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

PubMed Central

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

2015-01-01

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

Expression of beta 3-adrenoceptor mRNA in rat tissues.

PubMed

Evans, B A; Papaioannou, M; Bonazzi, V R; Summers, R J

1996-01-01

1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical beta-adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by beta 3-AR.

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

PubMed Central

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

2006-01-01

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

Effects of Thermal Stress on the mRNA Expression of SOD, HSP90, and HSP70 in the Spotted Sea Bass ( Lateolabrax maculatus)

NASA Astrophysics Data System (ADS)

Shin, Moon-Kyeong; Park, Ho-Ra; Yeo, Won-Jun; Han, Kyung-Nam

2018-03-01

The aim of this study was to elucidate the molecular mechanisms underlying the thermal stress response in the spotted sea bass ( Lateolabrax maculatus). Spotted sea basses were exposed to 4 different water temperatures (20, 22, 24, and 28°C) in increasing increments of 2°C/h from 18°C (control) for different time periods (0, 6, 12, 24, 48, 72, and 96 h). Subsequently, 3 tissues (liver, muscle, and gill) were isolated, and the levels of SOD, HSP90, and HSP70 mRNA were assessed. SOD mRNA expression was maintained at baseline levels of control fish at all water temperatures in the liver, while muscle and gill tissue showed an increase followed by a decrease over each certain time with higher water temperature. HSP90 mRNA expression increased in the liver at ≤ 24°C over time, but maintained baseline expression at 28°C. In muscle, HSP90 mRNA expression gradually increased at all water temperatures, but increased and then decreased at ≥ 24°C in gill tissue. HSP70 mRNA expression exhibited an increase and then a decrease in liver tissue at 28°C, but mainly showed similar expression patterns to HSP90 in all tissues. These results suggest the activity of a defense mechanism using SOD, HSP90, and HSP70 in the spotted sea bass upon rapid increases in water temperature, where the expression of these genes indicated differences between tissues in the extent of the defense mechanisms. Also, these results indicate that high water temperature and long-term thermal stress exposure can inhibit physiological defense mechanisms.

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

Expression of Leukemia/Lymphoma-Related Factor (LRF/POKEMON) in Human Breast Carcinoma and Other Cancers

PubMed Central

Aggarwal, Anshu; Hunter, William J.; Aggarwal, Himanshu; Silva, Edibaldo D.; Davey, Mary S.; Murphy, Richard F.; Agrawal, Devendra K.

2010-01-01

The POK family of proteins plays an important role in not only embryonic development and cell differentiation, but also in oncogenesis. Leukemia/lymphoma-related factor (LRF) belongs to the POK family of transcriptional repressors and is also known as POK erythroid myeloid ontogenic factor (POKEMON), which binds to short transcripts of HIV-1 (FBI-1) and TTF-1 interacting peptide (TIP21). Its oncogenic role is known only in lymphoma, non-small cell lung carcinoma, and malignant gliomas. The functional expression of LRF in human breast carcinoma has not yet been confirmed. The aim of this study was to investigate and compare the expression of LRF in human breast cancer tissues and other human tumors. The expression of LRF mRNA transcripts and protein was observed in twenty human benign and malignant breast biopsy tissues. Expression of LRF was observed in several formalin-fixed tissues by immunohistochemistry and immunofluorescence. All malignant breast tissues expressed mRNA transcripts and protein for LRF. However, 40% and 15% benign breast biopsy tissues expressed LRF mRNA transcripts and protein, respectively. The overall expression of LRF mRNA transcripts and total protein was significantly more in malignant breast tissues than the benign breast tissues. LRF expression was also observed in the nuclei of human colon, renal, lung, hepatocellular carcinomas and thymoma tumor cells. In general, a significantly higher expression of LRF was seen in malignant tissues than in the corresponding benign or normal tissue. Further studies are warranted to determine the malignant role of LRF in human breast carcinoma. PMID:20471975

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

[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.

Promoter methylation, mRNA expression of goat tumor‑associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

PubMed

Quan, Zifang; Ye, Ni; Hao, Zhongxiang; Wen, Caifang; Liao, Hong; Zhang, Manli; Luo, Lu; Cao, Sanjie; Wen, Xintian; Wu, Rui; Yan, Qigui

2015-10-01

The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor‑associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation‑specific polymerase chain reaction and SYBR Green reverse transcription‑quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6‑methylguanine‑DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto‑oncogenes KRAS, NRAS and C‑myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C‑myc increased by 2.9‑fold and CEBPA by 2‑fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C‑myc was the only gene identified to be methylated amongst proto‑oncogenes. The methylation expression rate of C‑myc was 87.5% in nasal tumor tissues and 15% in normal nasal epithelial tissues. The methylation expression rate of CEBPA was 100% in nasal tumor tissues and 40% in normal nasal epithelial tissues. The methylation expression rate of the EGFR gene was ~80% in the two tissues. In summary, the present study identified abnormal methylation of the C‑myc, CEBPA, GADD45G and THBS1 genes in nasal tumor tissues. The expression levels of DNMT1, C‑myc and CEBPA were upregulated and the expression of P53 and THBSI were downregulated in nasal tumor tissues, with a significant difference between the two groups (P<0.05). Therefore, it is suggested that these six genes may be used as diagnostic marker candidates for ENT. The results may serve as a foundation for screening of tumor‑specific markers for early diagnosis of ENT and further investigate the epigenetic mechanisms of enzootic nasal tumor virus (ENTV)‑induced nasal epithelium cell carcinoma.

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

PubMed

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

2016-12-01

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

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.

Growth hormone mRNA in mammary gland tumors of dogs and cats.

PubMed Central

Mol, J A; van Garderen, E; Selman, P J; Wolfswinkel, J; Rijinberk, A; Rutteman, G R

1995-01-01

We have shown recently that in the dog progestin administration results in mammary production of immunoreactive growth hormone (GH). At present we demonstrate the expression of the gene encoding GH in the mammary gland of dogs and cats using reverse-transcriptase PCR. GH mRNA was found in the great majority of normal mammary tissues as well as benign and malignant mammary tumors of the dog and was associated with the presence of immunoreactive GH in cryostat sections. The mammary PCR product proved to be identical to that of the pituitary. The highest expression levels were found after prolonged treatment with progestins. In carcinomas GH mRNA was also found in progesterone receptor-negative tissue samples, indicating that after malignant transformation GH gene expression may become progestin independent. GH mRNA was also present in mammary tissues of cats with progestin-induced fibroadenomatous changes. It is concluded that GH gene expression occurs in normal, hyperplastic, and neoplastic mammary tissue of the dog. The expression in normal tissue is stimulated by progestins and might mediate the progestin-stimulated development of canine mammary tumors. The demonstration of progestin-stimulated GH expression in mammary tissue of cats indicates that the phenomenon is more generalized among mammals. Images PMID:7738169

Comparison of the QuantiGene 2.0 Assay and Real-Time RT-PCR in the Detection of p53 Isoform mRNA Expression in Formalin-Fixed Paraffin-Embedded Tissues- A Preliminary Study

PubMed Central

Morten, Brianna C.; Scott, Rodney J.; Avery-Kiejda, Kelly A.

2016-01-01

p53 is expressed as multiple smaller isoforms whose functions in cancer are not well understood. The p53 isoforms demonstrate abnormal expression in different cancers, suggesting they are important in modulating the function of full-length p53 (FLp53). The quantification of relative mRNA expression has routinely been performed using real-time PCR (qPCR). However, there are serious limitations when detecting p53 isoforms using this method, particularly for formalin-fixed paraffin-embedded (FFPE) tissues. The use of FFPE tumours would be advantageous to correlate expression of p53 isoforms with important clinical features of cancer. One alternative method of RNA detection is the hybridization-based QuantiGene 2.0 Assay, which has been shown to be advantageous for the detection of RNA from FFPE tissues. In this pilot study, we compared the QuantiGene 2.0 Assay to qPCR for the detection of FLp53 and its isoform Δ40p53 in matched fresh frozen (FF) and FFPE breast tumours. FLp53 mRNA expression was detected using qPCR in FF and FFPE tissues, but Δ40p53 mRNA was only detectable in FF tissues. Similar results were obtained for the QuantiGene 2.0 Assay. FLp53 relative mRNA expression was shown to be strongly correlated between the two methods (R2 = 0.9927, p = 0.0031) in FF tissues, however Δ40p53 was not (R2 = 0.4429, p = 0.3345). When comparing the different methods for the detection of FLp53 mRNA from FFPE and FF samples, no correlation (R2 = 0.0002, p = 0.9863) was shown using the QuantiGene 2.0 Assay, and in contrast, the level of expression was highly correlated between the two tissues using qPCR (R2 = 0.8753, p = 0.0644). These results suggest that both the QuantiGene 2.0 Assay and qPCR methods are inadequate for the quantification of Δ40p53 mRNA in FFPE tissues. Therefore, alternative methods of RNA detection and quantification are required to study the relative expression of Δ40p53 in FFPE samples. PMID:27832134

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.

Effect of peritoneal dialysis on expression of vascular endothelial growth factor, basic fibroblast growth factor and endostatin of the peritoneum in peritoneal dialysis patients.

PubMed

Gao, Dan; Zhao, Zhan-Zheng; Liang, Xian-Hui; Li, Yan; Cao, Ying; Liu, Zhang-Suo

2011-11-01

The aim of this study is to investigate the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endostatin (ES) in human peritoneum and investigate the relationship between them and peritoneum neoangiogensis in the patients with uraemia and peritoneal dialysis (PD). Peritoneal biopsies were obtained from normal subjects (n = 8), uraemic predialysis patients (n = 12) and PD patients (n = 10). The mRNA expression of VEGF, bFGF and ES in peritoneal tissues were measured through real-time polymerase chain reaction. The protein expression of VEGF, bFGF and ES in peritoneal tissues were determined through western blot. Microvessel density (MVD) of peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody. The mRNA and protein of VEGF, bFGF and ES were expressed in all peritoneal samples. Compared with the normal control group, the mRNA and protein expression of VEGF and bFGF in peritoneal tissues were all significantly upregulated in the uraemic predialysis and PD group (all P < 0.05). Compared with the normal control group, the protein expression of ES were significantly upregulated in the uraemic predialysis and PD group (all (P < 0.05), but the mRNA expression of ES did not have obvious differences in the uraemic predialysis and PD group as compared to the normal control group (P > 0.05). MVD of peritoneal tissue were increased in the uraemic predialysis and PD group compared with the normal group (all P < 0.05). A significant positive correlation was found between VEGF mRNA expression and MVD, bFGF mRNA expression and MVD. The mRNA expression of VEGF and bFGF, the protein expression of VEGF, bFGF, and ES and microvessel density (MVD) are increased both in the uraemic predialysis and PD patients. These results show that uraemia circumstances and non-physiological compatibility of peritoneal dialysis solution might increase VEGF, bFGF and ES expression and MVD, which might participate in the increment of the peritoneum neoangiogensis and ultrafiltration failure in PD patients. © 2011 The Authors. Nephrology © 2011 Asian Pacific Society of Nephrology.

Early postnatal maternal separation causes alterations in the expression of β3-adrenergic receptor in rat adipose tissue suggesting long-term influence on obesity

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

Miki, Takanori, E-mail: mikit@med.kagawa-u.ac.jp; Liu, Jun-Qian; Ohta, Ken-ichi

Highlights: •High-fat diet intake following maternal separation did not cause body weight gain. •However, levels of metabolism-related molecules in adipose tissue were altered. •Increased levels of prohibitin mRNA in white fat were observed. •Attenuated levels of β3-adrenergic receptor mRNA were observed in brown fat. •Such alterations in adipose tissue may contribute to obesity later in life. -- Abstract: The effects of early postnatal maternal deprivation on the biological characteristics of the adipose tissue later in life were investigated in the present study. Sprague–Dawley rats were classified as either maternal deprivation (MD) or mother-reared control (MRC) groups. MD was achieved bymore » separating the rat pups from their mothers for 3 h each day during the 10–15 postnatal days. mRNA levels of mitochondrial uncoupling protein 1 (UCP-1), β3-adrenergic receptor (β3-AR), and prohibitin (PHB) in the brown and white adipose tissue were determined using real-time RT-PCR analysis. UCP-1, which is mediated through β3-AR, is closely involved in the energy metabolism and expenditure. PHB is highly expressed in the proliferating tissues/cells. At 10 weeks of age, the body weight of the MRC and MD rats was similar. However, the levels of the key molecules in the adipose tissue were substantially altered. There was a significant increase in the expression of PHB mRNA in the white adipose tissue, while the β3-AR mRNA expression decreased significantly, and the UCP-1 mRNA expression remained unchanged in the brown adipose tissue. Given that these molecules influence the mitochondrial metabolism, our study indicates that early postnatal maternal deprivation can influence the fate of adipose tissue proliferation, presumably leading to obesity later in life.« less

Expression of early growth response factor-1 in rats with cerulein-induced acute pancreatitis and its significance

PubMed Central

Gong, Lan-Bo; He, Li; Liu, Yang; Chen, Xue-Qing; Jiang, Bo

2005-01-01

AIM: To observe the expressions of early growth response factor-1 (Egr-1) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance. METHODS: A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesin-administered group were used for comparison. RESULTS: After the stimulation of a large dose of cerulein, the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1 protein reached its peak 2 h after the stimulation. Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein, which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF. CONCLUSION: Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF. PMID:16124058

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.

Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles (Trachemys scripta) using an external RNA control for normalization.

PubMed

Stecyk, Jonathan A W; Couturier, Christine S; Fagernes, Cathrine E; Ellefsen, Stian; Nilsson, Göran E

2012-03-01

The mRNA expression of heat-shock protein 90 (HSP90) and heat-shock cognate 70 (HSC70) was examined in cardiac chambers and telencephalon of warm- (21°C) and cold-acclimated (5°C) turtles (Trachemys scripta) exposed to normoxia, prolonged anoxia or anoxia followed by reoxygenation. Additionally, the suitability of total RNA as well as mRNA from β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclophilin A (PPIA) for normalizing gene expression data was assessed, as compared to the use of an external RNA control. Measurements of HSP90 and HSC70 mRNA expression revealed that anoxia and reoxygenation have tissue- and gene-specific effects. By and large, the alterations support previous investigations on HSP protein abundance in the anoxic turtle heart and brain, as well as the hypothesized roles of HSP90 and HSC70 during stress and non-stress conditions. However, more prominent was a substantially increased HSP90 and HSC70 mRNA expression in the cardiac chambers with cold acclimation. The finding provides support for the notion that cold temperature induces a number of adaptations in tissues of anoxia-tolerant vertebrates that precondition them for winter anoxia. β-actin, GAPDH and PPIA mRNA expression and total RNA also varied with oxygenation state and acclimation temperature in a tissue- and gene-specific manner, as well as among tissue types, thus disqualifying them as suitable for real-time RT-PCR normalization. Thus, the present data highlights the advantages of normalizing real-time RT-PCR data to an external RNA control, an approach that also allows inter-tissue and potentially inter-species comparisons of target gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Expression and significance of CD44s, CD44v6, and nm23 mRNA in human cancer.

PubMed

Liu, Yong-Jun; Yan, Pei-Song; Li, Jun; Jia, Jing-Fen

2005-11-14

To investigate the relationship between the expression levels of nm23 mRNA, CD44s, and CD44v6, and oncogenesis, development and metastasis of human gastric adenocarcinoma, colorectal adenocarcinoma, intraductal carcinoma of breast, and lung cancer. Using tissue microarray by immuhistochemical (IHC) staining and in situ hybridization (ISH), we examined the expression levels of nm23 mRNA, CD44s, and CD44v6 in 62 specimens of human gastric adenocarcinoma and 62 specimens of colorectal adenocarcinoma; the expression of CD44s and CD44v6 in 120 specimens of intraductal carcinoma of breast and 20 specimens of normal breast tissue; the expression of nm23 mRNA in 72 specimens of human lung cancer and 23 specimens of normal tissue adjacent to cancer. The expression of nm23 mRNA in the tissues of gastric and colorectal adenocarcinoma was not significantly different from that in the normal tissues adjacent to cancer (P>0.05), and was not associated with the invasion of tumor and the pathology grade of adenocarcinoma (P>0.05). However, the expression of nm23 mRNA was correlated negatively to the lymph node metastasis of gastric and colorectal adenocarcinoma (r = -0.49, P<0.01; r = -4.93, P<0.01). The expression of CD44s in the tissues of gastric and colorectal adenocarcinoma was significantly different from that in the normal tissues adjacent to cancer (P<0.05; P<0.01). CD44v6 was expressed in the tissues of gastric and colorectal adenocarcinoma only, the expression of CD44v6 was significantly associated with the lymph node metastasis, invasion and pathological grade of the tumor (r = 0.47, P<0.01; r = 5.04, P<0.01). CD44s and CD44v6 were expressed in intraductal carcinoma of breast, the expression of CD44s and CD44v6 was significantly associated with lymph node metastases and invasion (P<0.01). However, neither of them was expressed in the normal breast tissue. In addition, the expression of CD44v6 was closely related to the degree of cell differentiation of intraductal carcinoma of breast (c2 = 5.68, P<0.05). The expressional level of nm23 mRNA was closely related to the degree of cell differentiation (P<0.05) and lymph node metastasis (P<0.01), but the expression of nm23 gene was not related to sex, age, and type of histological classification (P>0.05). Patients with overexpression of CD44s and CD44v6 and low expression of nm23 mRNA have a higher lymph node metastatic rate and invasion. In addition, overexpression of CD44v6 is closely related to the degree of cell differentiation. Detection of the three genes is able to provide a reliable index to evaluate the invasion and metastasis of tumor cells.

