 patients were determined using immunohistochemistry and real-time quantitative PCR, respectively. Two HCC cell lines (Huh7 and SMMC-7721) and human umbilical vein endothelial cells (HUVECS) were used to investigate the pro-angiogenic effect of FABP5 by tube formation, CCK8 and Transwell migration assays. The expression levels of interleukin 6 (IL6) and vascular endothelial growth factor A (VEGFA) secreted from HCC cells were detected by enzyme-linked immunosorbent assay (ELISA). In 43 HCC patients, the expression of FABP5 mRNA was positively correlated with intratumoral VEGFA mRNA expression. FABP5 mRNA expression was also associated with adverse HCC characteristics. In vitro, cell viability, cell migration and tube formation in HUVECs were enhanced with increasing expression of FABP5 in HCC cells. Downregulation of FABP5 expression inhibited the IL6/STAT3/VEGFA pathway in HCC cells and inhibited tumor angiogenesis. FABP5 was shown to promote angiogenesis and activate the IL6/STAT3/VEGFA pathway in HCC. FABP5 may be a potential antiangiogenic target in the treatment of HCC. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

[Expression of BAG3 Gene in Acute Myeloid Leukemia and Its Prognostic Value].

PubMed

Zhu, Hua-Yuan; Fu, Yuan; Wu, Wei; Xu, Jia-Dai; Chen, Ting-Mei; Qiao, Chun; Li, Jian-Yong; Liu, Peng

2015-08-01

To investigate the expression of BAG3 gene in acue myeloid leukemia (AML) and its prognostic value. Real-time quantitative RT-PCR was used to detect the expression of BAG3 mRNA in 88 previously untreated AML patients. The corelation of BAG3 expression level with clinical characteristics and known prognostic markers of AML was analyzed. In 88 patients with AML, the expression of BAG3 mRNA in NPMI mutated AML patients was obviously lower than that in NPMI unmutated patients (P = 0.018). The expression level of BAG3 mRNA did not related to clinical parameters, such as age, sex, FAB subtype, WBC count, extra-modullary presentation, and to prognostic factors including cytogenetics, FLT3-ITD, c-kit and CEBPα mutation status (P > 0.05). The expression level of BAG3 had no obvious effect on complete remission (CR) of patients in first treatment. The expression level of BAG3 in non-M3 patients was higher than that in relapsed patients (P = 0.036). The expression level of BAG3 had no effect on overall survival (OS) of patients. The expression level of BAG3 does not correlated with known-prognostic markers of AML, only the expression level of BAG3 in NPM1 mutated patients is lower than that in NPM1 unmutated patients. The expression level of BAG3 has no effect on OS of AML patients, the BAG3 can not be difined as a prognostic marker in AML.

Up-regulation of visfatin expression in subjects with hyperthyroidism and hypothyroidism is partially relevant to a nonlinear regulation mechanism between visfatin and tri-iodothyronine with various concentrations.

PubMed

Han, Jing; Zhang, Tian-ou; Xiao, Wen-hua; Chang, Cui-qing; Ai, Hua

2012-03-01

Visfatin, a visceral fat-derived adipocytokine, plays a significant physiological function in lipid metabolism. However, the precise function of visfatin and its regulation by thyroid hormones are still unknown. This study observed the plasma visfatin concentrations in subjects with hyperthyroidism and hypothyroidism in vivo, and investigated the possible regulation mechanism between visfatin and tri-iodothyronine (T3) in vitro as a further interpretation. The experiment in vivo included clinical subjects (57 patients with thyroid dysfunction and 29 euthyroid healthy volunteers) and an animal model (24 Wistar rats). All subjects were divided into hyperthyroidism, hypothyroidism and euthyroidism groups, with plasma thyroid hormones, thyrotropin, visfatin and triglyceride concentrations determined. Visfatin mRNA expression in visceral fat and liver of rats was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The experiment in vitro studied 3T3-L1 cells and visfatin mRNA expression under nine different T3 concentrations (0, 0.1, 0.25, 0.5, 1, 5, 10, 20, 100 nmol/L) using quantitative real-time RT-PCR. Clinical subjects and animal models showed elevated plasma visfatin concentrations in the hyperthyroidism group (20.466 ng/ml (15.263, 26.795 ng/ml) and (1209.164±165.292) ng/L) and hypothyroidism group (12.457 ng/ml (11.115, 15.454 ng/ml) and (1205.425±109.200) ng/L) compared to euthyroidism group (6.891 ng/ml (5.888, 8.803 ng/ml) and (926.650±54.002) ng/L, P<0.001). For animal models, visfatin mRNA expression in visceral fat in the hyperthyroidism and hypothyroidism groups increased about 3.33-fold and 1.98-fold compared to the euthyroidism group (P<0.001), which was positively correlated with plasma visfatin concentrations (r=0.713, P<0.001). However, no significant group difference (P>0.05) and correlation (r=0.121, P=0.572) was found in the liver. T3 induced a remarkable increase of visfatin mRNA expression in 3T3-L1 cells at low concentrations (0-0.5 nmol/L T3) followed by a sharp decrease at higher concentrations (0.5-100 nmol/L T3), with an inflection point at 0.5 nmol/L T3. Elevated circulating visfatin levels in subjects with hyperthyroidism and hypothyroidism are possibly due to an increase of visfatin mRNA expression in visceral fat, and a nonlinear regulation mechanism on visfatin mRNA expression under various T3 concentrations might be involved.

Digestion of a single meal affects gene expression of ion and ammonia transporters and glutamine synthetase activity in the gastrointestinal tract of freshwater rainbow trout.

PubMed

Bucking, Carol; Wood, Chris M

2012-04-01

Experiments on freshwater rainbow trout, Oncorhynchus mykiss, demonstrated how digestion affected the transcriptional expression of gastrointestinal transporters following a single satiating meal (~3% body mass ration) after a 1-week fast. Quantitative real-time polymerase chain reaction was employed to measure the relative mRNA expression of three previously cloned and sequenced transporters [H(+)-K(+)-ATPase (HKA), Na(+)/HCO(3)(-) cotransporter (NBC), and the Rhesus glycoprotein (Rhbg1; an ammonia transporter)] over a 24-h time course following feeding. Plasma total ammonia increased about threefold from pre-feeding levels to 288 μmol l(-1), whereas total ammonia levels in chyme supernatant reached a sixfold higher value (1.8 mmol l(-1)) than plasma levels. Feeding did not appear to have a statistically significant effect on the relative mRNA expression of the gastric HKA or Rhbg1. However, the relative mRNA expression of gastric NBC was increased 24 h following the ingestion of a meal. Along the intestinal tract, feeding increased the relative mRNA expression of Rhbg1, but had no effect on the expression of NBC. Expression of the gastric HKA was undetectable in the intestinal tract of freshwater rainbow trout. Digestion increased the activity of glutamine synthetase in the posterior intestine at 12 and 24 h following feeding. This study is among the first to show that there are digestion-associated changes in gene expression and enzyme activity in the gastrointestinal tract of teleost fish illustrating the dynamic plasticity of this organ. These post-prandial changes occur over the relative short-term duration of digesting a single meal.

Cloning of HSP90, expression and localization of HSP70/90 in different tissues including lactating/non-lactating yak (Bos grunniens) breast tissue.

PubMed

Liu, Penggang; Yu, Sijiu; Cui, Yan; He, Junfeng; Yu, Chuan; Wen, Zexing; Pan, Yangyang; Yang, Kun; Song, Liangli; Yang, Xue

2017-01-01

The aim of this study is to investigate the expression and localization of HSP70/90 in different tissues and explore the regulation effects of HSP70/90 at lactation period of female yaks. HSP90 mRNA was cloned from the heart samples of female yaks, Quantitative real-time (qRT-PCR), Western blotting (WB), immunohistochemistry and immunofluorescence assays were utilized to analyze the expressions of HSP70/90 mRNA and protein in different tissues. Sequence analysis showed that HSP90 is a conserved molecular chaperone of female yaks. The qRT-PCR, WB results showed that the expressions of HSP70/90 mRNA and protein were significantly different in different tissues, and 3-fold higher expression during the lactation period than the non-lactation period of breast tissue (P < 0.01). Immunohistochemistry and immunofluorescence assays results showed that HSP70/90 were located in the cardiac muscle cells, cerebellar medulla, theca cells lining at the reproductive system, and the mammary epithelia of the breasts. In addition, the expression level of HSP70 was higher than those of HSP90 in all examined tissues. Therefore, our results strongly suggest that the expression and localization of HSP70/90 could provide significant evidence to further research in tissue specific expression, and lactation function of female yaks.

Longitudinal monitoring of bottlenose dolphins leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Effect of dietary betaine supplementation on lipogenesis gene expression and CpG methylation of lipoprotein lipase gene in broilers.

PubMed

Xing, Jinyi; Kang, Li; Jiang, Yunliang

2011-03-01

Experiments were conducted to investigate the effect of betaine supplementation on mRNA expression levels of lipogenesis genes and CpG methylation of lipoprotein lipase gene (LPL) in broilers. From 22 days of age, 78 broilers were feed basal diet without betaine and basal diet supplemented with 0.1% betaine, respectively, and at 56 and 66 days of age, the traits of 15 chickens (7 males and 8 females) of each group were recorded and abdominal fat pads were collected. The mRNA expression levels of several lipogenesis gene were analyzed by semi-quantitative RT-PCR and real-time quantitative RT-PCR (qPCR), respectively. The CpG methylation profile at the promoter region of LPL gene in 66-day-old broilers was determined by bisulfite sequencing. The average daily gain and percent abdominal fat traits were slightly improved in 56-day-old and 66-day-old broilers after dietary supplementation of betaine to diet. After adding 0.1% betaine to diet, the mRNA levels of fatty acid synthase (FAS) and adipocyte-type fatty acid-binding protein genes in abdominal adipose were significantly decreased in 56-day-old broilers, and those of LPL and FAS genes in abdominal adipose were significantly decreased in 66-day-old broilers comparing with the control group (P < 0.05 and P < 0.001). Moreover, in 66-day-old broilers fed 0.1% betaine diet, a different CpG methylation pattern was observed: the CpG dinucleotides of 1st, 6th, 7th, 8th and from 10th to 50th were less methylated; however, those of 2nd, 5th and 9th were more heavily methylated. The results suggest that transcription of some lipogenesis genes was decreased by betaine supplementation and betaine may decrease LPL mRNA expression by altering CpG methylation pattern on LPL promoter region.

Altered profile of mRNA expression in atrioventricular node of streptozotocin-induced diabetic rats

PubMed Central

Howarth, Frank Christopher; Parekh, Khatija; Jayaprakash, Petrilla; Inbaraj, Edward Samuel; Oz, Murat; Dobrzynski, Halina; Adrian, Thomas Edward

2017-01-01

Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)-induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ-induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription-quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization-activated cyclic-nucleotide 2 and 3, calcium channel voltage-dependent, β2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage-gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage-gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ-induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function. PMID:28731153

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

Altered Markers of Cortical γ-Aminobutyric Acid Neuronal Activity in Schizophrenia

PubMed Central

Kimoto, Sohei; Zaki, Mark M.; Bazmi, H. Holly; Lewis, David A.

2016-01-01

IMPORTANCE In schizophrenia, working memory deficits appear to reflect abnormalities in the generation of gamma oscillations in the dorsolateral prefrontal cortex. The generation of gamma oscillations requires the phasic excitation of inhibitory parvalbumin-containing interneurons. Thus, gamma oscillations depend, in part, on the number of synaptic glutamate receptors on parvalbumin interneurons. However, little is known about the molecular factors that regulate glutamate receptor–mediated excitation of parvalbumin interneurons in schizophrenia. OBJECTIVE To quantify in individuals with schizophrenia the expression of immediate early genes (NARP, ARC, and SGK1) regulating glutamate synaptic neurotransmission. DESIGN, SETTING, AND PARTICIPANTS Postmortem brain specimens (n = 206) were obtained from individuals with schizophrenia, bipolar disorder, or major depressive disorder and from well-matched healthy persons (controls). For a study of brain tissue, quantitative polymerase chain reaction, in situ hybridization, or microarray analyses were used to measure transcript levels in the dorsolateral prefrontal cortex at gray matter, laminar, and cellular levels of resolutions. This study was conducted between January 1, 2013, and November 30, 2014. MAIN OUTCOMES AND MEASURES Expression levels for NARP, ARC, and SGK1 messenger RNA (mRNA) were compared between specimens from individuals with schizophrenia and controls. Diagnostic specificity was assessed by quantifying NARP mRNA levels in specimens from individuals with mood disorders. RESULTS By quantitative polymerase chain reaction, levels of NARP mRNA were significantly lower by 25.6%in specimens from individuals with schizophrenia compared with the controls (mean [SD], 0.036 [0.018] vs 0.049 [0.015]; F1,114 = 21.0; P < .001). Levels of ARC (F1,112 = 0.93; P = .34) and SGK1 (F1,110 = 2.52; P = .12) were not significant. These findings were supported by in situ hybridization (NARP; individuals with schizophrenia vs controls: 40.1% lower [P = .003]) and microarray analyses (NARP; individuals with schizophrenia vs controls: 12.2%lower in layer 3 [P = .11] and 14.6%lower in layer 5 pyramidal cells [P = .001]). In schizophrenia specimens, NARP mRNA levels were positively correlated with GAD67 mRNA (r = 0.55; P < .001); the expression of GAD67 mRNA in parvalbumin interneurons is activity dependent. The NARP mRNA levels were also lower than healthy controls in bipolar disorder (−18.2%; F1,60 = 11.39; P = .001) and major depressive disorder (−21.7%; F1,30 = 5.36; P = .03) specimens, especially those from individuals with psychosis. In all 3 diagnostic groups, NARP mRNA levels were positively correlated (all r ≥ 0.53; all P ≤ .02) with somatostatin mRNA, the expression of which is activity dependent. CONCLUSIONS AND RELEVANCE Given the role of NARP in the formation of excitatory inputs to parvalbumin (and perhaps somatostatin) interneurons, our findings suggest that lower NARP mRNA expression contributes to lower excitatory drive onto parvalbumin interneurons in schizophrenia. This reduced excitatory drive may lead to lower synthesis of γ-aminobutyric acid in these interneurons, contributing to a reduced capacity to generate the gamma oscillations required for working memory. PMID:26038830

Altered Markers of Cortical γ-Aminobutyric Acid Neuronal Activity in Schizophrenia: Role of the NARP Gene.

PubMed

Kimoto, Sohei; Zaki, Mark M; Bazmi, H Holly; Lewis, David A

2015-08-01

In schizophrenia, working memory deficits appear to reflect abnormalities in the generation of gamma oscillations in the dorsolateral prefrontal cortex. The generation of gamma oscillations requires the phasic excitation of inhibitory parvalbumin-containing interneurons. Thus, gamma oscillations depend, in part, on the number of synaptic glutamate receptors on parvalbumin interneurons. However, little is known about the molecular factors that regulate glutamate receptor-mediated excitation of parvalbumin interneurons in schizophrenia. To quantify in individuals with schizophrenia the expression of immediate early genes (NARP, ARC, and SGK1) regulating glutamate synaptic neurotransmission. Postmortem brain specimens (n = 206) were obtained from individuals with schizophrenia, bipolar disorder, or major depressive disorder and from well-matched healthy persons (controls). For a study of brain tissue, quantitative polymerase chain reaction, in situ hybridization, or microarray analyses were used to measure transcript levels in the dorsolateral prefrontal cortex at gray matter, laminar, and cellular levels of resolutions. This study was conducted between January 1, 2013, and November 30, 2014. Expression levels for NARP, ARC, and SGK1 messenger RNA (mRNA) were compared between specimens from individuals with schizophrenia and controls. Diagnostic specificity was assessed by quantifying NARP mRNA levels in specimens from individuals with mood disorders. By quantitative polymerase chain reaction, levels of NARP mRNA were significantly lower by 25.6% in specimens from individuals with schizophrenia compared with the controls (mean [SD], 0.036 [0.018] vs 0.049 [0.015]; F1,114 = 21.0; P < .001). Levels of ARC (F1,112 = 0.93; P = .34) and SGK1 (F1,110 = 2.52; P = .12) were not significant. These findings were supported by in situ hybridization (NARP; individuals with schizophrenia vs controls: 40.1% lower [P = .003]) and microarray analyses (NARP; individuals with schizophrenia vs controls: 12.2% lower in layer 3 [P = .11] and 14.6% lower in layer 5 pyramidal cells [P = .001]). In schizophrenia specimens, NARP mRNA levels were positively correlated with GAD67 mRNA (r = 0.55; P < .001); the expression of GAD67 mRNA in parvalbumin interneurons is activity dependent. The NARP mRNA levels were also lower than healthy controls in bipolar disorder (-18.2%; F1,60 = 11.39; P = .001) and major depressive disorder (-21.7%; F1,30 = 5.36; P = .03) specimens, especially those from individuals with psychosis. In all 3 diagnostic groups, NARP mRNA levels were positively correlated (all r ≥ 0.53; all P ≤ .02) with somatostatin mRNA, the expression of which is activity dependent. Given the role of NARP in the formation of excitatory inputs to parvalbumin (and perhaps somatostatin) interneurons, our findings suggest that lower NARP mRNA expression contributes to lower excitatory drive onto parvalbumin interneurons in schizophrenia. This reduced excitatory drive may lead to lower synthesis of γ-aminobutyric acid in these interneurons, contributing to a reduced capacity to generate the gamma oscillations required for working memory.

Hydrogen sulfide upregulated mRNA expressions of sodium bicarbonate cotransporter1, trefoil factor1 and trefoil factor2 in gastric mucosa in rats.

PubMed

Cheraghi, Parisa; Mard, Seyyed Ali; Nagi, Tahereh

2016-01-01

Hydrogen sulfide (H 2 S) has been shown to protect the gastric mucosa through several protective mechanisms but till now its effect on mRNA expression of sodium bicarbonate cotransporter 1 (NBC1), trefoil factor1 (TFF1) and trefoil factor2 (TFF2) was not investigated. This study was aimed to evaluate the effect of H 2 S on mRNA expression of NBC1, TFF1 and TFF2 in rat gastric mucosa in response to gastric distention. Thirty two rats were randomly assigned into four equal groups. They were control (C), distention (D), propargylglycine (PAG)-, and NaHS-treated groups. To evaluate the effect of exogenous and endogenous H 2 S on gene expression of NBC1, TFF1 and TFF2, two groups of rats were received H 2 S donor, intra-peritoneal NaHS (80 µg Kg -1 ), and PAG (50 mg kg -1 ), accompanied to stimulate the gastric acid secretion, respectively. Under general anesthesia and laparotomy, a catheter was inserted into the stomach through duodenum for instillation of isotonic saline for gastric distention. Ninety min after beginning the experiment, animals were sacrificed and the gastric mucosa was collected to determine total acid content of gastric effluents and to quantify the mRNA expression of studied genes by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that A) gastric distention increased the level of mRNA expressions of NBC1, TFF1 and TFF2; B) these levels in NaHS-treated rats were significantly higher than those in Distention group; and C) PAG decreased the expression levels of NBC1 and TFF1. The Findings showed H 2 S upregulated gene expression of NBC1, TFF1 and TFF2 in gastric mucosa.

Tristetraprolin regulates the expression of the human inducible nitric-oxide synthase gene.

PubMed

Fechir, Marcel; Linker, Katrin; Pautz, Andrea; Hubrich, Thomas; Förstermann, Ulrich; Rodriguez-Pascual, Fernando; Kleinert, Hartmut

2005-06-01

The expression of human inducible NO synthase (iNOS) is regulated both by transcriptional and post-transcriptional mechanisms. Stabilization of mRNAs often depends on activation of p38 mitogen-activated protein kinase (p38 MAPK). In human DLD-1 cells, inhibition of p38 MAPK by the compound 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) or by overexpression of a dominant-negative p38 MAPKalpha protein resulted in a reduction of human iNOS mRNA and protein expression, whereas human iNOS promoter activity was not affected. An important RNA binding protein regulated by the p38 MAPK pathway and involved in the regulation of the stability of several mRNAs is tristetraprolin. RNase protection, quantitative real-time polymerase chain reaction, and Western blot experiments showed that cytokines used to induce iNOS expression in DLD-1 cells also enhanced tristetraprolin expression. SB203580 incubation reduced cytokine-mediated enhancement of tristetraprolin expression. Overexpression or down-regulation of tristetraprolin in stably transfected DLD-1- or A549/8 cells consistently resulted in enhanced or reduced iNOS expression by modulating iNOS-mRNA stability. In UV cross-linking experiments, recombinant tristetraprolin did not interact with the human iNOS mRNA. However, coimmunoprecipitation experiments showed interaction of tristetraprolin with the KH-type splicing regulatory protein (KSRP), which is known to recruit mRNAs containing AU-rich elements to the exosome for degradation. This tristetraprolin-KSRP interaction was enhanced by cytokines and reduced by SB203580 treatment. We conclude that tristetraprolin positively regulates human iNOS expression by enhancing the stability of human iNOS mRNA. Because tristetraprolin does not directly bind to the human iNOS mRNA but interacts with KSRP, tristetraprolin is likely to stabilize iNOS mRNA by capturing the KSRP-exosome complex.

Molecular characterization of Na+/K+/2Cl- cotransporter 1 alpha from Trachinotus ovatus (Linnaeus, 1758) and its expression responses to acute salinity stress.

PubMed

Zhao, Chao-Ping; Guo, Hua-Yang; Zhu, Ke-Cheng; Guo, Liang; Zhang, Nan; Liu, Bao-Suo; Yang, Jing-Wen; Liu, Bo; Jiang, Shi-Gui; Zhang, Dian-Chang

2018-06-06

Trachinotus ovatus is widely cultured in the ponds and gulf on the southeast coast of China. The dramatic salinity decrease caused by heavy rainfall could cause mass mortality of T. ovatus in aquaculture. It is very important to understand the osmoregulatory mechanism of T. ovatus. Na + /K + /2Cl - cotransporter 1a (NKCC1a) is involved in the osmoregulation of fish and plays a crucial role in cell volume homeostasis and maintenance of the electrolyte content. In this study, we characterized nkcc1a (designed as Tonkcc1a) from T. ovatus and investigated its expression responses to acute salinity changes. Tonkcc1a is approximately 70 kb in length and contains 26 exons and 25 introns. The phylogenetic analysis confirmed that ToNKCC1a belonged to the NKCC1a subclade. Quantitative real-time (qRT-PCR) analysis indicated that Tonkcc1a was ubiquitously expressed in all examined tissues, with the highest mRNA levels observed in gills, and the lowest level in liver. When T. ovatus were transferred from seawater (30‰) into fresh water, the expression levels of Tonkcc1a mRNA were significantly downregulated in gills and kidney, whereas its expression level was markedly upregulated in intestine. When transferred from seawater (30‰) to 10‰ sea water, the expression levels of Tonkcc1a mRNA were clearly increased in gills and kidney. When transferred from seawater (30‰) to 20‰ sea water, the expression of Tonkcc1a mRNA increased to some extent in gills, kidney, and intestine. When transferred from seawater (30‰) to 40‰ sea water, the expression levels of Tonkcc1a mRNA were dramatically upregulated in gills and intestine compared to that in the control. These results suggested that Tonkcc1a was involved in the response to acute salinity changes. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantitative mRNA expression analysis of selected genes in patients with early-stage hypothyroidism induced by treatment with iodine-131.

PubMed

Guo, Kun; Gao, Rui; Yu, Yan; Zhang, Weixiao; Yang, Yuxuan; Yang, Aimin

2015-11-01

The present study aimed to investigate the molecular markers indicative of early-stage hypothyroidism induced by treatment with iodine-131, in order to assist in further investigations of radio iodine‑induced hypothyroidism. A total of 59 patients diagnosed with hyperthyroidism (male/female, 16/43; median age, 46.4 years) and 27 healthy subjects (male/female, 7/21; median age, 44.6 years) were included in the present study. All patients were treated with appropriate doses of iodine‑131 and, three months following treatment, the patients were subdivided into two groups: A group with early‑stage hypothyroidism symptoms, and a group with non‑early‑stage hypothyroidism, including euthyroid patients and patients remaining with hyperthyroidism. Tissue samples from the patients and healthy subjects were collected by fine needle biopsies, and the mRNA expression levels of B-cell lymphoma 2 (Bcl‑2), nuclear factor (NF)‑κB, Ku70, epidermal growth factor receptor (EGFR), early growth response 1 (Egr‑1), TP53 and ataxia telangiectasia mutated were analyzed using reverse transcription‑quantitative polymerase chain reaction prior to iodine‑131 treatment. The association of the variation of target genes with susceptibility to early‑stage hypothyroidism was analyzed. Compared with normal subjects, the mRNA expression levels of Ku70 (0.768, vs. 3.304, respectively; P<0.001) and EGFR (0.859, vs. 1.752, respectively; P<0.05) were significantly higher, whereas those of NF‑κB (0.884, vs. 0.578, respectively; P<0.05) and Bcl‑2 (1.235, vs. 0.834, respectively; P<0.05) were lower in the hyperthyroid patients. Following treatment with iodine‑131, 30 of the 59 (50.8%) patients with hyperthyroidism were diagnosed with early‑stage hypothyroidism, and in the early‑stage hypothyroidism group, the mRNA expression levels of Bcl‑2 were significantly decreased (P<0.05), whereas those of Egr‑1 (P<0.05) were significantly increased, compared with the non‑early‑stage hypothyroidism group. The association between the changes in the expression levles of Bcl‑2 and Egr‑1 and susceptibility to early‑stage hypothyroidism was supported by multivariate regression analysis. No significant changes in the expression levels of the other target genes were detected. The opposing changes in the mRNA expression levels of Bcl‑2 and Egr‑1 in patients with early‑stage hypothyroidism indicates their potential as prognostic markers of early-stage hypothyroidism induced by iodine-131 treatment.

Attachment, proliferation and collagen type I mRNA expression of human gingival fibroblasts on different biodegradable membranes.

PubMed

Hakki, Sema S; Korkusuz, Petek; Purali, Nuhan; Bozkurt, Buket; Kus, Mahmut; Duran, Ismet

2013-01-01

The purpose of this study was to investigate adhesion, proliferation and type I collagen (COL I) mRNA expression of gingival fibroblasts on different membranes used in periodontal applications. Collagen (C), acellular dermal matrix (ADM) and polylactic acid; polyglycolic acid; lactide/glycolide copolymer (PLGA) biodegradable membranes were combined with gingival fibroblasts in culture and incubated for 48 h. Cell adhesion was examined with scanning electron and confocal microscopy. MTT assay was used to measure proliferation. COL I mRNA expression was assessed using quantitative-polymerase chain reaction (QPCR). The PLGA group exhibited the lowest cell survival on day 5 and 10, and lowest cell proliferation on days 5, 10 and 14. While cell proliferation was similar in C and ADM groups, the C membrane showed a slightly greater increase in viable cells to day 10. Confocal and scanning electron microscopy confirmed the results of proliferation and MTT assays. The highest COL I mRNA expression was noted in the PLGA membrane group when compared to the C (p < 0.01) and ADM (p < 0.05) membrane groups. These data revealed that adherence and proliferation of primary gingival fibroblasts on collagen-based C and ADM membranes is better than that seen with PLGA membranes, and thus may be preferable in the treatment of gingival recession defects.

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

Lundasen, Thomas; Molecular Nutrition Unit, Department of Biosciences and Nutrition, NOVUM, Karolinska Institutet, Huddinge, SE-141 86 Stockholm; Hunt, Mary C.

The metabolic regulator fibroblast growth factor 21 (FGF21) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF21 is regulated by the peroxisome proliferator-activated receptor alpha (PPAR{alpha}). Fasting or treatment of mice with the PPAR{alpha} agonist Wy-14,643 induced FGF21 mRNA by 10-fold and 8-fold, respectively. In contrast, FGF21 mRNA was low in PPAR{alpha} deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF21. Obese ob/ob mice, known to have increased PPAR{alpha} levels, displayed 12-fold increased hepatic FGF21 mRNA levels. The potential importance of PPAR{alpha} formore » FGF21 expression also in human liver was shown by Wy-14,643 induction of FGF21 mRNA in human primary hepatocytes, and PPAR{alpha} response elements were identified in both the human and mouse FGF21 promoters. Further studies on the mechanisms of regulation of FGF21 by PPAR{alpha} in humans will be of great interest.« less

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

PubMed

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

2010-01-01

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

Expression of Angiotensin II Types 1 and 2 Receptors in Endometriotic Lesions.

PubMed

Nakao, Takehiro; Chishima, Fumihisa; Sugitani, Masahiko; Tsujimura, Ryusuke; Hayashi, Chuyu; Yamamoto, Tatsuo

2017-01-01

The aim of this study was to evaluate the gene and protein expression of angiotensin type (AT) 1, AT2 receptors in endometriotic lesions and its relation to prostaglandin (PG) synthases. Endometriosis samples were obtained from 32 patients with endometriotic cysts. Endometrial tissues were obtained during operations for benign gynecological conditions. The expression of the AT1 and AT2 receptor mRNA and that of PG-endoperoxide synthase 2 and microsomal PGE2 synthase-1 (mPGES-1) was examined by quantitative RT-PCR. Immunohistochemical staining was performed for these receptors. AT1 and AT2 receptor proteins were mostly located in endometrial glandular epithelium and some stromal cells. Immunoreactivity of the receptor proteins was observed in both the eutopic endometrium and endometriotic lesions. The AT1/AT2 ratio in endometriotic cysts (median 7.29, range 1.88-187.60) was significantly increased compared with that in the eutopic endometrium in the proliferative-phase in controls (median 1.01, range 0.37-2.09, p < 0.001). There was a relationship between the AT1 mRNA expression and that of mPGES-1 mRNA in the endometriotic cysts (r = 0.394089, p < 0.05). There was a significant relationship between the mRNA expression of the AT2 receptor and that of mPGES-1 in eutopic endometrium of non-endometriotic control (r = 0.610714, p < 0.05). Renin-angiotensin system may play an important role in the pathophysiology of endometriosis. © 2016 S. Karger AG, Basel.

Biological significance of PinX1 telomerase inhibitor in esophageal carcinoma treatment

PubMed Central

Fan, Xiang-Kui; Yan, Rui-Hua; Geng, Xiang-Qun; Li, Jing-Shan; Chen, Xiang-Ming; Li, Jian-Zhe

2016-01-01

In the present study, to investigate the expression of PinX1 gene and its functional effects in human esophageal carcinoma (Eca)-109 cell line, expression vectors of human PinX1 (pEGFP-C3-PinX1) and its small interfering RNA (PinX1-FAM-siRNA) were constructed and transfected into Eca-109 cells using Lipofectamine 2000. Firstly, the mRNA expression level of PinX1 was examined using reverse transcription-polymerase chain reaction (RT-PCR). Once successful transfection was achieved, the effects on the mRNA level of human telomerase reverse transcriptase (hTERT), telomerase activity, cell proliferation and apoptosis were examined by semi-quantitative RT-PCR, stretch PCR, MTT assay and flow cytometry, respectively. Analysis of restriction and sequencing demonstrated that the recombining plasmids were successfully constructed. The results also indicated that transfection with pEGFP-C3-PinX1 and PinX1-FAM-siRNA into Eca-109 cells significantly increased PinX1 mRNA, decreased hTERT mRNA by 29.9% (P<0.05), and significantly reduced telomerase activity (P<0.05), inhibited cell growth, and increased the cell apoptotic index from 19.27±0.76 to 49.73±2%. The transfected PinX1-FAM-SiRNA exhibited PinX1 mRNA expression levels that were significantly decreased by 70% (P<0.05), whereas the remaining characteristics of Eca-109 cells, including cell growth, mRNA level of hTERT, telomerase activity and cell apoptotic index were not altered. Exogenous PinX1 has been demonstrated to be highly expressed in human Eca. PinX1 can inhibit human telomerase activity and the expression of hTERT mRNA, reduce tumor cell growth and induce apoptosis. Notably, these inhibitory functions were inhibited by silencing PinX1 in Eca with PinX1-FAM-siRNA. PinX1 was successfully increased and decreased in the present study, demonstrating that it may be a potential telomerase activity inhibitor. As PinX1 is an endogenous telomerase inhibitor, it may be used as a novel tumor-targeted gene therapy. PMID:27698711

Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression.

PubMed

Mack, D R; Michail, S; Wei, S; McDougall, L; Hollingsworth, M A

1999-04-01

Probiotic agents, live microorganisms with beneficial effects for the host, may offer an alternative to conventional antimicrobials in the treatment and prevention of enteric infections. The probiotic agents Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG quantitatively inhibited the adherence of an attaching and effacing pathogenic Escherichia coli to HT-29 intestinal epithelial cells but did not inhibit adherence to nonintestinal HEp-2 cells. HT-29 cells were grown under conditions that induced high levels of either MUC2 or MUC3 mRNA, but HEp-2 cells expressed only minimal levels of MUC2 and no MUC3 mRNA. Media enriched for MUC2 and MUC3 mucin were added exogenously to binding assays and were shown to be capable of inhibiting enteropathogen adherence to HEp-2 cells. Incubation of L. plantarum 299v with HT-29 cells increased MUC2 and MUC3 mRNA expression levels. From these in vitro studies, we propose the hypothesis that the ability of probiotic agents to inhibit adherence of attaching and effacing organisms to intestinal epithelial cells is mediated through their ability to increase expression of MUC2 and MUC3 intestinal mucins.

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

PubMed

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

1995-09-01

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

[Expression and significance of P-gp/mdr1 mRNA, MRP and LRP in non-Hodgkin's lymphoma].

PubMed

Li, Le; Su, Li-ping; Ma, Li; Zhao, Jin; Zhu, Lei; Zhou, Yong-an

2009-03-01

To explore the expression and clinical significance of P-glycoprotein (P-gp)/mdr1mRNA, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) in newly diagnosed non-Hodgkin's lymphoma. mdr1 mRNA of in 41 patients with non-Hodgkin's lymphoma was assayed by semi-quantitative RT-PCR. The expressions of P-gp, MRP and LRP proteins in lymph node viable blasts were identified by flow cytometry. The results were compared with those obtained from control cases, and the correlation of the changes with clinical outcomes was analyzed. (1) Among the 41 cases, the positive expression of P-gp protein was detected in 8 cases, MRP in 7 cases, LRP in 15 cases, and mdr 1 mRNA in 11 cases. (2) The P-gp and LRP levels in NHL were significantly higher than those in control group, but MRP wasn't. The P-gp over-expression was significantly associated with mdr1mRNA (r = 0.396, P = 0.01). No correlation was showed among the expressions of P-gp, MRP and LRP. (3) Patients with P-gp expression had a poorer outcome of chemotherapy than those with P-gp-negative (P = 0.005). P-gp expression was significantly associated with higher clinical stage (P = 0.046) and elevated serum lactate dehydrogenase level (P = 0.032), but not associated with malignant degree (P = 0.298). MRP had no impact on the outcome of chemotherapy (P = 0.212), and wasn't significantly associated with higher clinical stage (P = 0.369), elevated LDH (P = 0.762) and higher malignant degree (P = 0.451). Patients with LRP expression had a poorer outcome of chemotherapy than those LRP-negative (P = 0.012). LRP expression was significantly associated with higher clinical stage (P = 0.0019), elevated LDH (P = 0.02) and higher malignant degree (P = 0.01). The data of this study indicate that P-gp and LRP expressions but not MRP expression are important in the mechanism of drug resistance associated with a poor clinical outcome in previously untreated NHL.

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.

GDF-15 gene expression alterations in human lymphoblastoid cells and peripheral blood lymphocytes following exposure to ionizing radiation

PubMed Central

Li, Shuang; Zhang, Qing-Zhao; Zhang, De-Qin; Feng, Jiang-Bin; Luo, Qun; Lu, Xue; Wang, Xin-Ru; Li, Kun-Peng; Chen, De-Qing; Mu, Xiao-Feng; Gao, Ling; Liu, Qing-Jie

2017-01-01

The identification of rapid, sensitive and high-throughput biomarkers is imperative in order to identify individuals harmed by radiation accidents, and accurately evaluate the absorbed doses of radiation. DNA microarrays have previously been used to evaluate the alterations in growth/differentiation factor 15 (GDF15) gene expression in AHH-1 human lymphoblastoid cells, following exposure to γ-rays. The present study aimed to characterize the relationship between the dose of ionizing radiation and the produced effects in GDF-15 gene expression in AHH-1 cells and human peripheral blood lymphocytes (HPBLs). GDF-15 mRNA and protein expression levels following exposure to γ-rays and neutron radiation were assessed by reverse transcription-quantitative polymerase chain reaction and western blot analysis in AHH-1 cells. In addition, alterations in GDF-15 gene expression in HPBLs following ex vivo irradiation were evaluated. The present results demonstrated that GDF-15 mRNA and protein expression levels in AHH-1 cells were significantly upregulated following exposure to γ-ray doses ranging between 1 and 10 Gy, regardless of the dose rate. A total of 48 h following exposure to neutron radiation, a dose-response relationship was identified in AHH-1 cells at γ-ray doses between 0.4 and 1.6 Gy. GDF-15 mRNA levels in HPBLs were significantly upregulated following exposure to γ-ray doses between 1 and 8 Gy, within 4–48 h following irradiation. These results suggested that significant time- and dose-dependent alterations in GDF-15 mRNA and protein expression occur in AHH-1 cells and HPBLs in the early phases following exposure to ionizing radiation. In conclusion, alterations in GDF-15 gene expression may have potential as a biomarker to evaluate radiation exposure. PMID:28440431

Decreased placental and muscular expression of the fibroblast growth factor 19 in gestational diabetes mellitus.

PubMed

Wang, Dongyu; Xu, Shuqia; Ding, Wenjing; Zhu, Caixia; Deng, Songqing; Qiu, Xiwen; Wang, Zilian

2018-05-07

Fibroblast growth factor (FGF) 19 has been shown to improve glycaemic homeostasis and lipid metabolism in animal models. In humans, decreased FGF19 level has been described in diabetes. This study aimed to investigate the expression of FGF19 in gestational diabetes mellitus (GDM). Samples for measurement were obtained from 20 GDM women and 25 healthy controls. The mRNA and protein expression levels of FGF19, FGF21 and co-receptor β-klotho (KLB) in placenta, rectus muscle and subcutaneous fat tissues were quantified by real-time quantitative polymerase chain reaction, western-blot and immunohistochemistry, respectively. Women with GDM had significantly lower mRNA and protein expressions of FGF19 than control women had in placenta (mRNA: 0.33 ± 0.05 vs. 0.72 ± 0.09; protein: 0.34 ± 0.13 vs. 0.85 ± 0.20) and rectus muscle (mRNA: 0.83 ± 0.11 vs. 1.28 ± 0.19; protein: 0.78 ± 0.24 vs. 1.23 ± 0.39). However, there were no significant differences between GDM women and controls with respect to the expression levels of FGF21 and KLB in placenta and rectus muscle. There were almost no detectable FGF19 and FGF21 expressions in subcutaneous fat tissue. Moreover, KLB expression levels were not different between GDM and control group in subcutaneous fat. FGF19expressions are decreased in GDM women's placenta and rectus muscle. This may contribute to the pathophysiology or development of GDM. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Developmental expression and distribution of nesfatin-1/NUCB2 in the canine digestive system.

PubMed

Jiang, Shudong; Zhou, Weijuan; Zhang, Xingwang; Wang, Dengfeng; Zhu, Hui; Hong, Meizhen; Gong, Yajing; Ye, Jing; Fang, Fugui

2016-03-01

Nesfatin-1/NUCB2 is a neuropeptide that plays important roles in regulating food intake and energy homeostasis. The distribution of nesfatin-1/NUCB2 protein and mRNA has not been investigated in the canine digestive system. The present study was conducted to evaluate the expression of nesfatin-1/NUCB2 protein and NUCB2 mRNA in the canine digestive organs (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver and pancreas). The tissues of the digestive system were collected from dogs at different developmental stages (infantile, juvenile, pubertal and adult). Nesfatin-1/NUCB2 protein localization in the organs of adult dogs was detected by immunohistochemistry. The expression of NUCB2 mRNA at the four developmental stages was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). Nesfatin-1/NUCB2 protein was distributed in the fundic gland region of the stomach, and the islet area and exocrine portions of the pancreas. However, NUCB2 mRNA was found in all digestive organs, although the expression levels in the pancreas and stomach were higher than those in liver, duodenum and other digestive tract tissues (P<0.05) at the four different developmental stages of the dogs. In this study, nesfatin-1/NUCB2 was found to be present at high levels in the stomach and pancreas at both the protein and mRNA levels; however, NUCB2 expression was found at lower levels in all of the digestive organs. These findings provide the basis of further investigations to elucidate the functions of nefatin-1 in the canine digestive system. Copyright © 2015 Elsevier GmbH. All rights reserved.

Cytokine analysis in lesions refractory to endodontic treatment.

PubMed

Henriques, Luiz Carlos Feitosa; de Brito, Luciana Carla Neves; Tavares, Warley Luciano Fonseca; Vieira, Leda Quércia; Ribeiro Sobrinho, Antônio Paulino

2011-12-01

Failure in endodontic treatment is often caused by the persistence of microorganisms in the root canal after therapy. When treatment fails, an immune response develops that is characterized by an extensive network of immunologic mechanisms that lead to the production of cytokines and chemokines. The objective of this study was to determine the relative messenger RNA (mRNA) expression of IFN-γ, TNF-α, IL-1β, IL-17A, IL-10, and MCP-1 in periapical dental lesions refractory to treatment. Clinical samples were taken from teeth presenting periapical lesions refractory to endodontic treatment (the experimental group) or from healthy teeth with pulp vitality (the control group). Three paper points passing through the root apex (2 mm) were used to collect the samples. The total RNA was extracted from each sample, complementary DNA was synthesized, and quantitative polymerase chain reaction analysis was performed. The Mann-Whitney U test was used to determine the statistical significance of our findings (P < .05). Significant differences in the levels of IFN-γ, TNF-α, IL-17A, and MCP-1 mRNA expression were observed in cases refractory to endodontic treatment as compared with the control group. The expression of IL-1β mRNA was not significantly different between the groups. The expression of IL-10 mRNA was insignificant in both the experimental and control groups. A significantly increased expression of TNF-α, IFN-γ, IL-17A, and MCP-1 mRNA was observed in the periapical immune response in cases of endodontic failure. These results suggest that a proinflammatory cytokine profile predominates in these types of dental lesions. Copyright © 2011 American Association of Endodontists. All rights reserved.

Correlation of Cyfra 21-1 levels in saliva and serum with CK19 mRNA expression in oral squamous cell carcinoma.

PubMed

Malhotra, Rewa; Urs, Aadithya B; Chakravarti, Anita; Kumar, Suman; Gupta, V K; Mahajan, Bhawna

2016-07-01

Oral squamous cell carcinoma (OSCC) accounts for 90 % of malignant lesions of oral cavity. The study assessed the potential of Cyfra 21-1 as a tumor marker in OSCC. The study included 50 patients of OSCC to evaluate levels of Cyfra 21-1 in serum and saliva by electrochemiluminescent immunoassay (ECLIA) and CK19 messenger RNA (mRNA) expression in tissue by florescent quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) along with healthy individuals as control. The salivary and serum Cyfra 21-1 levels in patients of OSCC were significantly higher compared to controls (p value < 0.01). There was a 2.75-fold increase in CK19 mRNA expression in OSCC cases compared to controls. A significant positive correlation was found between serum and salivary Cyfra 21-1, serum Cyfra 21-1, and CK19 mRNA expression and between salivary Cyfra 21-1 and CK19 mRNA expression. Among these, correlation between serum and salivary Cyfra 21-1 was highly significant. Salivary and serum Cyfra 21-1 showed significantly elevated levels in grade II OSCC compared to grade I histopathologically. Elevated levels of salivary Cyfra 21-1 were associated with recurrence in OSCC patients. Reverse operating curve constructed using 3 ng/ml as a cutoff for serum Cyfra 21-1 revealed the sensitivity and specificity to be 88 and 78.2 %, respectively. Using a cutoff value of 8.5 ng/ml for salivary Cyfra 21-1, the sensitivity was found to be 93.8 % and specificity 84.3 %. We advocate salivary Cyfra 21-1 as a better diagnostic marker over serum Cyfra 21-1 as well as a potential marker in the prognosis of OSCC.

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning, tissue expression, and the function of feeding regulation.

PubMed

Wang, Tao; Zhou, Chaowei; Yuan, Dengyue; Lin, Fangjun; Chen, Hu; Wu, Hongwei; Wei, Rongbin; Xin, Zhiming; Liu, Ju; Gao, Yundi; Li, Zhiqiong

2014-10-01

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.

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

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.

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

PubMed

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

2017-01-01

In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. 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. 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. 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). 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.

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

Effects of low-level laser therapy on the expression of osteogenic genes related in the initial stages of bone defects in rats

NASA Astrophysics Data System (ADS)

Fernandes, Kelly Rossetti; Ribeiro, Daniel Araki; Rodrigues, Natália Camargo; Tim, Carla; Santos, Anderson Amaro; Parizotto, Nivaldo Antônio; de Araujo, Heloisa Selistre; Driusso, Patrícia; Rennó, Ana Claudia Muniz

2013-03-01

We evaluate the effects of low-level laser therapy (LLLT) on the histological modifications and temporal osteogenic genes expression during the initial phase of bone healing in a model of bone defect in rats. Sixty-four Wistar rats were divided into control and treated groups. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation (Ga-Al-As laser 830 nm, 30 mW, 0.028 cm2, 1.071 W/cm2, 1 min and 34 s, 2.8 Joules, 100 J/cm2) was performed for 1, 2, 3, and 5 sessions. Histopathology revealed that treated animals presented higher inflammatory cells recruitment, especially 12 and 36 h postsurgery. Also, a better tissue organization at the site of the injury, with the presence of granulation tissue and new bone formation was observed on days three and five postsurgery in the treated animals. The quantitative real time polymerase chain reaction showed that LLLT produced a significantly increase in mRNA expression of Runx-2, 12 h and three days post-surgery, a significant upregulation of alkaline phosphatase mRNA expression after 36 h and three days post-surgery and a significant increase of osteocalcin mRNA expression after three and five days. We concluded that LLLT modulated the inflammatory process and accelerated bone repair, and this advanced repair pattern in the laser-treated groups may be related to the higher mRNA expression of genes presented by these animals.

Effects of RNAi-Mediated Knockdown of Histone Methyltransferases on the Sex-Specific mRNA Expression of Imp in the Silkworm Bombyx mori

PubMed Central

Suzuki, Masataka G.; Ito, Haruka; Aoki, Fugaku

2014-01-01

Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to <10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity. PMID:24758924

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.

Cannabinoid receptor expression and phosphorylation are differentially regulated between male and female cerebellum and brain stem after repeated stress: implication for PTSD and drug abuse.

PubMed

Xing, Guoqiang; Carlton, Janis; Zhang, Lei; Jiang, Xiaolong; Fullerton, Carol; Li, He; Ursano, Robert

2011-09-08

Recent study demonstrated a close relationship between cerebellum atrophy and symptom severity of pediatric maltreatment-related posttraumatic stress disorder (PTSD). It has also been known that females are more vulnerable than males in developing anxiety disorders after exposure to traumatic stress. The mechanisms are unknown. Because cannabinoid receptors (CB₁ and CB₂) are neuroprotective and highly expressed in the cerebellum, we investigated cerebellar CB expression in stressed rats. Young male and female Sprague-Dawley rats were given 40 unpredictable electric tail-shocks for 2h daily on 3 consecutive days. CB₁ and CB₂ mRNA and protein levels in rat cerebellum and brain stem were determined using quantitative real-time PCR and Western blot, respectively. Two-way ANOVA revealed significant gender and stress effects on cerebellar CB₁ mRNA expression, with females and non-stressed rats exhibiting higher CB₁ mRNA levels than the males (3 fold, p<0.01) and stressed rats (30%, p<0.01), respectively. CB₁ and CB₂ mRNA levels in brain stem were also greater in female rats than males (p<0.01, p<0.05, respectively). Repeated stress increased the level of phosphorylated CB₁ receptors, the inactivated CB₁, in rat cerebellum (p<0.01), particularly in female rats as revealed by the significant gender × stress interaction. Thus, repeated severe stress caused greater CB₁ mRNA suppression and CB₁ receptor phosphorylation in female cerebellum that could lead to increased susceptibility to stress-related anxiety disorders including PTSD. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Elevated expression of the proto-oncogene c-kit in patients with mastocytosis.

PubMed

Nagata, H; Worobec, A S; Semere, T; Metcalfe, D D

1998-02-01

The stem cell factor (SCF)c-kit receptor interaction plays a critical role in the development and survival of mast cells. Several studies have also associated c-kit receptor mutations with the human diseases, mastocytosis and piebaldism. Overexpression of c-kit has been reported to be associated with myeloproliferative disorders and myelodysplastic syndromes. Using peripheral blood mononuclear cells (PBMCs) from 11 patients with indolent mastocytosis (category I), mastocytosis with an associated hematologic disorder (category II), or aggressive mastocytosis (category III); a patient with CMML unassociated with mastocytosis, and PBMCs from 13 normal subjects, we examined the level of expression of c-kit mRNA along with other c-kit isoforms to determine if overexpression of the c-kit receptor was associated with mastocytosis. Using quantitative competitive PCR, c-kit mRNA levels on average were found to be statistically elevated in the five patients with mastocytosis with an associated hematologic disorder and in the patient with aggressive mastocytosis as compared with controls, but not elevated in patients with indolent mastocytosis. The relative mRNA expression for the two c-kit isoforms was not significantly different in the mastocytosis patients compared with controls. This demonstration of the overexpression of c-kit mRNA in mastocytosis, and particularly those patients with clinical evidence of myelodysplastic syndrome, adds evidence to support the conclusion that mastocytosis, at least in some patients, is a feature of myelodysplasia; and suggests that determination of c-kit mRNA expression in PBMCs may provide an additional approach to assessing prognosis.

Putative presynaptic dopamine dysregulation in schizophrenia is supported by molecular evidence from post-mortem human midbrain

PubMed Central

Purves-Tyson, T D; Owens, S J; Rothmond, D A; Halliday, G M; Double, K L; Stevens, J; McCrossin, T; Shannon Weickert, C

2017-01-01

The dopamine hypothesis of schizophrenia posits that increased subcortical dopamine underpins psychosis. In vivo imaging studies indicate an increased presynaptic dopamine synthesis capacity in striatal terminals and cell bodies in the midbrain in schizophrenia; however, measures of the dopamine-synthesising enzyme, tyrosine hydroxylase (TH), have not identified consistent changes. We hypothesise that dopamine dysregulation in schizophrenia could result from changes in expression of dopamine synthesis enzymes, receptors, transporters or catabolic enzymes. Gene expression of 12 dopamine-related molecules was examined in post-mortem midbrain (28 antipsychotic-treated schizophrenia cases/29 controls) using quantitative PCR. TH and the synaptic dopamine transporter (DAT) proteins were examined in post-mortem midbrain (26 antipsychotic-treated schizophrenia cases per 27 controls) using immunoblotting. TH and aromatic acid decarboxylase (AADC) mRNA and TH protein were unchanged in the midbrain in schizophrenia compared with controls. Dopamine receptor D2 short, vesicular monoamine transporter (VMAT2) and DAT mRNAs were significantly decreased in schizophrenia, with no change in DRD3 mRNA, DRD3nf mRNA and DAT protein between diagnostic groups. However, DAT protein was significantly increased in putatively treatment-resistant cases of schizophrenia compared to putatively treatment-responsive cases. Midbrain monoamine oxidase A (MAOA) mRNA was increased, whereas MAOB and catechol-O-methyl transferase mRNAs were unchanged in schizophrenia. We conclude that, whereas some mRNA changes are consistent with increased dopamine action (decreased DAT mRNA), others suggest reduced dopamine action (increased MAOA mRNA) in the midbrain in schizophrenia. Here, we identify a molecular signature of dopamine dysregulation in the midbrain in schizophrenia that mainly includes gene expression changes of molecules involved in dopamine synthesis and in regulating the time course of dopamine action. PMID:28094812

TS and ERCC-1 mRNA expressions and clinical outcome in patients with metastatic colon cancer in CONFIRM-1 and -2 clinical trials.

PubMed

Grimminger, P P; Shi, M; Barrett, C; Lebwohl, D; Danenberg, K D; Brabender, J; Vigen, C L P; Danenberg, P V; Winder, T; Lenz, H-J

2012-10-01

To validate established cutoff levels of thymidylate synthase (TS) and excision repair cross-complementing (ERCC-1) intratumoral mRNA expressions in tumor samples from metastatic colorectal cancer (mCRC) patients treated with PTK787/ZK222584 (PTK/ZK). From 122 samples of patients with mCRC enrolled in CONFIRM-1 (Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases) or CONFIRM-2, mRNA was isolated of microdissected formalin-fixed paraffin-embedded samples and quantitated using TaqMan-based technology. Existing TS and ERCC-1 cutoff levels were tested for their prognostic value in first-line and second-line therapy. TS expression was associated with overall survival (OS) in first-line, but not second-line therapy. ERCC-1 was associated with OS in patients treated with first-line and second-line FOLFOX4. In first-line FOLFOX4, combination of high TS and/or high ERCC-1 was associated with shorter OS. A correlation was observed between ERCC-1 expression and benefit from PTK/ZK+FOLFOX4 treatment. TS and ERCC-1 expression is associated with clinical outcome in mCRC. Baseline TS and ERCC-1 levels may allow the selection of patients who benefit from FOLFOX4 chemotherapy.

Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

PubMed Central

XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

2015-01-01

The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P<0.01), but not in the Val-Lys-Gly-Ile-Leu-Ser group (P>0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

[Knock-down of ZEB1 inhibits the proliferation, invasion and migration of gastric cancer cells].

PubMed

Chen, Dengyu; Chu, Yifan; Zheng, Qingwei; Xu, Zhiben; Zhou, Ping; Li, Sheng

2017-08-01

Objective To down-regulate the expression of zinc-finger E-box binding homeobox 1 (ZEB1) gene by shRNA, and investigate its effect on invasion, migration and proliferation, as well as the related gene expressions of lncRNA HOTAIR and E-cadherin in human gastric cancer BGC823 cells. Methods RNA interfering (RNAi) was used to knock down ZEB1 in gastric cancer BGC823 cells. The recombinant plasmid shZEB1 was constructed and transfected into the gastric cancer BGC823 cells by Lipofectamine TM 2000, and the stably transfected cells were isolated by G418 selection and limited dilution. The expression of ZEB1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. Cell proliferation was determined by MTT assay, and the invasion and migration abilities of BGC823 cells were monitored by Transwell TM invasion assay and wound healing assay, respectively. The expressions of lncRNA HOTAIR and E-cadherin mRNA were detected by real-time quantitative PCR. Results After ZEB1 expression was successfully down-regulated in BGC823 cells by siRNA, the proliferation, invasion and migration rates in shZEB1 transfection group were significantly lower than those in control group; meanwhile, the expression of lncRNA HOTAIR was reduced and E-cadherin expression was enhanced. Conclusion Knock-down of ZEB1 expression by RNA interference can decease lncRNA HOTAIR expression and restrain cell proliferation, invasion and migration in gastric cancer BGC823 cells.

Anesthesia for euthanasia influences mRNA expression in healthy mice and after traumatic brain injury.

PubMed

Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin; Thal, Serge C

2014-10-01

Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real-time PCR data between studies or research groups and should therefore be considered for quantitative PCR data.

AMPK regulates energy metabolism through the SIRT1 signaling pathway to improve myocardial hypertrophy.

PubMed

Dong, H-W; Zhang, L-F; Bao, S-L

2018-05-01

We investigated the correlations of adenosine monophosphate-activated protein kinase (AMPK), Silence information regulator 1 (SIRT1) and energy metabolism with myocardial hypertrophy. Myocardial hypertrophy experimental model was established via transverse aortic constriction (TAC)-induced myocardial hypertrophy and phenylephrine (PE)-induced hypertrophic myocardial cell culture. After activation of AMPK, the messenger ribonucleic acid (mRNA) expressions in myocardial tissue- and myocardial cell hypertrophy-related genes, atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC), were detected. The production rate of 14C-labeled 14CO2 from palmitic acid was quantitatively determined to detect the fatty acid and glucose oxidation of hypertrophic myocardial tissues or cells, and the glucose uptake of myocardial cells was studied using [14C] glucose. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to detect the changes in SIRT1 mRNA and protein expressions in hypertrophic myocardial tissues. Moreover, SIRT1 small interfering ribonucleic acid (siRNA) was used to interfere in SIRT1 expression to further investigate the role of SIRT1 in the effect of AMPK activation on myocardial hypertrophy. AMPK activation could significantly reduce the mRNA expressions of ANP and β-MHC in vitro and in vivo. AMPK could increase the ejection fraction (EF) and decrease the protein synthesis rate in myocardial cells in mice with myocardial hypertrophy. Besides, AMPK activation could increase the fatty acid oxidation, improve the glucose uptake and reduce the glucose oxidation. After AMPK activation, both SIRT1 mRNA and protein expressions in hypertrophic myocardial tissues and myocardial cells were increased. After SIRT1 siRNA was further used to interfere in SIRT1 expression in myocardial cells, it was found that mRNA expressions and protein synthesis rates of ANP and β-MHC were increased. The activation of AMPK can inhibit the myocardial hypertrophy, which may be realized through regulating the myocardial energy metabolism via SIRT1 signaling pathway.

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

PubMed Central

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

2017-01-01

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

IL-17A alone weakly affects the transcriptome of intestinal epithelial cells but strongly modulates the TNF-α-induced expression of inflammatory mediators and inflammatory bowel disease susceptibility genes.

PubMed

Friedrich, Matthias; Diegelmann, Julia; Beigel, Florian; Brand, Stephan

2014-09-01

In contrast to anti-TNF-α antibodies, anti-IL-17A antibodies lacked clinical efficacy in a trial with patients suffering from Crohn's disease. We therefore analyzed how IL-17A modulates the inflammatory response elicited by TNF-α in intestinal epithelial cells (IEC). Target mRNA levels in IEC and colonic biopsies were assessed by RNA microarray and quantitative real-time PCR. Signaling pathways were analyzed using receptor neutralization and pharmacological inhibitors. Target protein levels were determined by immunoblotting. Microarray analysis demonstrated that IL-17A alone is a weak inducer of gene expression in IEC (29 regulated transcripts), but significantly affected the TNF-α-induced expression of 547 genes, with strong amplification of proinflammatory chemokines and cytokines (>200-fold increase of CCL20, CXCL1, and CXCL8). Interestingly, IL-17A differentially modulated the TNF-α-induced expression of several inflammatory bowel disease susceptibility genes in IEC (increase of JAK2 mRNA, decrease of FUT2, ICAM1, and LTB mRNA). Negative regulation of ICAM-1 by IL-17A was verified on protein level. The significance of these findings is emphasized by inflamed lesions of patients with inflammatory bowel disease demonstrating significant correlations (P < 0.01, Rho, 0.57-0.85) for JAK2, ICAM1, and LTB mRNA with IL17A and TNF mRNA. Our study demonstrates the modulation of inflammatory bowel disease susceptibility gene mRNA in IEC as a novel important property of IL-17A. Given the weak impact of sole IL-17A stimulation on IEC target gene expression, our study provides an important explanation for the lack of clinical efficacy of sole IL-17A neutralization, but suggests a beneficial effect of combined IL-17A/TNF-α that is currently in clinical development.

Caffeine modulates glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts.

PubMed

Fehrholz, Markus; Glaser, Kirsten; Speer, Christian P; Seidenspinner, Silvia; Ottensmeier, Barbara; Kunzmann, Steffen

2017-03-23

Although caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells. The human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-β1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition. Treatment with different glucocorticoids (1 μM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-β1 mRNA in H441 cells, increased expression of TGF-β2 and TGF-β3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression. In addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-β1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.

Lymphotoxin β receptor activation promotes mRNA expression of RelA and pro-inflammatory cytokines TNFα and IL-1β in bladder cancer cells.

PubMed

Shen, Mo; Zhou, Lianlian; Zhou, Ping; Zhou, Wu; Lin, Xiangyang

2017-07-01

The role of inflammation in tumorigenesis and development is currently well established. Lymphotoxin β receptor (LTβR) activation induces canonical and noncanonical nuclear factor (NF)‑κB signaling pathways, which are linked to inflammation‑induced carcinogenesis. In the present study, 5,637 bladder cancer cells were cultured and the activation of LTβR was induced by functional ligand, lymphotoxin (LT) α1β2, and silencing with shRNA. Reverse transcription‑quantitative polymerase chain reaction was utilized to detect the mRNA expression levels of NF‑κB family members RelA and RelB, cytokines including LTα, LTβ, tumor necrosis factor (TNF)α, TNF superfamily member 14, interleukin (IL)‑6 and IL‑1β, and proliferation‑related genes including CyclinD1 and Survivin. The expression of phospho‑p65 was determined by western blotting. Activation of LTβR on bladder cancer 5,637 cells was demonstrated to upregulate the mRNA expression levels of the RELA proto‑oncogene, RelA, by 2.5‑fold compared with unstimulated cells, while no significant change was observed in the RELB proto‑oncogene NF‑κB member mRNA levels. Expression of pro‑inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)‑1β mRNA levels were significantly increased nearly 5‑fold and 1.5‑fold, respectively, following LTβR activation compared with unstimulated cells. The LTβR‑induced upregulation of RelA, TNFα and IL‑1β was decreased by ~33, 27, and 26% respectively when LTβR was silenced via short hairpin RNA. Activation of LTβR had no effect on 5,637 cell growth, despite CyclinD1 and Survivin mRNA levels increasing by ~2.7 and 1.3‑fold, respectively, compared with unstimulated cells. In conclusion, activation of LTβR induced the expression of RelA mRNA levels. LTβR activation might be an important mediator in promoting an inflammatory microenvironment in bladder cancer, via the upregulation of TNFα and IL‑1β mRNA levels. LTβR may be a potential therapeutic target for bladder cancer.

Expression pattern of L-FABP gene in different tissues and its regulation of fat metabolism-related genes in duck.

PubMed

He, Jun; Tian, Yong; Li, Jinjun; Shen, Junda; Tao, Zhengrong; Fu, Yan; Niu, Dong; Lu, Lizhi

2013-01-01

Liver fatty acid binding protein (L-FABP) is a member of intracellular lipid-binding proteins responsible for the transportation of fatty acids. The expression pattern of duck L-FABP mRNA was examined in this study by quantitative RT-PCR. The results showed that duck L-FABP gene was expressed in many tissues, including heart, lung, kidney, muscle, ovary, brain, intestine, stomach and adipocyte tissues, and highly expressed in liver. Several lipid metabolism-related genes were selected to detect the regulation of L-FABP in duck. The expression of L-FABP and lipoprotein lipase was promoted by oleic acid. The L-FABP knockdown decreased the expression levels of peroxisome proliferator-activated receptor α (PPARα), fatty acid synthase and lipoprotein lipase by 61.1, 42.3 and 53.7 %, respectively (P < 0.05), but had no influences on the mRNA levels of PPARγ and leptin receptor. L-FABP might function through the PPARα to regulate the fat metabolism-related gene expression and play important roles in lipid metabolism in duck hepatocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is released by female mouse bladder urothelial cells and expressed by the urothelium as an early response to lipopolysaccharides (LPS).

PubMed

Li, Yan; Lu, Ming; Alvarez-Lugo, Lery; Chen, Gang; Chai, Toby C

2017-04-01

We studied in vitro and in vivo response of primary mouse bladder urothelial cells (mBUC) and bladder urothelium to lipopolysaccharides (LPS), focusing on granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. Female C57BL/6 mBUC were exposed for 12 hr to differing concentrations of LPS (100 ng/ml to 10 µg/ml). mBUC were also exposed to a single dose of LPS (1 µg/ml) for 3, 6, 12 hr. Neutralizing GM-CSF antibody (0.1 μg/ml) was used block GM-CSF activity in vitro. In vivo experiments were performed, whereby, LPS (1 mg/ml) was instilled intravesically and left to dwell for 30 min followed by harvest of bladder urothelium 3 to 18 hr later. ELISA measured GM-CSF. qPCR quantitated mRNA for GM-CSF, vascular endothelial growth factor-A (VEGF-A), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and tumor necrosis factor α (TNF-α). RT-PCR was used to detect mRNA for GM-CSF, GM-CSFRα, and β in bladder tissues. Immunohistofluorescence and Western blots for GM-CSFRα were performed on bladder tissues. LPS induced a dose-dependent release of GM-CSF by mBUC. Mouse bladder urothelium did not express GM-CSF mRNA at baseline, but expressed GM-CSF mRNA 3 hr after in vivo LPS exposure, with GM-CSF mRNA expression disappearing 18 hr later. GM-CSFRα expression was confirmed in bladder urothelium. GM-CSF neutralizing antibody significantly diminished LPS-induced increases of VEGF and COX-2 mRNA expression. Urothelium and mBUC secreted GM-CSF as an early response to LPS. GM-CSF mediated downstream expression of VEGF and COX-2. Urothelial GM-CSF may function as a signaling mediator for both inflammation and pain transduction. Neurourol. Urodynam. 36:1020-1025, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The expression of β3-adrenoceptors and their function in the human prostate.

PubMed

Suzuki, Takahisa; Otsuka, Atsushi; Matsumoto, Rikiya; Furuse, Hiroshi; Ozono, Seiichiro

2016-02-01

Little is known about β3-adrenoceptor (AR) expression and function in human prostate. We examined the expression and distribution of β-AR subtypes in normal prostate and benign prostatic hyperplasia (BPH) tissues, and investigated which selective β-AR subtype agonist was most involved in the relaxation of isolated human prostate strips. Messenger RNA (mRNA) expression for β1-, β2-, and β3 -ARs was investigated using reverse transcriptase-polymerase chain reactions (RT-PCR). Quantitative analysis of mRNA expression of β-AR subtypes between normal prostate and BPH tissues was performed using quantitative RT-PCR (qPCR). Distributions were examined by immunohistochemistry (IHC). Strips of human normal prostate or BPH were suspended in organ baths and exposed to isoproterenol, dobutamine, procaterol, and TRK-380 to investigate their relaxant effects on KCl-induced contractions, and their inhibitory effects on electrical field stimulation (EFS)-induced contractions. We confirmed the presence of mRNA for β1-, β2-, and β3-ARs both in normal prostate and in BPH tissues. For β3-AR, mRNA expression in BPH tissues was significantly higher than in normal prostate tissues, but there was no significant difference in β1- and β2-AR expression between normal and BPH tissues. IHC revealed differences in staining intensity between smooth muscle cells and glandular cells, with different proportions for different β-AR subtypes. Staining of β3-AR was particularly intense in smooth muscle cells as opposed to glandular cells. Isoproterenol and TRK-380 significantly decreased the tone of KCl-induced contractions of the normal prostate strips. The rank order of relaxant effects was isoproterenol > TRK-380 > procaterol > dobutamine. All selective β-AR agonists significantly decreased the amplitude of EFS-induced contractions of the normal prostate strips. The rank order of inhibitory effects was isoproterenol > dobutamine >TRK-380 > procaterol. In BPH strips, all selective β-AR agonists showed no significant relaxant or inhibitory effects on KCl- or EFS-induced contractions. β3 -AR is abundant in human prostate smooth muscle, whose relaxation is mediated by β1- and β3-AR stimulation. β3-AR agonists may have clinical use in the treatment of male non-BPH patients or neurogenic bladder patients with voiding dysfunction. © 2015 Wiley Periodicals, Inc.

Serum from pregnant women with gestational diabetes mellitus increases the expression of FABP4 mRNA in primary subcutaneous human pre-adipocytes

PubMed Central

Li, Lan; Lee, Se Jin; Kook, Song Yi; Ahn, Tae Gyu; Lee, Ji Yeon

2017-01-01

Objective Gestational diabetes mellitus (GDM) is defined as glucose intolerance first detected during pregnancy. It can result in pregnancy complications such as birth injury, stillbirth. Fatty acid-binding protein 4 (FABP4), found in adipose tissue, is associated with insulin resistance, and type 2 diabetes. The aim of this study was to investigate whether FABP4 in the placenta and decidua of pregnant women with GDM is higher than that in normal pregnant women, and whether serum from pregnant women with GDM may cause adipocytes to secrete more FABP4 than does serum from a normal pregnant group. Methods We obtained placentas, deciduas, and serum from 12 pregnant women with GDM and 12 normal pregnant women and performed enzyme-linked immunosorbent assay, real time quantitative-polymerase chain reaction. We cultured human pre-adipocytes for 17 days with GDM and non-GDM serum and performed western blot, real time quantitative-polymerase chain reaction, and oil red O staining. Results Expression of FABP4 in serum, placenta and decidua of pregnant women with GDM was significantly higher than that in normal pregnant women. Serum from pregnant women with GDM increased the expression of FABP4 mRNA and decreased the expression of adiponectin mRNA in human pre-adipocytes significantly. Adipocyte cultured in GDM serum showed significantly greater lipid accumulation than those cultured in normal serum. Conclusion Our results suggest that FABP4 is higher in placenta and decidua from pregnant women with GDM. Increased circulating FABP4 in maternal serum from pregnant women with GDM may originate from adipocytes and the placenta. Circulating FABP4 can induce increased insulin resistance and decreased insulin sensitivity. PMID:28534013

Serum from pregnant women with gestational diabetes mellitus increases the expression of FABP4 mRNA in primary subcutaneous human pre-adipocytes.

PubMed

Li, Lan; Lee, Se Jin; Kook, Song Yi; Ahn, Tae Gyu; Lee, Ji Yeon; Hwang, Jong Yun

2017-05-01

Gestational diabetes mellitus (GDM) is defined as glucose intolerance first detected during pregnancy. It can result in pregnancy complications such as birth injury, stillbirth. Fatty acid-binding protein 4 (FABP4), found in adipose tissue, is associated with insulin resistance, and type 2 diabetes. The aim of this study was to investigate whether FABP4 in the placenta and decidua of pregnant women with GDM is higher than that in normal pregnant women, and whether serum from pregnant women with GDM may cause adipocytes to secrete more FABP4 than does serum from a normal pregnant group. We obtained placentas, deciduas, and serum from 12 pregnant women with GDM and 12 normal pregnant women and performed enzyme-linked immunosorbent assay, real time quantitative-polymerase chain reaction. We cultured human pre-adipocytes for 17 days with GDM and non-GDM serum and performed western blot, real time quantitative-polymerase chain reaction, and oil red O staining. Expression of FABP4 in serum, placenta and decidua of pregnant women with GDM was significantly higher than that in normal pregnant women. Serum from pregnant women with GDM increased the expression of FABP4 mRNA and decreased the expression of adiponectin mRNA in human pre-adipocytes significantly. Adipocyte cultured in GDM serum showed significantly greater lipid accumulation than those cultured in normal serum. Our results suggest that FABP4 is higher in placenta and decidua from pregnant women with GDM. Increased circulating FABP4 in maternal serum from pregnant women with GDM may originate from adipocytes and the placenta. Circulating FABP4 can induce increased insulin resistance and decreased insulin sensitivity.

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.

Alterations in gene expression of proprotein convertases in human lung cancer have a limited number of scenarios.

PubMed

Demidyuk, Ilya V; Shubin, Andrey V; Gasanov, Eugene V; Kurinov, Alexander M; Demkin, Vladimir V; Vinogradova, Tatyana V; Zinovyeva, Marina V; Sass, Alexander V; Zborovskaya, Irina B; Kostrov, Sergey V

2013-01-01

Proprotein convertases (PCs) is a protein family which includes nine highly specific subtilisin-like serine endopeptidases in mammals. The system of PCs is involved in carcinogenesis and levels of PC mRNAs alter in cancer, which suggests expression status of PCs as a possible marker for cancer typing and prognosis. The goal of this work was to assess the information value of expression profiling of PC genes. Quantitative polymerase chain reaction was used for the first time to analyze mRNA levels of all PC genes as well as matrix metalloproteinase genes MMP2 and MMP14, which are substrates of PCs, in 30 matched pairs of samples of human lung cancer tumor and adjacent tissues without pathology. Significant changes in the expression of PCs have been revealed in tumor tissues: increased FURIN mRNA level (p<0.00005) and decreased mRNA levels of PCSK2 (p<0.007), PCSK5 (p<0.0002), PCSK7 (p<0.002), PCSK9 (p<0.00008), and MBTPS1 (p<0.00004) as well as a tendency to increase in the level of PCSK1 mRNA. Four distinct groups of samples have been identified by cluster analysis of the expression patterns of PC genes in tumor vs. normal tissue. Three of these groups covering 80% of samples feature a strong elevation in the expression of a single gene in cancer: FURIN, PCSK1, or PCSK6. Thus, the changes in the expression of PC genes have a limited number of scenarios, which may reflect different pathways of tumor development and cryptic features of tumors. This finding allows to consider the mRNAs of PC genes as potentially important tumor markers.

Up-regulation of IL-23 expression in human dental pulp fibroblasts by IL-17 via activation of the NF-κB and MAPK pathways.

PubMed

Wei, L; Liu, M; Xiong, H; Peng, B

2017-11-06

To investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-17 on HDPFs-mediated IL-23 production and the molecular mechanism involved. Interleukin (IL)-17R expression was determined by semi-quantitative reverse transcriptase-polymerase chain reaction and Western blot in cultured human dental pulp fibroblasts (HDPFs). Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to determine IL-23 mRNA and protein levels in IL-17-stimulated HDPFs, respectively. The nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signalling pathways that mediate the IL-17-stimulated production of IL-23 was investigated using Western blot and specific signalling inhibitor analyses. Statistical analyses were performed using Kruskal-Wallis tests followed by the Mann-Whitney U-test. Statistical significance was considered when the P value < 0.05. Primary HDPFs steadily expressed IL-17R mRNA and surface-bound protein. IL-17 stimulated the expression of IL-23 mRNA and protein in cultured human dental pulp fibroblasts, which was attenuated by IL-17 or IL-17R neutralizing antibodies. In accordance with the enhanced expression of IL-23, IL-17 stimulation resulted in rapid activation of p38 MAPK, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK) and NF-κB in HDPFs. Inhibitors of p38 MAPK, ERK 1/2 or NF-κB significantly suppressed, whereas blocking JNK substantially augmented IL-23 production from IL-17-stimulated HDPFs. HDPFs expressed IL-17R and responded to IL-17 to produce IL-23 via the activation of the NF-κB and MAPK signalling pathways. The findings provide insights into the cellular mechanisms of the participation of IL-17 in the activation of HDPFs in inflamed pulp tissue. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Pannus invasion and cartilage degradation in rheumatoid arthritis: involvement of MMP-3 and interleukin-1beta.

PubMed

Ainola, M M; Mandelin, J A; Liljeström, M P; Li, T F; Hukkanen, M V J; Konttinen, Y T

2005-01-01

Synovial inflammation in rheumatoid arthritis (RA) leads to pannus tissue invasion and destruction of cartilage/bone matrix by proteinases. Our intention was to analyze some of the key matrix metalloproteinases (MMPs) in pannus tissue overlying evolving cartilage erosions in RA. Frozen tissue samples of pannus and synovium from advanced RA and synovium from osteoarthritic patients were used for immunohistochemical, western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of MMP-1, -3, -13 and -14. Synovial fibroblast cultures, stimulated with tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), were analyzed with enzyme-linked immunosorbent assays (ELISA) and quantitative RT-PCR. MMP-3 was highly expressed in pannus tissue compared with significantly lower expression levels of MMP-1, -13 and -14. In fibroblast cultures IL-1beta was a potent stimulus for MMP-3, whereas TNF-alpha was more potent for MMP-1. This is the first study to demonstrate quantitatively in real time that MMP-3 mRNA expression is clearly higher in advanced RA pannus tissue compared to parallel RA or osteoarthritic synovium. MMP-3 mRNA levels were also clearly overexpressed in RA pannus compared to MMP-1, -13 and -14. Advanced RA has previously been found to overexpress IL-1beta. The high expression of MMP-3 in pannus and IL-1beta, mediated stimulation of MMP-3 suggest that MMP-3 plays a significant role in the progression of erosions through the proteoglycan-rich cartilage matrix.

Short communication: Growth hormone receptor expression in two dairy breeds during the periparturient period.

PubMed

Okamura, C S; Bader, J F; Keisler, D H; Lucy, M C

2009-06-01

The growth hormone receptor (GHR) 1A mRNA decreases after calving in the liver of Holstein dairy cows and may coordinate nutrient partitioning. The hypothesis that the decrease in GHR1A mRNA around the time of calving was characteristic of a second dairy breed was tested by examining Guernsey cows in addition to Holstein cows. Holstein and Guernsey cows were housed together and paired by parity and expected calving date. Liver biopsies and blood samples were collected prepartum (d -20 +/- 1) and postpartum on d 3, and d 14 +/- 1. The amounts of GHR1A, GHR1B, GHR1C, and insulin-like growth factor (IGF)1 mRNA were determined by quantitative reverse transcription polymerase chain reaction. Blood concentrations of growth hormone (GH) and IGF1 were measured by RIA. Both breeds underwent a decrease in GHR1A mRNA, a decrease in liver IGF1 mRNA, a decrease in blood IGF1, and an increase in blood GH after calving. The decrease in liver GHR1A and IGF1 mRNA after calving may be an inherent characteristic of dairy breeds that enables nutrient partitioning for greater milk production. Independent genetic selection in 2 dairy breeds seemingly exploited a similar mechanism, reduced GHR1A expression, to decrease blood IGF1 and increase blood GH, a key hormone involved in nutrient partitioning.

mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

PubMed

Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

2016-08-15

The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered.

Effect of retinoic acid on midkine gene expression in rat anterior pituitary cells.

PubMed

Maliza, Rita; Fujiwara, Ken; Azuma, Morio; Kikuchi, Motoshi; Yashiro, Takashi

2017-06-29

Retinoic acid (RA) is converted from retinal by retinaldehyde dehydrogenases (RALDHs) and is an essential signaling molecule in embryonic and adult tissue. We previously reported that RALDH1 was produced in the rat anterior pituitary gland and hypothesized that RA was generated in the gland. Midkine (MK) is an RA-inducible growth factor, and MK production in the rat anterior pituitary gland was recently reported. However, the mechanism that regulates gene expression of MK in the pituitary gland has not been determined. To investigate regulation of MK production in the anterior pituitary gland, we analyzed changes in MK mRNA in cultured rat anterior pituitary cells. We identified MK-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for RALDH1. MK mRNA was expressed in RALDH1-producing cells in the anterior pituitary gland. Using isolated anterior pituitary cells of rats, we examined the effect of RA on gene expression of MK. Quantitative real-time PCR revealed that 72 h exposure to a concentration of 10 -6 M of retinal and all-trans retinoic acid increased MK mRNA levels by about 2-fold. Moreover, the stimulatory effect of all-trans retinoic acid was mimicked by the RA receptor agonist Am80. This is the first report to show that RA is important in regulating MK expression in rat anterior pituitary gland.