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

PubMed

Jiang, Guangli; Qi, Yuxia

2015-05-01

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

Cytoskeleton structure and total methylation of mouse cardiac and lung tissue during space flight.

PubMed

Ogneva, Irina V; Loktev, Sergey S; Sychev, Vladimir N

2018-01-01

The purpose of this work was to evaluate the protein and mRNA expression levels of multiple cytoskeletal proteins in the cardiac and lung tissue of mice that were euthanized onboard the United States Orbital Segment of the International Space Station 37 days after the start of the SpaceX-4 mission (September 2014, USA). The results showed no changes in the cytoskeletal protein content in the cardiac and lung tissue of the mice, but there were significant changes in the mRNA expression levels of the associated genes, which may be due to an increase in total genome methylation. The mRNA expression levels of DNA methylases, the cytosine demethylases Tet1 and Tet3, histone acetylase and histone deacetylase did not change, and the mRNA expression level of cytosine demethylase Tet2 was significantly decreased.

Cytoskeleton structure and total methylation of mouse cardiac and lung tissue during space flight

PubMed Central

Loktev, Sergey S.; Sychev, Vladimir N.

2018-01-01

The purpose of this work was to evaluate the protein and mRNA expression levels of multiple cytoskeletal proteins in the cardiac and lung tissue of mice that were euthanized onboard the United States Orbital Segment of the International Space Station 37 days after the start of the SpaceX-4 mission (September 2014, USA). The results showed no changes in the cytoskeletal protein content in the cardiac and lung tissue of the mice, but there were significant changes in the mRNA expression levels of the associated genes, which may be due to an increase in total genome methylation. The mRNA expression levels of DNA methylases, the cytosine demethylases Tet1 and Tet3, histone acetylase and histone deacetylase did not change, and the mRNA expression level of cytosine demethylase Tet2 was significantly decreased. PMID:29768411

[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.

The expression characteristics of mt-ND2 gene in chicken.

PubMed

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

2016-09-01

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

Antimicrobial Barrier of an in vitro Oral Epithelial Model

PubMed Central

Kimball, Janet R.; Nittayananta, Wipawee; Klausner, Mitchell; Chung, Whasun O.; Dale, Beverly A.

2008-01-01

Objective Oral epithelia function as a microbial barrier and are actively involved in recognizing and responding to bacteria. Our goal was to examine a tissue engineered model of buccal epithelium for its response to oral bacteria and proinflammatory cytokines and compare the tissue responses with those of a submerged monolayer cell culture. Design The tissue model was characterized for keratin and β-defensin expression. Altered expression of β-defensins was evaluated by RT-PCR after exposure of the apical surface to oral bacteria and after exposure to TNF-α in the medium. These were compared to the response in traditional submerged oral epithelial cell culture. Results The buccal model showed expression of differentiation specific keratin 13, hBD1 and hBD3 in the upper half of the tissue; hBD2 was not detected. hBD1 mRNA was constitutively expressed, while hBD2 mRNA increased 2-fold after exposure of the apical surface to three oral bacteria tested and hBD3 mRNA increased in response to the non-pathogenic bacteria tested. In contrast, hBD2 mRNA increased 3–600 fold in response to bacteria in submerged cell culture. HBD2 mRNA increased over 100 fold in response to TNF-α in the tissue model and 50 fold in submerged cell culture. Thus, the tissue model is capable of upregulating hBD2, however, the minimal response to bacteria suggests that the tissue has an effective antimicrobial barrier due to its morphology, differentiation, and defensin expression. Conclusions The oral mucosal model is differentiated, expresses hBD1 and hBD3, and has an intact surface with a functional antimicrobial barrier. PMID:16815238

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.

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

PubMed

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

2002-01-01

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

Lysophosphatidic acid receptor mRNA levels in heart and white adipose tissue are associated with obesity in mice and humans.

PubMed

Brown, Amy; Hossain, Intekhab; Perez, Lester J; Nzirorera, Carine; Tozer, Kathleen; D'Souza, Kenneth; Trivedi, Purvi C; Aguiar, Christie; Yip, Alexandra M; Shea, Jennifer; Brunt, Keith R; Legare, Jean-Francois; Hassan, Ansar; Pulinilkunnil, Thomas; Kienesberger, Petra C

2017-01-01

Lysophosphatidic acid (LPA) receptor signaling has been implicated in cardiovascular and obesity-related metabolic disease. However, the distribution and regulation of LPA receptors in the myocardium and adipose tissue remain unclear. This study aimed to characterize the mRNA expression of LPA receptors (LPA1-6) in the murine and human myocardium and adipose tissue, and its regulation in response to obesity. LPA receptor mRNA levels were determined by qPCR in i) heart ventricles, isolated cardiomyocytes, and perigonadal adipose tissue from chow or high fat-high sucrose (HFHS)-fed male C57BL/6 mice, ii) 3T3-L1 adipocytes and HL-1 cardiomyocytes under conditions mimicking gluco/lipotoxicity, and iii) human atrial and subcutaneous adipose tissue from non-obese, pre-obese, and obese cardiac surgery patients. LPA1-6 were expressed in myocardium and white adipose tissue from mice and humans, except for LPA3, which was undetectable in murine adipocytes and human adipose tissue. Obesity was associated with increased LPA4, LPA5 and/or LPA6 levels in mice ventricles and cardiomyocytes, HL-1 cells exposed to high palmitate, and human atrial tissue. LPA4 and LPA5 mRNA levels in human atrial tissue correlated with measures of obesity. LPA5 mRNA levels were increased in HFHS-fed mice and insulin resistant adipocytes, yet were reduced in adipose tissue from obese patients. LPA4, LPA5, and LPA6 mRNA levels in human adipose tissue were negatively associated with measures of obesity and cardiac surgery outcomes. This study suggests that obesity leads to marked changes in LPA receptor expression in the murine and human heart and white adipose tissue that may alter LPA receptor signaling during obesity.

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

PubMed

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

2003-04-01

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

Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection.

PubMed

Zernova, Olga; Zhong, Wei; Zhang, Xing-Hai; Widholm, Jack

2008-11-01

This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3 kb promoter drives tissue culture specific expression and if it is strong enough to drive hpt (hygromycin phosphotransferase) gene expression at a level sufficient to allow selection of transformed soybean embryogenic culture lines. A number of transformed cell lines were selected showing that the promoter was strong enough. Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants. However, when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA. Embryogenic tissue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant. Friable callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant. Thus the ASA2 promoter is strong enough to drive sufficient hpt expression in soybean embryogenic cultures for hygromycin selection and only very low levels of expression were found in most plant tissues with none in mature seeds.

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

PubMed

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

2016-06-01

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

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 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.

Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats.

PubMed

Komar, Carolyn M; Curry, Thomas E

2002-05-01

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.

Mesenchymal stem cells cannot affect mRNA expression of toll-like receptors in different tissues during sepsis.

PubMed

Pedrazza, Leonardo; Pereira, Talita Carneiro Brandão; Abujamra, Ana Lucia; Nunes, Fernanda Bordignon; Bogo, Maurício Reis; de Oliveira, Jarbas Rodrigues

2017-07-01

Experimental animal models and human clinical studies support a crucial role for TLRs in infectious diseases. The aim of this study was to test the ability of MSCs, which have immunomodulatory effects, of altering the mRNA expression of toll-like receptors during a experimental model of sepsis in different tissues. Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 10 6 cells/animal). Lungs, cortex, kidney, liver and colon tissue were dissected after 12 h of sepsis induction and TLR2/3/4/9 mRNA were evaluated by RT-qPCR. We observed a decrease of TLR2 and 9 mRNA expression in the liver of the sepsis group, while TLR3 was decreased in the lung and liver. No change was found between the sepsis group and the sepsis + MSC group. In this model of experimental sepsis the MSCs were unable to modify the mRNA expression of the different toll-like receptors evaluated.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2016-08-05

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

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

PubMed

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

2015-01-01

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

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.

Expression of preoperative KISS1 gene in tumor tissue with epithelial ovarian cancer and its prognostic value.

PubMed

Cao, Fang; Chen, Liping; Liu, Manhua; Lin, Weiwei; Ji, Jinlong; You, Jun; Qiao, Fenghai; Liu, Hongbin

2016-11-01

Our study aimed to elucidate the role of Kisspeptin (KISS1) in tumor tissues of patients with epithelial ovarian cancer (EOC) and investigate the prognostic value of this biomarker.Forty EOC patients and 20 uterine fibroids female patients with healthy ovaries undergoing cytoreductive surgery between January 2010 and January 2014 in our hospital were enrolled in this study. KISS1 expression in tumor and normal tissues was detected. Correlations between clinic-pathologic variables and KISS1 expression in EOC tissues and the prognostic value of KISS1 for overall survival were evaluated.During the follow-up of 11.2 to 62.1 months, the overall survival rate and mean survival time were 28.9% (11/38) and 38.35 ± 2.84 months. Preoperative KISS1 mRNA was higher in tumor tissue than in normal tissue (P <0.001), and it was associated with histologic grade of tumor, surgical FIGO stage, metastasis, and residual tumor size (all P <0.05). Multivariate survival analysis indicated significant influence of residual tumor size (HR = 2.357, P = 0.039) and preoperative KISS1 mRNA (HR = 0.0001, P <0.001) on mean survival time. Patients with low KISS1 mRNA expression had shorter survival time than those with high expression (P = 0.001).Preoperative KISS1 mRNA was a potential prognostic biomarker for EOC, and high preoperative KISS1 expression indicated a favorable prognosis.

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes.

PubMed

Bruzzoni-Giovanelli, Heriberto; Fernandez, Plinio; Veiga, Lucía; Podgorniak, Marie-Pierre; Powell, Darren J; Candeias, Marco M; Mourah, Samia; Calvo, Fabien; Marín, Mónica

2010-02-09

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.

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

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.

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

PubMed Central

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

2010-01-01

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

Plasma concentrations and subcutaneous adipose tissue mRNA expression of clusterin in obesity and type 2 diabetes mellitus: the effect of short-term hyperinsulinemia, very-low-calorie diet and bariatric surgery.

PubMed

Kloučková, J; Lacinová, Z; Kaválková, P; Trachta, P; Kasalický, M; Haluzíková, D; Mráz, M; Haluzík, M

2016-07-18

Clusterin is a heterodimeric glycoprotein with wide range of functions. To further explore its possible regulatory role in energy homeostasis and in adipose tissue, we measured plasma clusterin and its mRNA expression in subcutaneous adipose tissue (SCAT) of 15 healthy lean women, 15 obese women (OB) and 15 obese women with type 2 diabetes mellitus (T2DM) who underwent a 2-week very low-calorie diet (VLCD), 10 obese women without T2DM who underwent laparoscopic sleeve gastrectomy (LSG) and 8 patients with T2DM, 8 patients with impaired glucose tolerance (IGT) and 8 normoglycemic patients who underwent hyperinsulinemic euglycemic clamp (HEC). VLCD decreased plasma clusterin in OB but not in T2DM patients while LSG and HEC had no effect. Clusterin mRNA expression in SCAT at baseline was increased in OB and T2DM patients compared with controls. Clusterin mRNA expression decreased 6 months after LSG and remained decreased 12 months after LSG. mRNA expression of clusterin was elevated at the end of HEC compared with baseline only in normoglycemic but not in IGT or T2DM patients. In summary, our data suggest a possible local regulatory role for clusterin in the adipose tissue rather than its systemic involvement in the regulation of energy homeostasis.

Tilmicosin does not inhibit interleukin-8 gene expression in the bovine lung experimentally infected with Mannheimia (Pasteurella) haemolytica.

PubMed Central

Goubau, S; Morck, D W; Buret, A

2000-01-01

The expression of the interleukin-8 (IL-8) gene was examined by in situ hybridization in lung tissues from calves experimentally infected with Mannheimia (Pasteurella) haemolytica and treated with tilmicosin. Interleukin-8 mRNA expression was detected in alveolar areas, particularly along interlobular septa, in the lumen, and in the epithelial cells of some bronchioles. In lesional lung tissues from animals that had received tilmicosin, we found large areas with limited inflammation. There was no staining for IL-8 mRNA in these areas. In contrast, in strongly inflamed areas, the same patterns and intensities of staining for IL-8 mRNA were detected in tilmicosin- and sham-treated animals. We conclude that tilmicosin does not affect the expression of IL-8 mRNA in tissue showing microscopic signs of inflammation. Together with previous reports, this supports the view that the pro-apoptotic properties of tilmicosin on neutrophils do not compromise the host defense mechanisms required to control the infection. Images Figure 1. PMID:11041503

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.

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.

[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.

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.

Regulation of LPS-induced tissue factor expression in human monocytic THP-1 cells by curcumin

USDA-ARS?s Scientific Manuscript database

Tissue factor (TF) is a transmembrane receptor, which initiates thrombotic episodes associated with various diseases. In addition to membrane-bound TF, we have discovered an alternatively spliced form of human TF mRNA. It was later confirmed that this form of TF mRNA expresses a soluble protein circ...

Prognostic value of decreased expression of RBM4 in human gastric cancer.

PubMed

Yong, Hongmei; Zhu, Huijun; Zhang, Shu; Zhao, Wei; Wang, Wei; Chen, Chen; Ding, Guipeng; Zhu, Lun; Zhu, Ziyuan; Liu, Huaidong; Zhang, Yongjie; Wen, Jinbo; Kang, Xing; Zhu, Jin; Feng, Zhenqing; Liu, Baorui

2016-06-21

RNA-binding motif 4 (RBM4) is a multifunctional protein that participates in regulating alternative splicing and mRNA translation. Its reduced expression has been associated with poor overall survival in lung cancer, breast cancer and ovarian cancer. We assessed RBM4 protein expression levels with immunohistochemistry in tissue microarrays containing malignant gastric cancer tissues and benign tissues from 813 patients. We also examined the expression levels of RBM4 mRNA in twenty-five paired gastric cancer samples and adjacent noncancerous tissues. Both RBM4 protein and mRNA expression levels were significantly lower in gastric cancer tissues compared with the adjacent noncancerous tissues. There was a significant association between reduced RBM4 protein expression and differentiation (P < 0.001), lymph node metastasis (P = 0.026), TNM state (P = 0.014) and distant metastasis (P = 0.036). Patients with reduced RBM4 expression (P < 0.001, CI = 0.315-0.710) and TNM stage III and IV (P < 0.001, CI = 4.757-11.166) had a poor overall survival. These findings suggest that RBM4 is a new biomarker in gastric cancer, as the reduced expression of this protein is correlated with poor differentiation, lymph node status and distant metastasis. Further, lower RBM4 expression is an independent prognostic marker for gastric cancer.

Ovarian Expression and Regulation of the Stromelysins During the Periovulatory Period in the Human and the Rat1

PubMed Central

McCord, Lauren A.; Li, Feixue; Rosewell, Katherine L.; Brännström, Mats; Curry, Thomas E.

2011-01-01

ABSTRACT The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization. PMID:22116802

Ovarian expression and regulation of the stromelysins during the periovulatory period in the human and the rat.

PubMed

McCord, Lauren A; Li, Feixue; Rosewell, Katherine L; Brännström, Mats; Curry, Thomas E

2012-03-01

The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.

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.

Effect of supplementing a fibrous diet with a xylanase and β-glucanase blend on growth performance, intestinal glucose uptake, and transport-associated gene expression in growing pigs.