Common variants of OPA1 conferring genetic susceptibility to leprosy in Han Chinese from Southwest China.

PubMed

Xiang, Yang-Lin; Zhang, Deng-Feng; Wang, Dong; Li, Yu-Ye; Yao, Yong-Gang

2015-11-01

Leprosy is an ancient chronic infection caused by Mycobacterium leprae. Onset of leprosy was highly affected by host nutritional condition and energy production, (partially) due to genomic loss and parasitic life style of M. leprae. The optic atrophy 1 (OPA1) gene plays an essential role in mitochondria, which function in cellular energy supply and innate immunity. To investigate the potential involvement of OPA1 in leprosy. We analyzed 7 common genetic variants of OPA1 in 1110 Han Chinese subjects with and without leprosy, followed by mRNA expression profiling and protein-protein interaction (PPI) network analysis. We observed positive associations between OPA1 variants rs9838374 (Pgenotypic=0.003) and rs414237 (Pgenotypic=0.002) with lepromatous leprosy. expression quantitative trait loci (eQTL) analysis showed that the leprosy-related risk allele C of rs414237 is correlated with lower OPA1 mRNA expression level. Indeed, we identified a decrease of OPA1 mRNA expression in both with patients and cellular model of leprosy. In addition, the PPI analysis showed that OPA1 protein was actively involved in the interaction network of M. leprae induced differentially expressed genes. Our results indicated that OPA1 variants confer risk of leprosy and may affect OPA1 expression, mitochondrial function and antimicrobial pathways. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

PubMed

da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

2016-08-01

Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

Differential mRNA expression of neuroinflammatory modulators in the spinal cord and thalamus of type 2 diabetic monkeys.

PubMed

Ding, Huiping; Kiguchi, Norikazu; Kishioka, Shiroh; Ma, Tao; Peters, Christopher M; Ko, Mei-Chuan

2018-05-11

Given that diabetes-associated complications are closely associated with neuroinflammation, it is imperative to study potential changes in neuroinflammatory modulators in the central nervous system of diabetic primates. The mRNA levels of pro- and anti-inflammatory cytokines, toll-like receptors (TLRs), growth factors, and cannabinoid receptors were compared in the spinal dorsal horn (SDH) and thalamus of naturally occurring type 2 diabetic monkeys and an age-matched control group using reverse transcription and quantitative real-time polymerase chain reaction. In the SDH of diabetic monkeys, mRNA levels of proinflammatory cytokines (i.e. interleukin [IL]-1β and tumor necrosis factor [TNF] α), TLR1, and TLR2 were increased, whereas mRNA levels of IL-10, an anti-inflammatory cytokine, were decreased. No changes were observed in the mRNA levels of growth factors and cannabinoid receptors. In line with the mRNA data, TNFα immunoreactivity was significantly increased in diabetic monkeys. Moreover, mRNA expression levels of IL-1β, TNFα, TLR1, and TLR2 in the SDH were positively correlated with plasma glucose concentrations in all monkeys. Several ligands and receptors involved in neuroinflammation are simultaneously dysregulated in the spinal cord of diabetic monkeys. This primate disease model will facilitate the design of novel treatment approaches to ameliorate neuroinflammation-driven adverse effects in diabetic patients. © 2018 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Increased expression of resistin in ectopic endometrial tissue of women with endometriosis.

PubMed

Oh, Yoon Kyung; Ha, Young Ran; Yi, Kyong Wook; Park, Hyun Tae; Shin, Jung-Ho; Kim, Tak; Hur, Jun-Young

2017-11-01

Inflammation is a key process in the establishment and progression of endometriosis. Resistin, an adipocytokine, has biological properties linked to immunologic functions, but its role in endometriosis is unclear. Resistin gene expression was examined in eutopic and ectopic endometrial tissues from women with (n=25) or without (n=25) endometriosis. Resistin mRNA and protein levels were determined in endometrial tissue using quantitative real-time reverse transcription PCR and Western blotting, following adipokine profiling arrays. Resistin protein was detected in human endometrial tissues using an adipokine array test. Resistin mRNA and protein levels were significantly higher in ectopic endometrial tissue of patients with endometriosis than in normal eutopic endometrial tissue. Our results indicate that resistin is differentially expressed in endometrial tissues from women with endometriosis and imply a role for resistin in endometriosis-associated pelvic inflammation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Association of High Cardiovascular Fitness and the Rate of Adaptation to Heat Stress

PubMed Central

Nowak-Zaleska, Alicja; Chruściński, Grzegorz; Zaleski, Ryszard; Tymański, Roman; Kochanowicz, Andrzej

2018-01-01

This study aimed to compare changes in genes expression associated with inflammation and apoptosis in response to heat stress caused by sauna between people with varying cardiorespiratory fitness levels. We hypothesis that high cardiorespiratory level caused higher positive changes after four weeks of sauna bathing. Blood samples were taken at rest before and after the first and last sauna sessions and 48 hours after the last sauna session and used to assay HSP70 (HSPA1A), HSP27 (HSPB1), interleukin 6 (IL6), and interleukin 10 (IL10) genes expression in blood with quantitative real-time qRT-PCR. Overall, small decreases in rest values of HSPA1A and IL6 mRNA, increase in HSPB1 mRNA, and a significant increase in IL10 mRNA were observed after four weeks of exposure to heat stress. Our findings suggest that an adaptive response to heat stress (an anti-inflammatory response) occurs faster in people with higher cardiorespiratory fitness. PMID:29682518

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

NASA Astrophysics Data System (ADS)

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

2017-06-01

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

In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups

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

Burbach, J.P.; Voorhuis, T.A.; van Tol, H.H.

1987-05-29

Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded /sup 35/S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at thesemore » various sites.« less

MEASUREMENT OF ESTROGEN-INDUCED VITELLOGENIN AND VITELLINE ENVELOPE PROTEIN MRNA IN SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS) USING QUANTITATIVE REAL-TIME PCR TECHNOLOGY

EPA Science Inventory

Many environmentally persistent chemicals found in both European and U.S. waterways can act as estrogens by binding to estrogen receptors and modifying the expression of genes regulated by endogenous estrogens. Synthesis of female-specific proteins (Vitellogenin [VTG], vitelline ...

Effects of temperature and melatonin on day-night expression patterns of arginine vasotocin and isotocin mRNA in the diencephalon of a temperate wrasse Halichoeres tenuispinis.

PubMed

Bouchekioua, Selma; Hur, Sung-Pyo; Takeuchi, Yuki; Lee, Young-Don; Takemura, Akihiro

2018-06-01

Most wrasses are protogynous species that swim to feed, reproduce during the daytime, and bury themselves under the sandy bottom at night. In temperate and subtropical wrasses, low temperature influences emergence from the sandy bottom in the morning, and induces a hibernation-like state in winter. We cloned and characterized the prohormone complementary DNAs (cDNAs) of arginine vasotocin (AVT) and isotocin (IT) in a temperate wrasse (Halichoeres tenuispinis) and examined the effects of day/night and temperature on their expression in the diencephalon, because these neurohypophysial peptides are related to the sex behavior of wrasses. The full-length cDNAs of pro-AVT and pro-IT were 938 base pairs (154 amino acids) and 759 base pairs (156 amino acids) in length, respectively. Both pro-peptides contained a signal sequence followed by the respective hormones and neurophysin connected by a Gly-Lys-Arg bridge. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that pro-AVT mRNA expression was specifically observed in the diencephalon, whereas pro-IT mRNA expression was seen in the whole brain. Quantitative RT-PCR revealed that the mRNA abundance of pro-AVT and pro-IT was higher at midday (zeitgeber time 6; ZT6) than at midnight (ZT18) under 12 h light and 12 h darkness (LD 12:12) conditions, but not under constant light. Intraperitoneal injection of melatonin decreased the mRNA abundance of pro-AVT, but not of pro-IT. When fish were reared under LD 12:12 conditions at 25, 20, and 15 °C, day high and night low mRNA expressions of pro-AVT and pro-IT were maintained. A field survey revealed seasonal variation in the number of swimming fish at observatory sites; many fish emerged from the sandy bottom in summer, but not in winter, suggesting a hibernation-like state under the sandy bottom under low temperature conditions. We conclude that the day-night fluctuation of pro-AVT and pro-IT mRNA abundance in the brain is not affected by temperature and repeated under the sandy bottom in winter.

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

PubMed

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

2011-02-17

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

p53, Bcl-2 and cox-2 are involved in berberine hydrochloride-induced apoptosis of HeLa229 cells.

PubMed

Wang, Hai-Yan; Yu, Hai-Zhong; Huang, Sheng-Mou; Zheng, Yu-Lan

2016-10-01

The present study aimed to investigate the effects of berberine hydrochloride on the proliferation and apoptosis of HeLa229 human cervical cancer cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to examine the cytotoxicity of berberine hydrochloride against HeLa229 cells. The effects of berberine hydrochloride on the apoptosis of HeLa229 cells was detected by immunofluorescence and flow cytometry, and the mRNA expression levels of p53, B‑cell lymphoma 2 (Bcl‑2) and cyclooxygenase‑2 (cox‑2) were analyzed by reverse transcription-quantitative polymerase chain reaction. Berberine hydrochloride inhibited the proliferation of HeLa229 cells in a dose‑dependent manner; minimum cell viability (3.61%) was detected following treatment with 215.164 µmol/l berberine hydrochloride and the half maximal inhibitory concentration value was 42.93 µmol/l following treatment for 72 h. In addition, berberine hydrochloride induced apoptosis in HeLa229 cells in a dose‑ and time‑dependent manner. Berberine hydrochloride upregulated the mRNA expression levels of p53, and downregulated mRNA expression levels of Bcl‑2 and cox‑2, in a dose‑dependent manner. In conclusion, berberine hydrochloride inhibited the proliferation and induced apoptosis of HeLa229 cells, potentially via the upregulation of p53 and the downregulation of Bcl‑2 and cox‑2 mRNA expression levels.

Modulation of renal CNG-A3 sodium channel in rats subjected to low- and high-sodium diets.

PubMed

Novaira, Horacio J; Botelho, Bruno F; Goldenberg, Regina C; Guggino, Sandra E; Morales, Marcelo M

2004-10-11

In this work, we studied the mRNA distribution of CNG-A3, an amiloride-sensitive sodium channel that belongs to the cyclic nucleotide-gated (CNG) family of channels, along the rat nephron. The possible involvement of aldosterone in this process was also studied. We also evaluated its expression in rats subjected to diets with different concentrations of sodium or to alterations in aldosterone plasma levels. Total RNA isolated from whole kidney and/or dissected nephron segments of Wistar rats subjected to low- and high-sodium diets, furosemide treatment, adrenalectomy, and adrenalectomy with replacement by aldosterone were analyzed by the use of Western blot, ribonuclease protection assay (RPA) and/or reverse transcription followed by semi-quantitative polymerase chain reaction (RT-PCR). CNG-A3 sodium channel mRNA and protein expression, in whole kidneys of rats subjected to high-Na+ diet, were lower than those in animals given a low-salt diet. Renal CNG-A3 mRNA expression was also decreased in adrenalectomized rats, and was normalized by aldosterone replacement. Moreover, a CNG-A3 mRNA expression study in different nephron segments revealed that aldosterone modulation is present in the cortical thick ascending loop (cTAL) and cortical collecting duct (CCD). This result suggests that CNG-A3 is responsive to the same hormone signaling as the amiloride sensitive sodium channel ENaC and suggests the CNG-A3 may have a physiological role in sodium reabsorption.

Comparison of Tight Junction Protein-Related Gene mRNA Expression Levels between Male and Female Gastroesophageal Reflux Disease Patients.

PubMed

Kim, Jin Joo; Kim, Nayoung; Park, Ji Hyun; Kim, Young Sun; Lee, Sun Min; Lee, Dong Ho; Jung, Hyun Chae

2018-03-21

Male predominance has been observed in the erosive reflux disease (ERD), but reverse finding in nonerosive reflux disease (NERD). This suggests sex-specific medicine approach is needed but its mechanism is remained to be elucidated. We aimed to compare clinical characteristics and mRNA expression levels of tight junction-related proteins between male and female gastroesophageal reflux disease (GERD) patients. Sixteen healthy controls, 45 ERD, and 14 NERD patients received upper endoscopies and completed questionnaires. Quantitative real-time polymerase chain reactions (qPCR) of occludin (OCLN), zonal occludens (ZO) 1, claudin1 (CDLN1) and claudin4 (CDLN4), and neurokinin 1 receptor (NK1R) were performed in the distal esophageal mucosal specimen. These results were analyzed by sex. Female GERD patients were affected more by reflux symptoms than males. The impairment of overall QoL was more prominent in female patients with reflux symptoms than male patients (5.6±0.2 vs. 4.9±0.6, p=0.009). The levels of OCLN mRNA expression were significantly lower in the male ERD group. On the other hand, those of CLDN1, CLDN4, and NK1R except ZO-1 were significantly higher in the male ERD group. We demonstrated that female ERD/NERD patients were affected more by GERD and male ERD patients showed significant changes of tight junction protein mRNA expression levels.

Adipocyte resistin mRNA levels are down-regulated by laparoscopic ovarian electrocautery in both obese and lean women with polycystic ovary syndrome.

PubMed

Seow, Kok-Min; Juan, Chi-Chang; Ho, Low-Tone; Hsu, Yung-Pei; Lin, Yu-Hung; Huang, Lee-Wen; Hwang, Jiann-Loung

2007-04-01

The aim of this study was to investigate serum and adipocyte mRNA expression of resistin in lean and obese women with polycystic ovary syndrome (PCOS) before and 3 months after laparoscopic ovarian electrocauterization (LOE). Adipose tissue obtained from 12 women with PCOS (six obese and six lean, body mass index > 27 kg m(-1) as threshold point) before and after LOE was analysed. Gene expression of resistin was measured by semi-quantitative RT-PCR. Ten lean, age-matched healthy women served as controls. Both lean and obese women with PCOS had significantly higher fasting and 2 h insulin and homeostasis model insulin resistance index (HOMA(IR)) values and lower fasting glucose-to-insulin ratios (G(0)/I(0)) than did the controls. The serum levels of glucose and insulin and HOMA(IR) were significantly decreased, and the G(0)/I(0) ratio was significantly increased 3 months after LOE. No difference was found in serum resistin levels between controls and either obese or lean women with PCOS before LOE, nor between PCOS patients before and after LOE. However, resistin mRNA expression levels in both lean and obese women with PCOS before LOE were significantly higher than that in controls and were decreased significantly after LOE back to control levels. Local resistin activity may be actively involved in the pathogenesis of PCOS. LOE reduces insulin resistance and down-regulates resistin mRNA expression in lean and obese women with PCOS.

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

[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.

Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.

PubMed

Pan, Yinbing; Sun, Xiaodi; Jiang, Lai; Hu, Liang; Kong, Hong; Han, Yuan; Qian, Cheng; Song, Chao; Qian, Yanning; Liu, Wentao

2016-11-17

Tolerance seriously impedes the application of morphine in clinical medicine. Thus, it is necessary to investigate the exact mechanisms and efficient treatment. Microglial activation and neuroinflammation in the spinal cord are thought to play pivotal roles on the genesis and maintaining of morphine tolerance. Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception. Metformin, a biguanide class of antidiabetic drugs and activator of AMPK, has a potential anti-inflammatory effect. The present study evaluated the effects and potential mechanisms of metformin in inhibiting microglial activation and alleviating the antinociceptive tolerance of morphine. The microglial cell line BV-2 cells and mouse brain-derived endothelial cell line bEnd3 cells were used. Cytokine expression was measured using quantitative polymerase chain reaction. Cell signaling was assayed by western blot and immunohistochemistry. The antinociception and morphine tolerance were assessed in CD-1 mice using tail-flick tests. We found that morphine-activated BV-2 cells, including the upregulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-inflammatory cytokines, and Toll-like receptor-4 (TLR-4) mRNA expression, which was inhibited by metformin. Metformin suppressed morphine-induced BV-2 cells activation through increasing AMPK phosphorylation, which was reversed by the AMPK inhibitor compound C. Additionally, in BV-2 cells, morphine did not affect the cell viability and the mRNA expression of anti-inflammatory cytokines. In bEnd3 cells, morphine did not affect the mRNA expression of interleukin-1β (IL-1β), but increased IL-6 and tumor necrosis factor-α (TNF-α) mRNA expression; the effect was inhibited by metformin. Morphine also did not affect the mRNA expression of TLR-4 and chemokine ligand 2 (CCL2). Furthermore, systemic administration of metformin significantly blocked morphine-induced microglial activation in the spinal cord and then attenuated the development of chronic morphine tolerance in mice. Metformin significantly attenuated morphine antinociceptive tolerance by suppressing morphine-induced microglial activation through increasing AMPK phosphorylation.

Differential Expression of Cytochrome P450 Enzymes in Normal and Tumor Tissues from Childhood Rhabdomyosarcoma

PubMed Central

Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

2014-01-01

Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

Effects of irradiation on human leukocyte antigen class I expression in human papillomavirus positive and negative base of tongue and mobile tongue squamous cell carcinoma cell lines.

PubMed

Haeggblom, Linnea; Nordfors, Cecilia; Tertipis, Nikolaos; Bersani, Cinzia; Ramqvist, Torbjörn; Näsman, Anders; Dalianis, Tina

2017-03-16

Human papillomavirus (HPV) infection is a risk factor for oropharyngeal cancer, besides smoking and alcohol. Patients with HPV-positive tumors have a better prognosis than those with HPV-negative tumors. Furthermore, patients with HPV-positive tumors, with high CD8+ tumor infiltrating lymphocyte counts or absent/low human leukocyte antigen (HLA) class I expression have the best outcome. The latter is paradoxical, since HLA class I expression is important for tumor recognition. Below, the hypothesis that radiation therapy increases HLA class I expression was tested. HPV16 positive head and neck cancer cell lines UPCI-SCC-154, UPCI-SCC-090 and UM-SCC-47, and the HPV-negative cancer cell line UT-SCC-14, were treated with 2-10 Gray (Gy) and tested for HLA class I expression, cell cycle changes and apoptosis by flow cytometry. HPV16 E5, E7 and HLA-A mRNA expression was tested by quantitative PCR. A dose of 10 Gy resulted in a tendency of increased HLA class I cell surface expression for all cell lines and reached statistical significance for UPCI-SCC-154 and UPCI-SCC-090. There were, however, no significant changes in HLA-A mRNA expression in any of the cell lines, or HPV16 E5, or E7 mRNA expression for UPCI-SCC-47 and UPCI-SCC-154, while for UPCI-SCC-090 HPV16 E5 mRNA decreased. In all cell lines there was a shift towards G2/M phase and increased apoptosis after irradiation with 10 Gy. To conclude, irradiation with 10 Gy increased HLA class I expression in the HPV-positive cell lines UPCI-SCC-154 and UPCI-SCC-090. A similar tendency was observed for HPV-positive UM-SCC-47 and HPV-negative UT-SCC-14.

Extent, Causes, and Consequences of Small RNA Expression Variation in Human Adipose Tissue

PubMed Central

Knights, Andrew J.; Abreu-Goodger, Cei; van de Bunt, Martijn; Guerra-Assunção, José Afonso; Bartonicek, Nenad; van Dongen, Stijn; Mägi, Reedik; Nisbet, James; Barrett, Amy; Rantalainen, Mattias; Nica, Alexandra C.; Quail, Michael A.; Small, Kerrin S.; Glass, Daniel; Enright, Anton J.; Winn, John; Deloukas, Panos; Dermitzakis, Emmanouil T.; McCarthy, Mark I.; Spector, Timothy D.; Durbin, Richard; Lindgren, Cecilia M.

2012-01-01

Small RNAs are functional molecules that modulate mRNA transcripts and have been implicated in the aetiology of several common diseases. However, little is known about the extent of their variability within the human population. Here, we characterise the extent, causes, and effects of naturally occurring variation in expression and sequence of small RNAs from adipose tissue in relation to genotype, gene expression, and metabolic traits in the MuTHER reference cohort. We profiled the expression of 15 to 30 base pair RNA molecules in subcutaneous adipose tissue from 131 individuals using high-throughput sequencing, and quantified levels of 591 microRNAs and small nucleolar RNAs. We identified three genetic variants and three RNA editing events. Highly expressed small RNAs are more conserved within mammals than average, as are those with highly variable expression. We identified 14 genetic loci significantly associated with nearby small RNA expression levels, seven of which also regulate an mRNA transcript level in the same region. In addition, these loci are enriched for variants significant in genome-wide association studies for body mass index. Contrary to expectation, we found no evidence for negative correlation between expression level of a microRNA and its target mRNAs. Trunk fat mass, body mass index, and fasting insulin were associated with more than twenty small RNA expression levels each, while fasting glucose had no significant associations. This study highlights the similar genetic complexity and shared genetic control of small RNA and mRNA transcripts, and gives a quantitative picture of small RNA expression variation in the human population. PMID:22589741

MET amplification, expression, and exon 14 mutations in colorectal adenocarcinoma.

PubMed

Zhang, Meng; Li, Guichao; Sun, Xiangjie; Ni, Shujuan; Tan, Cong; Xu, Midie; Huang, Dan; Ren, Fei; Li, Dawei; Wei, Ping; Du, Xiang

2018-04-08

MET amplification, expression, and splice mutations at exon 14 result in dysregulation of the MET signaling pathway. The aim of this study was to identify the relationship between MET amplification, protein or mRNA expression, and mutations in colorectal cancer (CRC). MET immunohistochemistry (IHC) was used for MET protein expression analysis and fluorescence in situ hybridization (FISH) was used for MET amplification detection. Both analyses were performed in tissue microarrays (TMA) containing 294 of colorectal adenocarcinoma tissue samples and 131 samples of adjacent normal epithelial tissue. MET mRNA expression was examined by real-time quantitative polymerase chain reaction (qRT-PCR) in 72 fresh colorectal adenocarcinoma tissue samples and adjacent normal colon tissue. PCR sequencing was performed to screen for MET exon 14 splice mutations in 59 fresh CRC tissue samples. Our results showed that MET protein expression was higher in colorectal tumor tissue than in adjacent normal intestinal epithelium. Positive MET protein expression was associated with significantly poorer overall survival (OS) and disease-free survival (DFS). Multivariate analysis revealed that positive MET protein expression was an independent risk factor for DFS, but not for OS. MET mRNA expression was upregulated in tumor tissues compared with the adjacent normal tissues. The incidence of MET amplification was 4.4%. None of the patients was positive for MET mutation. Collectively, MET was overexpressed in colorectal adenocarcinoma, and its positive protein expression predicted a poorer outcome in CRC patients. Furthermore, according to our results, MET amplification and 14 exon mutation are extremely rare events in colorectal adenocarcinoma. Copyright © 2018. Published by Elsevier Inc.

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

PubMed

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

2015-11-01

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

Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas.

PubMed

Fallo, F; Pezzi, V; Barzon, L; Mulatero, P; Veglio, F; Sonino, N; Mathis, J M

2002-12-01

The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

Quantitative Interaction Effects of Carbon Dioxide, Sodium Chloride, and Sodium Nitrite on Neurotoxin Gene Expression in Nonproteolytic Clostridium botulinum Type B

PubMed Central

Lövenklev, Maria; Artin, Ingrid; Hagberg, Oskar; Borch, Elisabeth; Holst, Elisabet; Rådström, Peter

2004-01-01

The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO2 in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO2 was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO2 concentration; the cntB mRNA level was fivefold greater in a 70% CO2 atmosphere than in a 10% CO2 atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng · ml−1 · unit of optical density−1 in the 10% CO2 atmosphere and 126 ng · ml−1 · unit of optical density−1 in the 70% CO2 atmosphere. PMID:15128553

In vivo oestrogenic modulation of Egr1 and Pitx1 gene expression in female rat pituitary gland.

PubMed

Gajewska, Alina; Herman, Andrzej P; Wolińska-Witort, Ewa; Kochman, Kazimierz; Zwierzchowski, Lech

2014-12-01

EGR1 and PITX1 are transcription factors required for gonadotroph cell Lhb promoter activation. To determine changes in Egr1 and Pitx1 mRNA levels in central and peripheral pituitary stimulations, an in vivo model based on i.c.v. pulsatile (1 pulse/0.5 h over 2 h) GnRH agonist (1.5 nM buserelin) or antagonist (2 nM antide) microinjections was used. The microinjections were given to ovariectomised and 17β-oestradiol (E2) (3×20 μg), ERA (ESR1) agonist propyl pyrazole triol (PPT) (3×0.5 mg), ERB (ESR2) agonist diarylpropionitrile (DPN) (3×0.5 mg) s.c. pre-treated rats 30 min after last pulse anterior pituitaries were excised. Relative mRNA expression was determined by quantitative RT-PCR (qRT-PCR). Results revealed a gene-specific response for GnRH and/or oestrogenic stimulations in vivo. Buserelin pulses enhanced Egr1 expression by 66% in ovariectomised rats, whereas the oestradiol-supplemented+i.c.v. NaCl-microinjected group showed a 50% increase in Egr1 mRNA expression. The oestrogenic signal was transmitted via ERA (ESR1) and ERB (ESR2) activation as administration of PPT and DPN resulted in 97 and 62%, respectively, elevation in Egr1 mRNA expression. A synergistic action of GnRH agonist and 17β-oestradiol (E2) stimulation of the Egr1 gene transcription in vivo were found. GnRHR activity did not affect Pitx1 mRNA expression; regardless of NaCl, buserelin or antide i.c.v. pulses, s.c. oestrogenic supplementation (with E2, PPT or DPN) consistently decreased (by -46, -48 and -41% respectively) the Pitx1 mRNA in the anterior pituitary gland. Orchestrated Egr1 and Pitx1 activities depending on specific central and peripheral regulatory inputs could be responsible for physiologically variable Lhb gene promoter activation in vivo. © 2014 Society for Endocrinology.

ECM1 and TMPRSS4 Are Diagnostic Markers of Malignant Thyroid Neoplasms and Improve the Accuracy of Fine Needle Aspiration Biopsy

PubMed Central

Kebebew, Electron; Peng, Miao; Reiff, Emily; Duh, Quan-Yang; Clark, Orlo H.; McMillan, Alex

2005-01-01

Objective: The objective of this study was to determine whether genes that regulate cellular invasion and metastasis are differentially expressed and could serve as diagnostic markers of malignant thyroid nodules. Summary and Background Data: Patients whose thyroid nodules have indeterminate or suspicious cytologic features on fine needle aspiration (FNA) biopsy require thyroidectomy because of a 20% to 30% risk of thyroid cancer. Cell invasion and metastasis is a hallmark of malignant phenotype; therefore, genes that regulate these processes might be differentially expressed and could serve as diagnostic markers of malignancy. Methods: Differentially expressed genes (2-fold higher or lower) in malignant versus benign thyroid neoplasms were identified by extracellular matrix and adhesion molecule cDNA array analysis and confirmed by real-time quantitative polymerase chain reaction (PCR). The area under the receiver operating characteristic (AUC) curve was calculated to determine diagnostic accuracy of gene expression level cutoffs established by logistic regression analysis. Results: By cDNA array analysis, ADAMTS8, ECM1, MMP8, PLAU, SELP, and TMPRSS4 were upregulated, and by quantitative PCR, ECM1, SELP, and TMPRSS4 mRNA expression was higher in malignant (n = 57) than in benign (n = 38) thyroid neoplasms (P< 0.002). ECM1 and TMPRSS4 mRNA expression levels were independent predictors of a malignant thyroid neoplasm (P < 0.003). The AUC was 0.956 for ECM1 and 0.926 for TMPRSS4. Combining both markers improved their diagnostic use (AUC 0.985; sensitivity, 91.7%; specificity, 89.8%; positive predictive value, 85.7%; negative predictive value, 82.8%). ECM1 and TMPRSS4 expression analysis improved the diagnostic accuracy of FNA biopsy in 35 of 38 indeterminate or suspicious results. The level of ECM1 mRNA expression was higher in TNM stage I differentiated thyroid cancers than in stage II and III tumors (P ≤ 0.031). Conclusions: ECM1 and TMPRSS4 are excellent diagnostic markers of malignant thyroid nodules and may be used to improve the diagnostic accuracy of FNA biopsy. ECM1 is also a marker of the extent of disease in differentiated thyroid cancers. PMID:16135921

[Effect of Epimedium extract on osteoprotegerin and RANKL mRNA expressions in glucocorticoid-induced femoral head necrosis in rats].

PubMed

Wang, Jian-zhong; Gao, Hong-yan; Wang, Kun-zheng; Zhou, Rong-xing; Li, Xiao-dong; Guo, Jun; Lv, Hui-cheng

2011-10-01

To investigate the effect of glucocorticoid on the expression levels of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) mRNAs in rat femoral head and the antagonistic effect of Epimedium, and explore the mechanism of Epimedium in preventing glucocorticoid-induced femoral head necrosis. Forty-eight adult SD rats were randomized into glucocorticoid group, Epimedium group and control group. In the former two groups, the rats received intramuscular injection of 12.5 mg prednisolone twice a week, and in Epimedium group, additional 1 ml/100 g aqueous Epimedium extract (equivalent to 0.1 g/ml of the crude drug) was administered intragastrically once daily. The control group received only intramuscular saline injection. After 4 weeks of treatment, osteonecrosis of the left femoral head was detected by HE staining, and the right femoral head was sampled for detection of OPG and RANKL mRNA expressions using real-time quantitative PCR. In glucocorticoid, Epimedium and control groups, the mortality rate of the rats was 12.5% (2/16), 6.25% (1/16), 0 (0/16), and femoral head necrosis occurred at a rate of 71.43% (10/14), 26.67% (4/15), and 0 (0/16), respectively. In glucocorticoid group, the expression level of OPG mRNA was significantly lower, RANKL expression significantly higher, and OPG/RANKL ratio significantly lower than those in Epimedium and control groups (P<0.05). OPG, RANKL and their ratios showed no significant differences between Epimedium group and the control group. Epimedium can prevent glucocorticoid-induced femoral head necrosis probably by antagonizing glucocorticiod-induced abnormal expressions of OPG and RANKL mRNA.

[The Impact of Electroacupuncture Intervention on Expression of 5-HTR 1 B/2 C Genes in Mice under Radiation Stimulation from Mobile Phone].

PubMed

Dai, Jian-yu; Chen, Yi-guo; Zhang, Xiao-qing

2015-08-01

To observe the effect of electroacupuncture (EA) stimulation of "Yifen" (TE 17), "Shenshu" (BL 23) on the expression of 5-hydroxytryptamine receptor 1 B (5-HTR 1 B) mRNA and 5-hydroxytryptamine receptor 2 C (5-HTR 2 C) mRNA in the cochlear nucleus tissue in mice experiencing radiation from mobile phone, so as to explore its mechanisms underlying improvement of tinnitus. Thirty Kunming mice were randomly divided into control group (n = 6) and modeling group (n = 24). The tinnitus model was established by giving the mice with mobile phone-radiation for 1 h in the morning and 1 h in the afternoon, continuously for 40 days. EA stimulation was applied to "Yifeng" (TE 17) group (n = 6) and "Shenshu" (BL 23) group (n = 6) for 20 min, once a day for 7 days. The expression of 5-THR 1 B/2 C mRNA in the cochlear nucleus was assayed by fluorescence quantitative polymerase chain reaction (real time-PCR). The expression level of 5-HTR 1 B was significantly lower in the model group than in the control group (P < 0.05), while that of 5-HTR 2 C mRNA significantly increased (P < 0.01). TE 17 group received a significant acupoint intervention effect (P < 0.01). Compared with TE 17 group, BL 23 group received a weaker effect (P < 0.05). EA of TE 17 can up-regulate expression level of 5-HTR 1 B and down-regulate expression level of 5-HTR 2 C in the cochlear nucleus in mice experiencing mobile-phone radiation.

Molecular cloning of heat shock protein 60 (PtHSP60) from Portunus trituberculatus and its expression response to salinity stress.

PubMed

Xu, Qianghua; Qin, Ye

2012-09-01

Heat shock protein 60 (HSP60) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Portunus trituberculatus is an important marine fishery and aquaculture species, and water salinity condition influenced its artificial propagations significantly. In order to investigate the function of P. trituberculatus HSP60 against osmotic stress, P. trituberculatus HSP60 gene was firstly cloned. The full-length cDNA of PtHSP60 contains 1,743 nucleotides encoding 577 amino acids with a calculated molecular weight of 61.25 kDa. Multiple alignments indicated that the deduced amino acid sequences of PtHSP60 shared a high level of identity with invertebrate and vertebrate HSP60 sequence including shrimp, fruit fly, zebrafish, and human. The expression profiles of PtHSP60 at mRNA and protein levels under salinity treatment were investigated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. It was found that the mRNA transcripts of PtHSP60 gene varied among different tissues under normal salinity conditions, and the antennal gland showed the highest expression level among the tissues tested. As for low salinity challenge, the mRNA expression of PtHSP60 gene was higher in the gill and appendicular muscle compared with other tissues, and gill and hypodermis represented the higher gene expressions during the hyperosmotic stress, which indicated that those tissues were salinity-sensitive tissues. In addition, salinity challenges significantly altered the expression of PtHSP60 at mRNA and protein level in a salinity- and time-dependent manner in P. trituberculatus gill tissue. The results indicate that PtHSP60 played important roles in mediating the salinity stress in P. trituberculatus.

Markers of hepatic regeneration associated with surgical attenuation of congenital portosystemic shunts in dogs.

PubMed

Tivers, Michael S; Lipscomb, Victoria J; Smith, Kenneth C; Wheeler-Jones, Caroline P D; House, Arthur K

2014-05-01

Dogs with congenital portosystemic shunts (CPSS) have liver hypoplasia and hepatic insufficiency. Surgical CPSS attenuation results in liver growth associated with clinical improvement. The mechanism of this hepatic response is unknown, although liver regeneration is suspected. This study investigated whether markers of liver regeneration were associated with CPSS attenuation. Dogs treated with CPSS attenuation were prospectively recruited. Residual liver tissue was collected for gene expression analysis (seven genes) from 24 CPSS dogs that tolerated complete attenuation, 25 dogs that tolerated partial attenuation and seven control dogs. Relative gene expression was measured using quantitative polymerase chain reaction (qPCR). Blood samples were collected before, 24 h and 48 h post-surgery from 36 CPSS dogs and from 10 control dogs. Serum hepatocyte growth factor (HGF) concentration was measured using a canine specific enzyme-linked immunosorbent assay (ELISA). HGF mRNA expression was significantly decreased in CPSS compared with control dogs (P = 0.046). There were significant increases in HGF (P = 0.050) and methionine adenosyltransferase 2 A (MAT2A; P = 0.002) mRNA expression following partial CPSS attenuation. Dogs with complete attenuation had significantly greater MAT2A (P = 0.024) mRNA expression compared with dogs with partial attenuation. Serum HGF concentration significantly increased 24 h following CPSS attenuation (P < 0.001). Hepatic mRNA expression of two markers of hepatocyte proliferation (HGF and MAT2A) was associated with the response to surgery in dogs with CPSS, and serum HGF significantly increased following surgery, suggesting hepatocyte proliferation. These findings support the concept that hepatic regeneration is important in the hepatic response to CPSS surgery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recombinant cell lines expressing shRNA targeting herpes simplex virus 2 VP16 inhibit virus replication.

PubMed

Zhang, Rui; Wang, Yan; Song, Bo; Han, Zhi Qiang; Xu, Yu Ming

2012-01-01

To establish HSV2 VP16 targeting shRNA-expressing cell lines and investigate the antiviral effect of shRNA targeting HSV2 VP16. The cell lines Vero-shRNAs and negative-control Vero-shCON were established. Their inhibition effects on VP16 mRNA expression were tested by real-time fluorescent quantitative polymerase chain reaction (PCR) and their antiviral effects were evaluated by yield reduction assay. The influence of passage numbers on the inhibition ability of cell lines was researched. Vero-shRNA24 targeting the upper stream, Vero-shRNA642 targeting the lower stream and Vero-shCON were established. Vero-shRNA24, Vero-shRNA642 and Vero-shRNA24 + 642 could reduce the VP16 mRNA significantly. Vero-shRNA24 was the most efficient. The HSV2 titers in Vero and Vero-shCON were the highest at 72 h after infection, and started decreasing thereafter. The viral titers of the Vero-shRNA groups reached a peak after 84 h and the highest titers were lower than in the Vero group. The inhibiting effect on VP16 mRNA expression and viral replication of Vero-shRNA24 cell lines of passages 10 and 20 were not significantly different from the primary cell line. Although of no statistical significance, the passage 50 cell line showed decreased inhibiting ability. Recombinant cell lines expressing shRNA targeting HSV2 VP16 were established. They can stably inhibit HSV2 VP16 mRNA expression and viral replication within passage 50. Copyright © 2012 S. Karger AG, Basel.

Does Harvey-Ras gene expression lead to oral squamous cell carcinoma? A clinicopathological aspect

PubMed Central

Krishna, Akhilesh; Singh, Shraddha; Singh, Vineeta; Kumar, Vijay; Singh, Uma Shankar; Sankhwar, Satya Narayan

2018-01-01

Background: Harvey-Ras (H-Ras) is an important guanosine triphosphatase protein for the regulation of cellular growth and survival. Altered Ras signaling has been observed in different types of cancer either by gene amplification and/or mutation. The H-Ras oncogene mutations are well reported, but expression of the H-Ras gene is still unknown. Objective: This study aimed to examine both protein and messenger-RNA (mRNA) expressions of H-Ras in oral squamous cell carcinoma (OSCC) and analyzed the association with risk habits and the clinicopathological profile of cases. Methodology: A total of 65 tissue specimens of OSCC (case group) and equal number of normal tissues (control group) were included in this study. H-Ras protein and mRNA expressions were analyzed using immunohistochemical and quantitative real time-polymerase chain reaction techniques, respectively. Results: The H-Ras protein was significantly overexpressed in the oral carcinoma group compared to the normal group (P = 0.03). Most of the OSCC cases showed positive staining with moderate expression, while negative and moderate staining was high in the control group. The majority of H-Ras positive cases were found in individuals with multiple risk habits including tobacco chewing. The risk of H-Ras positivity was 1.46 times higher in smokers than non-smokers. H-Ras positivity increased in cases affected with buccal mucosa site and higher grade of carcinoma. Relative mRNA level of H-Ras was significantly elevated in oral carcinoma as compared with the control group (P ≤ 0.001). Protein and mRNA levels of H-Ras in case group was poorly correlated. Conclusion: H-Ras oncogene expression was markedly higher in oral carcinoma, and it can be a prognostic marker and target for an effective molecular therapy. PMID:29731559

Perinatal asphyxia alters neuregulin-1 and COMT gene expression in the medial prefrontal cortex in rats.

PubMed

Wakuda, Tomoyasu; Iwata, Keiko; Iwata, Yasuhide; Anitha, Ayyappan; Takahashi, Taro; Yamada, Kohei; Vasu, Mahesh Mundalil; Matsuzaki, Hideo; Suzuki, Katsuaki; Mori, Norio

2015-01-02

Epidemiological studies suggest that perinatal complications, particularly hypoxia-related ones, increase the risk of schizophrenia. Recent genetic studies of the disorder have identified several putative susceptibility genes, some of which are known to be regulated by hypoxia. It can be postulated therefore that birth complications that cause hypoxia in the fetal brain may be associated with a dysregulation in the expression of some of the schizophrenia candidate genes. To test this, we used an animal model of perinatal asphyxia, in which rat pups were exposed to 15 min of intrauterine anoxia during Caesarean section birth, and examined the expression of mRNA of five of the putative susceptibility genes (NRG1, ErbB4, AKT1, COMT and BDNF) by real-time quantitative PCR in the medial prefrontal cortex (mPFC) and the hippocampus at 6 and 12 weeks after birth. The expression of NRG1 mRNA was significantly decreased in the mPFC, but not in the hippocampus, at 6 and 12 weeks after birth. In addition, a significant increase in COMT mRNA expression was observed in the mPFC at 12 weeks. The alteration in mRNA levels of NRG1 and COMT was not associated with a change in their protein levels. These results suggest that perinatal asphyxia may lead to disturbances in the PFC, which in turn may exert a long-lasting influence on the expression of specific genes, such as NRG1 and COMT. Our results also suggest that translational interruption may occur in this model of perinatal asphyxia. Copyright © 2014 Elsevier Inc. All rights reserved.

Decoy Oligonucleotide Rescues IGF1R Expression from MicroRNA-223 Suppression

PubMed Central

Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3’ untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5’, central or 3’ region of mature miR-223 suppressed miR-223 targeting the 3’UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3’UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3’UTRs have similar binding sites for miR-223 with IGF1R 3’UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting. PMID:24324762

Decoy oligonucleotide rescues IGF1R expression from MicroRNA-223 suppression.

PubMed

Wu, Li Hui; Cai, Qian Qian; Dong, Yi Wei; Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3' untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5', central or 3' region of mature miR-223 suppressed miR-223 targeting the 3'UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3'UTRs have similar binding sites for miR-223 with IGF1R 3'UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting.

Analysis of FMR1 gene expression in female premutation carriers using robust segmented linear regression models

PubMed Central

García-Alegría, Eva; Ibáñez, Berta; Mínguez, Mónica; Poch, Marisa; Valiente, Alberto; Sanz-Parra, Arantza; Martinez-Bouzas, Cristina; Beristain, Elena; Tejada, Maria-Isabel

2007-01-01

Fragile X syndrome is caused by the absence or reduction of the fragile X mental retardation protein (FMRP) because FMR1 gene expression is reduced. Alleles with repeat sizes of 55–200 are classified as premutations, and it has been demonstrated that FMR1 expression is elevated in the premutation range. However, the majority of the studies reported were performed in males. We studied FMR1 expression in 100 female fragile X family members from the northern region of Spain using quantitative (fluorescence) real-time polymerase chain reaction. Of these 100 women, 19 had normal alleles, 19 were full mutation carriers, and 62 were premutation carriers. After confirming differences between the three groups of females, and increased levels of the FMR1 transcript among premutation carriers, we found that the relationship between mRNA levels and repeat size is nonlinear. These results were obtained using a novel methodology that, based on the size of the CGG repeats, allows us to find out the most probable threshold from which the relationship between CGG repeat number and mRNA levels changes. Using this approach, a significant positive correlation between CGG repeats and total mRNA levels has been found in the premutation range <100 CGG, but this correlation diminishes from 100 onward. However, when correcting by the X inactivation ratio, mRNA levels increase as the number of CGG repeats increases, and this increase is highly significant over 100 CGG. We suggest that due to skewed X inactivation, mRNA levels tend to normalize in females when the number of CGG repeats increases. PMID:17449730

Effects of childbirth on podocyturia in women with normotensive, uncomplicated pregnancies.

PubMed

Furuta, Itsuko; Zhai, Tianyue; Umazume, Takeshi; Ishikawa, Satoshi; Nakagawa, Kinuko; Kojima, Takashi; Yamada, Takahiro; Morikawa, Mamoru; Minakami, Hisanori

2017-06-01

Changes in hemodynamics and blood pressure occur shortly before and after childbirth regardless of the mode of delivery. This study aimed to test the hypothesis that parturition induces a temporal increase in podocyturia monitored by podocyte-specific protein podocin mRNA expression levels (Pod-mRNA). A total of 105 urine specimens, consisting of 43 and 62 from 18 and 20 otherwise healthy women with vaginal delivery (VD) and elective cesarean delivery (ECS), respectively, were studied. Determination of urine protein and creatinine (Cr) concentrations and quantitative analyses of Pod-mRNA, nephrin mRNA (Nep-mRNA), synaptopodin mRNA (Syn-mRNA), and aquaporin 2 mRNA expression were performed using RT-PCR in pelleted urine samples. Levels of mRNA expression were corrected by urine Cr concentration. Podocyturia increased significantly, concomitant with a significantly decreased Nep:Pod-mRNA ratio (NPR) in the urine, collected immediately before or after childbirth regardless of the delivery mode compared with urine collected before commencement of labor or on postpartum day 3 or later. Podocyturia was significantly negatively correlated with NPR [correlation coefficient ( r ) = -0.614/-0.750 for VD/ECS women, respectively], as well as the Syn:Pod-mRNA ratio. Systolic blood pressure exceeded 140 mmHg during labor in 50% of VD women, and mean arterial pressure was significantly positively correlated with podocyturia during labor in VD women ( r  = 0.733). Thus parturition induces a transient increase in urine podocytes with reduced Nep- and Syn-mRNA expressions. Glomerular podocytes with reduced Nep- and Syn-mRNA levels were suggested to be likely to detach from the glomerular basement membrane around childbirth. Copyright © 2017 the American Physiological Society.

Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer.

PubMed

Elshafae, Said M; Hassan, Bardes B; Supsavhad, Wachiraphan; Dirksen, Wessel P; Camiener, Rachael Y; Ding, Haiming; Tweedle, Michael F; Rosol, Thomas J

2016-06-01

The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and β-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer. Prostate 76:796-809, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

Modeling RNA interference in mammalian cells

PubMed Central

2011-01-01

Background RNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers. Results We tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects. Conclusions Our model has a simple mathematical form, amenable to analytical investigations and a small set of parameters with an intuitive physical meaning, that makes it a unique and reliable mathematical tool. The findings here presented will be a useful instrument for better understanding RNAi biology and as modelling tool in Systems and Synthetic Biology. PMID:21272352

Fruit and vegetable consumption and proinflammatory gene expression from peripheral blood mononuclear cells in young adults: a translational study

PubMed Central

2010-01-01

Background Fruits and vegetables are important sources of fiber and nutrients with a recognized antioxidant capacity, which could have beneficial effects on the proinflammatory status as well as some metabolic syndrome and cardiovascular disease features. The current study assessed the potential relationships of fruit and vegetable consumption with the plasma concentrations and mRNA expression values of some proinflammatory markers in young adults. Methods One-hundred and twenty healthy subjects (50 men/70 women; 20.8 ± 2.6 y; 22.3 ± 2.8 kg/m2) were enrolled. Experimental determinations included anthropometry, blood pressure and lifestyle features as well as blood biochemical and inflammatory measurements. The mRNA was isolated from peripheral blood mononuclear cells (PBMC) and the gene expression concerning selected inflammatory markers was assessed by quantitative real-time PCR. Nutritional intakes were estimated by a validated semi-quantitative food-frequency questionnaire. Results The highest tertile of energy-adjusted fruit and vegetable consumption (>660 g/d) was associated with lower plasma concentrations of C-reactive protein (CRP) and homocysteine and with lower ICAM1, IL1R1, IL6, TNFα and NFκB1 gene expression in PBMC (P for trend < 0.05), independently of gender, age, energy intake, physical activity, smoking, body mass index, systolic blood pressure and circulating non-esterified fatty acids. In addition, plasma CRP, homocysteine and TNFα concentrations and ICAM1, TNFα and NFκB1 gene expression in PBMC showed a descending trend as increased fiber intake (>19.5 g/d) from fruits and vegetables (P for trend < 0.05). Furthermore, the participants within the higher tertile (>11.8 mmol/d) of dietary total antioxidant capacity showed lower plasma CRP and mRNA values of ICAM1, IL1R1, IL6, TNFα and NFκB1 genes (P for trend < 0.05). The inverse association between fruit and vegetable consumption and study proinflammatory markers followed the same trend and remained statistically significant, after the inclusion of other foods/nutrients in the linear regression models. Conclusion A higher fruit and vegetable consumption was independently associated not only with reduced CRP and homocysteine concentrations but also with a lower mRNA expression in PBMC of some relevant proinflammatory markers in healthy young adults. PMID:20465828

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

PubMed

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

2008-12-01

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

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

PubMed

Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

A circadian neuropeptide PDF in the honeybee, Apis mellifera: cDNA cloning and expression of mRNA.

PubMed

Sumiyoshi, Miho; Sato, Seiji; Takeda, Yukimasa; Sumida, Kazunori; Koga, Keita; Itoh, Tsunao; Nakagawa, Hiroyuki; Shimohigashi, Yasuyuki; Shimohigashi, Miki

2011-12-01

Pigment-dispersing factor (PDF) is a pacemaker hormone regulating the locomotor rhythm in insects. In the present study, we cloned the cDNAs encoding the Apis PDF precursor protein, and found that there are at least seven different pdf mRNAs yielded by an alternative splicing site and five alternative polyadenylation sites in the 5'UTR and 3'UTR regions. The amino acid sequence of Apis PDF peptide has a characteristic novel amino acid residue, aspargine (Asn), at position 17. Quantitative real-time PCR of total and 5'UTR insertion-type pdf mRNAs revealed, for the first time, that the expression levels change in a circadian manner with a distinct trough at the beginning of night in LD conditions, and at the subjective night under DD conditions. In contrast, the expression level of 5'UTR deletion-type pdf mRNAs was about half of that of the insertion type, and the expression profile failed to show a circadian rhythm. As the expression profile of the total pdf mRNA exhibited a circadian rhythm, transcription regulated at the promoter region was supposed to be controlled by some of the clock components. Whole mount in situ hybridization revealed that 14 lateral neurons at the frontal margin of the optic lobe express these mRNA isoforms. PDF expressing cells examined with a newly produced antibody raised against Apis PDF were also found to have a dense supply of axon terminals in the optic lobes and the central brain.

Transient receptor potential vanilloid type 2 (TRPV2) expression in normal urothelium and in urothelial carcinoma of human bladder: correlation with the pathologic stage.

PubMed

Caprodossi, Sara; Lucciarini, Roberta; Amantini, Consuelo; Nabissi, Massimo; Canesin, Giacomo; Ballarini, Patrizia; Di Spilimbergo, Adriana; Cardarelli, Marco Andrea; Servi, Lucilla; Mammana, Gabriele; Santoni, Giorgio

2008-09-01

To evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. Bladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal (n=6) and cancer bladder tissues (n=58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. Enhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. Normal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.

Differential expression of melanopsin mRNA and protein in Brown Norwegian rats.

PubMed

Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

2013-01-01

Melanopsin is expressed in a subpopulation of retinal ganglion cells rendering these cells intrinsically photosensitive (ipRGCs). The ipRGCs are the primary RGCs mediating light entrainment of the circadian clock and control of the pupillary light reflex, light regulated melatonin secretion and negative masking behaviour. Previous studies have demonstrated that melanopsin expression in albino rats is regulated by light and darkness. The present study was undertaken to study the influence of light and darkness during the circadian day and after extended periods of constant light and darkness on melanopsin expression in the pigmented retina of the Brown Norwegian rat (Rattus norvegicus). The diurnal and circadian expressions were examined in retinal extracts from rats euthanized every 4 h during a 24 h light/dark (LD) and a 24 h dark cycle (DD) using quantitative real-time PCR and Western blotting. To study whether light regulates melanopsin expression, rats were sacrificed after being placed in either constant light (LL) or darkness for 3 or 21 d. Flat mount retinas from animals kept during either LL or DD were also examined by immunohistochemistry. Melanopsin mRNA expression displayed a significant rhythmic change during the LD cycle with peak expression around dusk and nadir at dawn. Melanopsin protein also changed over the LD cycle with peak expression at the end of the night and nadir at dusk. Rhythmic expression of melanopsin mRNA but not melanopsin protein was found in constant darkness. After 3 or 21 d in either LL or DD melanopsin mRNA expression was unaltered. Melanopsin protein was at the same high level after 3 and 21 d in DD, whereas a significant decrease was found after prolonging the light period for 3 or 21 d. The change in melanopsin protein was primarily due to change in immunoreactivity in the dendritic processes. In conclusion we found that light and darkness are important for regulation of melanopsin protein expression whereas input from a retinal networks regulates melanopsin mRNA expression. It is likely to speculate that altered level of melanopsin is one way in which the retina adapts to environmental light and darkness conditions ensuring optimal light sensitivity for the transmission to the brain. Copyright © 2012 Elsevier Ltd. All rights reserved.

VEGF expression and the effect of NSAIDs on ascites cell proliferation in the hen model of ovarian cancer.

PubMed

Urick, M E; Giles, J R; Johnson, P A

2008-09-01

We aimed to determine the expression of vascular endothelial growth factor (VEGF) and the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the proliferation of cells isolated from ascites in the hen model of ovarian cancer. Ovarian tumor and normal ovary were collected from hens and ascites cells were isolated from hens with ovarian cancer. Quantitative real-time PCR was used to quantify mRNA expression. Immunohistochemical and/or Western blot analyses were used to localize protein expression in ovarian tumors, normal ovaries, and ascites cells. Cells were treated with a nonspecific, COX-1-specific, or COX-2-specific NSAID and proliferation was determined. VEGF mRNA was increased in ascites cells and there was a trend for a correlation between VEGF mRNA in ascites cells and ascites volume. VEGF protein was localized to theca cells of normal ovaries, in glandular areas of tumors, and to the cytoplasm of ascites cells. Aspirin and a COX-1-specific inhibitor decreased the proliferation of ascites cells, whereas a COX-2-specific inhibitor did not. VEGF may play a role in ovarian cancer progression in the hen and the proliferation of ascites cells can be decreased by targeting the COX-1 but not COX-2 pathway.

Decreased β-catenin expression in first-trimester villi and decidua of patients with recurrent spontaneous abortion.

PubMed

Li, Shuhong; Li, Ning; Zhu, Peng; Wang, Yanwei; Tian, Yongjie; Wang, Xietong

2015-06-01

We aimed to study the relation between Wnt/β-catenin signaling pathway and recurrent spontaneous abortion through investigating the expression of β-catenin and Dickkof-1 in first-trimester villi and decidua of recurrent spontaneous abortion patients. Villous and decidual tissues were collected from 40 women (20 patients with recurrent spontaneous abortion and 20 patients with normal, early pregnancy). Western blots were used to measure the protein levels of β-catenin in villi and decidua, and the localization of β-catenin was investigated by immunohistochemistry. Quantitative real-time reverse transcription polymerase chain reaction was used to quantify the mRNA levels of β-catenin and Dickkof-1 in villi and decidua, respectively. Our results indicated that β-catenin was expressed predominantly in plasma membranes of the villous cytotrophoblasts and glandular epithelium. What's more, its expression significantly decreased at both mRNA and protein levels, whereas the mRNA levels of Dickkof-1 significantly increased in villi and decidua of the recurrent spontaneous abortion group compared with the normal control group. We therefore speculated that the downregulated Wnt/β-catenin signaling pathway might be associated with the process of the pathogenesis of recurrent spontaneous abortion. © 2014 The Authors. Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology.

The comparison of skin rejuvenation effects of vitamin A, fractional laser, and their combination on rat.

PubMed

Qu, Yan; Wang, Ying; Zhang, Yan; Han, Chunyu; Gao, Dong; Jin, Waishu; Liang, Jinning; Xia, Xiujuan

2018-03-15

Because of long-term exposure of skin, skin aging is an unavoidable natural law with age. Traditional Vitamin A and novel ablative fractional laser technique both have the effects of skin rejuvenation, and studies have demonstrated both of them have apparent clinical efficacy and histology-improving effects on photo-aging skin. 45 female healthy Wistar rats were selected and the depilation areas of every rat were divided into four regions: control region(Region A), Vitamin A acid region(Region B), combination treatment region(Region C), and fractional laser region(Region D). 0.025% Vitamin A acid cream was applied to Region B and C every day for 3 weeks; Region C and D were irradiated once with 10600nm CO 2 fractional laser on the first day of the trail. The skin tissue was dissected and placed into liquid nitrogen according to the design. The real-time quantitative PCR and western blotting methods were taken to detect the expression changes of miR-29a, Akt, TGF-β, and mRNA of type III pre-collagen. It can be seen from the results of the real-time quantitative PCR that the mRNA expression levels of type III pre-collagen, Akt, and TGF-β in the treatment regions are up-regulated and the expression levels of miR-29a mRNA are down-regulated compared to the Region A. The hybridization tests showed that changes of the expression of type III pre-collagen, Akt gene, miR-29a gene, and TGF-β gene across the experiment regions are all significantly different in the third week, and the expression levels of them all achieve the highest value in the third week, the expression level of miR-29a gene achieves the lowest value in the third week, which are consistent with the results of real-time quantitative PCR. It is indicated that the combination region of Vitamin A acid and fractional laser may lead to low expression of miR-29a, thus the inhibition of downstream Akt activation is loss, Akt activation is enhanced, enhancement of the expression of TGF-β is induced, leading to proliferation of fibroblasts, and promotion of the collagen proteins' synthesis in skin. Therefore miR-29a/Akt/TGF-β signal pathway may participate in the skin rejuvenation mechanism of action Vitamin A acid and fractional laser. This may provide a new treatment approach for skin rejuvenation.

Increased FOXP3 expression in tumour-associated tissues of horses affected with equine sarcoid disease.

PubMed

Mählmann, K; Hamza, E; Marti, E; Dolf, G; Klukowska, J; Gerber, V; Koch, C

2014-12-01

Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. Bovine papilloma virus (BPV) is recognised as the most important aetiological factor in equine sarcoid (ES) disease. The aim of this study was to compare expression levels of Treg markers and associated cytokines in tissue samples of ES-affected equids with skin samples of healthy control horses. Eleven ES-affected, and 12 healthy horses were included in the study. Expression levels of forkhead box protein 3 (FOXP3), interleukin 10 (IL10), interleukin 4 (IL4) and interferon gamma (IFNG) mRNA in lesional and tumour-distant samples from ES-affected horses, as well as in dermal samples of healthy control horses were measured using quantitative reverse transcription polymerase chain reaction (PCR). Expression levels were compared between lesional and tumour-distant as well as between tumour-distant and control samples. Furthermore, BPV-1 E5 DNA in samples of ES-affected horses was quantified using quantitative PCR, and possible associations of viral load, disease severity and gene expression levels were evaluated. Expression levels of FOXP3, IL10 and IFNG mRNA and BPV-1 E5 copy numbers were significantly increased in lesional compared to tumour-distant samples. There was no difference in FOXP3 and cytokine expression in tumour-distant samples from ES- compared with control horses. In tumour-distant samples viral load was positively correlated with IL10 expression and severity score. The increased expression of Treg markers in tumour-associated tissues of ES-affected equids indicates a local, Treg-induced immune suppression. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

PubMed

Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

2017-10-01

Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

Low BMI is correlated with increased TGF-β and IL-10 mRNA levels in the peripheral blood of breast cancer patients.

PubMed

Liu, Chao; Wang, Qian; Sun, Bing; Meng, Xiangying; Li, Lan; Yang, Liuchun; Cong, Yang; Liu, Jiannan; Xuan, Liang; Huang, Yan; Wu, Shikai

2018-03-01

Transforming growth factor-β (TGF-β), interleukin-10 (IL-10), and forkhead box P3 (Foxp3) have important roles in breast cancer development. Previous studies confirmed a correlation between these immune molecules and tumor characteristics, but their association with nutritional status in breast cancer is largely unknown. We aimed to investigate the association between body mass index (BMI), hemoglobin, total protein, albumin, globulin (GLB), albumin/GLB ratio (AGR), pre-albumin, prognostic nutritional index, and TGF-β, IL-10, and Foxp3 mRNA expression in patients with breast cancer. Quantitative real-time PCR was used to detect the mRNA expression of TGF-β, IL-10, and Foxp3 in the peripheral blood of 107 patients with breast cancer and 21 healthy controls. We found that TGF-β mRNA levels were 2.6-fold, 3.2-fold, and 2.3-fold higher in patients with low BMI (<23), low AGR, and high GLB, respectively, than in their counterparts (P < 0.05). In addition, IL-10 mRNA expression levels in patients with normal BMI (<23) were 2.8-fold and 3.5-fold higher than in those who were overweight (23≤ BMI <25) and obese (BMI ≥ 25), respectively (P < 0.05). In addition, TGF-β, IL-10, and Foxp3 mRNA levels were significantly higher in patients with breast cancer than in healthy controls (P < 0.05). In summary, our results suggest that nutritional status, especially BMI, may strongly affect systematic immune function in patients with breast cancer. © 2018 IUBMB Life, 70(3):237-245, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae.

PubMed

Kortner, Trond M; Overrein, Ingrid; Oie, Gunvor; Kjørsvik, Elin; Bardal, Tora; Wold, Per-Arvid; Arukwe, Augustine

2011-10-01

We have profiled the expression of twelve genes, in order to provide an overview on the molecular ontogeny of digestive capability with the associated endocrine control during Atlantic cod (Gadus morhua) larval development. Enzyme activity levels for the key digestive enzyme, trypsin, was also measured. Specifically, transcripts for trypsin, amylase, lipolytic enzymes: bile salt activated lipase (BAL), phospholipase A2 (PLA2) and Acyl CoA dehydrogenase (ACADM), regulatory peptides: neuropeptide Y (NPY), orexin (OX) cholecystokinin (CCK) and cocaine and amphetamine-related transcript (CART), the somatotropic factors: growth hormone (GH), preprosomatostatin 1 (PPSS1) and thyroid hormone receptors (TRα and TRβ) were analyzed using quatitative (real-time) polymerase chain reaction (qPCR). Trypsin and BAL mRNA levels peaked at approximately day 17 and 25 post-hatch, respectively, and thereafter displayed a decreasing pattern until metamorphosis. GH mRNA levels decreased moderately from 3 to 33dph, and thereafter, an increase was observed until 46dph. TRα mRNA levels showed a fluctuating pattern peaking at day 39 post-hatch. TRβ mRNA levels were too low to obtain quantitative measurements. Amylase mRNA slightly increased from day 3 to 17 post-hatch, and thereafter showed a steady decrease until day 60. Interestingly, PLA2 mRNA expression showed a consistent increase throughout the study period, indicating an increasingly important role during larval development. Overall, data from this study indicate that cod larvae show differential developmental mode of expression patterns for key genes and endocrine factors that regulate digestive capability, growth and development. These data are discussed in relation to larval trypsin enzyme activity and previous reports for other teleost species. Copyright © 2011 Elsevier Inc. All rights reserved.

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

Four SNPs and Systemic Level of FOXP3 in Smokers and Patients with Chronic Obstructive Pulmonary Disease.

PubMed

Chu, Shuyuan; Zhong, Xiaoning; Zhang, Jianquan; Lai, Xiaoying; Xie, Jiajun; Li, Yu

2016-12-01

Forkhead box P3 (FOXP3) is the essential transcription factor for the function of regulatory T-cell (Treg). However, the gene mutation of FOXP3 in patients with chronic obstructive pulmonary disease (COPD) at different stages has not been reported. We aim to investigate four single nucleotide polymorphisms (SNPs) and the mRNA expression of FOXP3 in smokers with normal lung function and smokers with COPD at different stages. FOXP3 mRNA expression and SNPs in FOXP3 were assessed in nonsmokers with normal lung function (N), smokers with normal lung function (S), smokers with COPD in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 1 or 2 grade (COPD 1-2), and smokers with COPD in GOLD 3 or 4 grade (COPD 3-4). In peripheral blood sample, FOXP3 mRNA was assessed using real-time quantitative PCR and SNPs were analyzed by TaqMan PCR. FOXP3 mRNA level in peripheral blood sample was decreased when COPD was aggravated. The frequency of FOXP3 rs5902434 genotype del/del and allele del are lower in COPD 1-2 and COPD 3-4 than that in N or S. The rs5902434 genotype del/del and allele del were, respectively, associated with decreased risk of COPD and lung function decline. The rs5902434 genotypic distribution was correlated with FOXP3 mRNA level. In conclusion, both FOXP3 rs5902434 genotypes and alleles were differently distributed in COPD patients and smokers with normal lung function. The distribution of del/del genotype was associated with systemic expression of FOXP3 mRNA. More research is needed to explore the role of FOXP3 gene polymorphism in immunoinflammation of COPD.

Innate Immune Response to Viral Infections in Primary Bronchial Epithelial Cells is Modified by the Atopic Status of Asthmatic Patients.

PubMed

Moskwa, Sylwia; Piotrowski, Wojciech; Marczak, Jerzy; Pawełczyk, Małgorzata; Lewandowska-Polak, Anna; Jarzębska, Marzanna; Brauncajs, Małgorzata; Głobińska, Anna; Górski, Paweł; Papadopoulos, Nikolaos G; Edwards, Michael R; Johnston, Sebastian L; Kowalski, Marek L

2018-03-01

In order to gain an insight into determinants of reported variability in immune responses to respiratory viruses in human bronchial epithelial cells (HBECs) from asthmatics, the responses of HBEC to viral infections were evaluated in HBECs from phenotypically heterogeneous groups of asthmatics and in healthy controls. HBECs were obtained during bronchoscopy from 10 patients with asthma (6 atopic and 4 non-atopic) and from healthy controls (n=9) and grown as undifferentiated cultures. HBECs were infected with parainfluenza virus (PIV)-3 (MOI 0.1) and rhinovirus (RV)-1B (MOI 0.1), or treated with medium alone. The cell supernatants were harvested at 8, 24, and 48 hours. IFN-α, CXCL10 (IP-10), and RANTES (CCL5) were analyzed by using Cytometric Bead Array (CBA), and interferon (IFN)-β and IFN-λ1 by ELISA. Gene expression of IFNs, chemokines, and IFN-regulatory factors (IRF-3 and IRF-7) was determined by using quantitative PCR. PIV3 and RV1B infections increased IFN-λ1 mRNA expression in HBECs from asthmatics and healthy controls to a similar extent, and virus-induced IFN-λ1 expression correlated positively with IRF-7 expression. Following PIV3 infection, IP-10 protein release and mRNA expression were significantly higher in asthmatics compared to healthy controls (median 36.03-fold). No differences in the release or expression of RANTES, IFN-λ1 protein and mRNA, or IFN-α and IFN-β mRNA between asthmatics and healthy controls were observed. However, when asthmatics were divided according to their atopic status, HBECs from atopic asthmatics (n=6) generated significantly more IFN-λ1 protein and demonstrated higher IFN-α, IFN-β, and IRF-7 mRNA expressions in response to PIV3 compared to non-atopic asthmatics (n=4) and healthy controls (n=9). In response to RV1B infection, IFN-β mRNA expression was lower (12.39-fold at 24 hours and 19.37-fold at 48 hours) in non-atopic asthmatics compared to atopic asthmatics. The immune response of HBECs to virus infections may not be deficient in asthmatics, but seems to be modified by atopic status. Copyright © 2018 The Korean Academy of Asthma, Allergy and Clinical Immunology · The Korean Academy of Pediatric Allergy and Respiratory Disease

BAG3 promotes chondrosarcoma progression by upregulating the expression of β-catenin

PubMed Central

Shi, Huijuan; Chen, Wenfang; Dong, Yu; Lu, Xiaofang; Zhang, Wenhui; Wang, Liantang

2018-01-01

To investigate the roles of B-cell lymphoma-2 associated athanogene 3 (BAG3) in human chondrosarcoma and the potential mechanisms, the expression levels of BAG3 were detected in the present study, and the associations between BAG3 and clinical pathological parameters, clinical stage as well as the survival of patients were analyzed. The present study detected BAG3 mRNA and protein expression in the normal cartilage cell line HC-a and in SW1353 chondrosarcoma cells by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The BAG3 protein expression in 59 cases of chondrosarcoma, 30 patients with endogenous chondroma and 8 cases of normal cartilage was semi-quantitatively analyzed using the immunohistochemical method. In addition, the BAG3 protein expression level, the clinical pathological parameters, clinical stage and the survival time of patients with chondrosarcoma were analyzed. The plasmid transfection method was employed to upregulate the expression BAG3 and small RNA interference to downregulate the expression of BAG3 in SW1353 cells. The expression levels of BAG3 protein and mRNA were significantly increased in the chondrosarcoma cell line when compared with the normal cartilage cell line. The immunohistochemistry results indicated that BAG3 protein was overexpressed in the tissue of human chondrosarcoma. Statistical analysis showed that the expression level of BAG3 was significantly increased in the different Enneking staging of patients with chondrosarcoma and Tumor staging, and there were no statistical differences in age, gender, histological classification and tumor size. In the in vitro experiments, the data revealed that BAG3 significantly promoted chondrosarcoma cell proliferation, colony-formation, migration and invasion; however, it inhibited chondrosarcoma cell apoptosis. It was observed that BAG3 upregulated β-catenin expression at the mRNA and protein levels. In addition, BAG3 induced the expression of runt-related transcription factor 2 (RUNX2) in chondrosarcoma cells by upregulating β-catenin. These clinical analyses revealed a positive association between β-catenin and BAG3 in chondrosarcoma tumors. BAG3 was significantly increased in chondrosarcoma cells and tissues compared with the normal cartilage cells, tissue and cartilage benign tumors. Thus, BAG3 may serve as an oncogene in the development of chondrosarcoma via the induction of RUNX2 expression. The results of the present study contribute to further research on the biological development of chondrosarcoma. PMID:29484408

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

PubMed

Torelli, A; Soragni, E; Bolchi, A; Petrucco, S; Ottonello, S; Branca, C

1996-12-01

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

Rapid, High-Throughput, and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire-Incorporated and Pneumatic Pressure-Driven Microdevice.

PubMed

Kim, Kyung Hoon; Kim, Jung; Choi, Jong Seob; Bae, Sunwoong; Kwon, Donguk; Park, Inkyu; Kim, Do Hyun; Seo, Tae Seok

2015-12-01

Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real-time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire-incorporated and pneumatic pressure-driven microdevice for rapid, high-throughput, and direct MB delivery to human breast cancer MCF-7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump-associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire-patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB-coated nanowire array can be manipulated. The combination of the nanowire-mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Effects of disulfiram on apoptosis in PANC-1 human pancreatic cancer cell line.

PubMed

Dastjerdi, M Nikbakht; Babazadeh, Z; Rabbani, M; Gharagozloo, M; Esmaeili, A; Narimani, M

2014-01-01

Pancreatic carcinoma is currently considered as a rapidly progressive and fatal disease, and is typically diagnosed late in its natural course. It is characterized by a poor diagnosis and lack of response to conventional therapy. Recent studies have suggested that disulfiram (DSF), a member of the dithiocarbamate family, may have antitumor activity. This study aimed to evaluate the in vitro effect of DSF on apoptosis in human pancreatic cancerous cell line (PANC-1). PANC-1 cells were cultured and treated with DSF at doses of 5, 10, 13 μM for 24 h and apoptosis was measured. Methylation specific PCR (MS-PCR) and real-time quantitative PCR were carried out to detect the methylation pattern and to estimate the mRNA expression levels of RASSF1A, p21 and Bax. MS-PCR analysis demonstrated that no unmethylated band was apeared in PANC-1 cell line after DSF treatments. The real-time quantitative PCR results showed no significant mRNA expression for RASSF1A (p>0.05); whereas p21 and Bax expression were significantly (p<0.01) enhanced after treatment with DSF. The results of the current study indicated that DSF can induce appoptosis in PANC-1 through p21 and Bax pathway but not through RASSF1A.

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

[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.

Antihypertensive methyldopa, labetalol, hydralazine, and clonidine reversed tumour necrosis factor-α inhibited endothelial nitric oxide synthase expression in endothelial-trophoblast cellular networks.

PubMed

Xu, Bei; Bobek, Gabriele; Makris, Angela; Hennessy, Annemarie

2017-03-01

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor-α (TNF-α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose TNF-α (0.5 ng/mL) or TNF-α plus soluble fms-like tyrosine kinase-1 (sFlt-1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR-8/SVneo cells were co-cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF-α on eNOS mRNA expression. After pre-incubating endothelial cells with TNF-α and sFlt-1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF-α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF-α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF-α. The anti-angiogenic molecule sFlt-1 may antagonise the potential benefit of these medications by interfering with the NOS pathway. © 2016 John Wiley & Sons Australia, Ltd.

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

[Effects of 5-aza-2-deoxycytidine on methylation status of RECK gene and cancer cell invasion in tongue cancer SCC-4 cells].

PubMed

Jiang, Xv

2014-10-01

To investigate the effects of 5-aza-2-deoxycytidine on methylation status and invasion ability of RECK gene in tongue cancer SCC-4 cells. Tongue cancer cell line SCC-4 cells were treated with 5-aza-dC at different concentrations for 72 h. Methylation status of RECK gene of SCC-4 cells was detected by methylation specific PCR (MSP), the expression of RECK gene mRNA was detected by real-time quantitative PCR. The expression of RECK protein was detected by Western blot, and the invasion ability of SCC-4 cell was examined by Transwell assay. SPSS13.0 software package was used for statistical analysis. RECK gene of SCC-4 cells was in high methylation status in untreated group, abnormal methylation was effectively reversed by 5-aza-dC treatment. After treatment with different concentration of 5-aza-dC for 72 h, relative mRNA expression level increased gradually (P<0.05). The relative expression level of RECK protein in 5-aza-dC treated group was significantly higher than that in the control group,the invasion ability of SCC-4 cell was decreased gradually. 5-aza-dC treatment for tongue cancer SCC-4 cells can successfully reverse high methylation status of RECK gene and restore the expression of RECK gene mRNA and protein, and reduced the invasion ability.

Overexpression of dihydrofolate reductase is a factor of poor survival in acute lymphoblastic leukemia.

PubMed

Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Illades-Aguiar, Berenice; Rivera-Ramírez, Ana Bertha; Saavedra-Herrera, Mónica Virginia; Leyva-Vázquez, Marco Antonio

2018-06-01

Dihydrofolate reductase (DHFR) has an important function in DNA synthesis and is a target of methotrexate, which is a crucial treatment option for acute lymphoblastic leukemia (ALL). However, the number of studies conducted to date on DHFR expression in childhood ALL is limited. The aim of the present study was to determine whether the expression of DHFR is associated with survival in childhood ALL. The expression of DHFR in 96 children with ALL and 100 control individuals was determined using reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that the expression of DHFR mRNA in children with ALL was significantly increased (P<0.001), compared with that in the control group. In addition, increased levels of DHFR mRNA were observed in patients with B-cell lineage, compared with patients with T-cell lineage ALL (P<0.05). The Kaplan-Meier estimator analysis revealed that children with ALL who exhibited increased levels of DHFR mRNA had a decreased overall survival time (P<0.05). It was observed that certain patient prognostic features (including age, sex, white blood cell count and high DHFR expression), are associated with poor survival (log-rank test, P<0.05). Therefore, the results of the present study indicated that DHFR upregulation is a factor for poor survival in ALL.

Molecular cloning and gene expression analysis of Ercc6l in Sika deer (Cervus nippon hortulorum).

PubMed

Yin, Yupeng; Tang, Lina; Zhang, Jiabao; Tang, Bo; Li, Ziyi

2011-01-01

One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42-82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals.

Molecular Cloning and Gene Expression Analysis of Ercc6l in Sika Deer (Cervus nippon hortulorum)

PubMed Central

Zhang, Jiabao; Tang, Bo; Li, Ziyi

2011-01-01

Background One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. Methodology/Principal Findings In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42–82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. Conclusions/Significance We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals. PMID:21695076

Rapid Induction of Hypothalamic Iodothyronine Deiodinase Expression by Photoperiod and Melatonin in Juvenile Siberian Hamsters (Phodopus sungorus)

PubMed Central

Prendergast, Brian J.; Pyter, Leah M.; Kampf-Lassin, August; Patel, Priyesh N.

2013-01-01

Production of T3 in the mediobasal hypothalamus is critical for regulation of seasonal reproductive physiology. Type 2 iodothyronine deiodinase (DIO2) and DIO3 enzymes catalyze the prohormone T4 into biologically-active T3 and biologically-inactive rT3, respectively. In several seasonally-breeding vertebrates, DIO2 and DIO3 expression is implicated in photoperiod signal transduction in adulthood. These experiments tested the hypothesis that juvenile Siberian hamsters, which are highly responsive to photoperiod at weaning (postnatal day [PND]18), exhibit rapid and sustained changes in hypothalamic dio3 mRNA expression during photoperiod-induced and photoperiod-inhibited puberty. Hypothalamic dio2 and dio3 expression was measured via quantitative PCR in hamsters born and reared in a long-day photoperiod (15L:9D) and weaned on PND18 into short-day photoperiods (9L:15D). In SD males, hypothalamic dio3 mRNA was elevated 2.5-fold within 3 days (PND21) and continued to increase (>20-fold) through PND32; changes in dio3 mRNA preceded inhibition of gonadotropin (FSH) secretion and gonadal regression in SD. Females exhibited comparable dio3 responses to SD. In LD males, dio3 remained low and invariant from PND18–PND32. In contrast, dio2 mRNA rose conspicuously on PND21, independent of photoperiod, returning to basal levels thereafter. In LD, a single afternoon melatonin (MEL) injection on PND18 or PND20 was sufficient to increase hypothalamic dio3 mRNA, and dio3 increased in proportion to the number of successive days of MEL treatment. SD photoperiods and MEL exert rapid, sustained, and additive effects on hypothalamic dio3 mRNA, which may play a central role in inhibiting maturation of the peripubertal hypothalamo-pituitary-gonadal axis. PMID:23295738

Increased endothelin-1 vasoconstriction in mesenteric resistance arteries after superior mesenteric ischaemia-reperfusion

PubMed Central

Martínez-Revelles, S; Caracuel, L; Márquez-Martín, A; Dantas, AP; Oliver, E; D'Ocon, P; Vila, E

2012-01-01

BACKGROUND AND PURPOSE Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone. We aimed to evaluate the influence of superior mesenteric artery (SMA) ischaemia-reperfusion (I/R) on mesenteric resistance artery vasomotor function and the mechanism involved in the changes in vascular responses to ET-1. EXPERIMENTAL APPROACH SMA from male Sprague-Dawley rats was occluded (90 min) and following reperfusion (24 h), mesenteric resistance arteries were dissected. Vascular reactivity was studied using wire myography. Protein and mRNA expression, superoxide anion (O2•−) production and ET-1 plasma concentration were evaluated by immunofluorescence, real-time quantitative PCR, ethidium fluorescence and elisa, respectively. KEY RESULTS I/R increased ET-1 plasma concentration, ET-1-mediated vasoconstriction and ETB mRNA expression, and down-regulated ETA mRNA expression. Immunofluorescence confirmed mRNA results and revealed an increase in ETB receptors in the mesenteric resistance artery media layer after I/R. Therefore, the ETB receptor agonist sarafotoxin-6 induced a contraction that was inhibited by the ETB receptor antagonist BQ788 only in vessels, with and without endothelium, from I/R rats. Furthermore, BQ788 potentiated ET-1 vasoconstriction only in sham rats. Endothelium removal in rings from I/R rats unmasked the inhibition of ET-1 vasoconstriction by BQ788. Endothelium removal, Nω-nitro-L-arginine methyl ester and superoxide dismutase abolished the differences in ET-1 vasoconstriction between sham and I/R rats. We also found that I/R down-regulates endothelial NOS mRNA expression and concomitantly enhanced O2•− production by increasing NADPH oxidase 1 (NOX-1) and p47phox mRNA. CONCLUSIONS AND IMPLICATIONS Mesenteric I/R potentiated the ET-1-mediated vasoconstriction by a mechanism that involves up-regulation of muscular ETB receptors and decrease in NO bioavailability. PMID:21806604

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

Low-intensity infrared lasers alter actin gene expression in skin and muscle tissue

NASA Astrophysics Data System (ADS)

Fonseca, A. S.; Mencalha, A. L.; Campos, V. M. A.; Ferreira-Machado, S. C.; Peregrino, A. A. F.; Magalhães, L. A. G.; Geller, M.; Paoli, F.