PubMed

Agyekum, A K; Sands, J S; Regassa, A; Kiarie, E; Weihrauch, D; Kim, W K; Nyachoti, C M

2015-07-01

The present study evaluated supplemental carbohydrase effect on performance, intestinal nutrient uptake, and transporter mRNA expressions in growing pigs offered a high-fiber diet manufactured with distillers dried grains with solubles (DDGS). Twenty-four pigs (22.4 ± 0.7 kg BW) were randomly assigned to 1of 3 nutritionally adequate diets (8 pigs per diet) based on corn and soybean meal (SBM) with either 0 (control) or 30% DDGS (high fiber [HF]). The third diet was supplemented with a xylanase and β-glucanase blend (XB) in addition to the 30% DDGS (HF+XB). Parameters determined were ADFI, ADG, G:F, plasma glucose and plasma urea nitrogen (PUN) concentrations, jejunal tissue electrophysiological properties, and mRNA expressions of the sodium-dependent glucose transport 1 (SGLT1) and cationic AA transporter, bo,+AT, in the jejunal and ileal tissues. In addition, mRNA expressions of the short-chain fatty acid transporters, monocarboxylate transporter 1 (MCT1) and sodium-coupled monocarboxylate transporter, and mucin genes were quantified in the ileum. Feed intake, plasma glucose, and jejunal tissue electrophysiological properties were not affected (P > 0.05) by diet. However, control-fed pigs had superior growth rate and feed efficiency and higher PUN (P < 0.05) than HF- and HF+XB-fed pigs. The HF diet increased (P < 0.05) SGLT1 mRNA expression in the jejunum and decreased (P < 0.05) bo,+ mRNA expression in the ileum. The XB supplementation also increased bo,+ mRNA expression in the ileum relative to HF-fed pigs. Additionally, MCT1 mRNA expression was greater (P < 0.05) in the ileum of the HF- and HF+XB-fed pigs. In the present study, XB supplementation influenced nutrient transporter mRNA expression, although it was not accompanied by improved pig performance.

RANKL/RANK/OPG cytokine receptor system: mRNA expression pattern in BPH, primary and metastatic prostate cancer disease.

PubMed

Christoph, Frank; König, Frank; Lebentrau, Steffen; Jandrig, Burkhard; Krause, Hans; Strenziok, Romy; Schostak, Martin

2018-02-01

The cytokine system RANKL (receptor activator of NF-κB ligand), its receptor RANK and the antagonist OPG (osteoprotegerin) play a critical role in bone turnover. Our investigation was conducted to describe the gene expression at primary tumour site in prostate cancer patients and correlate the results with Gleason Score and PSA level. Seventy-one samples were obtained from prostate cancer patients at the time of radical prostatectomy and palliative prostate resection (n = 71). Patients with benign prostate hyperplasia served as controls (n = 60). We performed real-time RT-PCR after microdissection of the samples. The mRNA expression of RANK was highest in tumour tissue from patients with bone metastases (p < 0.001) as compared to BPH or locally confined tumours, also shown in clinical subgroups distinguished by Gleason Score (< 7 or ≥ 7, p = 0.028) or PSA level (< 10 or ≥ 10 µg/l, p = 0.004). RANKL and OPG mRNA expression was higher in tumour tissue from patients with metastatic compared to local disease. The RANKL/OPG ratio was low in normal prostate tissue and high tumours with bone metastases (p < 0.05). Expression of all three cytokines was high in BPH tissue but did not exceed as much as in the tumour tissue. We demonstrated that RANK, RANKL and OPG are directly expressed by prostate cancer cells at the primary tumour site and showed a clear correlation with Gleason Score, serum PSA level and advanced disease. In BPH, mRNA expression is also detectable, but RANK expression does not exceed as much as compared to tumour tissue.

Lysophosphatidic acid receptor mRNA levels in heart and white adipose tissue are associated with obesity in mice and humans

PubMed Central

Perez, Lester J.; Nzirorera, Carine; Tozer, Kathleen; D’Souza, Kenneth; Trivedi, Purvi C.; Aguiar, Christie; Yip, Alexandra M.; Shea, Jennifer; Brunt, Keith R.; Legare, Jean-Francois; Hassan, Ansar; Pulinilkunnil, Thomas

2017-01-01

Background Lysophosphatidic acid (LPA) receptor signaling has been implicated in cardiovascular and obesity-related metabolic disease. However, the distribution and regulation of LPA receptors in the myocardium and adipose tissue remain unclear. Objectives This study aimed to characterize the mRNA expression of LPA receptors (LPA1-6) in the murine and human myocardium and adipose tissue, and its regulation in response to obesity. Methods LPA receptor mRNA levels were determined by qPCR in i) heart ventricles, isolated cardiomyocytes, and perigonadal adipose tissue from chow or high fat-high sucrose (HFHS)-fed male C57BL/6 mice, ii) 3T3-L1 adipocytes and HL-1 cardiomyocytes under conditions mimicking gluco/lipotoxicity, and iii) human atrial and subcutaneous adipose tissue from non-obese, pre-obese, and obese cardiac surgery patients. Results LPA1-6 were expressed in myocardium and white adipose tissue from mice and humans, except for LPA3, which was undetectable in murine adipocytes and human adipose tissue. Obesity was associated with increased LPA4, LPA5 and/or LPA6 levels in mice ventricles and cardiomyocytes, HL-1 cells exposed to high palmitate, and human atrial tissue. LPA4 and LPA5 mRNA levels in human atrial tissue correlated with measures of obesity. LPA5 mRNA levels were increased in HFHS-fed mice and insulin resistant adipocytes, yet were reduced in adipose tissue from obese patients. LPA4, LPA5, and LPA6 mRNA levels in human adipose tissue were negatively associated with measures of obesity and cardiac surgery outcomes. This study suggests that obesity leads to marked changes in LPA receptor expression in the murine and human heart and white adipose tissue that may alter LPA receptor signaling during obesity. PMID:29236751

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.

[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.

Progesterone-dependent Regulation of Endometrial Cannabinoid Receptor Type 1 (CB1-R) Expression is Disrupted in Women with Endometriosis and in Isolated Stromal Cells Exposed to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)

PubMed Central

Resuehr, David; Glore, Dana R.; Taylor, Hugh S.; Bruner-Tran, Kaylon L.; Osteen, Kevin G.

2012-01-01

Objective To examine the differentiation-related expression of CB1-R mRNA and protein in endometrial tissue obtained from women with and without endometriosis and to determine the impact of acute TCDD exposure on CB1-R gene expression in isolated endometrial stromal cells. Design Laboratory-based study Setting University-affiliated medical center Patients Women with and without endometriosis undergoing volunteer endometrial biopsies after informed consent. Interventions None Main Outcome Measures Analysis of in vivo CB1-R mRNA and protein expression in human endometrial tissues and mRNA expression in isolated stromal cells following exposure to TCDD or a progesterone receptor antagonist (Onapristone). Results CB1-R mRNA and protein expression was highest during the progesterone-dominated secretory phase in control women, while expression was minimal in endometrial tissues acquired from women with endometriosis, regardless of the cycle phase. Although progesterone was found to induce CB1-R mRNA expression in endometrial stromal cells from control donors, steroid-induced expression of this gene was inhibited by co-treatment with either TCDD or Onapristone. Conclusions Our studies reveal a role for the anti-inflammatory actions of progesterone in regulating endometrial cannabinoid signaling, which is disrupted in women with endometriosis. Significantly, our studies demonstrate, for the first time, that acute TCDD exposure disrupts cannabinoid signaling in the human endometrium. PMID:22789143

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.

Correlation of HIWI and HILI Expression with Cancer Stem Cell Markers in Colorectal Cancer.

PubMed

Litwin, Monika; Dubis, Joanna; Arczyńska, Katarzyna; Piotrowska, Aleksandra; Frydlewicz, Anna; Karczewski, Maciej; Dzięgiel, Piotr; Witkiewicz, Wojciech

2015-06-01

Cancer stem cells (CSCs) constitute a sub-population of tumor cells that possess stem cell properties, such as self-renewal and the ability of differentiation. The presence of CSCs is associated with metastatic potential, treatment resistance and poor patient prognosis. Recently, aberrant expression of P-element induced wimpy testis proteins-PIWI (HIWI and HILI) has been identified in various types of tumors. The aim of the study was to evaluate the clinical significance of the HIWI and HILI expression and its relationship with cancer stem cells markers in 72 patients with colorectal carcinoma (CRC). The expression level of HIWI and HILI and cancer stem cells markers in paired cancerous and non-cancerous tissues was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. Immunohistochemistry was performed to confirm the observed changes on mRNA level and detect tissue localization of PIWI proteins. Significantly higher mRNA levels of HIWI and decreased HILI mRNA were measured in colorectal cancer tissues compared to corresponding non-cancerous samples. The changes in HIWI mRNA level in cancer tissues were correlated with OCT4 expression. Positive correlations between HILI level and SOX2 were also observed in cancerous tissues. Our results indicate a reciprocal regulation between HIWI, HILI and some CSCs markers in colorectal cancer. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

[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.

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.

Several genes encoding ribosomal proteins are over-expressed in prostate-cancer cell lines: confirmation of L7a and L37 over-expression in prostate-cancer tissue samples.

PubMed

Vaarala, M H; Porvari, K S; Kyllönen, A P; Mustonen, M V; Lukkarinen, O; Vihko, P T

1998-09-25

A cDNA library specific for mRNA over-expressed in prostate cancer was generated by subtractive hybridization of transcripts originating from prostatic hyperplasia and cancer tissues. cDNA encoding ribosomal proteins L4, L5, L7a, L23a, L30, L37, S14 and S18 was found to be present among 100 analyzed clones. Levels of ribosomal mRNA were significantly higher at least in one of the prostate-cancer cell lines, LNCaP, DU-145 and PC-3, than in hyperplastic tissue, as determined by slot-blot hybridization. Furthermore, L23a- and S14-transcript levels were significantly elevated in PC-3 cells as compared with those in the normal prostate epithelial cell line PrEC. Generally, dramatic changes in the mRNA content of the ribosomal proteins were not detected, the most evident over-expression being that of L37 mRNA, which was 3.4 times more abundant in LNCaP cells than in hyperplastic prostate tissue. The over-expression of L7a and L37 mRNA was confirmed in prostate-cancer tissue samples by in situ hybridization. Elevated cancer-related expression of L4 and L30 has not been reported, but levels of the other ribosomal proteins are known to be increased in several types of cancers. These results therefore suggest that prostate cancer is comparable with other types of cancers, in that a larger pool of some ribosomal proteins is gained during the transformation process, by an unknown mechanism.

Comparative study of Hsp27, GSK3β, Wnt1 and PRDX3 in Hirschsprung's disease.

PubMed

Gao, Hong; Liu, Xiaomei; Chen, Dong; Lv, Liangying; Wu, Mei; Mi, Jie; Wang, Weilin

2014-06-01

Hirschsprung's disease (HSCR) is a developmental disorder of the enteric nervous system characterized by aganglionosis in distal gut. In this study, we used two-dimensional gel electrophoresis (2-DE) technology coupled with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis to identify differentially expressed proteins in the aganglionic (stenotic) and ganglionic (normal) colon segment tissues from patients with HSCR. We identified 15 proteins with different expression levels between the stenotic and the normal colon segment tissues from patients with HSCR. Nine proteins were upregulated and six proteins downregulated in the stenotic colon segment tissues compared to the normal colon segment tissues. Based on the biological functions, we selected the Hsp27 upregulated proteins and the PRDX3 downregulated proteins to confirm their expression in 20 patients. The protein and mRNA expressions of Hsp27 were statistically higher in the stenotic colon segment tissues than in the normal colon segment tissues, whereas the protein and mRNA expressions of PRDX3 were statistically lower in the stenotic colon segment tissues than in the normal colon segment tissues. These findings of changes in mRNA and protein in tissues from patients with HSCR provide information which may be helpful in understanding the pathomechanism that is implicated in the disease. © 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.

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.

Metformin increases the novel adipokine adipolin/CTRP12: role of the AMPK pathway.

PubMed

Tan, Bee K; Chen, Jing; Adya, Raghu; Ramanjaneya, Manjunath; Patel, Vanlata; Randeva, Harpal S

2013-11-01

Adipolin is a novel adipokine with anti-inflammatory and glucose-lowering properties. Lower levels of adipolin are found in obese and diabetic mice. Polycystic ovary syndrome (PCOS) is a pro-inflammatory state associated with obesity and diabetes. To date, there are no human studies on adipolin. Therefore, we measured serum (ELISA) and adipose tissue adipolin mRNA expression (RT-PCR) and protein concentrations (western blotting) in PCOS and control subjects. We also investigated the ex vivo effect of glucose and metformin on adipolin protein production in human subcutaneous adipose tissue explants. We report novel data that serum and subcutaneous adipose tissue adipolin mRNA expression and protein concentrations were significantly lower in women with PCOS compared with control subjects. Furthermore, Spearman's rank analysis showed that serum adipolin concentrations were significantly negatively correlated with BMI, waist-to-hip ratio, and glucose (P<0.05). However, when subjected to multiple regression analysis, none of these variables were predictive of serum adipolin concentrations (P>0.05). Also, subcutaneous adipose tissue adipolin mRNA expression and protein concentrations were only significantly negatively correlated with glucose (P<0.05). No significant correlations were found with omental adipose tissue adipolin mRNA expression and protein concentrations (P>0.05). Moreover, glucose profoundly reduced and metformin significantly increased adipolin protein production in human adipose tissue explants respectively. Importantly, metformin's effects appear to be via the AMP-activated protein kinase signaling pathway.

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

ATP-binding cassette (ABC) transporters in caprine preantral follicles: gene and protein expression.

PubMed

Guerreiro, Denise Damasceno; de Lima, Laritza Ferreira; Mbemya, Gildas Tetaping; Maside, Carolina Mielgo; Miranda, André Marrocos; Tavares, Kaio César Simiano; Alves, Benner Geraldo; Faustino, Luciana Rocha; Smitz, Johan; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

2018-06-01

The multidrug resistance proteins ABCB1, ABCC2 and ABCG2 are an energy-dependent efflux pump that functions in systemic detoxification processes. Physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier and various stem cells, until now, these pumps were not identified in goat ovarian tissue. Therefore, the aim of this study is to analyze ABCB1, ABCC2, and ABCG2 mRNA and protein expression in goat preantral follicles. Fragments (3 × 3 × 1 mm) from five pairs of ovary (n = 10) obtained from five goat were collected and immediately submitted to qPCR, Western blot, and immunofluorescence assay for mRNA detection and identification and localization of the ABC transporters, respectively. mRNA for ABCB1, ABCC2, and ABCG2 and the presence of their proteins were observed on ovarian tissue samples. Positive marks were observed for the three transport proteins in all follicular categories studied. However, the marks were primarily localized in the oocyte of primordial, transition and primary follicle categories. In conclusion, goat ovarian tissue expresses mRNA for the ABCB1, ABCC2 and ABCG2 transporters and the expression of these proteins in the preantral follicles is a follicle-dependent stage.

Effects of heat stress on respiratory burst, oxidative damage and SERPINH1 (HSP47) mRNA expression in rainbow trout Oncorhynchus mykiss.

PubMed

Wang, Yanni; Liu, Zhe; Li, Zhen; Shi, Haina; Kang, Yujun; Wang, Jianfu; Huang, Jinqiang; Jiang, Li

2016-04-01

For rainbow trout Oncorhynchus mykiss, high temperature is a major abiotic stress that limits its growth and productivity. In this study, spleen macrophage respiratory burst (RB), serum superoxide dismutase (SOD), serum malondialdehyde (MDA) and mRNA expression of the SERPINH1 (HSP47) gene in different tissues (liver, spleen, head kidney and heart) were measured in unstressed (18 °C) and heat-stressed (25 °C) fish. Spleen macrophage RB activity, serum SOD activity and MDA content all increased significantly (P < 0.05) during heat shock, and peaked at 8, 12 and 4 h, respectively. SERPINH1 mRNA expression responded in a time- and tissue-specific manner to heat stress, which was mainly reflected in the significant up-regulation in all tissues (P < 0.05) and greater expression in the liver than the other tissues (P < 0.05). During the heat-shock recovery period, the MDA content returned to the unstressed level. These results indicate that heat shock causes cell injury, induces oxidative damage and promotes SERPINH1 mRNA expression, which plays an important protective function during heat stress in O. mykiss. In practice, close attention should be given to temperature changes in O. mykiss production to reduce the effects of high temperature.

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

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.