2013-02-01

The biostimulative effect of low-intensity lasers is the basis for treatment of diseases in soft tissues. However, data about the influence of biostimulative lasers on gene expression are still scarce. The aim of this work was to evaluate the effects of low-intensity infrared lasers on the expression of actin mRNA in skin and muscle tissue. Skin and muscle tissue of Wistar rats was exposed to low-intensity infrared laser radiation at different fluences and frequencies. One and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis and evaluation of actin gene expression by quantitative polymerase chain reaction. The data obtained show that laser radiation alters the expression of actin mRNA differently in skin and muscle tissue of Wistar rats depending of the fluence, frequency and time after exposure. The results could be useful for laser dosimetry, as well as to justify the therapeutic protocols for treatment of diseases of skin and muscle tissues based on low-intensity infrared laser radiation.

Reference genes for measuring mRNA expression.

PubMed

Dundas, Jitesh; Ling, Maurice

2012-12-01

The aim of this review is to find answers to some of the questions surrounding reference genes and their reliability for quantitative experiments. Reference genes are assumed to be at a constant expression level, over a range of conditions such as temperature. These genes, such as GADPH and beta-actin, are used extensively for gene expression studies using techniques like quantitative PCR. There have been several studies carried out on identifying reference genes. However, a lot of evidence indicates issues to the general suitability of these genes. Recent studies had shown that different factors, including the environment and methods, play an important role in changing the expression levels of the reference genes. Thus, we conclude that there is no reference gene that can deemed suitable for all the experimental conditions. In addition, we believe that every experiment will require the scientific evaluation and selection of the best candidate gene for use as a reference gene to obtain reliable scientific results.

Regulation of androgen receptor transactivity and mTOR-S6 kinase pathway by Rheb in prostate cancer cell proliferation.

PubMed

Kobayashi, Takashi; Shimizu, Yosuke; Terada, Naoki; Yamasaki, Toshinari; Nakamura, Eijiro; Toda, Yoshinobu; Nishiyama, Hiroyuki; Kamoto, Toshiyuki; Ogawa, Osamu; Inoue, Takahiro

2010-06-01

Ras homolog-enriched in brain (Rheb), a small GTP-binding protein, is associated with prostate carcinogenesis through activating mammalian target of rapamycin (mTOR) signaling pathway. This study aimed to elucidate whether Rheb promotes proliferation of prostate cancer cells and can act as a potent therapeutic target in prostate cancer. Prostate cancer cell lines and human prostatic tissues were examined for the expression of Rheb. The effects of forced expression or knockdown of Rheb on cell proliferation were also examined. Semi-quantitative and quantitative RT-PCR were performed to evaluate mRNA expression. Western blotting was used to examine protein expression. Cell count and WST-1 assay were used to measure cell proliferation. Fluorescence-activated cell sorting was used to assess the cell cycle. Rheb mRNA and protein expression was higher in more aggressive, androgen-independent prostate cancer cell lines PC3, DU145, and C4-2, compared with the less aggressive LNCaP. Rheb expression was higher in cancer tissues than in benign prostatic epithelia. Forced expression of Rheb in LNCaP cells accelerated proliferation without enhancing androgen receptor transactivity. Attenuation of Rheb expression or treatment with the mTOR inhibitor rapamycin decreased proliferation of PC3 and DU145 cells, with a decrease in the activated form of p70S6 kinase, one of the main targets of mTOR. Rheb potentiates proliferation of prostate cancer cells and inhibition of Rheb or mTOR can lead to suppressed proliferation of aggressive prostate cancer cell lines in vitro. Rheb and the mTOR pathway are therefore probable targets for suppressing prostate cancer.

Up-regulated ephrinB3/EphB3 expression in intractable temporal lobe epilepsy patients and pilocarpine induced experimental epilepsy rat model.

PubMed

Huang, Hao; Li, Ruohan; Yuan, Jinxian; Zhou, Xin; Liu, Xi; Ou, Shu; Xu, Tao; Chen, Yangmei

2016-05-15

EphB family receptor tyrosine kinases, in cooperation with cell surface-bound ephrinB ligands, play a critical role in maintenance of dendritic spine morphogenesis, axons guidance, synaptogenesis, synaptic reorganization and plasticity in the central nervous system (CNS). However, the expression pattern of ephrinB/EphB in intractable temporal lobe epilepsy (TLE) and the underlying molecular mechanisms during epileptogenesis remain poorly understood. Here we investigated the expression pattern and cellular distribution of ephrinB/EphB in intractable TLE patients and lithium chloride-pilocarpine induced TLE rats using real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry, double-labeled immunofluorescence and Western blot analysis. Compared to control groups, ephrinB3 and EphB3 mRNA expression were significantly up-regulated in intractable TLE patients and TLE rats, while the mRNA expression trend of ephrinB1/2 and EphB1/2/4/6 in intractable TLE patients and TLE rats were inconsistent. Western blot analysis and semi-quantitative immunohistochemistry confirmed that ephrinB3 and EphB3 protein level were up-regulated in intractable TLE patients and TLE rats. At the same time, double-labeled immunofluorescence indicate that ephrinB3 was expressed mainly in the cytoplasm and protrusions of glia and neurons, while EphB3 was expressed mainly in the cytoplasm of neurons. Taken together, up-regulated expression of ephrinB3/EphB3 in intractable TLE patients and experimental TLE rats suggested that ephrinB3/EphB3 might be involved in the pathogenesis of TLE. Copyright © 2016 Elsevier B.V. All rights reserved.

The ribosome uses two active mechanisms to unwind messenger RNA during translation.

PubMed

Qu, Xiaohui; Wen, Jin-Der; Lancaster, Laura; Noller, Harry F; Bustamante, Carlos; Tinoco, Ignacio

2011-07-06

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs. ©2011 Macmillan Publishers Limited. All rights reserved

Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

PubMed Central

Rueda-Martínez, Carmen; Lamas, Oscar; Mataró, María José; Robledo-Carmona, Juan; Sánchez-Espín, Gemma; Jiménez-Navarro, Manuel; Such-Martínez, Miguel; Fernández, Borja

2014-01-01

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples. PMID:24841551

Anesthesia for Euthanasia Influences mRNA Expression in Healthy Mice and after Traumatic Brain Injury

PubMed Central

Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin

2014-01-01

Abstract Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10–11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real-time PCR data between studies or research groups and should therefore be considered for quantitative PCR data. PMID:24945082

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

PubMed

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

2017-01-01

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

Expression profile of osteoprotegerin, RANK and RANKL genes in the femoral head of patients with avascular necrosis.

PubMed

Samara, Stavroula; Dailiana, Zoe; Chassanidis, Christos; Koromila, Theodora; Papatheodorou, Loukia; Malizos, Konstantinos N; Kollia, Panagoula

2014-02-01

Femoral head avascular necrosis (AVN) is a recalcitrant disease of the hip that leads to joint destruction. Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B (RANK) and RANK ligand (RANKL) regulate the balance between osteoclasts-osteoblasts. The expression of these genes affects the maturation and function of osteoblasts-osteoclasts and bone remodeling. In this study, we investigated the molecular pathways leading to AVN by studying the expression profile of OPG, RANK and RANKL genes. Quantitative Real Time-PCR was performed for evaluation of OPG, RANK and RANKL expression. Analysis was based on parallel evaluation of mRNA and protein levels in normal/necrotic sites of 42 osteonecrotic femoral heads (FHs). OPG and RANKL protein levels were estimated by western blotting. The OPG mRNA levels were higher (insignificantly) in the necrotic than the normal site (p > 0.05). Although the expression of RANK and RANKL was significantly lower than OPG in both sites, RANK and RANKL mRNA levels were higher in the necrotic part than the normal (p < 0.05). Protein levels of OPG and RANKL showed no remarkable divergence. Our results indicate that differential expression mechanisms for OPG, RANK and RANKL that could play an important role in the progress of bone remodeling in the necrotic area, disturbing bone homeostasis. This finding may have an effect on the resulting bone destruction and the subsequent collapse of the hip joint. Copyright © 2013. Published by Elsevier Inc.

[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.

Expression and regulation of anti-mullerian hormone in an oviparous species, the hen.

PubMed

Johnson, P A; Kent, T R; Urick, M E; Giles, J R

2008-01-01

Anti-mullerian hormone (AMH) has a critical role in regression of the mullerian duct system during development in male mammalian and avian species and in regression of the right oviduct in female avian species. AMH in adult female birds has not been investigated. Chicken-specific cDNA primers were used to isolate Amh by RT-PCR. This probe was used in Northern blot analysis to identify a 2.8-kb band with expression in total ovarian RNA and in granulosa cell RNA. Quantitative real-time PCR was used to assess Amh expression in follicles of different maturity (1, 3, 5, and 6-12 mm and the largest F1 follicle; n = 4-6 of each size). There was an increased amount of Amh mRNA in the granulosa layer of the smaller follicles and a lower amount in the granulosa layer of the larger follicles (P < 0.01). There was no difference in granulosa Amh expression between the germinal disc and non-germinal disc region of 6- to 12-mm follicles, although expression differed with follicle size (P < 0.01). To examine hormone regulation of Amh, granulosa cells (from 6- to 8-mm follicles) were cultured with various concentrations of estradiol (E(2)) and progesterone (P(4)), and Amh mRNA was assessed. Neither E(2) nor P(4) influenced Amh mRNA accumulation. Granulosa cells were also cultured in the presence of oocyte-conditioned medium (OCM), which decreased Amh mRNA expression in a dose-related manner (P < 0.05); FSH receptor expression was not affected. Heat treatment of OCM abolished the effect, but growth differentiation factor 9 antiserum did not block the suppression. Immunohistochemistry confirmed that the granulosa layer was the predominant source of AMH in the small follicles of the hen and indicated that AMH was present early in follicle development, with expression in very small follicles (approximately 150 mum).

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

Activation of the PI3K-Akt pathway promotes neuroprotection of the δ-opioid receptor agonist against cerebral ischemia-reperfusion injury in rat models.

PubMed

Lv, Mei-Rong; Li, Bin; Wang, Ming-Guang; Meng, Fan-Guo; Yu, Jian-Jun; Guo, Feng; Li, Ye

2017-09-01

The central objective was to identify the role of the PI3K-Akt activation pathway on the neuroprotection of δ-opioid receptor agonist (DADLE) against cerebral ischemia-reperfusion (I/R) injury in a rat model. Fifty-five male Sprague-Dawley (SD) rats were included to establish a middle cerebral artery occlusion (MCAO) model which were then divided into the sham, MCAO, LY294002 (MCAO+DADLE+LY294002 [inhibitor of PI3K-Akt pathway]), DADLE (MCAO+DADLE) and DMSO (MCAO+DADLE+DMSO [dimethyl sulphoxide]) groups. The cerebral infarction (CI) volume and nerve cell apoptosis was determined using TTC and TUNEL staining. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry staining were applied for the expressions of Bad, Bax, Bcl-2 and cleaved caspase-3. The MCAO group showed higher CI volume, nerve cell apoptosis and cleaved caspase-3 expressions than the DADLE and DMSO groups, which were also higher in the LY294002 group than the DADLE group. Compared with the MCAO group, the mRNA and protein expressions of PI3K and Bcl-2, and the protein expressions of p-Akt and p-Bad were elevated, while the mRNA and protein expressions of Bax were decreased in the DADLE and DMSO groups. Decreased mRNA and protein expressions of PI3K and Bcl-2, reduced protein expressions of p-Akt and p-Bad and elevated mRNA and protein expressions of Bax exhibited in the LY294002 group than the DADLE group. These results indicate that activation of PI3K-Akt pathway promotes the neuroprotection of DADLE against cerebral I/R injury in a rat model by decreasing nerve cells apoptosis. Copyright © 2017. Published by Elsevier Masson SAS.

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

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

PubMed

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

2013-03-01

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

Taurine restores the exploratory behavior following alcohol withdrawal and decreases BDNF mRNA expression in the frontal cortex of chronic alcohol-treated rats.

PubMed

Hansen, Alana Witt; Almeida, Felipe Borges; Bandiera, Solange; Pulcinelli, Rianne Remus; Fragoso, Ana Luiza Rodrigues; Schneider, Ricardo; Barros, Helena Maria Tannhauser; Gomez, Rosane

2017-10-01

Alcohol use disorder is an alarming health problem, and the withdrawal symptoms increase the risk of relapse. We have hypothesized that taurine, a multitarget substance acting as a gamma-aminobutyric acid A receptor (GABA A R) positive modulator and a partial inhibitor of N-methyl-d-aspartate (NMDA) glutamate receptors, may reduce the withdrawal symptoms or modify behaviors when combined with alcohol. Therefore, we investigated the effects of taurine on behavior in the open field test (OFT), the GABA A R α 2 subunit and BDNF mRNA expression in the frontal cortex of rats after chronic alcohol treatment or upon withdrawal. Rats received alcohol 2g/kg (alcohol and withdrawal groups) or water (control group) twice daily by oral gavage for 28days. On day 29, the withdrawal rats received water instead of alcohol, and all groups were reallocated to receive 100mg/kg taurine or vehicle intraperitoneally, once a day for 5days. On day 33, the rats were exposed to OFT; 18h later, they were euthanized, and the frontal cortex was dissected for GABA A R α 2 subunit detection and BDNF mRNA expression determination by real-time quantitative PCR. Taurine administration restored rearing behavior to the control levels in the withdrawal rats. Taurine also showed anxiolytic-like effects in control rats and did not change the behaviors in the chronic alcohol group. Chronic alcohol treatment or withdrawal did not change the GABA A R α 2 subunit or BDNF mRNA expression in the frontal cortex, but taurine decreased the α 2 subunit level in control rats and to the BDNF levels in the alcohol rat group. We conclude that taurine restored exploratory behavior after alcohol withdrawal but that this effect was not related to the GABA A R α 2 subunit or BDNF mRNA expression in the frontal cortex of the rats. Copyright © 2017 Elsevier Inc. All rights reserved.

Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

PubMed

Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing

2013-03-15

Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer

PubMed Central

Barrón, Eira Valeria; Roman-Bassaure, Edgar; Sánchez-Sandoval, Ana Laura; Espinosa, Ana María; Guardado-Estrada, Mariano; Medina, Ingrid; Juárez, Eligia; Alfaro, Ana; Bermúdez, Miriam; Zamora, Rubén; García-Ruiz, Carlos; Gomora, Juan Carlos; Kofman, Susana; Pérez-Armendariz, E. Martha; Berumen, Jaime

2015-01-01

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10−6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5–10, p = 3.3 x 10−6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC. PMID:26372210

CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.

PubMed

Barrón, Eira Valeria; Roman-Bassaure, Edgar; Sánchez-Sandoval, Ana Laura; Espinosa, Ana María; Guardado-Estrada, Mariano; Medina, Ingrid; Juárez, Eligia; Alfaro, Ana; Bermúdez, Miriam; Zamora, Rubén; García-Ruiz, Carlos; Gomora, Juan Carlos; Kofman, Susana; Pérez-Armendariz, E Martha; Berumen, Jaime

2015-01-01

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.

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

Evaluation of tyrosine-kinase receptor c-KIT (c-KIT) mutations, mRNA and protein expression in canine leukemia: might c-KIT represent a therapeutic target?

PubMed

Giantin, M; Aresu, L; Aricò, A; Gelain, M E; Riondato, F; Martini, V; Comazzi, S; Dacasto, M

2013-04-15

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8-11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials. Copyright © 2013 Elsevier B.V. All rights reserved.

Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle1

PubMed Central

Rosewell, Katherine L.; Li, Feixue; Puttabyatappa, Muraly; Akin, James W.; Brännström, Mats; Curry, Thomas E.

2013-01-01

ABSTRACT Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability. PMID:24048576

Ovarian expression, localization, and function of tissue inhibitor of metalloproteinase 3 (TIMP3) during the periovulatory period of the human menstrual cycle.

PubMed

Rosewell, Katherine L; Li, Feixue; Puttabyatappa, Muraly; Akin, James W; Brännström, Mats; Curry, Thomas E

2013-11-01

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

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

Exacerbated Glial Response in the Aged Mouse Hippocampus Following Controlled Cortical Impact Injury

PubMed Central

Sandhir, Rajat; Onyszchuk, Gregory; Berman, Nancy E. J.

2008-01-01

Old age is associated with enhanced susceptibility to and poor recovery from brain injury. An exacerbated microglial and astrocyte response to brain injury might be involved in poor outcomes observed in the elderly. The present study was therefore designed to quantitate the expression of markers of microglia and astrocyte activation using real-time RT-PCR, immunoblot and immunohistochemical analysis in aging brain in response to brain injury. We examined the hippocampus, a region that undergoes secondary neuron death, in aged (21–24 month) and adult (5–6 month) mice following controlled cortical impact (CCI) injury to the sensorimotor cortex. Basal mRNA expression of CD11b and Iba1, markers of activated microglia, was higher in aged hippocampus as compared to the adult. The mRNA expression of microglial markers increased and reached maximum 3 days post injury in both adult and aged mice, but was higher in the aged mice at all time points studied, and in the aged mice the return to baseline levels was delayed. Basal mRNA expression of GFAP and S100B, markers of activated astrocytes, was higher in aged mice. Both markers increased and reached maximum 7 days post injury. The mRNA expression of astrocyte markers returned to near basal levels rapidly after injury in the adult mice, whereas again in the aged mice return to baseline was delayed. Immunochemical analysis using Iba1 and GFAP antibodies indicate accentuated glial responses in the aged hippocampus after injury. The pronounced and prolonged activation of microglia and astrocytes in hippocampus may contribute to worse cognitive outcomes in the elderly following TBI. PMID:18692046

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

PubMed

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

2014-01-01

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

Sequence and expression analyses of porcine ISG15 and ISG43 genes.

PubMed

Huang, Jiangnan; Zhao, Shuhong; Zhu, Mengjin; Wu, Zhenfang; Yu, Mei

2009-08-01

The coding sequences of porcine interferon-stimulated gene 15 (ISG15) and the interferon-stimulated gene (ISG43) were cloned from swine spleen mRNA. The amino acid sequences deduced from porcine ISG15 and ISG43 genes coding sequence shared 24-75% and 29-83% similarity with ISG15s and ISG43s from other vertebrates, respectively. Structural analyses revealed that porcine ISG15 comprises two ubiquitin homologues motifs (UBQ) domain and a conserved C-terminal LRLRGG conjugating motif. Porcine ISG43 contains an ubiquitin-processing proteases-like domain. Phylogenetic analyses showed that porcine ISG15 and ISG43 were mostly related to rat ISG15 and cattle ISG43, respectively. Using quantitative real-time PCR assay, significant increased expression levels of porcine ISG15 and ISG43 genes were detected in porcine kidney endothelial cells (PK15) cells treated with poly I:C. We also observed the enhanced mRNA expression of three members of dsRNA pattern-recognition receptors (PRR), TLR3, DDX58 and IFIH1, which have been reported to act as critical receptors in inducing the mRNA expression of ISG15 and ISG43 genes. However, we did not detect any induced mRNA expression of IFNalpha and IFNbeta, suggesting that transcriptional activations of ISG15 and ISG43 were mediated through IFN-independent signaling pathway in the poly I:C treated PK15 cells. Association analyses in a Landrace pig population revealed that ISG15 c.347T>C (BstUI) polymorphism and the ISG43 c.953T>G (BccI) polymorphism were significantly associated with hematological parameters and immune-related traits.

Stage-dependent and temperature-controlled expression of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis activating neuropeptide in the silkworm, Bombyx mori.

PubMed

Xu, W H; Sato, Y; Ikeda, M; Yamashita, O

1995-02-24

Embryonic diapause and sex pheromone biosynthesis in the silkworm, Bombyx mori, are, respectively, induced by diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN), which are produced in the subesophageal ganglion from a common polyprotein precursor (DH-PBAN precursor) encoded by a single gene (DH-PBAN gene). Using DH-PBAN cDNA as a probe, we quantitatively measured DH-PBAN mRNA content throughout embryonic and postembryonic development and observed the effects of incubation temperature, which is a key factor for determination of diapause, on DH-PBAN gene expression. The silkworm, which is programmed to lay diapause eggs by being incubated at 25 degrees C, showed peaks of DH-PBAN mRNA content at five different stages throughout the life cycle: at the late embryonic stage, at the middle of the fourth and the fifth larval instars, and at early and late stages of pupal-adult development. In the non-diapause type silkworms programmed by a 15 degrees C incubation, only the last peak of DH-PBAN mRNA in pupal-adult development was found, and the other peaks were absent. Furthermore, interruption of the incubation period at 25 degrees C by incubation at 15 degrees C decreased both DH-PBAN mRNA content in mature embryos and in subesophageal ganglia of day 3 pupae and the incidence of diapause eggs. Thus, there were two types of regulatory mechanisms for DH-PBAN gene expression. One is a temperature-controlled expression that is responsible for diapause induction, and the other is a temperature-independent, stage-dependent expression related to pheromone production.

Animal serum-free expansion and differentiation of human mesenchymal stromal cells.

PubMed

Felka, Tino; Schäfer, Richard; De Zwart, Peter; Aicher, Wilhelm K

2010-04-01

Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products. MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation. Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.

[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.

Metabolic effects of dietary sugar beet pulp or wheat bran in growing female pigs.

PubMed

Weber, T E; Kerr, B J

2012-02-01

An experiment was conducted to determine the effects of feeding a moderate level of 2 different fiber sources on energy metabolites; mitochondrial biogenesis in the intestine, liver, and muscle; and the expression of some genes that regulate energy metabolism in intestine, liver, muscle, and adipose tissue. Female pigs (n = 36; BW = 15.0 ± 0.7 kg) were fed diets containing no added fiber, 12.5% sugar beet pulp (SBP), or 12.5% wheat bran (WB) for 24 d. Blood samples were collected on d 7 and 24 for cholesterol, glucose, NEFA, and triglyceride analyses. At completion of the experiment, ileum, colon, subcutaneous adipose, and LM samples were obtained from a subset (n = 6) of pigs fed each diet for analysis of tissue mitochondrial DNA (mtDNA) content and mRNA abundance by quantitative real-time reverse-transcription PCR. Glycogen and triglyceride content of liver and LM were determined, and colon content VFA was also determined. The addition of SBP or WB to the diet had no effect (P > 0.55) on ADG, ADFI, or G:F. Serum NEFA and triglycerides were increased (P < 0.05) in pigs fed SBP compared with pigs fed the control diet or WB on d 7, and NEFA remained increased (P < 0.05) on d 24 in pigs fed SBP. Dietary fiber had no effect (P > 0.24) on glycogen and triglyceride content of liver or LM, but colonic acetate concentrations were increased (P < 0.05) in pigs fed either SBP or WB. Pigs fed WB had an increased (P < 0.05) mtDNA content in ileum tissue and increased (P < 0.05) citrate synthase mRNA in colon tissue. In the liver, feeding either SBP or WB led to a decrease (P < 0.05) in mtDNA content, whereas feeding WB decreased (P < 0.05) mtDNA abundance in the LM, and feeding either SBP or WB decreased (P < 0.05) expression of citrate synthase mRNA. Quantitative reverse-transcription PCR revealed that feeding WB increased (P < 0.05) proliferating cell nuclear antigen mRNA abundance in the ileum and colon. Feeding WB increased (P < 0.05) mRNA abundance of a regulator of mitochondrial biogenesis, PPAR coactivator 1 α, in ileum tissue, and increased (P < 0.05) mRNA abundance of another mediator of mitochondrial biogensis, sirtuin 1, in colon tissue. Colonic mRNA expression of fasting-induced adipose factor was increased (P < 0.05) in pigs fed either SBP or WB, and adipose triglyceride lipase mRNA abundance was increased (P < 0.05) in adipose tissue of pigs fed SBP. These data indicate that increasing dietary fiber can increase the capacity of the intestine for oxidative metabolism and induce a repartitioning of energy metabolites depending on fiber source.

Imaging mRNA In Vivo, from Birth to Death.

PubMed

Tutucci, Evelina; Livingston, Nathan M; Singer, Robert H; Wu, Bin

2018-05-20

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

Activity-induced and developmental downregulation of the Nogo receptor.

PubMed

Josephson, Anna; Trifunovski, Alexandra; Schéele, Camilla; Widenfalk, Johan; Wahlestedt, Claes; Brené, Stefan; Olson, Lars; Spenger, Christian

2003-03-01

The three axon growth inhibitory proteins, myelin associated glycoprotein, oligodendrocyte-myelin glycoprotein and Nogo-A, can all bind to the Nogo-66 receptor (NgR). This receptor is expressed by neurons with high amounts in regions of high plasticity where Nogo expression is also high. We hypothesized that simultaneous presence of high levels of Nogo and its receptor in neurons confers a locked state to hippocampal and cortical microcircuitry and that one or both of these proteins must be effectively and temporarily downregulated to permit plastic structural changes underlying formation of long-term memory. Hence, we subjected rats to kainic acid treatment and exposed rats to running wheels and measured NgR mRNA levels by quantitative in situ hybridization at different time points. We also studied spinal cord injuries and quantified NgR mRNA levels in spinal cord and ganglia during a critical postnatal period using real-time PCR. Strikingly, kainic acid led to a strong transient downregulation of NgR mRNA levels in gyrus dentatus, hippocampus, and neocortex during a time when BDNF mRNA was upregulated instead. Animals exposed to running wheels for 3 and 7, but not 1 or 21, days showed a significant downregulation of NgR mRNA in cortex, hippocampus and the dentate gyrus. NgR mRNA levels decreased from high to low expression in spinal cord and ganglia during the first week of life. No robust regulation of NgR was observed in the spinal cord following spinal cord injury. Together, our data show that NgR levels in developing and adult neurons are regulated in vivo under different conditions. Strong, rapid and transient downregulation of NgR mRNA in response to kainic acid and after wheel running in cortex and hippocampus suggests a role for NgR and Nogo-A in plasticity, learning and memory.

[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.

The vitamin D system is deregulated in pancreatic diseases

PubMed Central

Hummel, Doris; Aggarwal, Abhishek; Borka, Katalin; Bajna, Erika; Kállay, Enikö; Horváth, Henrik Csaba

2014-01-01

The vitamin D system is deregulated during development and progression of several cancer types. Data on the expression of the vitamin D system in the diseased pancreas are missing. The aim of this study was to investigate the expression of the vitamin D receptor (VDR), 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1), and the calcium-sensing receptor (CaSR), a vitamin D target gene, in the different regions of the pancreas in patients with chronic pancreatitis (n = 6) and pancreatic ductal adenocarcinomas (PDAC) (n = 17). We analyzed the expression of these genes at mRNA and protein level with quantitative real-time RT-PCR and immunostaining. mRNA expression of CYP24A1 and VDR was significantly increased in tumors compared with the adjacent non-tumorous tissue (p < 0.01), while CaSR mRNA expression decreased. Both the VDR and the CaSR protein were highly expressed in the endocrine compared with the exocrine pancreas. In CP the CYP24A1 expression was highest in the endocrine pancreas, while in PDACs in the transformed ducts. In the PDAC patients CYP24A1 expression in the islets was significantly lower than in CP patients. Our data suggest that during ductal adenocarcinoma development the vitamin D system in the pancreas becomes deregulated on two levels: in the islets CYP24A1 expression decreases weakening the negative feedback regulation of the vitamin D-dependent insulin synthesis/secretion. In the transformed ducts CYP24A1 expression increases, impairing the antiproliferative effect of vitamin D in these cells. PMID:25090635

Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing.

PubMed

Etich, Julia; Bergmeier, Vera; Pitzler, Lena; Brachvogel, Bent

2017-03-01

Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing. Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs. Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome. RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.

Proteoglycans in Leiomyoma and Normal Myometrium

PubMed Central

Barker, Nichole M.; Carrino, David A.; Caplan, Arnold I.; Hurd, William W.; Liu, James H.; Tan, Huiqing; Mesiano, Sam

2015-01-01

Uterine leiomyoma are a common benign pelvic tumors composed of modified smooth muscle cells and a large amount of extracellular matrix (ECM). The proteoglycan composition of the leiomyoma ECM is thought to affect pathophysiology of the disease. To test this hypothesis, we examined the abundance (by immunoblotting) and expression (by quantitative real-time polymerase chain reaction) of the proteoglycans biglycan, decorin, and versican in leiomyoma and normal myometrium and determined whether expression is affected by steroid hormones and menstrual phase. Leiomyoma and normal myometrium were collected from women (n = 17) undergoing hysterectomy or myomectomy. In vitro studies were performed on immortalized leiomyoma (UtLM) and normal myometrial (hTERT-HM) cells with and without exposure to estradiol and progesterone. In leiomyoma tissue, abundance of decorin messenger RNA (mRNA) and protein were 2.6-fold and 1.4-fold lower, respectively, compared with normal myometrium. Abundance of versican mRNA was not different between matched samples, whereas versican protein was increased 1.8-fold in leiomyoma compared with myometrium. Decorin mRNA was 2.4-fold lower in secretory phase leiomyoma compared with proliferative phase tissue. In UtLM cells, progesterone decreased the abundance of decorin mRNA by 1.3-fold. Lower decorin expression in leiomyoma compared with myometrium may contribute to disease growth and progression. As decorin inhibits the activity of specific growth factors, its reduced level in the leiomyoma cell microenvironment may promote cell proliferation and ECM deposition. Our data suggest that decorin expression in leiomyoma is inhibited by progesterone, which may be a mechanism by which the ovarian steroids affect leiomyoma growth and disease progression. PMID:26423601

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

Prognostic significance of TRIM24/TIF-1α gene expression in breast cancer.

PubMed

Chambon, Monique; Orsetti, Béatrice; Berthe, Marie-Laurence; Bascoul-Mollevi, Caroline; Rodriguez, Carmen; Duong, Vanessa; Gleizes, Michel; Thénot, Sandrine; Bibeau, Frédéric; Theillet, Charles; Cavaillès, Vincent

2011-04-01

In this study, we have analyzed the expression of TRIM24/TIF-1α, a negative regulator of various transcription factors (including nuclear receptors and p53) at the genomic, mRNA, and protein levels in human breast tumors. In breast cancer biopsy specimens, TRIM24/TIF-1α mRNA levels (assessed by Real-Time Quantitative PCR or microarray expression profiling) were increased as compared to normal breast tissues. At the genomic level, array comparative genomic hybridization analysis showed that the TRIM24/TIF-1α locus (7q34) exhibited both gains and losses that correlated with mRNA levels. By re-analyzing a series of 238 tumors, high levels of TRIM24/TIF-1α mRNA significantly correlated with various markers of poor prognosis (such as the molecular subtype) and were associated with worse overall survival. By using a rabbit polyclonal antibody for immunochemistry, the TRIM24/TIF-1α protein was detected in nuclei of normal luminal epithelial breast cells, but not in myoepithelial cells. Tissue microarray analysis confirmed that its expression was increased in epithelial cells from normal to breast infiltrating duct carcinoma and correlated with worse overall survival. Altogether, this work is the first study that shows that overexpression of the TRIM24/TIF-1α gene in breast cancer is associated with poor prognosis and worse survival, and it suggests that this transcription coregulator may play a role in mammary carcinogenesis and represent a novel prognostic marker. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2016-06-01

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

Involvement of TLR2 and TLR9 in the anti-inflammatory effects of chlorogenic acid in HSV-1-infected microglia.

PubMed

Guo, Yuan-Jin; Luo, Tao; Wu, Fei; Mei, Yuan-Wu; Peng, Jun; Liu, Huan; Li, Hua-Rong; Zhang, Shu-Ling; Dong, Ji-Hua; Fang, Yuan; Zhao, Lei

2015-04-15

There is no effective medication to date for herpes simplex virus encephalitis (HSE). In this study, we investigated the anti-inflammatory effect of chlorogenic acid (CGA) on herpes simplex virus (HSV)-1-induced responses in BV2 microglia. The cellular model was established with BV2 cells stimulated by HSV-1 and then treated with CGA at different concentrations. Cell viability was assayed by the MTT assay. The mRNA expression of Toll-like receptor (TLR)-2, TLR9 and myeloid differentiation factor88 (Myd88) was assayed by real-time quantitative PCR, and the protein expression was assayed by flow cytometry or Western blotting. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were measured by ELISA as well as real-time quantitative PCR. Nuclear NF-κB p65 protein was assayed by Western blotting. The cell survival rate was significantly improved after CGA treatment, and CGA prevented increases in TLR2, TLR9 and Myd88 following HSV-1 challenge in BV2 cells both at the mRNA and protein levels. Moreover, CGA could attenuate HSV-induced TNF-α and IL-6 release into the supernatant. The mRNA levels of TNF-α and IL-6 were also significantly inhibited by CGA. The expression of NF-κB p65 increased significantly in the nucleus in HSV-1-stimulated microglia but could be reduced by CGA. CGA inhibits the inflammatory reaction in HSE via the suppression of TLR2/TLR9-Myd88 signaling pathways. CGA may serve as an anti-inflammatory agent and provide a new strategy for treating HSE. Copyright © 2015 Elsevier Inc. All rights reserved.

Associations between CD36 gene polymorphisms and susceptibility to coronary artery heart disease

PubMed Central

Zhang, Y.; Ling, Z.Y.; Deng, S.B.; Du, H.A.; Yin, Y.H.; Yuan, J.; She, Q.; Chen, Y.Q.

2014-01-01

Associations between polymorphisms of the CD36 gene and susceptibility to coronary artery heart disease (CHD) are not clear. We assessed allele frequencies and genotype distributions of CD36 gene polymorphisms in 112 CHD patients and 129 control patients using semi-quantitative polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Additionally, we detected CD36 mRNA expression by real-time quantitative PCR, and we quantified plasma levels of oxidized low-density lipoprotein (ox-LDL) using an enzyme-linked immunosorbent assay (ELISA). There were no significant differences between the two groups (P>0.05) in allele frequencies of rs1761667 or in genotype distribution and allele frequencies of rs3173798. The genotype distribution of rs1761667 significantly differed between CHD patients and controls (P=0.034), with a significantly higher frequency of the AG genotype in the CHD group compared to the control group (P=0.011). The plasma levels of ox-LDL in patients with the AG genotype were remarkably higher than those with the GG and AA genotypes (P=0.010). In a randomized sample taken from patients in the two groups, the CD36 mRNA expression of the CHD patients was higher than that of the controls. In CHD patients, the CD36 mRNA expression in AG genotype patients was remarkably higher than in those with an AA genotype (P=0.005). After adjusted logistic regression analysis, the AG genotype of rs1761667 was associated with an increased risk of CHD (OR=2.337, 95% CI=1.336-4.087, P=0.003). In conclusion, the rs1761667 polymorphism may be closely associated with developing CHD in the Chongqing Han population of China, and an AG genotype may be a genetic susceptibility factor for CHD. PMID:25118627

Quantification of multiple gene expression in individual cells.

PubMed

Peixoto, António; Monteiro, Marta; Rocha, Benedita; Veiga-Fernandes, Henrique

2004-10-01

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.

Modulation of intestinal gene expression by dietary zinc status: Effectiveness of cDNA arrays for expression profiling of a single nutrient deficiency

PubMed Central

Blanchard, Raymond K.; Moore, J. Bernadette; Green, Calvert L.; Cousins, Robert J.

2001-01-01

Mammalian nutritional status affects the homeostatic balance of multiple physiological processes and their associated gene expression. Although DNA array analysis can monitor large numbers of genes, there are no reports of expression profiling of a micronutrient deficiency in an intact animal system. In this report, we have tested the feasibility of using cDNA arrays to compare the global changes in expression of genes of known function that occur in the early stages of rodent zinc deficiency. The gene-modulating effects of this deficiency were demonstrated by real-time quantitative PCR measurements of altered mRNA levels for metallothionein 1, zinc transporter 2, and uroguanylin, all of which have been previously documented as zinc-regulated genes. As a result of the low level of inherent noise within this model system and application of a recently reported statistical tool for statistical analysis of microarrays [Tusher, V.G., Tibshirani, R. & Chu, G. (2001) Proc. Natl. Acad. Sci. USA 98, 5116–5121], we demonstrate the ability to reproducibly identify the modest changes in mRNA abundance produced by this single micronutrient deficiency. Among the genes identified by this array profile are intestinal genes that influence signaling pathways, growth, transcription, redox, and energy utilization. Additionally, the influence of dietary zinc supply on the expression of some of these genes was confirmed by real-time quantitative PCR. Overall, these data support the effectiveness of cDNA array expression profiling to investigate the pleiotropic effects of specific nutrients and may provide an approach to establishing markers for assessment of nutritional status. PMID:11717422

[Research progress in neuropsychopharmacology updated for the post-genomic era].

PubMed

Nakanishi, Toru

2009-11-01

Neuropsychopharmacological research in the post genomic (genomic sequence) era has been developing rapidly through the use of novel techniques including DNA chips. We have applied these techniques to investigate the anti-tumor effect of NSAIDs, isolate novel genes specifically expressed in rheumatoid arthritis, and analyze gene expression profiles in mesenchymal stem cells. Recently, we have developed a novel system of quantitative PCR for detection of BDNF mRNA isoforms. By using this system, we identified the exon-specific mode of expression in acute and chronic pain. In addition, we have made gene expression profiles of KO mice of beta2 subunits in acetylcholine receptors.

Effects of nitrite stress on mRNA expression of antioxidant enzymes, immune-related genes and apoptosis-related proteins in Marsupenaeus japonicus.

PubMed

Zheng, Jinbin; Mao, Yong; Su, Yongquan; Wang, Jun

2016-11-01

Nitrite accumulation in aquaculture systems is a potential risk factor that may trigger stress responses in aquatic organisms. However, the mechanisms regulating the responses of shrimp to nitrite stress remain unclear. In this study, full-length cDNA sequences of two apoptosis-related genes, caspase-3 and defender against apoptotic death (DAD-1), were cloned from Marsupenaeus japonicus for the first time, and their expression levels and tissue distribution were analyzed by quantitative real-time PCR (qRT-PCR). The full lengths of Mjcaspase-3 and MjDAD-1 were 1203 bp and 640 bp respectively, with deduced amino acid (AA) sequences of 321 and 114 AA. Mjcaspase-3 was predominantly expressed in haemocytes and weakly expressed in the seven other tissues tested. MjDAD-1 was mainly expressed in the defense and digestive tissues, especially in the hepatopancreas and hemocytes. To explore the influence of nitrite stress on the genetic response of antioxidant enzymes, immune-related genes and apoptosis-related proteins, the mRNA expression profiles of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 in response to nitrite stress were analyzed by qRT-PCR. The mRNA levels of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 show both time- and dose-dependent changes in response to nitrite stress. The mRNA expression levels of MjCAT and MjSOD peaked at 6 h for all nitrite concentrations tested (p < 0.05) and the up-regulated of MjCAT and MjSOD exhibited a positive correlation with the nitrite concentration. The mRNA expression levels of Mj-ilys and Mj-sty gradually decreased during the experiment period. Mjcaspase-3 mRNA level reached a maximum at 6 h (p < 0.05), and MjDAD-1 reached its peak at 12 h and 48 h in 10 mg/L and 20 mg/L nitrite, respectively. In addition, CAT and SOD activity showed changes in response to nitrite stress that mirrored the induced expression of MjCAT and MjMnSOD, and prolonged nitrite exposure reduced the activity of CAT. This study provided basic data for further elucidating the responses of shrimp to nitrite stress at the molecular level. Copyright © 2016. Published by Elsevier Ltd.