Identification of tumor-restricted antigens NY-BR-1, SCP-1, and a new cancer/testis-like antigen NW-BR-3 by serological screening of a testicular library with breast cancer serum.

PubMed

Jäger, Dirk; Unkelbach, Marc; Frei, Claudia; Bert, Florian; Scanlan, Matthew J; Jäger, Elke; Old, Lloyd J; Chen, Yao-Tseng; Knuth, Alexander

2002-06-28

Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of several categories of new tumor antigens. We analyzed a testicular cDNA expression library with serum obtained from a breast cancer patient and isolated 13 genes designated NW-BR-1 through NW-BR-13. Of these, 3 showed tumor-restricted expression (NW-BR-1, -2 and -3), the others being expressed ubiquitously. NW-BR-3, representing 9 of 24 primary clones, showed tissue-restricted mRNA expression, being expressed in normal testis but not in 15 other normal tissues tested by Northern blotting. RT-PCR analysis showed strong NW-BR-3 expression in normal testis, weak expression in brain, kidney, trachea, uterus and normal prostate, and was negative in liver, heart, lung, colon, small intestine, bone marrow, breast, thymus, muscle, spleen, and stomach. NW-BR-3 mRNA expression was found in different tumor tissues and tumor cell lines by RT-PCR, thus showing a 'cancer/testis' (CT)-like mRNA expression pattern. NW-BR-3 shares 71% nucleotide and amino acid homology to a mouse gene cloned from mouse testicular tissue. Based on the mRNA expression pattern, NW-BR-3 represents a new candidate target gene for cancer immunotherapy. NW-BR-1 and NW-BR-2 also showed tumor-restricted mRNA expression. NW-BR-1 is a partial clone of the breast differentiation antigen NY-BR-1 previously identified by SEREX. NY-BR-1 is expressed in normal breast, testis and 80% of breast cancers. NW-BR-2 is identical to the CT antigen SCP-1, initially isolated by SEREX analysis of renal cancer. This study provides further evidence that SEREX is a powerful tool to identify new tumor antigens potentially relevant for immunotherapy approaches.

Toll-Like Receptor and Accessory Molecule mRNA Expression in Humans and Mice as Well as in Murine Autoimmunity, Transient Inflammation, and Progressive Fibrosis

PubMed Central

Ramaiah, Santhosh Kumar Vankayala; Günthner, Roman; Lech, Maciej; Anders, Hans-Joachim

2013-01-01

The cell type-, organ-, and species-specific expression of the Toll-like receptors (TLRs) are well described, but little is known about the respective expression profiles of their accessory molecules. We therefore determined the mRNA expression levels of LBP, MD2, CD36, CD14, granulin, HMGB1, LL37, GRP94, UNC93b1, TRIL, PRAT4A, AP3B1, AEP and the respective TLRs in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. In addition, the expression profiles in transient tissue inflammation upon renal ischemia-reperfusion injury, in spleens and kidneys from mice with lupus-like systemic autoimmunity, and in progressive tissue fibrosis upon unilateral ureteral obstruction were studied. Several TLR co-factors were specifically regulated during the different phases of these disease entities, suggesting a functional involvement in the disease process. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to TLR-mediated innate immunity, which seems to be involved in the tissue injury phase, in the phase of tissue regeneration, and in progressive tissue remodelling. PMID:23803655

Expression of ceramide-metabolising enzymes in subcutaneous and intra-abdominal human adipose tissue

PubMed Central

2012-01-01

Background Inflammation and increased ceramide concentrations characterise adipose tissue of obese women with high liver fat content compared to equally obese women with normal liver fat content. The present study characterises enzymes involved in ceramide metabolism in subcutaneous and intra-abdominal adipose tissue. Methods Pathways leading to increased ceramide concentrations in inflamed versus non-inflamed adipose tissue were investigated by quantifying expression levels of key enzymes involved in ceramide metabolism. Sphingomyelinases (sphingomyelin phosphodiesterases SMPD1-3) were investigated further using immunohistochemistry to establish their location within adipose tissue, and their mRNA expression levels were determined in subcutaneous and intra-abdominal adipose tissue from both non-obese and obese subject. Results Gene expression levels of sphingomyelinases, enzymes that hydrolyse sphingomyelin to ceramide, rather than enzymes involved in de novo ceramide synthesis, were higher in inflamed compared to non-inflamed adipose tissue of obese women (with high and normal liver fat contents respectively). Sphingomyelinases were localised to both macrophages and adipocytes, but also to blood vessels and to extracellular regions surrounding vessels within adipose tissue. Expression levels of SMPD3 mRNA correlated significantly with concentrations of different ceramides and sphingomyelins. In both non-obese and obese subjects SMPD3 mRNA levels were higher in the more inflamed intra-abdominal compared to the subcutaneous adipose tissue depot. Conclusions Generation of ceramides within adipose tissue as a result of sphingomyelinase action may contribute to inflammation in human adipose tissue. PMID:22974251

The expression of ADAM12 (meltrin alpha) in human giant cell tumours of bone.

PubMed

Tian, B L; Wen, J M; Zhang, M; Xie, D; Xu, R B; Luo, C J

2002-12-01

To examine the expression of ADAM12 (meltrin alpha), a member of the disintegrin and metalloprotease (ADAM) family, in human giant cell tumours of the bone, skeletal muscle tissue from human embryos, and human adult skeletal muscle tissue. ADAM12 mRNA was detected by reverse transcription polymerase chain reaction and in situ hybridisation. ADAM12 mRNA was detected in 14 of the 20 giant cell tumours of bone and in three of the six tumour cell cultures. The expression of ADAM12 in cells cultured from the tumour was linked to the presence of multinucleated giant cells. ADAM12 mRNA could not be detected in the five adult skeletal muscle tissue samples, although it was found in the two embryonic skeletal muscle tissue samples. ADAM12 mRNA was localised to the cytoplasm of multinucleated giant cells and some mononuclear stromal cells. These results indicate that multinucleated giant cells are formed by the cell fusion of mononuclear stromal cells in giant cell tumours of bone and that ADAM12 is involved in the cell fusion process.

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

PubMed

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

2015-11-01

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

The effect of dietary carbohydrate on genes for fatty acid synthase and inflammatory cytokines in adipose tissues from lean and obese subjects.

PubMed

Hudgins, Lisa C; Baday, Aline; Hellerstein, Marc K; Parker, Thomas S; Levine, Daniel M; Seidman, Cynthia E; Neese, Richard A; Tremaroli, Jolanta D; Hirsch, Jules

2008-04-01

Hepatic de novo lipogenesis (DNL) is markedly stimulated in humans by low-fat diets enriched in simple sugars. However, the dietary responsiveness of the key enzyme controlling DNL in human adipose tissue, fatty acid synthase (FAS), is uncertain. Adipose tissue mRNA for FAS is increased in lean and obese subjects when hepatic DNL is elevated by a eucaloric, low-fat, high-sugar diet. Twelve lean and seven obese volunteers were given two eucaloric diets (10% vs. 30% fat; 75% vs. 55% carbohydrate; sugar/starch 60/40) each for 2 weeks by a random-order cross-over design. FAS mRNA in abdominal and gluteal adipose tissues was compared to hepatic DNL measured in serum by isotopic and nonisotopic methods. Adipose tissue mRNA for tumor necrosis factor-alpha and IL-6, which are inflammatory cytokines that modulate DNL, was also assayed. The low-fat high-sugar diet induced a 4-fold increase in maximum hepatic DNL (P<.001) but only a 1.3-fold increase in adipose tissue FAS mRNA (P=.029) and no change in cytokine mRNA. There was a borderline significant positive correlation between changes in FAS mRNA and hepatic DNL (P=.039). Compared to lean subjects, obese subjects had lower levels of FAS mRNA and higher levels of cytokine mRNA (P<.001). The results suggest that key elements of human adipose tissue DNL are less responsive to dietary carbohydrate than is hepatic DNL and may be regulated by diet-independent factors. Irrespective of diet, there is reduced expression of the FAS gene and increased expression of cytokine genes in adipose tissues of obese subjects.

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.

Low-level laser therapy (LLLT) reduces the COX-2 mRNA expression in both subplantar and total brain tissues in the model of peripheral inflammation induced by administration of carrageenan.

PubMed

Prianti, Antonio Carlos Guimarães; Silva, José Antonio; Dos Santos, Regiane Feliciano; Rosseti, Isabela Bueno; Costa, Maricilia Silva

2014-07-01

In the classical model of edema formation and hyperalgesia induced by carrageenan administration in rat paw, the increase in prostaglandin E2 (PGE2) production in the central nervous system (CNS) contributes to the severity of the inflammatory and pain responses. Prostaglandins are generated by the cyclooxygenase (COX). There are two distinct COX isoforms, COX-1 and COX-2. In inflammatory tissues, COX-2 is greatly expressed producing proinflammatory prostaglandins (PGs). Low-level laser therapy (LLLT) has been used in the treatment of inflammatory pathologies, reducing both pain and acute inflammatory process. Herein we studied the effect of LLLT on both COX-2 and COX-1 messenger RNA (mRNA) expression in either subplantar or brain tissues taken from rats treated with carrageenan. The experiment was designed as follows: A1 (saline), A2 (carrageenan-0.5 mg/paw), A3 (carrageenan-0.5 mg/paw + LLLT), A4 (carrageenan-1.0 mg/paw), and A5 (carrageenan-1.0 mg/paw + LLLT). Animals from the A3 and A5 groups were irradiated at 1 h after carrageenan administration, using a diode laser with an output power of 30 mW and a wavelength of 660 nm. The laser beam covered an area of 0.785 cm(2), resulting in an energy dosage of 7.5 J/cm(2). Both COX-2 and COX-1 mRNAs were measured by RT-PCR. Six hours after carrageenan administration, COX-2 mRNA expression was significantly increased both in the subplantar (2.2-4.1-fold) and total brain (8.65-13.79-fold) tissues. COX-1 mRNA expression was not changed. LLLT (7.5 J/cm(2)) reduced significantly the COX-2 mRNA expression both in the subplantar (~2.5-fold) and brain (4.84-9.67-fold) tissues. The results show that LLLT is able to reduce COX-2 mRNA expression. It is possible that the mechanism of LLLT decreasing hyperalgesia is also related to its effect in reducing the COX-2 expression in the CNS.

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

PubMed

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

1998-01-01

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

L-DOPA decarboxylase mRNA expression is associated with tumor stage and size in head and neck squamous cell carcinoma: a retrospective cohort study

PubMed Central

2012-01-01

Background Head and neck squamous cell carcinoma (HNSCC) represents one of the most commonly diagnosed malignancies worldwide. The DDC gene encodes L-DOPA decarboxylase, an enzyme catalyzing the decarboxylation of L-DOPA to dopamine. We have recently shown that DDC mRNA is a significant predictor of patients’ prognosis in colorectal adenocarcinoma and prostate cancer. The aim of the current study was to analyze the DDC mRNA expression in HNSCC patients. Methods 53 malignant tumors were resected from the larynx, pharynx, tongue, buccal mucosa, parotid glands, and nasal cavity, as well as from 34 adjacent non-cancerous tissues of HNSCC patients, and were homogenized. Total RNA was isolated and converted into first-strand cDNA. An ultrasensitive real-time PCR method based on the SYBR Green chemistry was used for DDC mRNA quantification in head and neck tissue specimens. Relative quantification was performed using the comparative Ct (2-ddCt) method. Results DDC mRNA levels were lower in squamous cell carcinomas (SCCs) of the larynx and tongue than in adjacent non-cancerous tissue specimens. Furthermore, low DDC mRNA expression was noticed in laryngeal and tongue tumors of advanced TNM stage or bigger size, compared to early-stage or smaller tumors, respectively. No statistically significant differences were observed between SCCs resected from pharynx, buccal mucosa, or nasal cavity, and their normal counterparts. Conclusion This is the first study examining the DDC mRNA expression in HNSCC. According to our results, DDC mRNA expression may constitute a potential prognostic biomarker in tongue and/or larynx SCCs, which principally represent the overwhelming majority of HNSCC cases. PMID:23083099

L-DOPA decarboxylase mRNA expression is associated with tumor stage and size in head and neck squamous cell carcinoma: a retrospective cohort study.

PubMed

Geomela, Panagiota-Aikaterini; Kontos, Christos K; Yiotakis, Ioannis; Fragoulis, Emmanuel G; Scorilas, Andreas

2012-10-20

Head and neck squamous cell carcinoma (HNSCC) represents one of the most commonly diagnosed malignancies worldwide. The DDC gene encodes L-DOPA decarboxylase, an enzyme catalyzing the decarboxylation of L-DOPA to dopamine. We have recently shown that DDC mRNA is a significant predictor of patients' prognosis in colorectal adenocarcinoma and prostate cancer. The aim of the current study was to analyze the DDC mRNA expression in HNSCC patients. 53 malignant tumors were resected from the larynx, pharynx, tongue, buccal mucosa, parotid glands, and nasal cavity, as well as from 34 adjacent non-cancerous tissues of HNSCC patients, and were homogenized. Total RNA was isolated and converted into first-strand cDNA. An ultrasensitive real-time PCR method based on the SYBR Green chemistry was used for DDC mRNA quantification in head and neck tissue specimens. Relative quantification was performed using the comparative Ct (2-ddCt) method. DDC mRNA levels were lower in squamous cell carcinomas (SCCs) of the larynx and tongue than in adjacent non-cancerous tissue specimens. Furthermore, low DDC mRNA expression was noticed in laryngeal and tongue tumors of advanced TNM stage or bigger size, compared to early-stage or smaller tumors, respectively. No statistically significant differences were observed between SCCs resected from pharynx, buccal mucosa, or nasal cavity, and their normal counterparts. This is the first study examining the DDC mRNA expression in HNSCC. According to our results, DDC mRNA expression may constitute a potential prognostic biomarker in tongue and/or larynx SCCs, which principally represent the overwhelming majority of HNSCC cases.

Expression profiles of aquaporins in rat conjunctiva, cornea, lacrimal gland and Meibomian gland.

PubMed

Yu, Dongfang; Thelin, William R; Randell, Scott H; Boucher, Richard C

2012-10-01

The aim of the study was to elucidate aquaporin (AQP) family member mRNA expression and protein expression/localization in the rat lacrimal functional unit. The mRNA expression of all rat AQPs (AQP0-9, 11-12) in palpebral, fornical, and bulbar conjunctiva, cornea, lacrimal gland, and Meibomian gland was measured by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and real time RT-PCR. Antibodies against AQP1, 3, 4, 5, 9, and 11 were used in Western blotting and immunohistochemistry to determine protein expression and distribution. Our study demonstrated characteristic AQP expression profiles in rat ocular tissues. AQP1, 3, 4, 5, 8, 9, 11, and 12 mRNA were detected in conjunctiva. AQP0, 1, 2, 3, 4, 5, 6, 11, and 12 mRNA were expressed in cornea. AQP0, 1, 2, 3, 4, 5, 7, 8, and 11 mRNA were detected in lacrimal gland. AQP1, 3, 4, 5, 7, 8, 9, 11, and 12 mRNA were identified in Meibomian gland. By Western blot, AQP1, 3, 5, and 11 were detected in conjunctiva; AQP1, 3, 5, and 11 were identified in cornea; AQP1, 3, 4, 5, and 11 were detected in lacrimal gland; and AQP1, 3, 4, 5, 9, and 11 were present in Meibomian gland. Immunohistochemistry localized AQPs to distinct sites in the various tissues. This study rigorously analyzed AQPs expression and localization in rat conjunctiva, cornea, lacrimal gland, and Meibomian gland tissues. Our findings provide a comprehensive platform for further investigation into the physiological or pathophysiological relevance of AQPs in ocular surface. Copyright © 2012 Elsevier Ltd. All rights reserved.

Relative IGF-1 and IGF-2 gene expression in maternal and fetal tissues from diabetic swine

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

Wolverton, C.K.; Leaman, D.W.; White, M.E.