Effects and mechanism of integrin-β1 gene expression inhibited by shRNA in invasion of pancreatic carcinoma PANC-1 cells.

PubMed

Yu, Feng; Li, Hua; Bu, Xuefeng; Zhang, Yongjun

2012-01-01

To investigate the effects of integrin-β1 gene expression inhibited by shRNA on invasion of pancreatic carcinoma PANC-1 cells in vitro. The eukaryotic expression plasmid of short hairpin RNA (shRNA) targeting integrin-β1 gene (integrin-β1-shRNA) was constructed and transfected into PANC-1 cells. The expressions of integrin-β1 mRNA and protein were detected by real-time quantitative polymerase chain reaction (PCR) and western blot assay, respectively. The invasive ability of PANC-1 cells was observed with a transwell cell culture chamber and the expressions of MMP-2 and MMP-9 were assayed. Compared to the untransfected group, recombinant expression plasmid integrin-β1-shRNA resulted in reduction of integrin-β1 mRNA and protein by 78.58%±7.24% and 92.88%±3.18%, respectively and the average number of invading PANC-1 cells were decreased from 52±5 to 21±4 (p<0.01) and the expressions of MMP-2 and MMP-9 were inhibited. Recombinant expression plasmid integrin-β1- shRNA can inhibit the expression of integrin-β1 gene and suppress the invasion of PANC-1 cells in vitro significantly.

Effect of cigarette smoke extract and nicotine on the expression of thrombomodulin and endothelial protein C receptor in cultured human umbilical vein endothelial cells

PubMed Central

Wei, Yujie; Lai, Bin; Liu, Huiliang; Li, Yi; Zhen, Wang; Fu, Ling

2018-01-01

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5–5%) and nicotine (10-3-10-9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription-quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6-h exposure, TM protein and mRNA expression levels decreased in a dose-dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking-associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences. PMID:29257196

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

PubMed Central

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

2014-01-01

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

Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

PubMed Central

Travis, G H; Sutcliffe, J G

1988-01-01

To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA. Images PMID:2894033

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

PubMed Central

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

2006-01-01

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

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

PubMed

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

2006-02-21

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

Expression of TIGIT and FCRL3 is Altered in T Cells from Patients with Distinct Patterns of Chronic Autoimmune Thyroiditis.

PubMed

Štefanić, Mario; Tokić, Stana; Suver-Stević, Mirjana; Glavaš-Obrovac, Ljubica

2018-06-11

Co-inhibitory receptors (IR), such as TIGIT and FCRL3, provide a checkpoint against highly destructive immune responses. Co-expression of TIGIT and FCRL3, in particular, has been linked to the HELIOS + subset of regulatory CD4 + FOXP3 + T-cells. Of these, CD4 + FOXP3-exon(E)2 + cells have higher expression of IR and exhibit strongest suppressive properties. Nevertheless, how the expression of TIGIT, FCRL3, HELIOS, and FOXP3E2 is regulated in chronic autoimmune thyroiditis (AT), is not known. Thirty patients with AT [encompassing spontaneously euthyroid (euAT), hypothyroid-untreated and L-thyroxine-treated cases)] and 10 healthy controls (HC) were recruited. FCRL3, TIGIT, HELIOS and FOXP3E2 mRNA expression levels in peripheral blood (PB) T cells were measured via quantitative real-time PCR and compared to clinicopathological factors. The TIGIT and FCRL3 expression levels from T cells of AT cases were inversely related to the thyroid volume, and were significantly increased in hypothyroid patients (on+off L-thyroxine), but not euAT cases. The FCRL3 expression in PB T cells positively correlated with thyroid-peroxidase autoantibody levels; by contrast, T cells from aged AT patients and combined samples (AT+HC) accumulated more TIGIT mRNA. The patients with higher TIGIT mRNA levels had a greater prevalence of hypothyroidism, showing higher peak thyrotropin levels at diagnosis or at follow-up. Multiple IR, namely FCRL3 and TIGIT, but not the transcription factors HELIOS and FOXP3E2, showed increased mRNA levels in PB T cells from end-stage, long-standing and/or more aggressive AT, in proportion to disease severity. A relation with major clinical subphenotypes was observed, thereby identifying IR as potentially important players in AT. © Georg Thieme Verlag KG Stuttgart · New York.

[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.

Anatomical and histological profiling of orphan G-protein-coupled receptor expression in gastrointestinal tract of C57BL/6J mice.

PubMed

Ito, Junko; Ito, Masahiko; Nambu, Hirohide; Fujikawa, Toru; Tanaka, Kenichi; Iwaasa, Hisashi; Tokita, Shigeru

2009-11-01

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors and regulate a variety of physiological and disease processes. Although the roles of many non-odorant GPCRs have been identified in vivo, several GPCRs remain orphans (oGPCRs). The gastrointestinal (GI) tract is the largest endocrine organ and is a promising target for drug discovery. Given their close link to physiological function, the anatomical and histological expression profiles of benchmark GI-related GPCRs, such as the cholecystokinin-1 receptor and GPR120, and 106 oGPCRs were investigated in the mucosal and muscle-myenteric nerve layers in the GI tract of C57BL/6J mice by quantitative real-time polymerase chain reaction. The mRNA expression patterns of these benchmark molecules were consistent with previous in situ hybridization and immunohistochemical studies, validating the experimental protocols in this study. Of 96 oGPCRs with significant mRNA expression in the GI tract, several oGPCRs showed unique expression patterns. GPR85, GPR37, GPR37L1, brain-specific angiogenesis inhibitor (BAI) 1, BAI2, BAI3, and GPRC5B mRNAs were preferentially expressed in the muscle-myenteric nerve layer, similar to GPCRs that are expressed in both the central and enteric nerve systems and that play multiple regulatory roles throughout the gut-brain axis. In contrast, GPR112, trace amine-associated receptor (TAAR) 1, TAAR2, and GPRC5A mRNAs were preferentially expressed in the mucosal layer, suggesting their potential roles in the regulation of secretion, immunity, and epithelial homeostasis. These anatomical and histological mRNA expression profiles of oGPCRs provide useful clues about the physiological roles of oGPCRs in the GI tract.

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene

PubMed Central

Kapanadze, Bagrat; Morris, Erin; Smith, Edwin; Trojanowska, Maria

2010-01-01

Abstract Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-βRI indicative of altered TGF-β signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis. PMID:19432820

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene.

PubMed

Kapanadze, Bagrat; Morris, Erin; Smith, Edwin; Trojanowska, Maria

2010-05-01

Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-betaRI indicative of altered TGF-beta signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

A quantitative framework for the forward design of synthetic miRNA circuits.

PubMed

Bloom, Ryan J; Winkler, Sally M; Smolke, Christina D

2014-11-01

Synthetic genetic circuits incorporating regulatory components based on RNA interference (RNAi) have been used in a variety of systems. A comprehensive understanding of the parameters that determine the relationship between microRNA (miRNA) and target expression levels is lacking. We describe a quantitative framework supporting the forward engineering of gene circuits that incorporate RNAi-based regulatory components in mammalian cells. We developed a model that captures the quantitative relationship between miRNA and target gene expression levels as a function of parameters, including mRNA half-life and miRNA target-site number. We extended the model to synthetic circuits that incorporate protein-responsive miRNA switches and designed an optimized miRNA-based protein concentration detector circuit that noninvasively measures small changes in the nuclear concentration of β-catenin owing to induction of the Wnt signaling pathway. Our results highlight the importance of methods for guiding the quantitative design of genetic circuits to achieve robust, reliable and predictable behaviors in mammalian cells.

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis

PubMed Central

Bohonyi, Noémi; Pohóczky, Krisztina; Szalontai, Bálint; Perkecz, Anikó; Kovács, Krisztina; Kajtár, Béla; Orbán, Lajos; Varga, Tamás; Szegedi, Sarolta; Bódis, József; Koppán, Miklós

2017-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis (n = 15), uterine fibroid-induced moderate dysmenorrhoea (n = 7) and tubal infertility with no pain (n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease. PMID:28478727

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis.

PubMed

Bohonyi, Noémi; Pohóczky, Krisztina; Szalontai, Bálint; Perkecz, Anikó; Kovács, Krisztina; Kajtár, Béla; Orbán, Lajos; Varga, Tamás; Szegedi, Sarolta; Bódis, József; Helyes, Zsuzsanna; Koppán, Miklós

2017-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis ( n = 15), uterine fibroid-induced moderate dysmenorrhoea ( n = 7) and tubal infertility with no pain ( n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease.

[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.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Effects of manganese deficiency on the microstructure of proximal tibia and OPG/RANKL gene expression in chicks.

PubMed

Liu, Ran; Jin, Cong; Wang, Zhenyong; Wang, Zhaojun; Wang, Jian; Wang, Lin

2015-03-01

Manganese (Mn) deficiency can result in perosis in chicks, but the mechanism of Mn deficiency on tibia development remains poorly understood. Ninety one-day-old Arbor Acres male broiler chickens administered with control diet (60 mg Mn/kg) and Mn-deficient diets (40 mg Mn/kg, 8.7 mg Mn/kg) to investigate the effects of Mn deficiency on morphology of tibia and related signal transduction pathways in broiler chickens. At the age of 42 days, the bone trabecula, damaged osteoblasts and OPG/RANKL mRNA expression levels were investigated by histological assessment, electron microscopic examination and real-time quantitative PCR analysis, respectively. Results of histological observations showed that decreased trabecular thickness, trabecular number and trabecular bone area (%) together with increased trabecular bone separation were involved in perosis induced by Mn deficiency. The most striking ultrastructural modifications involved disruption of nuclear membrane and mitochondria outer membrane, loss of mitochondrion cristae and alteration in endoplasmic reticulum in osteoblasts of the Mn-deficient groups. Likewise, Mn deficiency results in a significant (P < 0.05) decrease in the relative mRNA expression levels of OPG and RANKL with a significantly higher RANKL/OPG ratio (P < 0.05). In conclusion, Mn deficiency can affect the development of tibia in broiler chickens, leading to metaphyseal osteoporosis which may be due to decreased OPG/RANKL mRNA expression.

Effects of tiletamine on the adenosine monophosphate-activated protein kinase signaling pathway in the rat central nervous system.

PubMed

Su, Li-Xue; Shi, Xing-Xing; Yang, Peng; Chen, Hao; Li, Xin; Fan, Hong-Gang; Wang, Hong-Bin

2017-10-01

The dissociative anesthetic tiletamine, which acts on the central nervous system (CNS), is widely used in veterinary medicine and animal experiments. Recent studies indicate that adenosine 5'-monophosphate activated protein kinase (AMPK) plays a key role in the analgesic action of tiletamine. In the present study, the effects of tiletamine on the AMPK signaling pathway in rats were investigated. Sprague-Dawley rats were injected intraperitoneally with tiletamine and executed at 10, 20, 40 and 60min post injection. The cerebral cortex, hippocampus, thalamus, cerebellum and brainstem were immediately taken out to evaluate the mRNA and protein phosphorylation levels of liver kinase B1 (LKB1), AMPKα and eIF4E-binding protein 1 (4EBP1) using quantitative real-time polymerase chain reaction and western blot analysis. Tiletamine increased AMPK mRNA expression in the rat brain (P<0.01). Increased mRNA expression of AMPK was accompanied by an increase in phosphorylation of LKB1, resulting in significant decreases in the phosphorylation levels of 4EBP1 in the corresponding brain regions (P<0.01). In summary, the findings indicate that tiletamine regulates the mRNA expression and protein phosphorylation levels of LKB1, AMPK and 4EBP1 in the CNS, suggesting that the analgesic effect of the anesthetic is mediated, at least in part, by the AMPK signaling pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

The protection of selenium against cadmium-induced cytotoxicity via the heat shock protein pathway in chicken splenic lymphocytes.

PubMed

Chen, Xi; Zhu, Yi-Hao; Cheng, Xin-Yue; Zhang, Zi-Wei; Xu, Shi-Wen

2012-12-07

Cadmium (Cd) is a heavy metal that poses a hazard to animal health due to its toxicity. Selenium (Se) is an important nutritional trace element. However, the potential protective effects of Se against Cd-induced toxicity remain to be elucidated. To investigate the cytotoxicity of Cd on bird immunocytes in vitro and the protective effects of Se against exposure to Cd, chicken splenic lymphocytes received Cd (10⁻⁶ mol/L), Se (10⁻⁷ mol/L), and the mixture of 10⁻⁷ mol/L Se and 10⁻⁶ mol/L Cd and were incubated for 12 h, 24 h, 36 h, 48 h, respectively. The transcription of heat shock protein (HSP) 27, HSP40, HSP60, HSP70 and HSP90 mRNA was tested by fluorescence quantitative PCR. The results showed that the mRNA expression of HSPs exposed to 10⁻⁶ mol/L Cd showed a sustained decrease at 12-48 h exposure. A statistically significant increase in the mRNA expression of HSPs in the case of Se group was observed, as compared to the control group of chicken splenic lymphocytes. Concomitantly, treatment of chicken splenic lymphocytes with Se in combination with Cd enhanced the mRNA expression of HSPs which were reduced by Cd treatment. This indicated that the protective effect of Se against the toxicity of Cd might, at least partially, be attributed to stimulation of the level of HSPs.

The influence of PSA-RNA yield on the analysis of expressed prostatic secretions (EPS) for prostate cancer diagnosis.

PubMed

Whelan, Christopher; Crocitto, Laura; Kawachi, Mark; Chan, Kevin; Smith, David; Wilson, Timothy; Smith, Steven

2013-02-01

In patients with prostate cancer, luminal prostate-specific antigen (PSA) enters the circulation because the basement membrane and glandular epithelium are damaged. Given that excess mobilization of prostate cells during prostatic massage can influence normalization in diagnostic testing, we studied PSA mRNA levels in expressed prostatic secretions (EPS) from patients undergoing biopsy for prostate cancer to determine if prostate cells are preferentially mobilized from patients with prostate cancer during prostatic massage. Quantitative Reverse-Transcription PCR (qRT-PCR) was used to measure the RNA levels of GAPDH, PSA, TMPRSS2:ERG and PCA3 in EPS specimens obtained from patients undergoing biopsy for prostate cancer. The level of PSA mRNA is significantly elevated in EPS specimens obtained from patients with a subsequent diagnosis of prostate cancer. This correlation influenced diagnostic testing results from EPS in two ways. First, when used as an exclusion parameter it appears to improve the diagnostic performance of TMPRSS2:ERG in EPS. Second, when used as a normalization parameter it appears to decrease the performance of these same tests. When comparing the results of mRNA based prostate cancer diagnostics in EPS it will be essential to consider PSA mRNA as a prostate specific gene and not a housekeeping gene.

Expression of the tachykinin receptor mRNAs in healthy human colon.

PubMed

Jaafari, Nadia; Hua, Guoqiang; Adélaïde, José; Julé, Yvon; Imbert, Jean

2008-12-03

Tachykinins are a family of neuropeptides, involved in a variety of physiological and pathological processes occurring in the gastrointestinal tract. They act via three distinct types of receptors, tachykinin NK(1), NK(2), and NK(3) receptors, which belong to the family of G protein-coupled receptors. The aim of the present study was to characterize, for the first time in the healthy human colon, the TACR(1), TACR(2) and TACR(3) mRNAs encoding the three different tachykinin receptors and to measure their relative expression by quantitative reverse transcription-PCR assay. Our results confirm the broad distribution of the tachykinin receptors but evidenced significant differences in the expression level of their respective mRNAs. A higher expression level of the TACR2 mRNA alpha isoform, the gene encoding the functional tachykinin NK(2) receptor, was observed in comparison to TACR1 and TACR3 mRNAs genes encoding for NK(1) and NK(3) receptors respectively. The prevalence of the TACR2 mRNA alpha isoform strongly suggests a major involvement of tachykinin NK(2) receptor in the regulation of human colonic functions.

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

PubMed Central

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

2014-01-01

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

EMMPRIN (basigin/CD147) is involved in the morphogenesis of tooth germ in mouse molars.

PubMed

Xie, Ming; Jiao, Ting; Chen, Yuqin; Xu, Chun; Li, Jing; Jiang, Xinquan; Zhang, Fuqiang

2010-05-01

The pattern of gene expression for extracellular matrix metalloproteinase inducer (EMMPRIN) was revealed in the tooth germ of mouse mandibular molars using quantitative real-time PCR. In situ hybridization and immunohistochemical study demonstrated the characteristic distribution of EMMPRIN in the different stages of tooth germ development. To investigate the functional role played by EMMPRIN in tooth germ development, EMMPRIN siRNA interference approach was carried out in cultured mouse mandibles at embryonic day 11.0 (E11.0). The results showed that EMMPRIN siRNA-treated explants exhibited a marked growth inhibition of tooth germ compared to the control and scrambled siRNA-treated explants. Meanwhile, a significant increase in MT1-MMP mRNA expression and a reduction in MMP-2, MMP-3, MMP-9, MMP-13 and MT2-MMP mRNA expression were observed in the mouse mandibles following EMMPRIN abrogation. The current results indicate that EMMPRIN could thus be involved in the early stage of tooth germ development and morphogenesis, possibly by regulating the expression of MMP genes.

Transepithelial Ion Transport is Suppressed in Hypoxic Sinonasal Epithelium

PubMed Central

Blount, Angela; Zhang, Shaoyan; Chestnut, Michael; Hixon, Brian; Skinner, Daniel; Sorscher, Eric J.; Woodworth, Bradford A.

2011-01-01

Objectives/Hypothesis Sinonasal respiratory epithelial mucociliary clearance (MCC) is dependent on the transepithelial transport of ions such as Cl−. The objectives of the present study were to investigate the role of oxygen restriction in 1) Cl− transport across primary sinonasal epithelial monolayers, 2) expression of the apical Cl− channels CFTR and TMEM16A, and 3) the pathogenesis of chronic rhinosinusitis (CRS). Study Design In vitro investigation. Methods Murine nasal septal epithelial (MNSE, wild type) and human sinonasal epithelial (HSNE) cultures were incubated under hypoxic conditions (1% O2, 5% CO2). Cultures were mounted in Ussing chambers for ion transport measurements. CFTR and TMEM16A expression were measured using quantitative RT-PCR. Results The change in short-circuit current (ΔISC (µA/cm2) attributable to CFTR (forskolin-stimulated) was significantly decreased due to a 12 hour hypoxia exposure in both MNSE (13.55+/− 0.46 vs. 19.23+/−0.18) and HSNE (19.55+/−0.56 vs. 25.49+/−1.48 (control); p<0.05. TMEM16A (UTP-stimulated transport) was inhibited by 48 hours of hypoxic exposure in MNSE (15.92+/−2.87 vs. 51.44+/−3.71(control) p<0.05] and by 12 hours of hypoxic exposure in HSNE (16.75+/−0.68 vs. 24.15+/−1.35 (control). Quantitative RT-PCR (reported as relative mRNA levels+/−S.D.) demonstrated significant reductions in both CFTR and TMEM16A mRNA expression in MNSE and HSNE due to airway epithelial hypoxia. Conclusions Sinonasal epithelial CFTR and TMEM16A-mediated Cl− transport and mRNA expression were robustly decreased in an oxygen restricted environment. The findings in the present study indicate persistent hypoxia may lead to acquired defects in sinonasal Cl− transport in a fashion likely to confer mucociliary dysfunction in CRS. Level of Evidence 1b PMID:22024847

[Effects of Salvianolate on Myosin Heavy Chain in Cardiomyocytes of Congestive Heart Failure Rats].

PubMed

Chen, Cheng; Zou, Xiang-gu; Qiu, Shan-dong; Chen, Hui; Chen, Yong-zhong; Lin, Xiu-ming

2015-07-01

To explore the effect of Salvianolate on myosin heavy chain (MHC) in cardiomyocytes of congestive heart failure (CHF) rats. Sixty male SD rats were divided into 6 groups according to random digit table, i.e., the normal control group (NCG), the model group, the Captopril group (CAG), the low dose Salvianolate group (LSG), the high dose Salvianolate group (HSG), the Captopril and high dose Salvianolate group (CSG), 10 in each group. CHF rat model was established with peritoneal injection of adriamycin in all rats except those in the NCG. Equal volume of normal saline was peritoneally injected to rats in the NCG, once per week for 6 successive weeks. Corresponding medication was started from the 5th week of injecting adriamycin. Rats in the CAG were administered with Captopril solution at the daily dose of 10 mg/kg by gastrogavage. Rats in the LSG and the HSG were administered with Salvianolate solution at the daily dose of 24.219 mg/kg and 48.438 mg/kg respectively by gastrogavage. Salvianolate was dissolved in 2 mL 5% glucose solution and administered by peritoneal injection. Rats in the CSG were peritoneally injected with high dose Salvianolate solution and administered with Captopril solution by gastrogavage. Two mL normal saline was peritoneally injected to rats in the model group, once per day for 8 successive weeks. Eight weeks later, the cardiac function and myocardial hypertrophy indices were detected by biological signal collecting and processing system. mRNA expression levels of alpha-MHC and beta-MHC in cardiac muscle were detected by fluorescence quantitative PCR. Expressions of protein kinase C (PKC) in cardiac muscle were detected by Western blot. Compared with the normal control group, heart mass index (HMI) and left ventricular mass index (LVMI) obviously increased in the model group (P < 0.01). Compared with the model group, HMI and LVMI decreased in HSG, CAG, and CSG groups (P < 0.05, P < 0.01). It was more obviously lowered in the CSG group than in the CAG group (P < 0.05). Compared with the NCG, the mRNA expression level of alpha-MHC in cardiac muscle decreased, the mRNA expression level of p-MHC and the expression of PKC in cardiac muscle increased in the model group (P < 0.01). Compared with the model group, the mRNA expression level of alpha-MHC in cardiac muscle was increased, and the mRNA expression level of beta-MHC and the expression of PKC in cardiac muscle were decreased in HSG, CAG, and CSG groups (P < 0.05, P < 0.01). There was statistical difference between the CSG group and the CAG group (P < 0.05). Salvianolate could up-regulate the mRNA expression level of alpha-MHC, and down-regulate the mRNA expression level of beta-MHC in cardiac muscle. Its mechanism might be related to decreasing the expression of PKC.

[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.

Correlation between ZBED6 Gene Upstream CpG Island methylation and mRNA expression in cattle.

PubMed

Huang, Yong-Zhen; Zhang, Zi-Jing; He, Hua; Cao, Xiu-Kai; Song, Cheng-Chuang; Liu, Kun-Peng; Lan, Xian-Yong; Lei, Chu-Zhao; Qi, Xing-Lei; Bai, Yue-Yu; Chen, Hong

2017-04-03

DNA methylation is essential for the regulation of gene expression and important roles in muscle development. To assess the extent of epigenetic modifications and gene expression on the differentially methylated region (DMR) in ZBED6, we simultaneously examined DNA methylation and expression in six tissues from two different developmental stages (fetal bovine and adult bovine). The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The result of quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution and is highly expressed in adult bovine (P < 0.05 or P < 0.01). The DNA methylation level was significantly different in liver, lung and spleen between the two cattle groups (P < 0.05 or P < 0.01). The adult bovine group exhibited a significantly higher mRNA level and lower DNA methylation level than the fetal bovine group in liver, lung, and spleen. No significant association was detected between DNA methylation level and muscle, heart, and kidney at two different stages. In this study, the statistical analyses indicated that DNA methylation patterns are associated with mRNA level in some tissues, these results may be a useful parameter to investigate muscle developmental in cattle and as a model for studies in other species, potentially contributing to an improvement of growth performance selection in beef cattle breeding program.

Transcuprein is a Macroglobulin Regulated by Copper and Iron Availability

PubMed Central

Liu, Nanmei; Lo, Louis Shi-li; Askary, S. Hassan; Jones, LaTrice; Kidane, Theodros Z.; Nguyen, Trisha Trang Minh; Goforth, Jeremy; Chu, Yu-Hsiang; Vivas, Esther; Tsai, Monta; Westbrook, Terence; Linder, Maria C.

2009-01-01

SUMMARY Transcuprein is a high affinity copper carrier in the plasma involved in the initial distribution of copper entering the blood from the digestive tract. To identify and obtain cDNA for this protein, it was purified from rat plasma by size exclusion and copper chelate affinity chromatography, and amino acid sequences were obtained. These revealed a 190 kDa glycosylated protein identified as the macroglobulin, α1inhibitorIII, the main macroglobulin of rodent blood plasma. Albumin (65 kDa) co-purified in variable amounts and was concluded to be a contaminant (although it transiently can bind the macroglobulin). The main macroglobulin in human blood plasma (α2-macroglobulin), homologous to α1inhibitorIII, also bound copper tightly. Expression of α1I3 (transcuprein) mRNA by the liver was examined in rats with and without copper deficiency, using quantitative PCR and Northern analysis. Protein expression was examined by Western blotting. Deficient rats with 40% less ceruloplasmin oxidase activity and liver copper concentrations expressed about twice as much α1I3 mRNA, but circulating levels of transcuprein did not differ. Iron deficiency, which increased liver copper concentrations 3-fold, reduced transcuprein mRNA expression and 7circulating levels of transcuprein relative to what occurred in rats with normal or excess iron. We conclude that transcupreins are specific macroglobulins that not only carry zinc but also transport copper in the blood; and that their expression can be modulated by copper and iron availability. PMID:17363239

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma.

PubMed

Ratajczak, Philippe; Leboeuf, Christophe; Wang, Li; Brière, Josette; Loisel-Ferreira, Irmine; Thiéblemont, Catherine; Zhao, Wei-Li; Janin, Anne

2012-06-01

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.

Paraoxonase 1 and oxidative stress in paediatric non-alcoholic steatohepatitis.

PubMed

Desai, Sonal; Baker, Susan S; Liu, Wensheng; Moya, Diana A; Browne, Richard W; Mastrandrea, Lucy; Baker, Robert D; Zhu, Lixin

2014-01-01

Non-alcoholic steatohepatitis (NASH) in children is a significant public health concern. Oxidative stress is an important component in the pathophysiology of NASH. Several enzymatic antioxidant mechanisms protect the liver from oxidative injury. Examination of the expression of these enzymes in NASH livers may provide insight on the roles for these antioxidant mechanisms in the pathophysiology of NASH. The mRNA expression of catalase, glutathione peroxidase 1 (GPX1), glutathione reductase (GSR), paraoxonase 1 (PON1) and other reactive oxygen species-related genes was evaluated by microarray and quantitative real-time PCR analyses. The PON1 protein levels were evaluated in liver and serum by Western blot analyses. Serum enzymatic activities of GPX, GSR and PON1 (paraoxonase and arylesterase activities) were examined. NASH livers exhibited elevated mRNA expression of catalase and PON1, but not GPX1 or GSR. No difference in serum GPX or GSR activity was detected between NASH patients and controls. Elevated expression of PON1 mRNA and protein was detected in NASH livers, but serum PON1 protein and activities were not elevated. Elevated expression of catalase and PON1 suggests protective roles for these antioxidants in NASH livers. Given the importance of oxidative stress in the pathophysiology of NASH, future studies focusing on these enzymes could identify important targets for therapeutic or preventive interventions for NASH patients. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

miR-21 promotes the differentiation of hair follicle-derived neural crest stem cells into Schwann cells

PubMed Central

Ni, Yuxin; Zhang, Kaizhi; Liu, Xuejuan; Yang, Tingting; Wang, Baixiang; Fu, Li; A, Lan; Zhou, Yanmin

2014-01-01

Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair follicles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA (miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist (agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist (antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regulating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA. PMID:25206896

The HGF Receptor c-Met Is Overexpressed in Esophageal Adenocarcinoma1

PubMed Central

Herrera, Luis J; El-Hefnawy, Talal; Queiroz de Oliveira, Pierre E; Raja, Siva; Finkelstein, Sydney; Gooding, William; Luketich, James D; Godfrey, Tony E; Hughes, Steven J

2005-01-01

Abstract The hepatocyte growth factor (HGF) receptor, Met, has established oncogenic properties; however, its expression and function in esophageal adenocarcinoma (EA) remain poorly understood. We aimed to determine the expression and potential alterations in Met expression in EA. Met expression was investigated in surgical specimens of EA, Barrett's esophagus (BE), and normal esophagus (NE) using immunohistochemistry (IHC) and quantitative reverse transcriptase polymerase chain reaction. Met expression, phosphorylation, and the effect of COX-2 inhibition on expression were examined in EA cell lines. IHC demonstrated intense Met immunoreactivity in all (100%) EA and dysplastic BE specimens. In contrast, minimal immunostaining was observed in BE without dysplasia or NE specimens. Met mRNA and protein levels were increased in three EA cell lines, and Met protein was phosphorylated in the absence of serum. Sequence analysis found the kinase domain of c-met to be wild type in all three EA cell lines. HGF mRNA expression was identified in two EA cell lines. In COX-2-overexpressing cells, COX-2 inhibition decreased Met expression. Met is consistently overexpressed in EA surgical specimens and in three EA cell lines. Met dysregulation occurs early in Barrett's dysplasia to adenocarcinoma sequence. Future study of Met inhibition as a potential biologic therapy for EA is warranted. PMID:15720819

Global Phosphoproteomics Identifies a Major Role for AKT and 14-3-3 in Regulating EDC3*

PubMed Central

Larance, Mark; Rowland, Alexander F.; Hoehn, Kyle L.; Humphreys, David T.; Preiss, Thomas; Guilhaus, Michael; James, David E.

2010-01-01

Insulin plays an essential role in metabolic homeostasis in mammals, and many of the underlying biochemical pathways are regulated via the canonical phosphatidylinositol 3-kinase/AKT pathway. To identify novel metabolic actions of insulin, we conducted a quantitative proteomics analysis of insulin-regulated 14-3-3-binding proteins in muscle cells. These studies revealed a novel role for insulin in the post-transcriptional regulation of mRNA expression. EDC3, a component of the mRNA decay and translation repression pathway associated with mRNA processing bodies, was shown to be phosphorylated by AKT downstream of insulin signaling. The major insulin-regulated site was mapped to Ser-161, and phosphorylation at this site led to increased 14-3-3 binding. Functional studies indicated that induction of 14-3-3 binding to EDC3 causes morphological changes in processing body structures, inhibition of microRNA-mediated mRNA post-transcriptional regulation, and alterations in the protein- protein interactions of EDC3. These data highlight an important new arm of the insulin signaling cascade in the regulation of mRNA utilization. PMID:20051463

Droplet Microfluidic and Magnetic Particles Platform for Cancer Typing.

PubMed

Ferraro, Davide; Champ, Jérôme; Teste, Bruno; Serra, M; Malaquin, Laurent; Descroix, Stéphanie; de Cremoux, Patricia; Viovy, Jean-Louis

2017-01-01

Analyses of nucleic acids are routinely performed in hospital laboratories to detect gene alterations for cancer diagnosis and treatment decision. Among the different possible investigations, mRNA analysis provides information on abnormal levels of genes expression. Standard laboratory methods are still not adapted to the isolation and quantitation of low mRNA amounts and new techniques needs to be developed in particular for rare subsets analysis. By reducing the volume involved, time process, and the contamination risks, droplet microfluidics provide numerous advantages to perform analysis down to the single cell level.We report on a droplet microfluidic platform based on the manipulation of magnetic particles that allows the clinical analysis of tumor tissues. In particular, it allows the extraction of mRNA from the total-RNA sample, Reverse Transcription, and cDNA amplification, all in droplets.

Allocation of Interferon Gamma mRNA Positive Cells in Caecum Hallmarks a Protective Trait Against Histomonosis.

PubMed

Kidane, Fana Alem; Mitra, Taniya; Wernsdorf, Patricia; Hess, Michael; Liebhart, Dieter

2018-01-01

Histomonosis is a parasitic disease of gallinaceous birds characterized by necrotic lesions in cacum and liver that usually turns fatal in turkeys while it is less severe in chickens. Vaccination using in vitro attenuated Histomonas meleagridis has been experimentally shown to confer protection against histomonosis. The protective mechanisms that underpin the vaccine-induced immune response are not resolved so far. Therefore, the actual study aimed to evaluate the location and quantitative distribution patterns of signature cytokines of type 1 [interferon gamma (IFN-γ)] or type 2 [interleukin (IL)-13] immune responses in vaccinated or infected hosts. An intergroup and interspecies difference in the spatial and temporal distribution patterns of cytokine mRNA positive cells was evident. Quantification of cells showed a significantly decreased percentage of IFN-γ mRNA positive cells at 4 days post-inoculation (DPI) in caeca of turkeys inoculated exclusively with the attenuated or the virulent inocula, compared to control birds. The decrement was followed by a surge of cells expressing mRNA for IFN-γ or IL-13, reaching a peak of increment at 10 DPI. By contrast, turkeys challenged following vaccination showed a slight increment of cecal IFN-γ mRNA positive cells at 4 DPI after which positive cell counts became comparable to control birds. The increase in infected birds was accompanied by an extensive distribution of positively stained cells up to the muscularis layer of cecal tissue whereas the vaccine group maintained an intact mucosal structure. In chickens, the level of changes of positive cells was generally lower compared to turkeys. However, control chickens were found with a higher percentage of IFN-γ mRNA positive cells in cecum compared to their turkey counterparts indicating a higher resistance to histomonosis, similar to the observation in immunized turkeys. In chickens, it could be shown that the changes of cytokine-positive cells were related to variations of mononuclear cells quantified by immunofluorescence. Furthermore, gene expression measured by reverse transcription quantitative real time PCR confirmed variations in organs between the different groups of both bird species. Overall, it can be concluded that a proportionally increased, yet controlled, allocation of IFN-γ mRNA positive cells in caeca hallmarks a protective trait against histomonosis.

Correlative mRNA and Protein Expression of Middle and Inner Ear Inflammatory Cytokines during Mouse Acute Otitis Media

PubMed Central

Trune, Dennis R.; Kempton, Beth; Hausman, Frances A.; Larrain, Barbara E.; MacArthur, Carol J.

2015-01-01

Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 hours. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 hour samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2 to 122 fold higher at 18 hours, declining slightly from there at 24 hours. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media. PMID:25922207

Correlative mRNA and protein expression of middle and inner ear inflammatory cytokines during mouse acute otitis media.

PubMed

Trune, Dennis R; Kempton, Beth; Hausman, Frances A; Larrain, Barbara E; MacArthur, Carol J

2015-08-01

Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 h. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 h samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2-122 fold higher at 18 h, declining slightly from there at 24 h. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media. Copyright © 2015 Elsevier B.V. All rights reserved.

The role of fructose‑1,6‑bisphosphatase 1 in abnormal development of ovarian follicles caused by high testosterone concentration.

PubMed

Liu, Tao; Zhao, Han; Wang, Jianfeng; Shu, Xin; Gao, Yuan; Mu, Xiaoli; Gao, Fei; Liu, Hongbin

2017-11-01

The present study aimed to identify the molecular mechanisms underlying the effects of the fructose‑1,6‑bisphosphatase 1 (FBP1) signaling pathway within normal follicle development and in hyperandrogenism‑induced abnormal follicle growth. To achieve this, murine primary follicles, granulosa cells (GCs) and theca‑interstitial cells (TICs) were isolated, cultured in vitro and treated with a high concentration of androgens. A concentration of 1x10‑5 mol/l testosterone was considerable to induce hyperandrogenism by MTT assay. All cells were divided into four groups, as follows: Control group, testosterone group, androgen receptor antagonist‑flutamide group and flutamide + testosterone group. Flutamide was used in the present study as it blocks the effects of the androgen receptor. The mRNA expression levels of FBP1 were detected using reverse transcription‑quantitative polymerase chain reaction. The expression levels and localization of FBP1 were analyzed by western blot analysis and immunofluorescence staining. The experimental results demonstrated that androgen presence stimulated follicle development, whereas excessive testosterone inhibited development. FBP1 was identified as being mainly expressed in follicles; FBP1 protein was significantly expressed in GCs of the 14‑day‑cultured follicle, as well as in the cytoplasm and nuclei of GCs and TICs in vitro. Testosterone increased FBP1 expression during a specific range of testosterone concentrations. Testosterone increased the expression of FBP1 within GCs. Furthermore, FBP1 and phosphoenolpyruvate carboxykinase 1 (PCK1) mRNA expression was increased in GCs treated with testosterone, whereas forkhead box protein O1 (FOXO1) and peroxisome proliferator‑activated receptor γ coactivator‑1α mRNA expression was significantly decreased in the testosterone group. In TICs, testosterone and flutamide inhibited the mRNA expression levels of FOXO1 and glucose‑6‑phosphatase enzyme, and promoted the expression of PCK1. These results suggested that the FBP1 signaling pathway may serve an important role in normal follicle growth and hyperandrogenism‑induced abnormal development, which may be associated with abnormal glucose metabolism induced by high concentrations of testosterone.

Increased Expression and Altered Methylation of HERVWE1 in the Human Placentas of Smaller Fetuses from Monozygotic, Dichorionic, Discordant Twins

PubMed Central

Wang, Zilian; Luo, Yanmin; Sun, Hongyu; Zhou, Yi; Huang, Linhuan; Li, Manchao; Fang, Qun; Jiang, Shiwen

2012-01-01

Background The human endogenous retroviral family W, Env(C7), member 1 gene (HERVWE1) is thought to participate in trophoblast cell fusion, and its expression is diminished in the placentas of singleton intrauterine growth-retarded pregnancies. However, there is limited information about the role of HERVWE1 in discordant fetal growth in twins. This study was to compare HERVWE1 gene expression between the placentas of discordant monozygotic twins and to identify its regulation by methylation. Methodology/Principal Findings Fetuses from twenty-one pairs of monozygotic, dichorionic, discordant twins were marked as “smaller” or “larger” according to birth weight. Placental HERVWE1 mRNA and protein expression profiles were analyzed using quantitative RT-PCR and immunohistochemistry (IHC) staining. Methylation profiles of the HERVWE1 promoter region were analyzed using a pyrosequencing assay. DNA methyltransferase (DNMT) transcript levels were analyzed by RT-PCR. 5-methyl cytosine (5-MC) was stained using an immunohistochemical assay. There was a significant negative correlation between HERVWE1 mRNA levels and birth weight in twins (P<0.01). Whereas the mean methylation level of the HERVWE1 promoter region was diminished in the smaller group in discordant twins(P<0.01), increased mRNA and protein levels of HERVWE1 were found in smaller fetuses compared with larger fetuses in discordant twins(P<0.01). There was no significant difference in 5-MC staining intensity between discordant twins (P>0.05). The DNMT3b3 mRNA levels in the smaller group were significantly downregulated compared with the larger group in discordant twins(P<0.05), whereas the DNMT3b7 mRNA levels in the smaller group were significantly upregulated compared with the larger group in discordant twins(P<0.05). Conclusions/Significance In discordant, monozygotic, dichorionic twins, HERVWE1 expression was higher in smaller fetuses and lower in larger fetuses. Methylation of the HERVWE1 gene promoter region may participate in the regulation of HERVWE1 gene expression in discordant twin pregnancies. PMID:22457770

In ovo leptin administration accelerates post-hatch muscle growth and changes myofibre characteristics, gene expression and enzymes activity in broiler chickens.

PubMed

Liu, P; Hu, Y; Grossmann, R; Zhao, R

2013-10-01

To evaluate the effect of maternal leptin on muscle growth, we injected 0 μg (control, CON), 0.5 μg (low leptin dose, LL) or 5.0 μg (high leptin dose, HL) of recombinant murine leptin dissolved in 100 μl of PBS into the albumen of broiler eggs prior to incubation. The newly hatched chicks were all raised under the same conditions until 21 days of age (D21), when body weight was measured and samples of gastrocnemius muscle were collected and weighed. Myosin ATPase staining was applied to identify myofibre types and measure the cross-sectional area (CSA) of myofibres. Real-time PCR was performed to quantify leptin receptor (LEPR), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor (GHR) and myostatin (MSTN) mRNA expression in the gastrocnemius muscle. The activity of calpains (CAPNs) in the gastrocnemius muscle was measured using a quantitative fluorescence detection kit. Male chickens treated with both high and low doses of leptin had significantly higher (p < 0.05) body weight on D21. The high leptin significantly increased the CSA (p < 0.05) of gastrocnemius muscle in male chickens, which coincided with a 93% increase (p < 0.05) in IGF-1 mRNA expression. Likewise, the LL dose increased the weight of gastrocnemius muscle in male chickens (p < 0.05), which was accompanied by a 41% down-regulation (p < 0.05) of MSTN mRNA expression and a decreased activity of CAPNs. However, all these changes were not observed in female chickens. The proportion of myofibre types did not altered. No significant change was detected for LEPR and GHR mRNA expression. These results indicate that in ovo leptin treatment affects skeletal muscle growth in chickens in a dose-dependent and sex-specific manner. The altered expression of IGF-1, MSTN mRNA and activity of CAPNs in skeletal muscle may be responsible for such effects. © 2012 Blackwell Verlag GmbH.

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

PubMed

Sun, Bing; Zheng, Yun-Ling

2018-01-01

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo

PubMed Central

Coles, Andrew H.; Osborn, Maire F.; Alterman, Julia F.; Turanov, Anton A.; Godinho, Bruno M.D.C.; Kennington, Lori; Chase, Kathryn; Aronin, Neil

2016-01-01

Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene® branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo. PMID:26595721

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells.

PubMed

Di Simone, Nicoletta; Di Nicuolo, Fiorella; Marzioni, Daniela; Castellucci, Mario; Sanguinetti, Maurizio; D'lppolito, Silvia; Caruso, Alessandro

2009-02-01

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [(3)H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50-100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.

Molecular-biological analysis of the effect of methamphetamine on the heart in restrained mice.

PubMed

Shinone, Kotaro; Tomita, Masafumi; Inoue, Hiromasa; Nakagawa, Yasuhisa; Ikemura, Mayumi; Nata, Masayuki

2010-03-01

In order to investigate the interaction in the heart between the administration of methamphetamine (MAP) and restraint of the body following it, we administrated MAP intraperitoneally to mice and restrained them, and then determined the level of mRNA expression of 22 genes in the heart using quantitative RT-PCR method. The mRNA expressions of Nfkbiz, Nr4a1 and Dusp1 changed significantly after the administration of MAP, suggesting the induction of an inflammatory condition such as damage to the myocardium. Moreover, the serum concentrations of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1 beta and IL-6 were significantly increased by the administration of MAP. On the other hand, the mRNA expressions of Rgs2 and Rasd1 were changed by both the administration of MAP and body restraint without interaction, which indicated that these insults affected the circulatory system additively or synergistically. From these results, it is likely that the administration of MAP, followed by body restraint, might cause acute myocardial damage due to the direct myocardial toxic effect of MAP, myocardial hypoxia and/or severe hypertension, which is one of the mechanisms for sudden death in MAP abusers who were restrained due to their excited state. (c) 2010. Published by Elsevier Ireland Ltd.

Expression of tomato prosystemin gene in Arabidopsis reveals systemic translocation of its mRNA and confers necrotrophic fungal resistance.

PubMed

Zhang, Haiyan; Yu, Pengli; Zhao, Jiuhai; Jiang, Hongling; Wang, Haiyang; Zhu, Yingfang; Botella, Miguel A; Šamaj, Jozef; Li, Chuanyou; Lin, Jinxing

2018-01-01

Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

cDNA nucleotide sequence coding for stearoyl-CoA desaturase and its expression in the zebrafish (Danio rerio) embryo.

PubMed

Hsieh, S L; Liu, R W; Wu, C H; Cheng, W T; Kuo, Ching-Ming

2003-12-01

A cDNA sequence of stearoyl-CoA desaturase (SCD) was determined from zebrafish (Danio rerio) and compared to the corresponding genes in several teleosts. Zebrafish SCD cDNA has a size of 1,061 bp, encodes a polypeptide of 325 amino acids, and shares 88, 85, 84, and 83% similarities with tilapia (Oreochromis mossambicus), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio), and milkfish (Chanos chanos), respectively. This 1,061 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains three histidine boxes, believed to be involved in catalysis. These observations suggested that SCD genes are highly conserved. In addition, an oligonucleotide probe complementary to zebrafish SCD mRNA was hybridized to mRNA of approximately 396 bases with Northern blot analysis. The Northern blot and RT-PCR analyses showed that the SCD mRNA was expressed predominantly in the liver, intestine, gill, and muscle, while a lower level was found in the brain. Furthermore, we utilized whole-mount in situ hybridization and real-time quantitative RT-PCR to identify expression of the zebrafish SCD gene at five different stages of development. This revealed that very high levels of transcripts were found in zebrafish at all stages during embryogenesis and early development. Copyright 2003 Wiley-Liss, Inc.

Expression of mRNAs encoding dopamine receptors in striatal regions is differentially regulated by midbrain and hippocampal neurons.

PubMed

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

1994-02-01

The glutamate analogue kainic acid was injected into the hippocampus of intact or 6-hydroxydopamine deafferented rats to investigate the influence of hippocampal neurons on the expression of dopamine D1 and D2 receptor mRNAs in subregions of the striatal complex and possible modulation by dopaminergic neurons. Quantitative in situ hybridization using 35S-labeled oligonucleotide probes specific for dopamine D1 and D2 receptor mRNAs, respectively, were used. It was found that an injection of kainic acid into the hippocampal formation had alone no significant effect on dopamine D1 or D2 receptor mRNA levels in any of the analyzed striatal subregions in animals analyzed 4 h after the injections. Kainic acid stimulation in the hippocampus ipsilateral to the dopamine lesion produced an increase in D1 receptor mRNA levels in the ipsilateral medial caudate-putamen, and a bilateral increase in core and shell of nucleus accumbens (ventral striatal limbic regions). A unilateral 6-hydroxydopamine lesion alone caused an increase in D2 receptor mRNA in the lateral caudate-putamen (dorsal striatal motor region) ipsilateral to the lesion and an increase in D1 receptor mRNA in the accumbens core ipsilateral to the lesion. However, in dopamine-lesioned animals, dopamine D1 receptor mRNA levels were increased bilaterally in nucleus accumbens core and shell and in the ipsilateral medial caudate-putamen following kainic acid stimulation in the hippocampus ipsilateral to the dopamine lesion. These results indicate a differential regulation of the expression of dopamine D1 and D2 receptor mRNAs by midbrain and hippocampal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

miR-205-5p negatively regulates hepatic acetyl-CoA carboxylase β mRNA in lipid metabolism of Oreochromis niloticus.

PubMed

Tao, Yi-Fan; Qiang, Jun; Bao, Jing-Wen; Li, Hong-Xia; Yin, Guo-Jun; Xu, Pao; Chen, De-Ju

2018-06-20

MicroRNAs (miRNAs) are non-coding RNAs that function as post-transcriptional gene regulators and that play vital roles controlling lipid metabolism. miR-205 is an important miRNA related to adipogenesis and lipid metabolism. However, little is known about the potential role of miR-205-5p in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). In this study, we used miRanda software to search for potential miR-205-5p target genes and found a lipid-metabolism-related gene called acetyl-CoA carboxylase β (ACACβ). Quantitative real-time polymerase chain reaction data indicated that there may be a negative regulation relationship between miR-205-5p and ACACβ gene expression under HFD rearing. Using luciferase reporter assays, we verified the binding site of miR-205-5p in the 3'-untranslated region of the ACACβ mRNA. Furthermore, an in vivo functional analysis of miR-205-5p was performed by injecting GIFT juveniles with a miR-205-5p antagomir. Reduced levels of miR-205-5p in GIFT liver increased ACACβ mRNA expression 12 h post-injection. miR-205-5p suppression also increased fatty acid synthase and peroxisome proliferator-activated receptor-α mRNA levels 48 h and 120 h post-injection, respectively. Taken together, our results indicate that miR-205-5p negative regulates hepatic ACACβ mRNA expression, and may serve as an important regulator in controlling hepatic lipid metabolism in GIFT. Copyright © 2018. Published by Elsevier B.V.

Susceptibility loci of CNOT6 in the general mRNA degradation pathway and lung cancer risk - a re-analysis of eight GWASs

PubMed Central

Zhou, Fei; Wang, Yanru; Liu, Hongliang; Ready, Neal; Han, Younghun; Hung, Rayjean J.; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S.; Caporaso, Neil; Landi, Maria Teresa; Brüske, Irene; Risch, Angela; Ye, Yuanqing; Wu, Xifeng; Christiani, David C.; Goodman, Gary; Chen, Chu; Amos, Christopher I.; Qingyi, Wei

2017-01-01

Purpose mRNA degradation is an important regulatory step for controlling gene expression and cell functions. Genetic abnormalities of the genes involved in mRNA degradation were found to be associated with cancer risks. Therefore, we systematically investigated the roles of genetic variants of genes in the general mRNA degradation pathway in lung cancer risk. Experimental design Meta-analyses were conducted in six lung cancer genome-wide association studies (GWASs) from the Transdisciplinary Research in Cancer of the Lung and additional two GWASs from Harvard University and deCODE in the International Lung Cancer Consortium. Expression quantitative trait loci analysis (eQTL) was used for in silico functional validation of the identified significant susceptibility loci. Results This pathway-based analysis included 4,603 single nucleotide polymorphisms (SNP) in 68 genes in 14,463 lung cancer cases and 44,188 controls, of which 20 SNPs were found to be associated with lung cancer risk with a false discovery rate threshold of <0.05. Among the 11 newly identified SNPs in CNOT6, which were in high linkage disequilibrium, the rs2453176 with a RegulomDB score “1f” was chosen as the tag SNP for further analysis. We found that the rs2453176 T allele was significantly associated with lung cancer risk (odds ratio=1.11, 95% confidence interval=1.04–1.18, P=0.001) in the eight GWASs. In the eQTL analysis, we found that levels of CNOT6 mRNA expression were significantly correlated with the rs2453176 T allele, which provided additional biological basis for the observed positive association. Conclusion The CNOT6 rs2453176 SNP may be a new functional susceptible locus for lung cancer risk. PMID:27805284

Susceptibility loci of CNOT6 in the general mRNA degradation pathway and lung cancer risk-A re-analysis of eight GWASs.

PubMed

Zhou, Fei; Wang, Yanru; Liu, Hongliang; Ready, Neal; Han, Younghun; Hung, Rayjean J; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S; Caporaso, Neil; Landi, Maria Teresa; Brüske, Irene; Risch, Angela; Ye, Yuanqing; Wu, Xifeng; Christiani, David C; Goodman, Gary; Chen, Chu; Amos, Christopher I; Wei, Qingyi

2017-04-01

mRNA degradation is an important regulatory step for controlling gene expression and cell functions. Genetic abnormalities involved in mRNA degradation genes were found to be associated with cancer risks. Therefore, we systematically investigated the roles of genetic variants in the general mRNA degradation pathway in lung cancer risk. Meta-analyses were conducted using summary data from six lung cancer genome-wide association studies (GWASs) from the Transdisciplinary Research in Cancer of the Lung and additional two GWASs from Harvard University and deCODE in the International Lung Cancer Consortium. Expression quantitative trait loci analysis (eQTL) was used for in silico functional validation of the identified significant susceptibility loci. This pathway-based analysis included 6816 single nucleotide polymorphisms (SNP) in 68 genes in 14 463 lung cancer cases and 44 188 controls. In the single-locus analysis, we found that 20 SNPs were associated with lung cancer risk with a false discovery rate threshold of <0.05. Among the 11 newly identified SNPs in CNOT6, which were in high linkage disequilibrium, the rs2453176 with a RegulomDB score "1f" was chosen as the tagSNP for further analysis. We found that the rs2453176 T allele was significantly associated with lung cancer risk (odds ratio = 1.11, 95% confidence interval = 1.04-1.18) in the eight GWASs. In the eQTL analysis, we found that levels of CNOT6 mRNA expression were significantly correlated with the rs2453176 T allele, which provided additional biological basis for the observed positive association. The CNOT6 rs2453176 SNP may be a new functional susceptible locus for lung cancer risk. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs.

PubMed

Yu, Hong; Bao, En-Dong; Zhao, Ru-Qian; Lv, Qiong-Xia

2007-11-01

To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs. 24 pigs (mean weight, 20 +/- 1 kg). Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA. No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs. Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.

Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.

PubMed

Mascarenhas, Roshan; Pietrzak, Maciej; Smith, Ryan M; Webb, Amy; Wang, Danxin; Papp, Audrey C; Pinsonneault, Julia K; Seweryn, Michal; Rempala, Grzegorz; Sadee, Wolfgang

2015-01-01

mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs) in lymphoblast cell lines (LCL) and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T) in ABCB1 (MDR1) on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq) in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs) affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.

Identification and Migration of Primordial Germ Cells in Atlantic Salmon, Salmo salar: Characterization of Vasa, Dead End, and Lymphocyte Antigen 75 Genes

PubMed Central

Nagasawa, Kazue; Fernandes, Jorge MO; Yoshizaki, Goro; Miwa, Misako; Babiak, Igor

2013-01-01

No information exists on the identification of primordial germ cells (PGCs) in the super-order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full-length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi-quantitative RT-PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole-mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid-blastula stage, vasa-expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp-rt-vasa 3′-UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. PMID:23239145

Effects of triclosan (TCS) on hormonal balance and genes of hypothalamus-pituitary- gonad axis of juvenile male Yellow River carp (Cyprinus carpio).

PubMed

Wang, Fan; Liu, Fei; Chen, Wanguang; Xu, Ruijie; Wang, Wei

2018-02-01

Triclosan (TCS) is a broad spectrum antimicrobial agent which has been widely dispersed and determinated in the aquatic environment. However, the effects of TCS on reproductive endocrine in male fish are poorly understood. In this study, male Yellow River carp (Cyprinus carpio) were exposed to 0, 1/5, 1/10 and 1/20 LC 50 (96 h LC 50 of TCS to carp) TCS under semi-static conditions for 42 d. Vitellogenin (Vtg), 17β-estradiol (E 2 ), testosterone(T), gonadotropin (GtH), and gonadotropin-releasing hormone (GnRH) levels were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also examined the mRNA expressions of aromatase, GtHs-β, GnRH, estrogen receptor (Er), and androgen receptor (Ar) by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). TCS induced Vtg levels of hepatopancreas, E 2 levels of serum, and inhibited Ar and Er mRNA levels, suggesting that the induction of Vtg production by TCS was indirectly caused by non-Er pathways. TCS-induced Vtg levels by interfering with the reproductive axis at plenty of latent loci of male carps: (a) TCS exposure increased the aromatase mRNA expression of hypothalamus and gonad aromatase, consequently increasing serum concentrations of E 2 to induce Vtg in hepatopancreas; (b) TCS treatment changed GtH-β and GnRH mRNA expression and secretion, causing the disturbance of reproductive endocrine; (c) TCS exposure decreased Ar mRNA levels, indicating potential Ar-mediated antiandrogen action. These mechanisms showed that TCS may induce Vtg production in male carp by non-Er-mediated pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation of natriuretic peptide receptor A and B expression by transforming growth factor-beta 1 in cultured aortic smooth muscle cells.

PubMed

Fujio, N; Gossard, F; Bayard, F; Tremblay, J

1994-06-01

Two types of natriuretic peptide receptors (NPR-A and NPR-B) are membrane guanylate cyclases whose relative expression varies in different tissues. Because natriuretic peptides have been shown to inhibit aortic smooth muscle proliferation, we investigated the regulation of NPR-A and NPR-B in these cells under different proliferative conditions. NPR subtype mRNA levels were measured by our newly developed quantitative reverse transcription-polymerase chain reaction assay using mutated NPR-A and NPR-B cRNA as internal standards. The functional impact of their expression was determined by atrial natriuretic peptide (ANP)- and C-type natriuretic peptide (CNP)-induced stimulation of cyclic GMP production. In the intact aorta, NPR-B mRNA levels were found to be 10-fold higher than those of NPR-A. This dominance was further amplified (1000-fold) in long-term cultures (10 to 15 passages) of aortic smooth muscle cells (ASMC). Higher cyclic GMP production with CNP than with ANP was observed in cultured ASMC from Wistar-Kyoto (WKY) rats. Similar stimulation by the two agonists was noted in spontaneously hypertensive rat (SHR) cells, paralleled by a 10-fold increase in NPR-A mRNA levels and ANP stimulation of cyclic GMP in hypertensive cells. The present study also evaluated NPR-A and NPR-B mRNA control by transforming growth factor-beta 1 (TGF-beta 1), an important regulator of cell proliferation that is overexpressed in SHR ASMC. TGF-beta 1 decreased both NPR-A and NPR-B mRNA levels with a predominant effect in SHR cells at high cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

Uromodulin mRNA from Urinary Extracellular Vesicles Correlate to Kidney Function Decline in Type 2 Diabetes Mellitus.

PubMed

Yamamoto, Cindy M; Murakami, Taku; Oakes, Melanie L; Mitsuhashi, Masato; Kelly, Colleen; Henry, Robert R; Sharma, Kumar

2018-05-18

Extracellular vesicles (EVs) enclose mRNA derived from their cell of origin and are considered a source of potential biomarkers. We examined urinary EV mRNA from individuals with diabetic kidney disease (DKD), chronic kidney disease, type 2 diabetes (T2DM), and obese and healthy controls to determine if such biomarkers had the potential to classify kidney disease and predict patients at higher risk of renal function decline. A total of 242 participants enrolled in this study. Urinary EV mRNA from all subjects were isolated by a filter-based platform, and the expression of 8 target genes were determined by quantitative polymerase chain reaction (qPCR). Changes in estimated glomerular filtration rate (eGFR) in 161 T2DM patients were evaluated for 2 consecutive years and compared with EV RNA profiles at baseline. We observe that mild and severe DKD groups show a significant 3.2- and -4.4-fold increase in UMOD compared to healthy controls and expression increases linearly from healthy, diabetic, and DKD subjects. UMOD expression is significantly correlated to albumin creatinine ratio (ACR), eGFR, and HbA1c. Using linear discriminant analyses with mRNA from severe DKD and T2DM as training data, a multi-gene signature classified DKD and -non-DKD with a sensitivity of 93% and specificity of 73% with area under the receiver operating characteristic (ROC) curve (AUC) = 0.90. Although 6% of T2DM were determined to have a > 80% posterior probability of developing DKD based on this mRNA profile, eGFR changes observed within the 2-year follow-up did not reveal a decline in kidney function. Urinary EV UMOD mRNA levels are progressively elevated from T2DM to DKD groups and correlate with widely used eGFR and ACR diagnostic criteria. An EV mRNA signature could identify DKD with greater than 90% sensitivity and 70% specificity. © 2018 S. Karger AG, Basel.

IL-4 mRNA Is Downregulated in the Liver of Pancreatic Cancer Patients Suffering from Cachexia.

PubMed

Prokopchuk, Olga; Steinacker, Jürgen M; Nitsche, Ulrich; Otto, Stephanie; Bachmann, Jeannine; Schubert, Elaine C; Friess, Helmut; Martignoni, Marc E

2017-01-01

Interleukin-4 (IL-4) together with interleukin-13 (IL-13) play an important role in inflammation and wound repair, and are known to be upregulated in human skeletal muscle after strenuous physical exercise. Additionally, these cytokines may act as autocrine growth factors in pancreatic cancer cells. We hypothesize that IL-4, IL-13, and their corresponding receptors are involved in mechanism of cancer cachexia. Tissue samples from human skeletal muscle, white fat, liver, healthy pancreas, and pancreatic ductal adenocarcinoma were analyzed by quantitative real-time polymerase chain reaction for mRNA expression levels of IL-4, IL-13, IL-4 receptor α, and IL-13 receptor α1. We demonstrate for the first time that liver IL-4 mRNA is downregulated in vivo in patients with pancreatic cancer and cachexia. Additionally, IL-4 mRNA in the liver inversely correlated with musculus psoas thickness. We speculate that suppression of IL-4 is involved in cancer cachexia, although the exact mechanisms have to be further elucidated.

S100A8/A9 mRNA induction in an ex vivo model of endotoxin tolerance: roles of IL-10 and IFNγ.

PubMed

Fontaine, Mathieu; Planel, Séverine; Peronnet, Estelle; Turrel-Davin, Fanny; Piriou, Vincent; Pachot, Alexandre; Monneret, Guillaume; Lepape, Alain; Venet, Fabienne

2014-01-01

Septic syndromes are the leading cause of death in intensive care units. They are characterized by the development of immune dysfunctions such as endotoxin tolerance (ET), whose intensity and duration are associated with increased risk of nosocomial infections and mortality. Alarmins S100A8 and S100A9 have been shown to be increased after septic shock. Importantly, a delayed S100A9 mRNA increase predicts hospital-acquired infection in patients. The aim of this study was to investigate the regulation of S100A8 and S100A9 mRNA expression in an ex vivo model of ET. ET was reproduced ex vivo by priming healthy peripheral blood mononuclear cells (number of donors  = 9 to 10) with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. ET was established by observing decreased TNFα and increased IL-10 transcriptomic responses to two subsequent endotoxin challenges. Interestingly, ET was associated with increased S100A8 and S100A9 mRNA expression ex vivo. We showed that IL-10 played a role in this process, since S100A8 and S100A9 mRNA increases were significantly abrogated by IL-10 blockade in the model. Conversely, treatment with rIFN-γ, a pro-inflammatory and immunostimulating molecule known to block ET induction, was able to restore normal S100A8 and S100A9 mRNA in this model. In this ex vivo model, we observed that S100A8 and S100A9 mRNA expression was significantly increased during ET. This reproduced ex vivo the observations we had previously made in septic shock patients. Interestingly, IL-10 blockade and rIFN-γ treatment partially abrogated S100A8/A9 mRNA increases in this model. Pending confirmation in larger, independent clinical studies, these preliminary results suggest that S100A8 and S100A9 mRNA levels might be used as surrogate markers of ET and as stratification tools for personalized immunotherapy in septic shock patients.

Expression of platelet-derived growth factor BB, erythropoietin and erythropoietin receptor in canine and feline osteosarcoma.

PubMed

Meyer, F R L; Steinborn, R; Grausgruber, H; Wolfesberger, B; Walter, I

2015-10-01

The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Expression of platelet-derived growth factor BB, erythropoietin and erythropoietin receptor in canine and feline osteosarcoma

PubMed Central

Meyer, F.R.L.; Steinborn, R.; Grausgruber, H.; Wolfesberger, B.; Walter, I.

2015-01-01

The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation. PMID:26189892

Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

PubMed

Wise, Lyn M; Bodaan, Christa J; Mercer, Andrew A; Riley, Christopher B; Theoret, Christine L

2016-10-01

Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses. © 2016 ESVD and ACVD.

Increased expression of placental growth factor in high-grade endometrial carcinoma

PubMed Central

COENEGRACHTS, LIEVE; SCHRAUWEN, STEFANIE; VAN BREE, RITA; DESPIERRE, EVELYN; LUYTEN, CATHERINE; JONCKX, BART; STASSEN, JEAN MARIE; VERGOTE, IGNACE; AMANT, FRÉDÉRIC

2013-01-01

Placental growth factor (PlGF), a homolog of vascular endothelial growth factor (VEGF), exerts pleiotropic functions in cancer by affecting tumor cells as well as endothelial and inflammatory cells. Moreover, PlGF expression correlates with tumor stage, recurrence, metastasis and patient outcome in different types of cancer. Recently, administration of anti-PlGF therapy reduced tumor growth and metastasis in preclinical tumor models. In the present study, we evaluated the diagnostic and prognostic value of systemic and local expression of PlGF in primary endometrial carcinomas. PlGF levels in tumor lysates (n=128) and serum (n=88) of patients with primary endometrial cancer were determined using ELISA. PlGF mRNA expression in endometrial carcinoma tissues was quantified by quantitative qRT-PCR. Results were compared to endometrial cancer stage and grade. Systemic PlGF levels were not altered in patients with endometrial cancer (FIGO stage I-II-III) as compared to healthy controls. Only in FIGO stage IV patients, serum PlGF levels were slightly increased. Local PlGF mRNA and protein expression in endometrial tumors progressively increased with tumor grade. In endometrioid carcinomas, PlGF mRNA expression was significantly increased in endometrioid grade 3 tumors as compared to normal endometrial tissue. PlGF protein expression was significantly increased in endometrioid grade 2 and 3 carcinomas and in serous carcinomas as compared to normal endometrial tissue. Our study showed that systemic/serum PlGF levels cannot be used as a diagnostic or prognostic marker in endometrial cancer. However, the increased local expression of PlGF, primarily in high-grade carcinomas, underscores the possibility for preclinical assessment of anti-PlGF therapy in endometrial cancer. PMID:23232836

Increased expression of placental growth factor in high-grade endometrial carcinoma.

PubMed

Coenegrachts, Lieve; Schrauwen, Stefanie; Van Bree, Rita; Despierre, Evelyn; Luyten, Catherine; Jonckx, Bart; Stassen, Jean Marie; Vergote, Ignace; Amant, Frédéric

2013-02-01

Placental growth factor (PlGF), a homolog of vascular endothelial growth factor (VEGF), exerts pleiotropic functions in cancer by affecting tumor cells as well as endothelial and inflammatory cells. Moreover, PlGF expression correlates with tumor stage, recurrence, metastasis and patient outcome in different types of cancer. Recently, administration of anti-PlGF therapy reduced tumor growth and metastasis in preclinical tumor models. In the present study, we evaluated the diagnostic and prognostic value of systemic and local expression of PlGF in primary endometrial carcinomas. PlGF levels in tumor lysates (n=128) and serum (n=88) of patients with primary endometrial cancer were determined using ELISA. PlGF mRNA expression in endometrial carcinoma tissues was quantified by quantitative qRT-PCR. Results were compared to endometrial cancer stage and grade. Systemic PlGF levels were not altered in patients with endometrial cancer (FIGO stage I-II-III) as compared to healthy controls. Only in FIGO stage IV patients, serum PlGF levels were slightly increased. Local PlGF mRNA and protein expression in endometrial tumors progressively increased with tumor grade. In endometrioid carcinomas, PlGF mRNA expression was significantly increased in endometrioid grade 3 tumors as compared to normal endometrial tissue. PlGF protein expression was significantly increased in endometrioid grade 2 and 3 carcinomas and in serous carcinomas as compared to normal endometrial tissue. Our study showed that systemic/serum PlGF levels cannot be used as a diagnostic or prognostic marker in endometrial cancer. However, the increased local expression of PlGF, primarily in high-grade carcinomas, underscores the possibility for preclinical assessment of anti-PlGF therapy in endometrial cancer.

Xeroderma pigmentosum, complementation group D expression in H1299 lung cancer cells following benzo[a]pyrene exposure as well as in head and neck cancer patients.

PubMed

Lin, Chang-Shen; Chiou, Wen-Yen; Lee, Ka-Wo; Chen, Tzu-Fen; Lin, Yuan-Jen; Huang, Jau-Ling

2016-01-01

DNA repair genes play critical roles in response to carcinogen-induced and anticancer therapy-induced DNA damage. Benzo[a]pyrene (BaP), the most carcinogenic polycyclic aromatic hydrocarbon (PAH), is classified as a group 1 carcinogen by International Agency for Research on Cancer. The aims of this study were to (1) evaluate the effects of BaP on DNA repair activity and expression of DNA repair genes in vitro and (2) examine the role of xeroderma pigmentosum, complementation group D (XPD) mRNA expression in human head and neck cancers. Host cell reactivation assay showed that BaP inhibited nucleotide excision repair in H1299 lung cancer cells. DNA repair through the non-homologous end-joining pathway was not affected by BaP. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot demonstrated that XPD was downregulated by BaP treatment. BaP exposure did not apparently affect expression of another 11 DNA repair genes. BaP treatment increased the DNA damage marker γ-H2AX and ultraviolet (UV) sensitivity, supporting an impairment of DNA repair in BaP-treated cells. XPD expression was also examined by quantitative RT-PCR in 68 head and neck cancers, and a lower XPD mRNA level was found in smokers' cancer specimens. Importantly, reduced XPD expression was correlated with patient 5-year overall survival rate (35 vs. 56%) and was an independent prognostic factor (hazard ratio: 2.27). Data demonstrated that XPD downregulation was correlated with BaP exposure and human head and neck cancer survival.

ARHGAP42 promotes cell migration and invasion involving PI3K/Akt signaling pathway in nasopharyngeal carcinoma.

PubMed

Hu, Qian; Lin, Xiao; Ding, Linxiaoxiao; Zeng, Yinduo; Pang, Danmei; Ouyang, Nengtai; Xiang, Yanqun; Yao, Herui

2018-06-24

Rho GTPase-activating protein 42 was identified as an inhibitor of RhoA to maintain normal blood pressure homeostasis. However, the effect of ARHGAP42 in promoting cell malignancy in nasopharyngeal carcinoma is demonstrated in this study. Microarray and real-time quantitative PCR were used for a mRNA profiling of ARHGAP42 in nasopharyngeal primary and metastatic carcinoma tissues. Western blot and immunohistochemical staining were used for detecting the expression of ARHGAP42 protein in nasopharyngeal carcinoma tissues and cell lines. The overexpression and silence experiments of ARHGAP42 were performed in NPC cell lines using siRNA and expressive plasmid for evaluating cancer cell migration and invasion in vitro. Real-time quantitative PCR, western blot, and transwell test were employed for with the function of ARHGAP42 and its antisense lncRNA uc010rul. We confirmed the elevated expression of ARHGAP42 in metastatic NPC tissues of mRNA and protein for the first time. Immunohistochemical analysis indicated that NPC patients with highly ARHGAP42 expression were significantly associated with shorter metastasis-free survival. Knockdown of ARHGAP42 resulted in significant inhibition of nasopharyngeal cancer cell migration and invasion in vitro, and the overexpression of ARHGAP42 showed the opposite effects. In addition, the silence of uc010rul resulted in ARHGAP42 expression decrease and significant inhibition of nasopharyngeal cancer cell migration and invasion. High expression of ARHGAP42 is associated with poor metastasis-free survival of nasopharyngeal carcinoma patients. ARHGAP42 promotes migration and invasion of nasopharyngeal carcinoma cells in vitro; the antisense lncRNA may be involved in this effect. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Glucocorticoids decrease body weight and food intake and inhibit appetite regulatory peptide expression in the hypothalamus of rats

PubMed Central

LIU, XIAO-YAN; SHI, JIAN-HUA; DU, WEN-HUA; FAN, YAN-PING; HU, XIAO-LEI; ZHANG, CHEN-CHEN; XU, HUAN-BAI; MIAO, YAN-JUN; ZHOU, HAI-YAN; XIANG, PING; CHEN, FENG-LING

2011-01-01

The aim of the present study was to investigate the effects of glucocorticoids (GCs) on appetite and gene expression of the hypothalamic appetite regulatory peptides, neuropeptide Y (NPY), agouti-related protein (AGRP) and cocaine and amphetamine-regulated transcript (CART), in non-obese and obese rats. Both non-obese and obese rats were randomly assigned to three groups: normal saline, low- and high-dose GC groups (NSG, LDG and HDG, respectively), which received an intraperitoneal injection with normal saline (0.2 ml/100 g) or hydrocortisone sodium succinate at 5 and 15 mg/kg, respectively, for 20 days. The expression levels of NPY, AGRP and CART mRNA in the hypothalamus were measured by real-time quantitative PCR. Non-obese and obese rats were found to undergo weight loss after GC injection, and a higher degree of weight loss was observed in the HDG rats. The average and cumulative food intakes in the obese and non-obese rats injected with high-dose GC were lower compared to that in the NSG (p<0.05). mRNA expression levels of the orexigenic neuropeptides, NPY and AGRP, and the anorexigenic neuropeptide, CART, were significantly lower in the HDG than levels in the NSG for both the obese and non-obese rats (p<0.05). GC treatment decreased appetite and body weight, induced apparent glucolipid metabolic disturbances and hyperinsulinemia, while down-regulated mRNA expression levels of the orexigenic neuropeptides, NPY and AGRP, and anorexigenic neuropeptide, CART, in the hypothalamus in the rats. The mechanism which induces this neuropeptide expression requires further study. PMID:22977608

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

PubMed

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

2009-03-01

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

SOX15 regulates proliferation and migration of endometrial cancer cells.

PubMed

Rui, Xiaohui; Xu, Yun; Jiang, Xiping; Guo, Caixia; Jiang, Jingting

2017-10-31

The study aimed to investigate the effects of Sry-like high mobility group box 15 ( SOX15 ) on proliferation and migration of endometrial cancer (EC) cells. Immunohistochemistry (IHC) was applied to determine the expression of SOX15 in EC tissues and adjacent tissues. We used cell transfection method to construct the HEC-1-A and Ishikawa cell lines with stable overexpression and low expression SOX15 Reverse-transcription quantitative real-time PCR (RT-qPCR) and Western blot were performed to examine expression of SOX15 mRNA and SOX15 protein, respectively. By conducting a series of cell proliferation assay and migration assay, we analyzed the influence of SOX15 overexpression or low expression on EC cell proliferation and migration. The expression of SOX15 mRNA and protein in EC tissues was significantly lower than that in adjacent tissues. After lentivirus-transfecting SOX15 , the expression level of SOX15 mRNA and protein was significantly increased in cells of SOX15 group, and decreased in sh- SOX15 group. Overexpression of SOX15 could suppress cell proliferation, while down-regulation of SOX15 increased cell proliferation. Flow cytometry results indicated that overexpression of SOX15 induced the ratio of cell-cycle arrest in G 1 stage. In addition, Transwell migration assay results showed that SOX15 overexpression significantly inhibited cell migration, and also down-regulation of SOX15 promoted the migration. As a whole, SOX15 could regulate the proliferation and migration of EC cells and up- regulation of SOX15 could be valuable for EC treatment. © 2017 The Author(s).

Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis.

PubMed

Carrarelli, Patrizia; Yen, Chih-Fen; Arcuri, Felice; Funghi, Lucia; Tosti, Claudia; Wang, Tzu-Hao; Huang, Joseph S; Petraglia, Felice

2015-09-01

To evaluate the expression pattern of activins and related growth factor messenger RNA (mRNA) levels in adenomyotic nodules and in their endometrium. Prospective study. University hospital. Symptomatic premenopausal women scheduled to undergo hysterectomy for adenomyosis. Samples from adenomyotic nodules and homologous endometria were collected. Endometrial tissue was also obtained from a control group. Quantitative real-time polymerase chain reaction (PCR) analysis and immunohistochemical localization of activin-related growth factors (activin A, activin B, and myostatin), binding protein (follistatin), antagonists (inhibin-α, cripto), and receptors (ActRIIa, ActRIIb) were performed. Myostatin mRNA levels in adenomyotic nodule were higher than in eutopic endometrium and myostatin, activin A, and follistatin concentrations were higher than in control endometrium. No difference was observed for inhibin-α, activin B, and cripto mRNA levels. Increased mRNA levels of ActRIIa and ActRIIb were observed in adenomyotic nodules compared with eutopic endometrium and control endometrium. Immunofluorescent staining for myostatin and follistatin confirmed higher protein expression in both glands and stroma of patients with adenomyosis than in controls. The present study showed for the first time that adenomyotic tissues express high levels of myostatin, follistatin, and activin A (growth factors involved in proliferation, apoptosis, and angiogenesis). Increased expression of their receptors supports the hypothesis of a possible local effect of these growth factors in adenomyosis. The augmented expression of ActRIIa, ActRIIb, and follistatin in the endometrium of these patients may play a role in adenomyosis-related infertility. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Gene expression during skeletal development in three osteopetrotic rat mutations. Evidence for osteoblast abnormalities.

PubMed

Shalhoub, V; Jackson, M E; Lian, J B; Stein, G S; Marks, S C

1991-05-25

Osteopetrosis is a group of metabolic bone diseases characterized by reductions in osteoclast development and/or function. These aspects of osteoclast biology are known to be influenced by osteoblasts and their products. To ascertain whether osteoblast dysfunction contributes to aberrations in the structural and functional properties of osteoclasts in osteopetrosis, we systematically examined gene expression as reflected by mRNA levels for a series of cell growth- and tissue-related genes associated with the osteoblast phenotype during skeletal development in normal and mutant rats of three different osteopetrotic stocks. We show that the methods used permit the reproducible isolation of undegraded total cellular RNA from bone and that mRNA levels can be reliably quantitated in these preparations. Each osteopetrotic mutation exhibits a distinct aberrant pattern of osteoblast gene expression that may be correlated with and explain some abnormalities in extracellular matrix composition, mineralization, osteoclast development, and effects of elevated serum levels of 1 alpha,25-dihydroxyvitamin D3, depending upon the mutation. Normal rats show minor variations in gene expression that reflect the genetic background (stock). This, the first comprehensive molecular analysis of osteoblast gene expression in osteopetrosis, suggests that some osteopetroses, particularly in the toothless rat, are associated with and potentially related to mechanisms associated with aberrations in osteoblast function. More generally, the present studies demonstrate alterations in gene expression as reflected by mRNA levels that are associated with functional properties of the osteoblast, particularly those contributing to the recruitment and/or differentiation of osteoclasts, thereby influencing skeletal modeling.

The distribution of the orphan bombesin receptor subtype-3 in the rat CNS.

PubMed

Jennings, C A; Harrison, D C; Maycox, P R; Crook, B; Smart, D; Hervieu, G J

2003-01-01

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor that shares between 47 and 51% homology with other known bombesin receptors. The natural ligand for BRS-3 is currently unknown and little is known about the mechanisms regulating BRS-3 gene expression. Unlike other mammalian bombesin receptors that have been shown to be predominantly expressed in the CNS and gastrointestinal tract, expression of the BRS-3 receptor in the rat brain has previously not been observed. To gain further understanding of the biology of BRS-3, we have studied the distribution of BRS-3 mRNA and protein in the rat CNS. The mRNA expression pattern was studied using reverse transcription followed by quantitative polymerase chain reaction. Using immunohistological techniques, the distribution of BRS-3 protein in the rat brain was investigated using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The BRS-3 receptor was found to be widely expressed in the rat brain at both mRNA and protein levels. Particularly strong immunosignals were observed in the cerebral cortex, hippocampal formation, hypothalamus and thalamus. Other regions of the brain such as the basal ganglia, midbrain and reticular formation were also immunopositive for BRS-3. In conclusion, our neuroanatomical data provide evidence that BRS-3 is as widely expressed in the rat brain as other bombesin-like peptide receptors and suggest that this receptor may also have important roles in the CNS, mediating the functions of a so far unidentified ligand.

BAG3 promotes chondrosarcoma progression by upregulating the expression of β-catenin.