1990-02-26

Fourteen pregnant, crossbred gilts were utilized in this study. Seven gilts were injected with alloxan (50 mg/kg) at day 75 of gestation to induce diabetes. Gilts underwent caesarean section on day 105 of gestation. Samples were collected from maternal skeletal muscle, adipose tissue, uterus and endometrium; and from fetal skeletal muscle, adipose tissue, placenta, liver, lung, kidney, heart, brain and spleen. Tissues were frozen in liquid nitrogen for later analysis of IGF-1 and IGF-2 gene expression. Samples were pooled and total RNA was isolated using the guanidine isothiocynate method. Total mRNA was analyzed by dot blot hybridization. Blots were probedmore » with {sup 32}P-cDNA for porcine IGF-1 and rat IGF-2. IGF-1 gene expression in maternal tissues was unaffected by diabetes. Maternal diabetes increased IGF-2 mRNA in maternal adipose tissue but exhibited no effect in muscle or uterus. Expression of IGF-2 by maternal endometrium was decreased by diabetes. Maternal diabetes induced an increase in IGF-1 gene expression in muscle and placenta while causing an increase in IGF-2 expression in fetal liver and placenta. IGF-2 mRNA was lower in lung from fetuses of diabetic mothers than in controls. These results suggest that maternal diabetes alters IGF-1 and IGF-2 gene expression in specific tissues and differential regulation of these genes appears to exist in the mother and developing fetus.« less

Inducible expression of A Disintegrin and Metalloproteinase 8 in chronic periodontitis and gingival epithelial cells.

PubMed

Aung, W P P; Chotjumlong, P; Pata, S; Montreekachon, P; Supanchart, C; Khongkhunthian, S; Sastraruji, T; Krisanaprakornkit, S

2017-06-01

The expression of A Disintegrin and Metalloproteinase 8 (ADAM8) is associated with several inflammatory diseases. Elevated ADAM8 levels have been shown in gingival crevicular fluid of patients with chronic periodontitis. The objective of this study was to investigate ADAM8 expression in chronic periodontitis tissues compared with that in normal tissues. ADAM8 expression and its inductive mechanism were examined in human gingival epithelial cells (HGECs) and human gingival fibroblasts. Total RNA and protein were extracted from gingival biopsies of 33 patients with chronic periodontitis and those of 23 healthy volunteers. ADAM8 mRNA and protein expression was analyzed by real-time polymerase chain reaction, immunoblotting and immunohistochemistry. ADAM8 expression in control and stimulated cells in the presence or absence of specific inhibitors for mitogen-activated protein kinase pathways was assayed by real-time polymerase chain reaction, immunoblotting, flow cytometry and immunofluorescence. ADAM8 mRNA and protein expression in chronic periodontitis tissues was significantly greater than that in normal tissues (p < 0.01). Significantly increased ADAM8 expression was detected in the gingival epithelium of chronic periodontitis tissues (p < 0.001). ADAM8 mRNA expression in HGECs, but not in human gingival fibroblasts, was significantly induced by stimulation with Fusobacterium nucleatum (p < 0.05), partially via the p44/42 mitogen-activated protein kinase pathway. ADAM8 expression in the cell lysates and on the surface of HGECs was induced by stimulation with F. nucleatum. ADAM8 expression is increased in inflamed chronic periodontitis tissues and localized within gingival epithelium, consistent with an upregulation of ADAM8 expression in F. nucleatum-stimulated HGECs, suggesting a possible role of ADAM8 in innate immunity of periodontal tissue. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Heparanase mRNA expression and point mutation in hepatocellular carcinoma

PubMed Central

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

2004-01-01

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

[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.

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes

PubMed Central

2010-01-01

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed. PMID:20144232

Development of a quantitative assay to measure expression of transforming growth factor ß (TGF-ß) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples

USGS Publications Warehouse

Robertson, Laura S.; Ottinger, Christopher A.; Burdick, Summer M.; VanderKooi, Scott P.

2012-01-01

The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes brevirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-β mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-β mRNA expression level has been correlated with disease status in several laboratory studies and TGF-β mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-β and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-β mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-β and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-β mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker.

Matrix metalloprotease-3 expression in the medial plica and pannus-like tissue in knees from patients with medial compartment osteoarthritis.

PubMed

Wang, Hwai-Shi; Kuo, Pei-Yin; Yang, Chih-Chang; Lyu, Shaw-Ruey

2011-03-01

The severity of cartilage degeneration is positively correlated with the severity of the pathologic change of medial plica. However, knowledge of the pathogenic mechanisms and the impact of plica on cartilage destruction is limited. The aim of the present study was therefore to investigate matrix metalloprotease-3 (MMP-3) expression in the plica isolated from patients with medial compartment osteoarthritis of the knee. Immunohistochemistry showed that MMP-3 was highly expressed in pannus-like tissue and the plica. Western blotting of culture supernatants showed that interleukin-1β (IL-1β) treatment induced MMP-3 release by cells isolated from pannus tissue or the plica. Furthermore, reverse transcriptase polymerase chain reaction and real-time polymerase chain reaction analysis showed that MMP-3 mRNA levels were increased after IL-1β treatment of the cultured cells. MMP-3 and IL-1β mRNAs were expressed in the plica and pannus-like tissue, with MMP-3 mRNA being expressed at significantly higher levels in the plica than in normal synovial membrane and highly expressed in the plica at different stages in osteoarthritis (OA) patients. Pannus-like tissue and the plica express IL-1β and MMP-3. Moreover, MMP-3 mRNA and protein expression in the plica may contribute to the pathogenesis of OA. © 2011 Blackwell Publishing Limited.

[Comparative study of expression of homeobox gene Msx-1, Msx-2 mRNA during the hard tissue formation of mouse tooth development].

PubMed

Wang, Y; Wang, J; Gao, Y

2001-07-01

To observe and compare the expression pattern of Msx-1, Msx-2 mRNA during the different stages of hard tissue formation in the first mandibular molar of mouse and investigate the relationship between the two genes. First mandibular molar germs from 1, 3, 7 and 14-days old mouse were separated and reverse transcription-polymerase chain reaction was performed on the total RNA of them using Msx-1, Msx-2 specific primers separately. Expression of both genes were detected during the different stages of hard tissue formation in the mouse first mandibular molars, but there was some interesting differences in the quantitiy between the two genes. Msx-1 transcripts appeared at the 1 day postnatally, and increase through 3 day, 7 day, then maximally expressed at 14 days postnatally; while Msx-2 mRNA was seen and expressed maximally at the 3 days postnatally, then there was a gradual reduction at 7 days, and 14 days postnatally. The homeobox gene Msx-1, Msx-2 may play a role in the events of the hard tissue formation. The complementary expression pattern of them during the specific stage of hard tissue formation indicates that there may be some functional redundancy between them during the biomineralization.

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

Identification of CRASH, a gene deregulated in gynecological tumors.

PubMed

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

2004-01-01

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

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.

Effects of breed, parity, and folic Acid supplement on the expression of folate metabolism genes in endometrial and embryonic tissues from sows in early pregnancy.

PubMed

Vallée, Maud; Guay, Frédéric; Beaudry, Danièle; Matte, Jacques; Blouin, Richard; Laforest, Jean-Paul; Lessard, Martin; Palin, Marie-France

2002-10-01

Folic acid and glycine are factors of great importance in early gestation. In sows, folic acid supplement can increase litter size through a decrease in embryonic mortality, while glycine, the most abundant amino acid in the sow oviduct, uterine, and allantoic fluids, is reported to act as an organic osmoregulator. In this study, we report the characterization of cytoplasmic serine hydroxymethyltransferase (cSHMT), T-protein, and vT-protein (variant T-protein) mRNA expression levels in endometrial and embryonic tissues in gestating sows on Day 25 of gestation according to the breed, parity, and folic acid + glycine supplementation. Expression levels of cSHMT, T-protein, and vT-protein mRNA in endometrial and embryonic tissues were performed using semiquantitative reverse transcription-polymerase chain reaction. We also report, for the first time, an alternative splicing event in the porcine T-protein gene. Results showed that a T-protein splice variant, vT-protein, is present in all the tested sow populations. Further characterizations revealed that this T-protein splice variant contains a coding intron that can adopt a secondary structure. Results demonstrated that cSHMT mRNA expression levels were significantly higher in sows receiving the folic acid + glycine supplementation, independently of the breed or parity and in both endometrial and embryonic tissues. Upon receiving the same treatment, the vT-protein and T-protein mRNA expression levels were significantly reduced in the endometrial tissue of Yorkshire-Landrace sows only. These results indicate that modulation of specific gene expression levels in endometrial and embryonic tissues of sows in early gestation could be one of the mechanism involved with the role of folic acid on improving swine reproduction traits.

Predominant mucosal expression of 5-HT4(+h) receptor splice variants in pig stomach and colon

PubMed Central

Priem, Evelien KV; De Maeyer, Joris H; Vandewoestyne, Mado; Deforce, Dieter; Lefebvre, Romain A

2013-01-01

AIM: To investigate cellular 5-HT4(-h/+h) receptor distribution, particularly in the epithelial layer, by laser microdissection and polymerase chain reaction (PCR) in porcine gastrointestinal (GI) tissues. METHODS: A stepwise approach was used to evaluate RNA quality and to study cell-specific 5-HT4 receptor mRNA expression in the porcine gastric fundus and colon descendens. After freezing, staining and laser microdissection and pressure catapulting (LMPC), RNA quality was evaluated by the Experion automated electrophoresis system. 5-HT4 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were examined by endpoint reverse transcription (RT)-PCR in mucosal and muscle-myenteric plexus (MMP) tissue fractions, in mucosal and MMP parts of hematoxylin and eosin (HE) stained tissue sections and in microdissected patches of the epithelial and circular smooth muscle cell layer in these sections. Pig gastric fundus tissue sections were also stained immunohistochemically (IHC) for enterochromaffin cells (EC cells; MAB352); these cells were isolated by LMPC and examined by endpoint RT-PCR. RESULTS: After HE staining, the epithelial and circular smooth muscle cell layer of pig colon descendens and the epithelial cell layer of gastric fundus were identified morphologically and isolated by LMPC. EC cells of pig gastric fundus were successfully stained by IHC and isolated by LMPC. Freezing, HE and IHC staining, and LMPC had no influence on RNA quality. 5-HT4 receptor and GAPDH mRNA expressions were detected in mucosa and MMP tissue fractions, and in mucosal and MMP parts of HE stained tissue sections of pig colon descendens and gastric fundus. In the mucosa tissue fractions of both GI regions, the expression of h-exon containing receptor [5-HT4(+h) receptor] mRNA was significantly higher (P < 0.01) compared to 5-HT4(-h) receptor expression, and a similar trend was obtained in the mucosal part of HE stained tissue sections. Large microdissected patches of the epithelial and circular smooth muscle cell layer of pig colon descendens and of the epithelial cell layer of pig gastric fundus, also showed 5-HT4 receptor and GAPDH mRNA expression. No 5-HT4 receptor mRNA expression was detected in gastric LMPC-isolated EC cells from IHC stained tissues, which cells were positive for GAPDH. CONCLUSION: Porcine GI mucosa predominantly expresses 5-HT4(+h) receptor splice variants, suggesting their contribution to the 5-HT4 receptor-mediated mucosal effects of 5-HT. PMID:23840113

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

LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

PubMed Central

Ying, Lihua; Lau, Agatha; Alvira, Cristina M.; West, Robert; Cann, Gordon M.; Zhou, Bin; Kinnear, Caroline; Jan, Eric; Sarnow, Peter; Van de Rijn, Matt; Rabinovitch, Marlene

2009-01-01

Summary Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3′ UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types. PMID:19366727

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues

PubMed Central

Blazie, Stephen M.; Geissel, Heather C.; Wilky, Henry; Joshi, Rajan; Newbern, Jason; Mangone, Marco

2017-01-01

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3′untranslated region (3′UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3′UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3′UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis. PMID:28348061

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues.

PubMed

Blazie, Stephen M; Geissel, Heather C; Wilky, Henry; Joshi, Rajan; Newbern, Jason; Mangone, Marco

2017-06-01

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3'untranslated region (3'UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3'UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3'UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis. Copyright © 2017 Blazie et al.

Desiccation and osmotic stress increase the abundance of mRNA of the tonoplast aquaporin BobTIP26-1 in cauliflower cells.

PubMed

Barrieu, F; Marty-Mazars, D; Thomas, D; Chaumont, F; Charbonnier, M; Marty, F

1999-07-01

Changes in vacuolar structure and the expression at the RNA level of a tonoplast aquaporin (BobTIP26-1) were examined in cauliflower (Brassicaoleracea L. var. botrytis) under water-stress conditions. Gradual drying out of slices of cauliflower floret tissue caused its collapse, with a shrinkage in tissue and cell volumes and an apparent vesiculation of the central vacuole, whereas osmotic stress resulted in plasmolysis with a collapse of the cytoplasm and the central vacuole within. Osmotic stress caused a rapid and substantial increase in BobTIP26 mRNA in slices of floret tissue. Exposure of tissue slices to a regime of desiccation showed a slower but equally large rise in BobTIP26 mRNA followed by a rapid decline upon rehydration. In situ hybridization showed that BobTIP26-2 mRNA is expressed most highly in meristematic and expanding cells of the cauliflower florets and that desiccation strongly increased the expression in those cells and in differentiated cells near the xylem vessels. These data indicate that under water-deficit conditions, expression of the tonoplast aquaporin gene in cauliflower is subject to a precise regulation that can be correlated with important cytological changes in the cells.

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow.

PubMed

Llewellyn, S; Fitzpatrick, R; Kenny, D A; Patton, J; Wathes, D C

2008-05-01

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14+/-0.4 postpartum (n=12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow☆

PubMed Central

Llewellyn, S.; Fitzpatrick, R.; Kenny, D.A.; Patton, J.; Wathes, D.C.

2008-01-01

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling. PMID:18258405

Exercise-induced changes of MCT1 in cardiac and skeletal muscles of diabetic rats induced by high-fat diet and STZ.

PubMed

Nikooie, Rohollah; Rajabi, Hamid; Gharakhanlu, Reza; Atabi, Fereshteh; Omidfar, Kobra; Aveseh, Malihe; Larijani, Bagher

2013-12-01

We hypothesized that a part of therapeutic effects of endurance training on insulin resistance is mediated by increase in cardiac and skeletal muscle mitochondrial lactate transporter, monocarboxylate transporter 1 (MCT1). Therefore, we examined the effect of 7 weeks endurance training on the mRNA and protein expression of MCT1 and MCT4 and their chaperon, CD147, on both sarcolemmal and mitochondrial membrane, separately, in healthy and type 2 diabetic rats. Diabetes was induced by injection of low dose of streptozotocin and feeding with high-fat diet. Insulin resistance was confirmed by homeostasis model assessment-estimated insulin resistance index and accuracy of two membranes separation was confirmed by negative control markers (glucose transporter 1 and cytochrome c oxidase. Real-time PCR and western blotting were used for mRNA and protein expression, respectively. Diabetes dramatically reduced MCT1 and MCT4 mRNA and their expression on sarcolemmal membrane whereas the reduction in MCT1 expression was less in mitochondrial membrane. Training increased the MCT1 mRNA and protein expression in both membranes and decreased insulin resistance as an adaptive consequence. In both tissues increase in CD147 mRNA was only parallel to MCT1 expression. The response of MCT1 on sarcolemmal and mitochondrial membranes was different between cardiac and skeletal muscles which indicate that intracellular lactate kinetic is tissue specific that allows a tissue to coordinate whole organism metabolism.

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

Zhang, Jie; Zheng, Fangxia; Yu, Gang

Highlights: •miR-196a was overexpressed in cervical cancer tissue compared to normal tissue. •miR-196a expression elevated proliferation and migration of cervical cancer cells. •miR-196a inhibited NTN4 expression by binding 3′-UTR region of NTN4 mRNA. •NTN4 inversely correlated with miR-196a expression in cervical tissue and cell line. •NTN4 expression was low in cervical cancer tissue compared to normal tissue. -- Abstract: Recent research has uncovered tumor-suppressive and oncogenic potential of miR-196a in various tumors. However, the expression and mechanism of its function in cervical cancer remains unclear. In this study, we assess relative expression of miR-196a in cervical premalignant lesions, cervical cancermore » tissues, and four cancer cell lines using quantitative real-time PCR. CaSki and HeLa cells were treated with miR-196a inhibitors, mimics, or pCDNA/miR-196a to investigate the role of miR-196a in cancer cell proliferation and migration. We demonstrated that miR-196a was overexpressed in cervical intraepithelial neoplasia 2–3 and cervical cancer tissue. Moreover, its expression contributes to the proliferation and migration of cervical cancer cells, whereas inhibiting its expression led to a reduction in proliferation and migration. Five candidate targets of miR-196a chosen by computational prediction and Cervical Cancer Gene Database search were measured for their mRNA in both miR-196a-overexpressing and -depleted cancer cells. Only netrin 4 (NTN4) expression displayed an inverse association with miR-196a. Fluorescent reporter assays revealed that miR-196a inhibited NTN4 expression by targeting one binding site in the 3′-untranslated region (3′-UTR) of NTN4 mRNA. Furthermore, qPCR and Western blot assays verified NTN4 expression was downregulated in cervical cancer tissues compared to normal controls, and in vivo mRNA level of NTN4 inversely correlated with miR-196a expression. In summary, our findings provide new insights about the functional role of miR-196a in cervical carcinogenesis and suggested a potential use of miR-196a for clinical diagnosis and as a therapeutic target.« less

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

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

PubMed Central

Ren, H; Stiles, G L

1994-01-01

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

L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

Expressions of apoptosis-regulating factors in bovine retained placenta.

PubMed

Kamemori, Y; Wakamiya, K; Nishimura, R; Hosaka, Y; Ohtani, S; Okuda, K

2011-01-01

The aim of the present study was to evaluate the relationship between the retention of fetal membranes (RFM) and apoptosis of the cells in fetal membranes. The present study investigated mRNA and protein expressions of apoptosis-regulating factors: FAS, cellular FLICE-like inhibiting protein (cFLIP), BAX, BCL2, caspase-8 (CASP8), and CASP3 in fetal membranes. Placentomes were manually collected from the uterus immediately after parturition and classified into two groups (RFM; n = 8 and non-RFM; n = 8) according to whether placental membranes were expelled or not within 12 h after delivery. FAS mRNA expression in maternal placental tissue was less in RFM cows than in non-RFM cows (P < 0.05). cFLIP mRNA expression in maternal and fetal placental tissue was greater in RFM cows than in non-RFM cows (P < 0.05). CASP3 mRNA expression in maternal placental tissue was greater in RFM cows than in non-RFM cows (P < 0.05). However, the protein expressions of FAS, cFLIP and cleaved CASP3 were not significantly different between the two groups. mRNA and protein expressions of BAX, BCL2 and CASP8 were also not significantly different between the two groups. In the immunohistochemical study, single-stranded DNA, which appears specifically in the apoptotic cells, was mainly found in the maternal placenta of non-RFM cows. Together these results suggest that RFM occurs at least in part due to a dysfunctional apoptotic process caused by the inhibition of FAS expression in the maternal placenta, and the increase of cFLIP expression in the maternal and fetal placenta. Copyright © 2010 Elsevier Ltd. All rights reserved.

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.

MAP3K8 (TPL2/COT) affects obesity-induced adipose tissue inflammation without systemic effects in humans and in mice.

PubMed

Ballak, Dov B; van Essen, Peter; van Diepen, Janna A; Jansen, Henry; Hijmans, Anneke; Matsuguchi, Tetsuya; Sparrer, Helmut; Tack, Cees J; Netea, Mihai G; Joosten, Leo A B; Stienstra, Rinke

2014-01-01

Chronic low-grade inflammation in adipose tissue often accompanies obesity, leading to insulin resistance and increasing the risk for metabolic diseases. MAP3K8 (TPL2/COT) is an important signal transductor and activator of pro-inflammatory pathways that has been linked to obesity-induced adipose tissue inflammation. We used human adipose tissue biopsies to study the relationship of MAP3K8 expression with markers of obesity and expression of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8). Moreover, we evaluated obesity-induced adipose tissue inflammation and insulin resistance in mice lacking MAP3K8 and WT mice on a high-fat diet (HFD) for 16 weeks. Individuals with a BMI >30 displayed a higher mRNA expression of MAP3K8 in adipose tissue compared to individuals with a normal BMI. Additionally, high mRNA expression levels of IL-1β, IL-6 and IL-8, but not TNF -α, in human adipose tissue were associated with higher expression of MAP3K8. Moreover, high plasma SAA and CRP did not associate with increased MAP3K8 expression in adipose tissue. Similarly, no association was found for MAP3K8 expression with plasma insulin or glucose levels. Mice lacking MAP3K8 had similar bodyweight gain as WT mice, yet displayed lower mRNA expression levels of IL-1β, IL-6 and CXCL1 in adipose tissue in response to the HFD as compared to WT animals. However, MAP3K8 deficient mice were not protected against HFD-induced adipose tissue macrophage infiltration or the development of insulin resistance. Together, the data in both human and mouse show that MAP3K8 is involved in local adipose tissue inflammation, specifically for IL-1β and its responsive cytokines IL-6 and IL-8, but does not seem to have systemic effects on insulin resistance.

Molecular characterization, mRNA expression of prolactin receptor (PRLR) gene during pregnancy, nonpregnancy in the yak (Bos grunniens).

PubMed

Zi, Xiang-Dong; Chen, Da-Wen; Wang, Hong-Mei

2012-02-01

Prolactin (PRL) plays central roles in a wide range of body functions in mammals, and the actions are mediated by the specific cell surface receptor, the prolactin receptor (PRLR). To better understand the role of PRL in the yak (Bos grunniens), in the present study, we first cloned yak PRLR cDNA, and compared its mRNA expression in several tissues with cattle (Bos taurus). By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length of yak PRLR cDNA sequence comprised of an open reading frame of 1746bp encoding a 581 amino acid protein, and contained a signal sequence and a transmembrane region. The intracellular domain had two pairs of cysteine residues and a WSXWS motif. The cytoplasmic domain comprised 323 residues and contained box 1 sequence. The yak PRLR shared 66.0-98.5% protein sequence identity with mammalian homologs. Real-time PCR analysis revealed that PRLR mRNA was higher in mammary tissue than in ovary and endometrium (P<0.01). During pregnancy, the ovary and mammary PRLR mRNA expression increased by 33- and 2.9-fold in yak, respectively, and increased by 46- and 3.8-fold in cattle, respectively. PRLR mRNA expression was higher (P<0.05) in mammary tissue and ovary of pregnant cow than that of pregnant yak. It is proposed that the increased ovarian and mammary PRLR mRNA expression during pregnancy may be associated with corpus luteum function for maintenance of pregnancy and mammary development for subsequent lactation. Copyright © 2011 Elsevier Inc. All rights reserved.

Stimulation of S14 mRNA and lipogenesis in brown fat by hypothyroidism, cold exposure, and cafeteria feeding: evidence supporting a general role for S14 in lipogenesis and lipogenesis in the maintenance of thermogenesis

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

Freake, H.C.; Oppenheimer, J.H.

1987-05-01

In liver, thyroid hormone rapidly induces S14 mRNA, which encodes a small acidic protein. This sequence is abundantly expressed only in lipogenic tissues and is thought to have some function in fat metabolism. In the euthyroid rat, we measured 20-fold higher levels of S14 mRNA in interscapular brown adipose tissue than liver. Furthermore, whereas in liver or epididymal fat, hypothyroidism resulted in an 80% fall in S14 mRNA, in brown fat the level of this sequence increased a further 3-fold. In all three tissues, the expression of S14 mRNA correlated well with lipogenesis, as assessed by /sup 3/H/sub 2/O incorporation.more » Physiological activation of brown fat by chronic cold exposure or cafeteria feeding increased the concentration of S14 mRNA in this tissue and again this was accompanied by a greater rate of fatty acid synthesis. Overall, in liver and white and brown adipose tissue, S14 mRNA and lipogenesis were well correlated and strongly suggest a function of the S14 protein related to fat synthesis. These studies suggest that the S14 protein and lipogenesis may be important for thyroid hormone-induced and brown adipose tissue thermogenesis and that stimulation of these functions in hypothyroid brown fat is a consequence of decreased thyroid hormone-induced thermogenesis elsewhere.« less

Circular RNA and gene expression profiles in gastric cancer based on microarray chip technology.

PubMed

Sui, Weiguo; Shi, Zhoufang; Xue, Wen; Ou, Minglin; Zhu, Ying; Chen, Jiejing; Lin, Hua; Liu, Fuhua; Dai, Yong

2017-03-01

The aim of the present study was to screen gastric cancer (GC) tissue and adjacent tissue for differences in mRNA and circular (circRNA) expression, to analyze the differences in circRNA and mRNA expression, and to investigate the circRNA expression in gastric carcinoma and its mechanism. circRNA and mRNA differential expression profiles generated using Agilent microarray technology were analyzed in the GC tissues and adjacent tissues. qRT-PCR was used to verify the differential expression of circRNAs and mRNAs according to the interactions between circRNAs and miRNAs as well as the possible existence of miRNA and mRNA interactions. We found that: i) the circRNA expression profile revealed 1,285 significant differences in circRNA expression, with circRNA expression downregulated in 594 samples and upregulated in 691 samples via interactions with miRNAs. The qRT-PCR validation experiments showed that hsa_circRNA_400071, hsa_circRNA_000543 and hsa_circRNA_001959 expression was consistent with the microarray analysis results. ii) 29,112 genes were found in the GC tissues and adjacent tissues, including 5,460 differentially expressed genes. Among them, 2,390 differentially expressed genes were upregulated and 3,070 genes were downregulated. Gene Ontology (GO) analysis of the differentially expressed genes revealed these genes involved in biological process classification, cellular component classification and molecular function classification. Pathway analysis of the differentially expressed genes identified 83 significantly enriched genes, including 28 upregulated genes and 55 downregulated genes. iii) 69 differentially expressed circRNAs were found that might adsorb specific miRNAs to regulate the expression of their target gene mRNAs. The conclusions are: i) differentially expressed circRNAs had corresponding miRNA binding sites. These circRNAs regulated the expression of target genes through interactions with miRNAs and might become new molecular biomarkers for GC in the future. ii) Differentially expressed genes may be involved in the occurrence of GC via a variety of mechanisms. iii) CD44, CXXC5, MYH9, MALAT1 and other genes may have important implications for the occurrence and development of GC through the regulation, interaction, and mutual influence of circRNA-miRNA-mRNA via different mechanisms.

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.

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

PubMed Central

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

2007-01-01

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

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.

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

PubMed Central

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

2015-01-01

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

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.

Characterization of glucagon-like peptide 2 pathway member expression in bovine gastrointestinal tract.

PubMed

Connor, E E; Baldwin, R L; Capuco, A V; Evock-Clover, C M; Ellis, S E; Sciabica, K S

2010-11-01

Glucagon-like peptide 2 (GLP-2), secreted by enteroendocrine cells, has several physiological effects on the intestine of monogastric species, including promotion of growth of intestinal epithelium, reduction of epithelial cell apoptosis, and enhancement of intestinal blood flow, nutrient absorption, and epithelial barrier function. The regulatory functions of GLP-2 in the ruminant gastrointestinal tract (GIT) have not been well studied. The objectives of this investigation were to characterize the mRNA expression of 4 members of the GLP-2 pathway throughout the bovine GIT, including (1) proglucagon (GCG), the parent peptide from which GLP-2 is derived through cleavage by prohormone convertase; (2) prohormone convertase (PCSK1); (3) GLP-2 receptor (GLP2R); and (4) dipeptidyl peptidase IV (DPP4), the enzyme that inactivates GLP-2. Gene expression was evaluated in rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, and rectum collected at slaughter from prepubertal heifers, mature cows in early, mid, and late lactation, and nonlactating cows (n=3 per stage) by a gene expression profiling assay. In addition, mRNA expression of 14 genes involved in nutrient transport, enzyme activity, blood flow, apoptosis, and proliferation were evaluated in the 9 GIT tissues for their association with GCG and GLP2R mRNA expression. Immunohistochemistry was used to localize GLP2R protein in tissues of the lower GIT. Results indicated that mRNA expression of GCG, PCSK1, GLP2R, and DPP4 varies across the 9 GIT tissues, with greatest expression in small and large intestines, and generally nondetectable levels in forestomachs. Expression of DPP4 and GLP2R mRNA varied by developmental stage or lactational state in intestinal tissues. Expression of GCG or GLP2R mRNA was correlated with molecular markers of proliferation, apoptosis, blood flow, enzyme activity, and urea transport, depending on the tissue examined, which suggests a potential for involvement of GLP-2 in these physiological processes in the ruminant GIT. The GLP2R protein was expressed in intestinal crypts of the bovine GIT, which is consistent with the distribution in monogastric species. Our findings support a functional role of the GLP-2 pathway in bovine GIT and the potential for use of GLP-2 as a therapy to improve intestinal function and nutrient absorption in ruminants. Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Relationship between transmembrane serine protease expression and prognosis of esophageal squamous cell carcinoma.

PubMed

Liu, G T; Shen, C; Ren, X H; Yang, L; Yu, Y M; Xiu, Y X; Li, R H; Jiang, L; Zhang, C L; Li, Y W

2017-01-01

Esophageal squamous cell carcinoma is the most common type of esophageal cancer in Eastern Europe and Asia, being the 6th most common cause of cancer deaths worldwide. The aim of this study was to analyze the expression of transmembrane serine protein in esophageal squamous cell carcinoma, and to correlate it with the clinical biological features of esophageal cancer. The expression of transmembrane protease serine 4 (TMPRSS4) mRNA and protein in carcinoma tissues and corresponding adjacent tissues and non-tumorous esophageal tissues was determined using PCR (qRT-PCR). The results show that both TMPRSS4 mRNA and protein expression were remarkably lower in adjacent normal tissues than in tumorous tissues. TMPRSS4 protein expression in esophageal carcinoma was correlated with patient demographic characteristics, tumor type, high TNM stages and overall survival (OS). Based on the experimental results, we conclude that TMPRSS4 is closely related to the occurrence, development and metastasis of esophageal squamous cell carcinoma.

Overexpression and localization of heat shock proteins mRNA in pancreatic carcinoma.

PubMed

Ogata, M; Naito, Z; Tanaka, S; Moriyama, Y; Asano, G

2000-06-01

In the present study we examined the localization and overexpression of heat shock proteins (hsps), mainly hsp90, in pancreatic carcinoma tissue compared with control tissue (including chronic pancreatitis and normal pancreas tissue), with the aid of immunohistochemical staining, in situ hybridization and reverse transcriptase polymerase chain reaction. Hsp90 alpha mRNA was overexpressed more highly in pancreatic carcinoma than in the control tissue. The proliferating-cell-nuclear-antigen labeling index was also high in pancreatic carcinoma tissue compared with the other tissue. These findings suggest that the overexpression of hsp90 alpha mRNA in carcinomas may be correlated with cell proliferation. However, hsp90 beta was constitutively overexpressed almost equally in all groups of pancreatic tissue including pancreatic carcinoma, chronic pancreatitis and normal pancreas tissue. Immunohistochemical staining demonstrated a differentiation in the expression of hsp90 between histological types of pancreatic carcinoma. These findings suggest that hsp90 alpha is involved in carcinogenesis and that hsp90 beta is correlated to structural conformation. Hsp90 alpha and hsp90 beta seem to perform different functions in tissue containing malignant cells. P53, MDM2 and WAF1, that were cell-cycle-related oncogene product were more strongly expressed in the nuclei of the cancer cells of the cancer tissue. Especially, MDM2 was more strongly expressed in mucinous carcinoma and the mucin secreting tissues surrounding pancreatic carcinoma tissue. The expression of MDM2 protein might also be correlated to secretion systems during structural conformation and be correlated to hsp90 beta.

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

PubMed

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

2014-02-01

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

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.

Cancer testis antigen OY-TES-1 expression and serum immunogenicity in colorectal cancer: its relationship to clinicopathological parameters.

PubMed

Luo, Bin; Yun, Xiang; Fan, Rong; Lin, Yong-Da; He, Shu-Jia; Zhang, Qing-Mei; Mo, Fa-Rong; Chen, Fang; Xiao, Shao-Wen; Xie, Xiao-Xun

2013-01-01

Cancer testis (CT) antigens are attractive targets for cancer immunotherapy because their expression is restricted in normal germ line tissues but frequently detected in variety of tumors. OY-TES-1 is identified as a member of CT antigens. Current knowledge about OY-TES-1 expression in colorectal cancer (CRC) is solely based on mRNA analysis. None of previous researches has studied OY-TES-1 at protein level. In this study, OY-TES-1 polyclonal antibody was generated. The expression of OY-TES-1 mRNA and protein was detected by RT-PCR and immunohistochemistry in 60 CRC and paired adjacent non-tumor tissues, 24 colorectal adenoma and 3 normal colon tissues, respectively. Sera from 73 CRC patients were also tested for OY-TES-1 antibody by ELISA. Our results showed that the frequency of OY-TES-1 mRNA expression was statistically higher in CRC (73.3%, 44/60) than that in adjacent non-tumor tissue (55.0%, 33/60) and colorectal adenoma (45.8%, 11/24). For the first time, OY-TES-1 protein expression was found in (43.3%, 26/60) of CRC tissues, but absent in any of adjacent non-tumor and colorectal adenoma tissues. No OY-TES-1 expression was found in normal colon by either RT-PCR or immunohistochemistry. Furthermore, OY-TES-1 protein expression was correlated with tumor invasion stage (P=0.004) and histological grade (P=0.040). Anti-OY-TES-1 antibody was detected in (9.6%, 7/73) of CRC patients' sera but not in 76 healthy donors. This finding demonstrates that OY-TES-1 is frequently expressed in CRC and is able to induce humoral immune response spontaneously in CRC patients, suggesting that it might be a promising immunotherapy target for CRC.