PubMed

Shi, Huijuan; Chen, Wenfang; Dong, Yu; Lu, Xiaofang; Zhang, Wenhui; Wang, Liantang

2018-04-01

To investigate the roles of B‑cell lymphoma‑2 associated athanogene 3 (BAG3) in human chondrosarcoma and the potential mechanisms, the expression levels of BAG3 were detected in the present study, and the associations between BAG3 and clinical pathological parameters, clinical stage as well as the survival of patients were analyzed. The present study detected BAG3 mRNA and protein expression in the normal cartilage cell line HC‑a and in SW1353 chondrosarcoma cells by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The BAG3 protein expression in 59 cases of chondrosarcoma, 30 patients with endogenous chondroma and 8 cases of normal cartilage was semi-quantitatively analyzed using the immunohistochemical method. In addition, the BAG3 protein expression level, the clinical pathological parameters, clinical stage and the survival time of patients with chondrosarcoma were analyzed. The plasmid transfection method was employed to upregulate the expression BAG3 and small RNA interference to downregulate the expression of BAG3 in SW1353 cells. The expression levels of BAG3 protein and mRNA were significantly increased in the chondrosarcoma cell line when compared with the normal cartilage cell line. The immunohistochemistry results indicated that BAG3 protein was overexpressed in the tissue of human chondrosarcoma. Statistical analysis showed that the expression level of BAG3 was significantly increased in the different Enneking staging of patients with chondrosarcoma and Tumor staging, and there were no statistical differences in age, gender, histological classification and tumor size. In the in vitro experiments, the data revealed that BAG3 significantly promoted chondrosarcoma cell proliferation, colony‑formation, migration and invasion; however, it inhibited chondrosarcoma cell apoptosis. It was observed that BAG3 upregulated β‑catenin expression at the mRNA and protein levels. In addition, BAG3 induced the expression of runt‑related transcription factor 2 (RUNX2) in chondrosarcoma cells by upregulating β‑catenin. These clinical analyses revealed a positive association between β‑catenin and BAG3 in chondrosarcoma tumors. BAG3 was significantly increased in chondrosarcoma cells and tissues compared with the normal cartilage cells, tissue and cartilage benign tumors. Thus, BAG3 may serve as an oncogene in the development of chondrosarcoma via the induction of RUNX2 expression. The results of the present study contribute to further research on the biological development of chondrosarcoma.

OXTR and ZEB1 expression before and after progesterone dosing in pregnant women with threatened premature labor.

PubMed

Xu, Y-J; Ren, L-D; Zhai, S-S; Ran, L-M; Hu, L-L; Luo, X-H; Hong, T; Liu, R; Yu, Y-R; Ban, Y-J

2017-07-01

To investigate changes in the peripheral blood mRNA levels of oxytocin receptor (OXTR) and Zinc finger E-box-binding homeobox 1 (ZEB1) before and after progesterone dosing in pregnant women with threatened premature labor. Blood samples were collected from 30 healthy pregnant women with 28- to 33+6-week gestational age and singleton pregnancy (group A) and from 30 pregnant women with singleton pregnancy and threatened premature labor before and 48 hours after progesterone dosing (groups B and C, respectively) for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay to assess the OXTR and ZEB1 mRNA levels. The OXTR mRNA level was higher in the group B than in the groups A and C, and the ZEB1 mRNA level was lower in the group B than in the groups A and C. Notably, no significant difference was found in the mRNA level of OXTR or ZEB1 between group A and group C. The peripheral blood mRNA level of OXTR was increased, and that of ZEB1 was decreased in pregnant women with threatened premature labor. Progesterone helped to maintain pregnancy by readjusting the mRNA levels of OXTR and ZEB1.

[Aging-related ionic remodeling of L-type voltage dependent calcium channel in left atria of canine].

PubMed

Zhou, Xian-hui; Zhang, Jian; Gan, Tian-yi; Xu, Guo-jun; Tang, Bao-peng

2012-04-01

To investigate aging-related ionic remodeling of L-type voltage dependent calcium channel (LVDCC) in left atria of canine. Seven adult (2.0 - 2.5 years) and 10 aged (> 8 years) dogs were used. The current of LVDCC was recorded by patch clamp technique in the whole cell mode. The action potential duration (APD(90)), amplitude of action potential plateau (APA), I(Ca-L) peak current density of LVDCC were recorded. The mRNA and protein expressions of α1c subunit (Ca(V1.2)), sarcoplasmic reticulum Ca(2+)-ATPase (SECRA(2)), Calpain-I, ryanodine receptor (RYR(2)) were detected by quantitative RT-PCR and Western blot, respectively. I(Ca-L) peak current density [(-8.11 ± 0.54) pA/pF vs. (-14.04 ± 0.82) pA/pF, P < 0.05] was significantly reduced and action potential duration to 90% repolarization (APD(90)) significantly prolonged [(340.5 ± 10.1) ms vs. (320.0 ± 7.9) ms, P < 0.05] in aged group than in adult group. The mRNA gene expression level of Ca(V1.2) was significantly lower (0.90 ± 0.35 vs. 2.38 ± 0.40, P < 0.05) while mRNA expression of RYR(2) was significantly higher (4.39 ± 4.68 vs. 1.49 ± 1.69, P < 0.05) in the aged dogs than in the adult dogs. mRNA expression of SECRA(2) and Calpain-I was similar between the two groups. Similarly, the protein expression level of Ca(V1.2) was significantly lower (0.13 ± 0.10 vs. 0.29 ± 0.12, P < 0.05) while the protein expression level of RYR(2) was significantly higher (0.18 ± 0.21 vs. 0.08 ± 0.36, P < 0.05) in the aged dogs than in the adult dogs. Again, protein expression of SECRA(2), PLN(1) and Calpain-I was similar between the two groups. These data suggest that aging could induce mRNA and protein expression changes of Ca(V1.2) and RYR(2) of LVDCC which might serve as the molecular basis of I(Ca-L) remodeling in aged dogs and might be linked to the increased likelihood of developing atrial fibrillation (AF) in aged dogs.

Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes.

PubMed

Spalenza, Veronica; Girolami, Flavia; Bevilacqua, Claudia; Riondato, Fulvio; Rasero, Roberto; Nebbia, Carlo; Sacchi, Paola; Martin, Patrice

2011-09-01

Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Study of the expression and function of ACY1 in patients with colorectal cancer.

PubMed

Yu, Bing; Liu, Xuezhong; Cao, Xiuzhen; Zhang, Mingyue; Chang, Hong

2017-04-01

Aminoacylase 1 (ACY1) is important for regulating the proliferation of numerous types of cancer. However, the expression and mechanisms underlying the function of ACY1 in colorectal cancer remain unclear. In order to investigate the expression and function of ACY1 in colorectal cancer, tumor tissue and blood samples were collected for analysis from 132 patients diagnosed with colorectal cancer. Reverse transcription-quantitative polymerase chain reaction analysis and western blotting identified significantly increased expression of ACY1 mRNA in colorectal tumor tissue (P<0.05 vs. adjacent normal tissue) and notably increased ACY1 protein levels. This ACY1 mRNA expression was found to be positively correlated with tumor stage. In addition, plasma ACY1 concentration was increased in patients with colorectal cancer compared with healthy controls. Furthermore, in vitro knockdown of ACY1 in human colorectal cancer HT-29 cells was shown to inhibit proliferation and increase apoptosis. This effect was found to be associated with the activation of ERK1 and TGF-β1 signaling. In conclusion, the results of the present study suggest that ACY1 promotes tumor progression, and thus may be a potential target for the diagnosis and treatment of colorectal cancer.

Low intensity infrared laser affects expression of oxidative DNA repair genes in mitochondria and nucleus

NASA Astrophysics Data System (ADS)

Fonseca, A. S.; Magalhães, L. A. G.; Mencalha, A. L.; Geller, M.; Paoli, F.

2014-11-01

Practical properties and physical characteristics of low intensity lasers have made possible their application to treat soft tissue diseases. Excitation of intracellular chromophores by red and infrared radiation at low energy fluences with increase of mitochondrial metabolism is the basis of the biostimulation effect but free radicals can be produced. DNA lesions induced by free radicals are repaired by the base excision repair pathway. In this work, we evaluate the expression of POLγ and APEX2 genes related to repair of mitochondrial and nuclear DNA, respectively. Skin and muscle tissue of Wistar rats were exposed to low intensity infrared laser at different fluences. One hour and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of POLγ and APEX2 mRNA expression by real time quantitative polymerase chain reaction. Skin and muscle tissue of Wistar rats exposed to laser radiation show different expression of POLγ and APEX2 mRNA depending of the fluence and time after exposure. Our study suggests that a low intensity infrared laser affects expression of genes involved in repair of oxidative lesions in mitochondrial and nuclear DNA.

Transcriptional profiling of the parr–smolt transformation in Atlantic salmon

USGS Publications Warehouse

Robertson, Laura S.; McCormick, Stephen D.

2012-01-01

The parr–smolt transformation in Atlantic salmon (Salmo salar) is a complex developmental process that culminates in the ability to migrate to and live in seawater. We used GRASP 16K cDNA microarrays to identify genes that are differentially expressed in the liver, gill, hypothalamus, pituitary, and olfactory rosettes of smolts compared to parr. Smolts had higher levels of gill Na+/K+-ATPase activity, plasma cortisol and plasma thyroid hormones relative to parr. Across all five tissues, stringent microarray analyses identified 48 features that were differentially expressed in smolts compared to parr. Using a less stringent method we found 477 features that were differentially expressed at least 1.2-fold in smolts, including 172 features in the gill. Smolts had higher mRNA levels of genes involved in transcription, protein biosynthesis and folding, electron transport, oxygen transport, and sensory perception and lower mRNA levels for genes involved in proteolysis. Quantitative RT-PCR was used to confirm differential expression in select genes identified by microarray analyses and to quantify expression of other genes known to be involved in smolting. This study expands our understanding of the molecular processes that underlie smolting in Atlantic salmon and identifies genes for further investigation.

Mitochondrial superoxide dismutase activation with 17 β-estradiol-treated human lens epithelial cells

PubMed Central

Gottipati, Srinivas

2008-01-01

Purpose 17 β-estradiol (17β-E2) protects human lens epithelial cells against oxidative stress by preserving mitochondrial function in part via the non-genomic rapid activation of prosurvival signal transduction pathways. The study described herein examined whether 17β-E2 also elicits genomic protection by influencing the expression (and activity) of mitochondrial-associated manganese superoxide dismutase (MnSOD) as a possible parallel mechanism by which 17β-E2 protects against oxidative stress. Methods Virally-transformed human lens epithelial cells (HLE-B3) were pre-incubated with 17β-E2, and mRNA or protein lysates were collected over a time course ranging from 90 min to 24 h. Positive expression of lens epithelial cell MnSOD mRNA was determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT–PCR), and its levels were monitored by real-time PCR up to 24 h after 17β-E2 administration. Western blot analysis was used to examine the pattern of protein expression as influenced by 17β-E2 treatment. MnSOD activity as influenced by 17β-E2 was determined by measuring enzymatic activity. Results A significant rapid increase in the activity of MnSOD was observed with HLE-B3 cells by 90 min post-bolus addition of 17β-E2, which returned to control level by 240 min. Neither an increase in MnSOD mRNA nor in protein expression was detected up through 24 h. Conclusions These data demonstrate that 17β-E2 rapidly and transiently increases the activity of MnSOD but influences neither its mRNA expression nor its protein expression. The results suggest that (estrogen-activated) MnSOD plays an important role against mitochondrial oxidative stress by diminishing reactive oxygen species, thus promoting cell survival. PMID:18490963

mRNA Expression of Platelet-Derived Growth Factor Receptor-{beta} and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

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

Erben, Philipp; Horisberger, Karoline; Muessle, Benjamin

2008-12-01

Purpose: Deviant expression of platelet-derived growth factor receptor-{beta} (PDGFR{beta}) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFR{beta} and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Methods and Materials: Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFR{beta} and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG,more » 0-1). Results: The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFR{beta} ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFR{beta} ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFR{beta} and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFR{beta} ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFR{beta} and c-kit mRNA expression and the pathologic regression rate. Conclusion: Cetuximab-based chemoradiotherapy increased PDGFR{beta} levels even further compared with the pretreatment samples and deserves further investigation.« less

Correlation between messenger RNA expression and protein expression of immune checkpoint-associated molecules in bladder urothelial carcinoma: A retrospective study.

PubMed

Le Goux, Constance; Damotte, Diane; Vacher, Sophie; Sibony, Mathilde; Delongchamps, Nicolas Barry; Schnitzler, Anne; Terris, Benoit; Zerbib, Marc; Bieche, Ivan; Pignot, Géraldine

2017-05-01

Immunotherapy for bladder cancer seems to have promising results. Here, we evaluated the association between messenger RNA (mRNA) and protein levels and possible prognostic value of the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) immune checkpoint pathways during bladder carcinogenesis. Tumor samples were obtained from 155 patients (84 with muscle-invasive bladder cancer [MIBC], and 71 non-muscle-invasive bladder cancer [NMIBC]) and normal bladder tissue from 15 patients. We evaluated the mRNA expression of 3 genes in the PD-1 pathway (PD-1, PD-L1, and PD-L2) and 4 in the CTLA4 pathway (CTLA4, CD28, CD80, and CD86) in normal and tumoral human bladder samples by quantitative real-time reverse transcription polymerase chain reaction, with immunohistochemistry used to evaluate the protein expression of PD-1 and PD-L1 in tumor and immune cells. Results of molecular analyses were compared with survival analyses. As compared with normal bladder tissue, MIBC tissue showed PD-1, PD-L1, CTLA4, and CD80 overexpression (59.5%, 60.7%, 84.5%, and 92.9%, respectively), whereas overexpression was lower in NMIBC tissue (22.5%, 4.2%, 35.2%, and 46.5%, respectively). The results of reverse transcription polymerase chain reaction analysis were confirmed by immunohistochemistry, with a high correlation between mRNA and protein expression. On multivariate analyses, overexpression of the studied genes was not associated with prognosis in relapse or progression of NMIBC or in recurrence-free and overall survival of MIBC. The CTLA4 pathway appears to be deregulated along with the PD-1/PD-L1 pathway in bladder carcinogenesis, with good correlation between mRNA and protein expression endorsing the useful role of immune checkpoints, especially for a large subgroup of MIBC. Copyright © 2017 Elsevier Inc. All rights reserved.

[Expressions and significance of TLR2 and TLR4 in Kupffer cells of tree shrews chronically infected with hepatitis B virus].

PubMed

Ruan, Ping; Yang, Chun; Su, Jianjia; Ou, Chao; Cao, Ji; Luo, Chengpiao; Tang, Yanping; Qin, Hong; Sun, Wen; Li, Yuan

2014-07-01

To investigate the mRNA expression levels of Toll-like receptors 2 (TLR2) and TLR4 in Kupffer cells of tree shrews that were chronically infected with hepatitis B virus (HBV), and the effects of these receptors on the function of Kupffer cells. The tree shrews were divided into tree shrews proved with chronic HBV infection, tree shrews suspected with chronic HBV infection, and normal control tree shrews without hepatitis B vaccination. The samples of serum and liver biopsy were collected periodically, and the levels of HBV DNA in serum and liver tissues were detected by fluorescence-based quantitative real-time PCR (qRT-PCR). Meanwhile, Kupffer cells were isolated from the biopsied liver tissues, and then purified and primarily cultured. Afterwards, qRT-PCR was applied to detect the mRNA expression levels of TLR2, TLR4 and TNF-α in the Kupffer cells. Cell migration assay and lysosome-specific fluorescent probe were adopted to analyze the effects of TLR2 and TLR4 on the migration capacity of Kupffer cells and the quantity of lysosomes in these cells. The mRNA expression levels of TLR2 and TLR4 in tree shrews proved with chronic HBV infection were lower than those in the ones suspected with chronic HBV infection and normal controls without hepatitis B vaccination (P<0.05), and these expression levels were all negatively correlated with the level of HBV DNA in liver tissues of the animals (P<0.05), but were positively correlated with the number of migrated Kupffer cells, the density of lysosomes and the mRNA expression level of TNF-α (P<0.05). TLR2 and TLR4 in Kupffer cells may play important roles in the chronic process of hepatic pathological changes in tree shrews infected with HBV through their influence on the function of Kupffer cells.

Aberrant Epigenetic Alterations of Glutathione-S-Transferase P1 in Age-Related Nuclear Cataract.

PubMed

Chen, Jia; Zhou, Jing; Wu, Jian; Zhang, Guowei; Kang, Lihua; Ben, Jindong; Wang, Yong; Qin, Bai; Guan, Huaijin

2017-03-01

Oxidative damage of lens tissue contributes to the formation of age-related cataract. Pi-class glutathione-S-transferase (GSTP1) plays a role in the removal of oxidative adducts by transferring them to glutathione. To assess epigenetic regulation of GSTP1 and its potential role in age-related nuclear cataract (ARNC) pathogenesis, we evaluated GSTP1 mRNA expression, methylation, and chromatin modifications in lenses from ARNC patients. The mRNA and protein of lens GSTP1 were assayed by relative quantitative real-time polymerase chain reaction (qRT-PCR) and Western blots. Methylation of the GSTP1 promoter was determined by bisulfite genomic sequencing. Chromatin modification was detected by chromatin immunoprecipitation. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities were also assayed by enzyme-linked immunosorbent assay (ELISA)-like reaction. To assess the effect of DNA methylation on the mRNA expression of GSTP1, human lens epithelium HLE-B3 cells were treated with the demethylation compound 5-aza-dC, followed by qRT-PCR assay. GSTP1 mRNA and protein levels were significantly reduced in lens epithelium and cortex of ARNC cases versus age-matched controls. The changes corresponded to hypermethylation of the GSTP1 promoter CpG islands. The loss of GSTP1 mRNA and protein and the increased DNA promoter methylation might be correlated with the severity of the ARNC. ARNC lenses also had lower acetylation of histone proteins H3, H4, and lower methylation of H3K4, and higher methylation of H3K9. Histone modifications were not correlated with the severity of the ARNCs. DNMT and HDAC were elevated in lenses from ARNCs compared with controls. Demethylation treatment of HLE-B3 cells with 5-aza-dC enhanced the expression of GSTP1. Epigenetic alteration of GSTP1 regulates its expression in lens epithelial and cortical tissues. These changes likely contribute to the pathogenesis of ARNC.

[Expression of HIF1-alpha on myocardium and lung in rats model of asphyxia death].

PubMed

Zhang, Geng-qian; Zhou, Bin; Du, Bing; Yang, Zhi-hui; Zhang, Bei-lei; Zhu, Yin-hua; Zhang, Lin

2006-12-01

To investigate the expression of HIF1-alpha in heart and lung tissue died from asphyxia. The rats model of asphyxia death was constructed by hanging, different asphyxia groups and control group sets were made according the postmortem time (0,2,6,24 h), immunohistochemistry and half-quantitative RT-PCR methods were used to investigate expression of HIF1-alpha and mRNA changes on heart and lung tissue. The positive staining of HIF1-alpha could be observed in the myocardium and lung tissue. Significant differences were found between the groups of asphyxia and their corresponding control group. HIF1-alpha expression was found in all the asphyxia groups while it was only expressed in the control groups of 2 h, 6 h and 24 h. Nucleic positive staining could be detected in all the asphyxia groups but none was found in the control groups. RT-PCR showed that the expression of mRNA between 0 h asphyxia group and 0 h control group were equal in both cardic muscle and lung, but elevated expression in groups of 2,6,24h compared to their control groups. The nuclear positive staining of HIF1-alpha in heart and lung can be a special character of suffocation death.

Expression Signatures of Long Noncoding RNAs in Adolescent Idiopathic Scoliosis

PubMed Central

Wang, Liang; Yu, Bin; Zhuang, Qian-yu; Wang, Yi-Peng

2015-01-01

Purpose. Adolescent idiopathic scoliosis (AIS), the most common pediatric spinal deformity, is considered a complex genetic disease. Causing genes and pathogenesis of AIS are still unclear. This study was designed to identify differentially expressed long noncoding RNAs (lncRNAs) involving the pathogenesis of AIS. Methods. We first performed comprehensive screening of lncRNA and mRNA in AIS patients and healthy children using Agilent human lncRNA + mRNA Array V3.0 microarray. LncRNAs expression in different AIS patients was further evaluated using quantitative PCR. Results. A total of 139 lncRNAs and 546 mRNAs were differentially expressed between AIS patients and healthy control. GO and Pathway analysis showed that these mRNAs might be involved in bone mineralization, neuromuscular junction, skeletal system morphogenesis, nucleotide and nucleic acid metabolism, and regulation of signal pathway. Four lncRNAs (ENST00000440778.1, ENST00000602322.1, ENST00000414894.1, and TCONS_00028768) were differentially expressed between different patients when grouped according to age, height, classification, severity of scoliosis, and Risser grade. Conclusions. This study demonstrates the abnormal expression of lncRNAs and mRNAs in AIS, and the expression of some lncRNAs was related to clinical features. This study is helpful for further understanding of lncRNAs in pathogenesis, treatment, and prognosis of AIS. PMID:26421281

The expression and function of epithelial membrane protein 1 in laryngeal carcinoma.

PubMed

Li, Hong; Zhang, Xiaowen; Jiang, Xuejun; Ji, Xu

2017-01-01

In this study, we compared the expression of epithelial membrane protein 1 (EMP1) on the steady-state mRNA level (by quantitative real-time PCR) and on the protein level (by western immunoblot and immunohistochemistry) in 51 pairs of laryngeal carcinoma tissues and matched cancer-free peritumor tissues, and we analyzed the correlation between EMP1 expression and different clinicopathological factors. Furthermore, we ectopically expressed EMP1 in human laryngeal carcinoma Hep-2 cells and examined the effects on cell viability, apoptosis, colonogenicity, and motility, by MTT assay, flow cytometry, colony formation assay and Transwell migration assay, respectively. EMP1 expression (on both the mRNA and protein levels) was significantly lower in the cancer tissues than in matched peritumor tissues (P<0.05). In laryngeal cancers, the level of EMP1 protein was correlated with histological grade (P<0.05), but not with age, gender, clinical stage, cancer subtype or lymph node metastasis (P>0.05). Functionally, ectopic expression of EMP1 in Hep-2 cells significantly reduced cell viability, colony formation, and migration, but enhanced apoptosis. Therefore, EMP1 is a tumor suppressor in laryngeal carcinoma. Boosting EMP1 expression in laryngeal carcinoma initiates multiple anticancer phenotypes and thus presents a promising therapeutic strategy for laryngeal cancer.

Effect of transient receptor potential vanilloid 6 gene silencing on the expression of calcium transport genes in chicken osteoblasts.

PubMed

Zhang, Jie; Deng, Yifeng; Ma, Huijie; Hou, Jiafa; Zhou, ZhenLei

2015-03-01

Ca2+ plays a major role in the regulation of signal transduction. Transient receptor potential vanilloid 6 is a Ca2+-selective channel that serves as an important rate-limiting step in the facilitation of Ca2+ entry into cells, but little is known about the regulation of transient receptor potential vanilloid 6 in chickens. In this study, we evaluated the effects of transient receptor potential vanilloid 6 gene interference on the expression of calbindin-D28K, Na+/Ca2+ exchangers, and plasma membrane Ca2+ ATPase 1b to investigate the mechanism underlying the regulation of transient receptor potential vanilloid 6. Three hairpin siRNA expression vectors targeting transient receptor potential vanilloid 6 (pSIREN- transient receptor potential vanilloid 6) and a negative control (pSIREN-control) were constructed and transfected into chicken osteoblasts. The mRNA and protein expression levels were evaluated by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. The mRNA expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 45.7% (P<0.01) and 27.9% (P<0.01), respectively, 48 h after transfection with one of the three constructs (pSIREN- transient receptor potential vanilloid 6-3) compared with the level obtained in the untreated group. There was no significant difference in the mRNA expression levels of Na+/Ca2+ exchangers and plasma membrane Ca2+ ATPase 1b. The protein expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 40.2% (P<0.01) and 29.8% (P<0.01), respectively, 48 h after transfection with pSIREN-transient receptor potential vanilloid 6-3 compared with the level obtained in the untreated group. In conclusion, the vector-based transient receptor potential vanilloid 6-shRNA can efficiently suppress the mRNA and protein expression of transient receptor potential vanilloid 6 in chicken osteoblasts, and transient receptor potential vanilloid 6 regulates the expression of calbindin-D28K during Ca2+ transport. © 2015 Poultry Science Association Inc.

Proteoglycans in Leiomyoma and Normal Myometrium: Abundance, Steroid Hormone Control, and Implications for Pathophysiology.

PubMed

Barker, Nichole M; Carrino, David A; Caplan, Arnold I; Hurd, William W; Liu, James H; Tan, Huiqing; Mesiano, Sam

2016-03-01

Uterine leiomyoma are a common benign pelvic tumors composed of modified smooth muscle cells and a large amount of extracellular matrix (ECM). The proteoglycan composition of the leiomyoma ECM is thought to affect pathophysiology of the disease. To test this hypothesis, we examined the abundance (by immunoblotting) and expression (by quantitative real-time polymerase chain reaction) of the proteoglycans biglycan, decorin, and versican in leiomyoma and normal myometrium and determined whether expression is affected by steroid hormones and menstrual phase. Leiomyoma and normal myometrium were collected from women (n = 17) undergoing hysterectomy or myomectomy. In vitro studies were performed on immortalized leiomyoma (UtLM) and normal myometrial (hTERT-HM) cells with and without exposure to estradiol and progesterone. In leiomyoma tissue, abundance of decorin messenger RNA (mRNA) and protein were 2.6-fold and 1.4-fold lower, respectively, compared with normal myometrium. Abundance of versican mRNA was not different between matched samples, whereas versican protein was increased 1.8-fold in leiomyoma compared with myometrium. Decorin mRNA was 2.4-fold lower in secretory phase leiomyoma compared with proliferative phase tissue. In UtLM cells, progesterone decreased the abundance of decorin mRNA by 1.3-fold. Lower decorin expression in leiomyoma compared with myometrium may contribute to disease growth and progression. As decorin inhibits the activity of specific growth factors, its reduced level in the leiomyoma cell microenvironment may promote cell proliferation and ECM deposition. Our data suggest that decorin expression in leiomyoma is inhibited by progesterone, which may be a mechanism by which the ovarian steroids affect leiomyoma growth and disease progression. © The Author(s) 2015.

Biostimulation with diode laser positively regulates cementoblast functions, in vitro.

PubMed

Bozkurt, Serife Buket; Hakki, Erdogan E; Kayis, Seyit Ali; Dundar, Niyazi; Hakki, Sema S

2017-05-01

The aim of this study was to evaluate the effects of diode laser biostimulation on cementoblasts (OCCM.30). A total of 40 root plates were obtained from healthy third molar teeth and assigned to the following two groups: (1) control group and (2) laser-treated group. Root plates were placed into the cell culture inserts, and OCCM.30 cells were seeded onto root plates. Cells were irradiated with a low level of diode laser (power: 0.3 W in continuous wave, 60 s/cm 2 ). Proliferation and mineralized tissue-associated gene's and BMP's messenger RNA (mRNA) expressions of cementoblasts were evaluated. Total RNAs were isolated on day 3 and integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap), Type I collagen (Col1a1), osteoblastic transcription factor, runt-related transcription factor (Runx2), and Bone Morphogenetic Protein (BMP)-2, 3, 4, 6, and 7 mRNA expressions were determined using quantitative RT-PCR. von Kossa staining was performed to evaluate biomineralization of OCCM.30 cells. In the proliferation experiment, while there was no significant difference until 96 h, laser irradiation retarded the decrease in cell proliferation trend after 96 h compared to the untreated control group. Statistically significant increase in Ibsp, Bglap, and BMP-2,3,6,7 mRNA expressions were noted in the laser groups when compared to the untreated control group (p < 0.05). Laser irradiation induced mineralized nodule formation of cementoblasts. The results of this study reveal that the biostimulation setting of diode laser modulates the behavior of cementoblasts inducing mineralized tissue-associated gene's mRNA expressions and mineralization. Therefore, biostimulation can be used during regenerative periodontal therapies to trigger cells with periodontal attachment apparatus.

Prenatal Exposure to Bisphenol A Disrupts Naturally Occurring Bimodal DNA Methylation at Proximal Promoter of fggy, an Obesity-Relevant Gene Encoding a Carbohydrate Kinase, in Gonadal White Adipose Tissues of CD-1 Mice.

PubMed

Taylor, Julia A; Shioda, Keiko; Mitsunaga, Shino; Yawata, Shiomi; Angle, Brittany M; Nagel, Susan C; Vom Saal, Frederick S; Shioda, Toshi

2018-02-01

Exposure of mammalian fetuses to endocrine disruptors can increase the risk of adult-onset diseases. We previously showed that exposure of mouse fetuses to bisphenol A (BPA) caused adult-onset obesity. To examine roles of epigenetic changes in this delayed toxicity, we determined the effects of fetal mouse exposure to BPA on genome-wide DNA methylation and messenger RNA (mRNA) expression in gonadal white adipose tissues (WATs) by deep sequencing, bisulfite pyrosequencing, and real-time quantitative polymerase chain reaction. Pregnant CD-1 mice (F0) were dosed daily with 0, 5, or 500 μg/kg/d BPA during gestational days 9 to 18, and the weaned F1 animals were fed ad libitum with standard chow until they were euthanized at 19 weeks old. In the vehicle-exposed F1 animals, fggy promoter showed a clear bimodal pattern of very strong (55% to 95%) or very weak (5% to 30%) DNA methylation occurring at nearly equal incidence with no intermediate strength. Promoter hypermethylation completely suppressed mRNA expression. BPA exposure eliminated this naturally occurring dichotomy, shifting fggy promoter toward the hypomethylation state to release transcriptional suppression. The strength of Fggy mRNA expression significantly correlated with increased whole body weight and gonadal fat weight of males but not females. Bioinformatics studies showed that expression of Fggy mRNA is stronger in mouse WATs than in brown adipose tissues and enhanced in gonadal fat by diet-induced obesity. These observations suggest that prenatal exposure to BPA may disrupt the physiological bimodal nature of epigenetic regulation of fggy in mouse WATs, possibly contributing to the adult-onset obesity phenotype. Copyright © 2018 Endocrine Society.

Chronic nicotine-induced changes in gene expression of delta and kappa-opioid receptors and their endogenous ligands in the mesocorticolimbic system of the rat.

PubMed

Ugur, Muzeyyen; Kaya, Egemen; Gozen, Oguz; Koylu, Ersin O; Kanit, Lutfiye; Keser, Aysegul; Balkan, Burcu

2017-09-01

Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)-derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real-time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied. © 2017 Wiley Periodicals, Inc.

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.

Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording.

PubMed

Faragó, Nóra; Kocsis, Ágnes K; Lovas, Sándor; Molnár, Gábor; Boldog, Eszter; Rózsa, Márton; Szemenyei, Viktor; Vámos, Enikő; Nagy, Lajos I; Tamás, Gábor; Puskás, László G

2013-06-01

Whole-cell patch-clamp recording enables detection of electrophysiological signals from single neurons as well as harvesting of perisomatic RNA through the patch pipette for subsequent gene expression analysis. Amplification and profiling of RNA with traditional quantitative real-time PCR (qRT-PCR) do not provide exact quantitation due to experimental variation caused by the limited amount of nucleic acid in a single cell. Here we describe a protocol for quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using high-density nanocapillary digital PCR (dPCR). Expression of a known cell-type dependent marker gene (gabrd), as well as oxidative-stress related induction of hspb1 and hmox1 expression, was quantified in individual neurogliaform and pyramidal cells, respectively. The miRNA mir-132, which plays a role in neurodevelopment, was found to be equally expressed in three different types of neurons. The accuracy and sensitivity of this method were further validated using synthetic spike-in templates and by detecting genes with very low levels of expression.

Temporal profiles of age-dependent changes in cytokine mRNA expression and glial cell activation after status epilepticus in postnatal rat hippocampus.

PubMed

Järvelä, Juha T; Lopez-Picon, Francisco R; Plysjuk, Anna; Ruohonen, Saku; Holopainen, Irma E

2011-04-08

Status epilepticus (SE) is proposed to lead to an age-dependent acute activation of a repertoire of inflammatory processes, which may contribute to neuronal damage in the hippocampus. The extent and temporal profiles of activation of these processes are well known in the adult brain, but less so in the developing brain. We have now further elucidated to what extent inflammation is activated by SE by investigating the acute expression of several cytokines and subacute glial reactivity in the postnatal rat hippocampus. SE was induced by an intraperitoneal (i.p.) injection of kainic acid (KA) in 9- and 21-day-old (P9 and P21) rats. The mRNA expression of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), matrix metalloproteinase-9 (MMP-9), glial-derived neurotrophic factor (GDNF), interferon gamma (IFN-γ), and transforming growth factor-beta 1 (TGF-β1) were measured from 4 h up to 3 days after KA injection with real-time quantitative PCR (qPCR). IL-1β protein expression was studied with ELISA, GFAP expression with western blotting, and microglial and astrocyte morphology with immunohistochemistry 3 days after SE. SE increased mRNA expression of IL-1β, TNF-α and IL-10 mRNA in hippocampus of both P9 and P21 rats, their induction being more rapid and pronounced in P21 than in P9 rats. MMP-9 expression was augmented similarly in both age groups and GDNF expression augmented only in P21 rats, whereas neither IFN-γ nor TGF-β1 expression was induced in either age group. Microglia and astrocytes exhibited activated morphology in the hippocampus of P21 rats, but not in P9 rats 3 d after SE. Microglial activation was most pronounced in the CA1 region and also detected in the basomedial amygdala. Our results suggest that SE provokes an age-specific cytokine expression in the acute phase, and age-specific glial cell activation in the subacute phase as verified now in the postnatal rat hippocampus. In the juvenile hippocampus, transient increases in cytokine mRNA expression after SE, in contrast to prolonged glial reactivity and region-specific microglial activity after SE, suggest that the inflammatory response is changed from a fulminant and general initial phase to a more moderate and specific subacute response.

Equine insulin receptor and insulin-like growth factor-1 receptor expression in digital lamellar tissue and insulin target tissues.

PubMed

Kullmann, A; Weber, P S; Bishop, J B; Roux, T M; Norby, B; Burns, T A; McCutcheon, L J; Belknap, J K; Geor, R J

2016-09-01

Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horses and examine the effect of dietary nonstructural carbohydrate (NSC) on mRNA expression of these receptors in LT, skeletal muscle, liver and two adipose tissue (AT) depots of lean and obese ponies. In vivo experiment. Lamellar tissue samples were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for receptor mRNA expression (n = 8) and immunoblotting for protein expression (n = 3). Archived LT, skeletal muscle, liver and AT from lean and obese mixed-breed ponies fed either a low (~7% NSC as dry matter; 5 lean, 5 obese) or high NSC diet (~42% NSC as dry matter; 6 lean, 6 obese) for 7 days were evaluated by RT-qPCR to determine the effect of body condition and diet on expression of the receptors in different tissues. Significance was set at P≤0.05. Lamellar tissue expresses both InsR isoforms, IGF-1R and Hybrid. LT IGF-1R gene expression was greater than either InsR isoform and InsR-A expression was greater than InsR-B (P≤0.05). Obesity significantly lowered IGF-1R, InsR-A and InsR-B mRNA expression in LT and InsR-A in tailhead AT. High NSC diet lowered expression of all three receptor types in liver; IGF-1R and InsR-A in LT and InsR-A in tailhead AT. Lamellar tissue expresses IGF-1R, InsR isoforms and Hybrids. The functional characteristics of these receptors and their role in endocrinopathic laminitis warrants further investigation. © 2015 EVJ Ltd.

Suppression of microRNA-135b-5p protects against myocardial ischemia/reperfusion injury by activating JAK2/STAT3 signaling pathway in mice during sevoflurane anesthesia.

PubMed

Xie, Xiao-Juan; Fan, Dong-Mei; Xi, Kai; Chen, Ya-Wei; Qi, Peng-Wei; Li, Qian-Hui; Fang, Liang; Ma, Li-Gang

2017-06-30

The study aims to explore the effects of miR-135b-5p on myocardial ischemia/reperfusion (I/R) injuries by regulating Janus protein tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription (STAT) signaling pathway by mediating inhalation anesthesia with sevoflurane. A sum of 120 healthy Wistar male mice was assigned into six groups. Left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVSF) were detected. Cardiomyocyte apoptosis was determined by terminal dexynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay. MiR-135b-5p expression, mRNA and protein expression of p-STAT3, p-JAK2, STAT3, JAK2, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein B (Bax) were detected by quantitative real-time PCR (qRT-PCR) and Western blotting. Target relationship between miR-135b-5p and JAK2 was confirmed by dual-luciferase reporter assay. The other five groups exhibited increased cardiomyocyte necrosis, apoptosis, miR-135b-5p and Bax expression, mRNA expression of JAK2 and STAT3, and protein expression of p-STAT3 and p-JAK2 compared with the sham group, but showed decreased LVEF, LVFS, and Bcl-2 expression. Compared with the model and AG490 + Sevo groups, the Sevo, inhibitor + Sevo and inhibitor + AG490 + Sevo groups displayed reduced cardiomyocyte necrosis, apoptosis, miR-135b-5p and Bax expression, but displayed elevated mRNA expression of JAK2 and STAT3, protein expression of p-STAT3 and p-JAK2, LVEF, LVFS and Bcl-2 expression. Compared with the Sevo and inhibitor + AG490 + Sevo groups, the AG490 + Sevo group showed decreased LVEF, LVFS, Bcl-2 expression, mRNA expressions of JAK2 and STAT3, and protein expressions of p-STAT3 and p-JAK2, but increased cardiomyocyte necrosis, apoptosis, and Bax expressions. MiR-135b-5p negatively targetted JAK2. Inhibition of miR-135b-5p can protect against myocardial I/R injury by activating JAK2/STAT3 signaling pathway through mediation of inhalation anesthesia with sevoflurane. © 2017 The Author(s).

Notch-1 regulates pulmonary neuroendocrine cell differentiation in cell lines and in transgenic mice.

PubMed

Shan, Lin; Aster, Jon C; Sklar, Jeffrey; Sunday, Mary E

2007-02-01

The notch gene family encodes transmembrane receptors that regulate cell differentiation by interacting with surface ligands on adjacent cells. Previously, we demonstrated that tumor necrosis factor-alpha (TNF) induces neuroendocrine (NE) cell differentiation in H82, but not H526, undifferentiated small cell lung carcinoma lines. We now test the hypothesis that TNF mediates NE cell differentiation in part by altering Notch gene expression. First, using RT-PCR, we determined that TNF treatment of H82, but not H526, transiently decreases notch-1 mRNA in parallel with induction of gene expression for the NE-specific marker DOPA decarboxylase (DDC). Second, we treated H82 and H526 with notch-1 antisense vs. sense oligodeoxynucleotides. Using quantitative RT-PCR and Western analyses we demonstrate that DDC mRNA and protein are increased in H82 by notch-1 antisense, whereas notch-1 mRNA and activated Notch-1 protein are decreased. mRNA for Hes1, a transcription factor downstream from activated Notch, is also decreased by Notch-1 antisense in H82 but not H526. After 7 days of Notch-1 antisense treatment, neural cell adhesion molecule (NCAM) immunoreactivity is induced in H82 but not H526. Third, we generated transgenic mice bearing notch-1 driven by the neural/NE-specific calcitonin promoter, which express activated Notch-1 in developing lung epithelium. Newborn NotchCal mouse lungs have high levels of hes1 mRNA, reflecting increased activated Notch, compared with wild-type. NotchCal lungs have decreased CGRP-positive NE cells, decreased protein gene product 9.5 (PGP9.5)-positive NE cells, and decreased gastrin-releasing peptide (GRP), CGRP, and DDC mRNA levels compared with normal littermates. Cumulatively, these observations provide further support for a role for Notch-1 signaling in regulating pulmonary NE cell differentiation.