The effect of very-low-calorie diet on mRNA expression of inflammation-related genes in subcutaneous adipose tissue and peripheral monocytes of obese patients with type 2 diabetes mellitus.

PubMed

Mraz, M; Lacinova, Z; Drapalova, J; Haluzikova, D; Horinek, A; Matoulek, M; Trachta, P; Kavalkova, P; Svacina, S; Haluzik, M

2011-04-01

Low-grade inflammation links obesity, type 2 diabetes mellitus (T2DM), and cardiovascular diseases. To explore the expression profile of genes involved in inflammatory pathways in adipose tissue and peripheral monocytes (PM) of obese patients with and without T2DM at baseline and after dietary intervention. Two-week intervention study with very-low-calorie diet (VLCD). University hospital. Twelve obese females with T2DM, 8 obese nondiabetic females (OB) and 15 healthy age-matched females. Two weeks of VLCD (2500 kJ/d). Metabolic parameters, circulating cytokines, hormones, and mRNA expression of 39 genes in sc adipose tissue (SCAT) and PM. Both T2DM and OB group had significantly increased serum concentrations of circulating proinflammatory factors (C-reactive protein, TNFα, IL-6, IL-8), mRNA expression of macrophage antigen CD68 and proinflammatory chemokines (CCL-2, -3, -7, -8, -17, -22) in SCAT and complementary chemokine receptors (CCR-1, -2, -3, -5) and other proinflammatory receptors (toll-like receptor 2 and 4, TNF receptor superfamily 1A and 1B, IL-6R) in PM, with OB group showing less pronounced chemoattracting and proinflammatory profile compared to T2DM group. In T2DM patients VLCD decreased body weight, improved metabolic profile, and decreased mRNA expression of up-regulated CCRs in PM and chemokines [CCL 8, chemokine (C-X-C motif) ligand 10] in SCAT. VLCD markedly increased mRNA expression of T-lymphocyte attracting chemokine CCL-17 in SCAT. Obese patients with and without T2DM have increased mRNA expression of chemotactic and proinflammatory factors in SCAT and expression of corresponding receptors in PM. Two weeks of VLCD significantly improved this profile in T2DM patients.

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

PubMed

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

2016-06-25

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

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.

Dexamethasone enhances agonist induction of tissue factor in monocytes but not in endothelial cells.

PubMed

Bottles, K D; Morrissey, J H

1993-06-01

Stimulation of monocytic cells by inflammatory agents such as bacterial lipopolysaccharide or tumour necrosis factor-alpha leads to the rapid and transient expression of tissue factor, the major cellular initiator of the extrinsic coagulation cascade in both haemostasis and tissue inflammation. In this study we investigated whether the synthetic anti-inflammatory glucocorticoid, dexamethasone, would inhibit agonist induction of tissue factor expression in both monocytes and endothelial cells. Surprisingly, dexamethasone significantly enhanced the induction of tissue factor expression by peripheral blood mononuclear cells and an established monocytic cell line, THP-1, in response to lipopolysaccharide or tumour necrosis factor-alpha. However, unlike monocytic cells, dexamethasone did not enhance agonist induction of tissue factor in endothelial cells. Synergistic enhancement of tissue factor expression by dexamethasone was also reflected in tissue factor mRNA levels in THP-1 cells, but was not the result of improved TF mRNA stability. Synergism between bacterial lipopolysaccharide and glucocorticoid in the induction of monocyte effector function is extremely unusual and may help to explain the variable outcome of glucocorticoid treatment of septic shock.

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.

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.

Modification of tissue-factor mRNA and protein response to thrombin and interleukin 1 by high glucose in cultured human endothelial cells.

PubMed

Boeri, D; Almus, F E; Maiello, M; Cagliero, E; Rao, L V; Lorenzi, M

1989-02-01

Because diabetic vascular disease is accompanied by a state of hypercoagulability, manifested by increased thrombin activity and foci of intravascular coagulation, we investigated whether a specific procoagulant property of the endothelium--production and surface expression of tissue factor--is modified by elevated glucose concentrations. In unperturbed human vascular endothelial cells, tissue factor mRNA and expression of the functional protein were undetectable and were not induced by 10-12 days of exposure to 30 mM glucose. In thrombin-stimulated cultures, tissue-factor expression was related inversely to cellular density, with confluent cultures producing (per 10(5) cells) half the amount of tissue factor measured in sparse cultures. Cells exposed to high glucose and studied when cell number and thymidine incorporation were identical to control cells manifested increased tissue-factor mRNA level and functional protein production in response to thrombin (P = .002). This effect was not attributable to hypertonicity and was not observed after short exposure to high glucose. In contrast, the tissue-factor response to interleukin 1, a modulator of endothelial function in the context of host defense, was decreased in cells cultured in high glucose (P = .04). These findings indicate that exposure to high glucose can alter tissue-factor gene expression in perturbed vascular endothelium. The reciprocal effects of high glucose on the tissue-factor response to thrombin and interleukin 1 points to different pathways of tissue-factor stimulation by the two agents and suggests functional consequences pertinent to the increased thrombin activity and compromised host-defense mechanisms observed in diabetes.

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

PubMed Central

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

1997-01-01

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

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

PubMed

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

1997-07-01

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

Tumor 5-FU-related mRNA Expression and Efficacy of Oral Fluoropyrimidines in Adjuvant Chemotherapy of Colorectal Cancer.

PubMed

Koda, Keiji; Miyauchi, Hideaki; Kosugi, Chihiro; Kaiho, Takashi; Takiguchi, Nobuhiro; Kobayashi, Susumu; Maruyama, Takashi; Matsubara, Hisahiro

2016-10-01

It has not been elucidated whether the clinical efficacy of oral fluoropyrimidines for adjuvant chemotherapy of colorectal cancer varies with tumor biological characteristics. A multicenter randomized trial was performed comparing oral tegafur/gimeracil/oteracil (S-1) and uracil-tegafur/ leucovorin (UFT/LV) as adjuvant therapy for stage III colorectal cancer. Postoperative survival was compared based on the 5-FU-related mRNA levels in cancer tissues. Among patients with tumor expressing dihydropyrimidine dehydrogenase (DPD) mRNA within the 66.7th percentile (lower 2/3) of all cases, overall survival (OS) was significantly better in the S-1 than in the UFT/LV group. In the S-1 group, patients with low DPD-expressing tumors had significantly better OS than those with highly expressing tumors. Patients with low thymidine synthase (TS)-expressing tumors had significantly better OS than those with highly expressing tumors. The efficacy of oral fluoropyrimidines as adjuvant chemotherapy for colorectal cancer may be influenced by the level of 5-FU-related mRNA in cancer tissues. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

PubMed Central

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

1989-01-01

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

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

PubMed

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

2014-01-01

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

A novel whole tooth-in-jaw-bone culture of rat molars: morphological, immunohistochemical, and laser capture microdissection analysis.

PubMed

Chokechanachaisakul, Uraiwan; Kaneko, Tomoatsu; Yamanaka, Yusuke; Okiji, Takashi; Suda, Hideaki

2012-10-01

In conventional whole-tooth culture systems, limitation exists regarding maintenance of the vitality of the dental pulp, because this tissue is encased in rigid dentin walls that hinder nutrition supply. We here report a whole tooth-in-jaw-bone culture system of rat mandibular first molars, where transcardiac perfusion with culture medium was carried out before placement of the jaw bone into culture medium, aiming to facilitate longer time preservation of the dental pulp tissue. Following 7 days of culture, the pulp tissues were analyzed by histology and immunohistochemistry to ED2 (antiresident macrophage). ED2-positive macrophages were also analyzed for their Class II MHC, interleukin-6 (IL-6), and p53 mRNA expression levels by means of immune-laser capture microdissection (immune-LCM). Dentin sialophosphoprotein (DSPP) mRNA expression in odontobalstic layer was also examined by LCM. Teeth cultured following saline-perfusion and nonperfusion served as cultured controls. Normal teeth also served as noncultured controls. Histological examination demonstrated that the structure of the pulp tissue was well preserved in the medium-perfused explants in contrast to the cultured control groups. The Class II MHC, IL-6, and p53 mRNA expression levels of ED2-positive cells and DSPP expression levels of odontoblastic layer tissues in the pulp of medium-perfused explants were not significantly different from those in the noncultured normal teeth. In conclusion, the structural integrity and mRNA expression in the pulp were maintained at the in vivo level in the ex vivo whole tooth-in-jaw-bone culture system. The system may lay the foundation for studies aiming at defining further histological and molecular mechanism of the pulp. Copyright © 2012 Wiley Periodicals, Inc.

Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men.

PubMed Central

Fried, S K; Russell, C D; Grauso, N L; Brolin, R E

1993-01-01

There are marked variations in the activity of lipoprotein lipase (LPL) among adipose depots, particularly in women. Consistent with data on LPL activity, the level of expression of LPL mRNA was lower in omental (OM) than subcutaneous (SQ) adipose tissue of women. To investigate the cellular basis of these differences, OM and SQ adipose tissues obtained at surgery from obese men and women were placed in organ culture for 7 d with varying concentrations of insulin and dexamethasone. Insulin increased levels of LPL mRNA and LPL activity in abdominal SQ but not OM adipose tissue. Dexamethasone also increased LPL mRNA and LPL activity, and these effects were more marked in the OM adipose tissue, particularly in men. When insulin and dexamethasone were added together, synergistic increases in LPL activity were seen in both depots, and this was in part explained at the level of LPL mRNA. The SQ depot was more sensitive to the effects of submaximal doses of dexamethasone in the presence of insulin. The maximum activity of LPL induced by insulin or insulin plus dexamethasone was higher in the SQ than in the OM depot of women, and this was associated with higher levels of LPL mRNA. Rates of LPL synthesis paralleled LPL mRNA levels. These data show that insulin and glucocorticoids influence human adipose tissue LPL activity at the level of LPL gene expression, as well as posttranslationally, and that responsiveness to these hormonal effects is dependent on adipose depot and gender. Images PMID:8227334

Olive Leaf Extract Elevates Hepatic PPAR α mRNA Expression and Improves Serum Lipid Profiles in Ovariectomized Rats.

PubMed

Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin; Kim, Hyun-Sook

2015-07-01

We hypothesized that olive leaf extract might alleviate dyslipidemia resulting from estrogen deficiency. Serum lipid profile and mRNA expression of the related genes in the liver and adipose tissue were analyzed after providing olive leaf extract (200 or 400 mg/kg body weight; n=7 for each group) to ovariectomized rats for 10 weeks. After 10 weeks' administration, the rats in the olive leaf extract-administered groups showed significantly lower levels of serum triglyceride and very-low-density lipoprotein (VLDL)-cholesterol compared with the rats in the control group, whereas the administration of olive leaf extract did not significantly change the elevated low-density lipoprotein cholesterol levels. In addition, administration of high dose of olive leaf extract significantly decreased the liver triglyceride and increased serum estradiol levels. mRNA expressions of peroxisome proliferator-activated receptor alpha (PPAR α) and acyl-CoA oxidase (ACO) were not affected by ovariectomy, however, administration of olive leaf extract significantly increased both PPAR α and ACO mRNA expression. Expression of adiponectin mRNA in adipose tissue was significantly decreased in the ovariectomized control group. Rats administered low-dose olive leaf extract showed significantly elevated adiponectin mRNA expression compared with rats in the ovariectomized control group. Even though dose-dependent effects were not observed in most of the measurements, these results suggest that genes involved in lipid metabolism may be regulated by olive leaf extract administration in ovariectomized rats.

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex.

PubMed

Grandclément, B; Morel, G

1998-06-01

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, -B and -C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.

Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin. I. Excisional wound model.

PubMed

Quaglino, D; Nanney, L B; Kennedy, R; Davidson, J M

1990-09-01

The effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix gene expression has been investigated during the process of wound repair, where the formation of new connective tissue represents a critical step in restoring tissue integrity. Split-thickness excisional wounds in the pig were studied by in situ hybridization in order to obtain subjective findings on the activity and location of cells involved in matrix gene expression after the administration of recombinant TGF-beta 1. Data focus on the stimulatory role of this growth factor in granulation tissue formation, on the enhanced mRNA content of collagen types I and III, fibronectin, TGF-beta 1 itself, and on the reduction in stromelysin mRNA, suggesting that increased matrix formation measured after treatment with TGF-beta 1 is due to fibroplasia regulated by the abundance of mRNAs for several different structural, matrix proteins as well as inhibition of proteolytic phenomena elicited by metalloproteinases. These studies reveal elastin mRNA early in the repair process, and elastin mRNA expression is enhanced by administration of TGF-beta 1. Moreover, we show that TGF-beta 1 was auto-stimulating in wounds, accounting, at least in part, for the persistent effects of single doses of this multipotential cytokine.

Polysaccharides from Enteromorpha prolifera Improve Glucose Metabolism in Diabetic Rats

PubMed Central

Lin, Wenting; Wang, Wenxiang; Liao, Dongdong; Chen, Damiao; Zhu, Pingping; Cai, Guoxi; Kiyoshi, Aoyagi

2015-01-01

This study investigated the effects of polysaccharides from Enteromorpha prolifera (PEP) on glucose metabolism in a rat model of diabetes mellitus (DM). PEP (0, 150, 300, and 600 mg/kg) was administered intragastrically to rats for four weeks. After treatment, fasting blood glucose (FBG) and insulin (INS) levels were measured, and the insulin sensitivity index (ISI) was calculated. The morphopathological changes in the pancreas were observed. Serum samples were collected to measure the oxidant-antioxidant status. The mRNA expression levels of glucokinase (GCK) and insulin receptor (InsR) in liver tissue and glucose transporter type 4 (GLUT-4) and adiponectin (APN) in adipose tissue were determined. Compared with the model group, the FBG and INS levels were lower, the ISI was higher, and the number of islet β-cells was significantly increased in all the PEP groups. In the medium- and high-dose PEP groups, MDA levels decreased, and the enzymatic activities of SOD and GSH-Px increased. The mRNA expression of InsR and GCK increased in all the PEP groups; APN mRNA expression increased in the high-dose PEP group, and GLUT-4 mRNA expression increased in adipose tissue. These findings suggest that PEP is a potential therapeutic agent that can be utilized to treat DM. PMID:26347892

Expression of 11beta-hydroxysteroid dehydrogenase 1 and 2 in subcutaneous adipose tissue of lean and obese women with and without polycystic ovary syndrome.

PubMed

Svendsen, P F; Madsbad, S; Nilas, L; Paulsen, S K; Pedersen, S B

2009-11-01

To investigate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 and 2 and hexose-6-phosphate dehydrogenase (H6PDH) mRNA in subcutaneous abdominal tissue from lean and obese women with and without polycystic ovary syndrome (PCOS), and to investigate the association between these enzymes and different measures of insulin sensitivity. Cross-sectional study. A total of 60 women, 36 women with PCOS, 17 lean (lean PCOS, LP) and 19 obese (obese PCOS, OP) and 24 age- and weight-matched control women, 8 lean (lean controls, LC) and 16 obese (obese controls, OC). Subcutaneous adipose tissue was collected from the abdomen. Peripheral insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp and determined as glucose disposal rate and insulin sensitivity index. Whole-body insulin sensitivity was calculated using homeostasis model assessment insulin resistance index. Body composition was evaluated by dual X-ray absorptiometry. Adipose mRNA expression of leptin and adiponectin were determined by real-time PCR. Polycystic ovary syndrome (P<0.05) and obesity (P<0.05) were independently associated with increased expression of 11beta-HSD1 mRNA. The subgroups LP and OC had increased 11beta-HSD1 and 11beta-HSD2 mRNA expression compared with LC (P<0.05, P<0.05). There were no effects of PCOS or obesity on11beta-HSD2 or H6PDH mRNA expression. Decreased peripheral insulin sensitivity (P<0.001) and increased upper body fat distribution (P<0.01) were associated with increased expression of 11beta-HSD1, but neither 11beta-HSD2 nor H6PDH. Polycystic ovary syndrome and obesity are independently associated with increased expression of 11beta-HSD1. This may lead to increased conversion of cortisone to cortisol in the peripheral adipose tissue and subsequently increased glucocorticoid activity. Decreased peripheral insulin sensitivity and central obesity was associated with increased expression of 11beta-HSD1.

Behaviour of human mesenchymal stem cells on a polyelectrolyte-modified HEMA hydrogel for silk-based ligament tissue engineering.

PubMed

Bosetti, M; Boccafoschi, F; Calarco, A; Leigheb, M; Gatti, S; Piffanelli, V; Peluso, G; Cannas, M

2008-01-01

The aim of this study was to design a functional bio-engineered material to be used as scaffold for autologous mesenchymal stem cells in ligament tissue engineering. Polyelectrolyte modified HEMA hydrogel (HEMA-co-METAC), applied as coating on silk fibroin fibres, has been formulated in order to take advantage of the biocompatibility of the polyelectrolyte by increasing its mechanical properties with silk fibres. Human bone marrow mesenchymal stem cells behaviour on such reinforced polyelectrolyte has been studied by evaluating cell morphology, cell number, attachment, spreading and proliferation together with collagen matrix production and its mRNA expression. Silk fibroin fibres matrices with HEMA-co-METAC coating exhibited acceptable mechanical behaviour compared to the natural ligament, good human mesenchymal stem cell adhesion and with mRNA expression studies higher levels of collagen types I and III expression when compared to control cells on polystyrene. These data indicate high expression of mRNA for proteins responsible for the functional characteristics of the ligaments and suggest a potential for use of this biomaterial in ligament tissue-engineering applications.

Effects of varying dietary iodine supplementation levels as iodide or iodate on thyroid status as well as mRNA expression and enzyme activity of antioxidative enzymes in tissues of grower/finisher pigs.

PubMed

Li, Qimeng; Mair, Christiane; Schedle, Karl; Hellmayr, Isabella; Windisch, Wilhelm

2013-02-01

The objective of this study was to investigate the influence of high dietary iodine supply and different iodine sources on thyroid status and oxidative stress in target tissues of the thyroid hormones in fattening pigs. Eighty castrates (body weight: 33.3 ± 0.4 kg) were randomly allotted into five different treatments: The control diet contained 150 μg I/kg as KI, the other feeding groups were supplemented with 4,000 μg I/kg (as KI and KIO(3)) and 10,000 μg I/kg (as KI and KIO(3)), respectively. The mRNA expression levels of sodium/iodide symporter (NIS) and key antioxidant enzymes (Cu/Zn SOD, CAT, GPx) were analyzed in thyroid gland, liver, kidney, muscle, and adipose tissue sampled during slaughter. Furthermore, antioxidant enzyme activities and the effect on lipid peroxidation (MDA) were determined in liver and muscle. In thyroid gland, a significant downregulation of NIS and Cu/Zn SOD mRNA expression was observed in high-iodine groups. In liver, a source effect on the mRNA expression of Cu/Zn SOD between KI and KIO(3) at 4,000 μg I/kg was shown. In contrast, not SOD but GPx activity was affected by iodine source with strongest downregulation in high KIO(3) group. In muscle, GPx activity was affected by both iodine source and dose, showing stronger downregulation in KI groups. In kidney and adipose tissue, oxidative stress parameters showed no or only unsystematic changes. However, variation in iodine supply had no effect on MDA concentrations. NIS expression was significantly decreased with increased iodine supplementation, which is to ensure the thyroid gland function. However, the alleviating effect of iodine supplementation observed in antioxidant enzyme mRNA expression and activity did not reflect on the lipid peroxide level.

Comparison of serum vaspin levels and vaspin expression in adipose tissue and smooth muscle tissue in pregnant women with and without gestational diabetes.

PubMed

Tang, Yuping; Qiao, Ping; Qu, Xiaoxian; Bao, Yirong; Li, Yuhong; Liao, Yini; Ying, Hao

2017-10-01

Vaspin is associated with metabolic parameters and insulin resistance. However, the expression of vaspin in visceral adipose tissue (VAT) in pregnant women with gestational diabetes mellitus (GDM) has not been fully explored, and the contribution of vaspin to the biological mechanisms underlying GDM remains unclear. This study aimed to compare circulating vaspin levels and its expression in different insulin target tissues including subcutaneous adipose tissue (SAT), VAT and smooth muscle tissue (SMT) in pregnant women with and without GDM. A total of 37 women with GDM (GDM group) and 37 normal pregnant women (control group) were selected. Fasting plasma glucose (FPG), fasting insulin (FINS) and serum vaspin levels were quantified at term, and homeostasis model of assessment2-insulin resistance (HOMA2-IR) values were calculated. RT-qPCR and Western blotting were used to measure mRNA and protein levels of vaspin in VAT, SAT and SMT of 15 GDM women and normal pregnant women. In the GDM group, serum vaspin concentrations were significantly higher than in the control group. Serum vaspin levels were positively correlated with HOMA2-IR in the GDM group but not in the control group. In the GDM group, vaspin mRNA and protein expression levels in SAT and VAT were both significantly higher than in controls, but no difference was found in SMT. Moreover, relative mRNA but not protein expression levels of vaspin in SAT were highest among the three tissues in both groups. Circulating vaspin levels and expression of vaspin in SAT and VAT were higher in GDM women than in normal pregnant women. However, the specific role of vaspin from SAT and VAT in the pathogenesis of GDM needs further study. © 2017 John Wiley & Sons Ltd.

PPAR expression throughout the oestrous cycle in the bovine endometrium.

PubMed

Socha, B M; Łupicka, M; Szczepańska, A A; Korzekwa, A J

2017-09-15

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that is composed of three isoforms: PPARα, PPARβ/δ and PPARγ. The ratio of two isoforms of PPARγ (1 and 2) varies among both species and tissues. The activity of PPARs can be modified by a number of endogenous compounds, including arachidonic acid (AA), its eicosanoid metabolites and synthetic ligands. Many studies have revealed that PPARs are important in reproduction. We hypothesized that the profiles of PPARs expression vary in the bovine endometrium during certain days of the oestrous cycle. The aim of this study was to determine the immunolocalization, mRNA expression and protein expression of PPARα, PPARβ/δ and PPARγ in the bovine endometrium throughout the oestrous cycle. Endometrial tissues were obtained post mortem from heifers on days 0 (oestrus phase, n = 6), 2-5 (early luteal phase, n = 6), 8-12 (mid-luteal phase, n = 6), 15-17 (late luteal phase, n = 6) and 19-21 (follicular phase, n = 6) of the oestrous cycle. PPARs immunolocalization was determined in the endometrium using immunohistochemistry. The mRNA and protein expression were evaluated by real-time PCR and Western blotting, respectively. The results were statistically analysed by one-way ANOVA followed by a Bonferroni test. Immunolocalization revealed the protein expression of PPARα, PPARβ/δ and PPARγ in bovine endometrial structures throughout the oestrous cycle. PPARγ 1 mRNA and protein expression fluctuated in the tissue depending on the studied days of the oestrous cycle, whereas the transcript and protein levels of PPARα and PPARβ/δ did not display significant differences during the oestrous cycle. We observed the highest PPARγ 1 mRNA expression at the oestrus phase and the lowest expression at the mid-luteal phase. During the late luteal and follicular phases, the mRNA and protein expression of PPARγ 1 were detectable at similar levels compared to the early luteal and mid-luteal phases of the oestrous cycle. The overall results indicate the presence of PPARα, PPARβ/δ and PPARγ in endometrial tissues, but the mRNA and protein expression of only PPARγ 1 changed throughout the oestrous cycle, especially during the oestrus and mid-luteal phases. Our findings suggest an association between the expression of PPARs in the bovine endometrium and stage of the oestrous cycle that may be a consequence of changes in ovarian steroids. Copyright © 2017. Published by Elsevier Inc.

Quantification of STAT3 and VEGF expression for molecular diagnosis of lymph node metastasis in breast cancer

PubMed Central

Chen, Yujuan; Liu, Ya; Wang, Yu; Li, Wen; Wang, Xiaolu; Liu, Xuejuan; Chen, Yao; Ouyang, Chibin; Wang, Jing

2017-01-01

Abstract Background: Axillary lymph node metastasis is associated with increased risk of regional recurrence, distant metastasis, and poor survival in breast malignant neoplasm. Expression of signal transducer and activator of transcription 3 (STAT3) is significantly associated with tumor formation, migration, and invasion in various cancers. In addition, vascular endothelial growth factor (VEGF) expression could promote angiogenesis and increase the risk of tumorigenesis. To determine correlations among STAT3 expression, VEGF, and clinicopathological data on lymph node involvement in breast cancer patients after surgery. Methods: The mRNA expression levels of STAT3 and VEGFs were measured in 45 breast invasive ductal carcinoma tissues, 45 peritumoral tissues, and 45 adjacent nontumor tissues by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Postoperative pathological examination revealed explicit axillary lymph node involvement in all patients. Results: Average mRNA levels of STAT3 and VEGFs were the highest in breast invasive ductal carcinoma tissues, followed by peritumoral tissues. High expression of STAT3 showed significant positive correlation with high axillary lymph node involvement and progesterone receptor (PR), VEGF-C, VEGF-D, and vascular endothelial growth factor receptor (VEGFR)-3 expression. The expression levels of STAT3, VEGF-C, and VEGFR-3 were significantly higher in the tumor tissues of patients with axillary lymph node metastasis than in those of patients without the metastasis. Expression levels of VEGF-C and VEGFR-3 were also significantly higher in peritumoral tissues of patients with axillary lymph node metastasis. Positive correlations were found between STAT3 and VEGF-C/-D mRNA levels. Conclusion: These data suggest that STAT3/VEGF-C/VEGFR-3 signaling pathway plays an important role in carcinogenesis and lymph-angiogenesis. Our findings suggest that STAT3 may be a potential molecular biomarker for predicting the involvement of axillary lymph nodes in breast cancer, and therapies targeting STAT3 may be important for preventing breast cancer metastasis. PMID:29137038

Differential Expression of Glypican-3 and Insulin–Like Growth Factor-II mRNAs and Alpha-Fetoprotein and Ki-67 Markers in HCV Related Hepatocellular Carcinomas In Egyptian Patients

PubMed

Saber, Mohamed A; MM AbdelHafiz, Samah; Khorshed, Fatma E; Aboushousha, Tarek S; Hamdy, Hussam EM; Seleem, Mohamed I; Soliman, Amira H

2017-01-01

Background: Increasing evidence indicates that in hepatocellular carcinomas (HCCs) abnormal gene expression, for example of glypican-3 (GPC-3) and insulin-like growth factor-II (IGF-II), are associated with the occurrence and progression of HCC. The objective of this study was to evaluate the differential expression of GPC-3 and IGF-II mRNAs in HCC tissues with a background of chronic hepatitis C virus (HCV) genotype 4 cirrhosis, in relation to Ki-67 and alpha-feto protein (AFP) tissue markers. Methods: One hundred and five patients with HCCs who had undergone hepatectomy, were included, after obtaining informed consent. Total RNA was extracted from malignant and corresponding peri-malignant liver tissues, and GPC-3 and IGF-II mRNAs in addition to beta-actin mRNA as an internal control, were evaluated in all samples by reverse transcriptase-polymerase chain reactions (RT-PCR). Routine histopathological diagnosis as well as immunohistochemical (IHC) staining using monoclonal antibodies for Ki-67 and AFP were also performed. Result: Expression of GPC-3 mRNA was positive in all HCC malignant tissue, with overexpression in 86/105 (81.9%); in respect to the grade of the tumor (1-3 grades), while in peri-malignant tissue it was over expressed only in 20/105 (19%). The IGF-II mRNA was over expressed in only 10/105 (9.5%) malignant and peri-malignant samples. AFP was expressed in 33.3% of malignant samples but absent in peri-malignant tissues. Ki-67 expression was significantly increased in malignant compared to peri-malignant tissue. Conclusion: GPC-3 and IGF II mRNAs may be good molecular markers for HCC, especially with a background of cirrhosis due to chronic HCV infection. Significant correlations were noted with the pattern of AFP and Ki-67 expression. Creative Commons Attribution License

Serum concentrations and subcutaneous adipose tissue mRNA expression of omentin in morbid obesity and type 2 diabetes mellitus: the effect of very-low-calorie diet, physical activity and laparoscopic sleeve gastrectomy.

PubMed

Urbanová, M; Dostálová, I; Trachta, P; Drápalová, J; Kaválková, P; Haluzíková, D; Matoulek, M; Lacinová, Z; Mráz, M; Kasalický, M; Haluzík, M

2014-01-01

Omentin is a novel adipokine with insulin-sensitizing effects expressed predominantly in visceral fat. We investigated serum omentin levels and its mRNA expression in subcutaneous adipose tissue (SCAT) of 11 women with type 2 diabetes mellitus (T2DM), 37 obese non-diabetic women (OB) and 26 healthy lean women (C) before and after various weight loss interventions: 2-week very-low-calorie diet (VLCD), 3-month regular exercise and laparoscopic sleeve gastrectomy (LSG). At baseline, both T2DM and OB groups had decreased serum omentin concentrations compared with C group while omentin mRNA expression in SCAT did not significantly differ among the groups. Neither VLCD nor exercise significantly affected serum omentin concentrations and its mRNA expression in SCAT of OB or T2DM group. LSG significantly increased serum omentin levels in OB group. In contrast, omentin mRNA expression in SCAT was significantly reduced after LSG. Baseline fasting serum omentin levels in a combined group of the studied subjects (C, OB, T2DM) negatively correlated with BMI, CRP, insulin, LDL-cholesterol, triglycerides and leptin and were positively related to HDL-cholesterol. Reduced circulating omentin levels could play a role in the etiopathogenesis of obesity and T2DM. The increase in circulating omentin levels and the decrease in omentin mRNA expression in SCAT of obese women after LSG might contribute to surgery-induced metabolic improvements and sustained reduction of body weight.

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.

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

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

PubMed Central

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

1999-01-01

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

RELATIONSHIP BETWEEN BRAIN AND OVARY AROMATASE ACTIVITY AND ISOFORM-SPECIFIC AROMATASE MRNA EXPRESSION IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

EPA Science Inventory

There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead min...

RELATIONSHIP BETWEEN BRAIN AND OVARY AROMATASE ACTIVITY AND ISOFORM-SPECIFIC AROMATASE MRNA EXPRESSION IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS) - JOURNAL ARTICLE

EPA Science Inventory

There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead minn...

Effect of Ginkgo biloba extract on apoptosis of brain tissues in rats with acute cerebral infarction and related gene expression.

PubMed

Wu, C; Zhao, X; Zhang, X; Liu, S; Zhao, H; Chen, Y

2015-06-11

We investigated the effect of Ginkgo biloba extract on apoptosis of brain tissues in rats with acute cerebral infarction and apoptosis-related gene expression. Rat models of acute cerebral infarction were constructed using the suture method, and randomly divided into the control group, model, and treatment groups. In the treatment group, 4 mg/kg G. biloba extract was intravenously injected into the rat tail vein. Phosphate-buffered saline solution was injected in the model group. Seventy-two hours after treatment, rats were euthanized, and brain tissues were removed to analyze the changes in caspase-3, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) mRNA and protein levels, and variation in brain tissue cells' apoptosis indices was measured. Compared with the control group, the model and treatment groups showed significantly upregulated caspase-3, Bcl-2, and Bax mRNA and protein levels in brain tissues, but remarkably downregulated Bcl-2 mRNA and protein levels (P < 0.05). After treatment, in treatment group brain tissues, caspase-3 and Bax mRNA and protein levels were significantly lower than those in the model group, while Bcl-2 mRNA and protein levels were higher than that in the model group (P < 0.05). The model and treatment groups showed increased cell apoptosis indices of brain tissues compared to the control group; after treatment, the apoptosis index in the treatment group was significantly downregulated compared with that in the model group (P < 0.05). In conclusion, G. biloba extract significantly reduced apoptosis in rat brain tissue cells with acute cerebral infarction and thus protected brain tissues.

Vitamin D Receptor gene (VDR) transcripts in bone, cartilage, muscles and blood and microarray analysis of vitamin D responsive genes expression in paravertebral muscles of Juvenile and Adolescent Idiopathic Scoliosis patients

PubMed Central

2012-01-01

Background VDR may be considered as a candidate gene potentially related to Idiopathic Scoliosis susceptibility and natural history. Transcriptional profile of VDR mRNA isoforms might be changed in the structural tissues of the scoliotic spine and potentially influence the expression of VDR responsive genes. The purpose of the study was to determine differences in mRNA abundance of VDR isoforms in bone, cartilage and paravertebral muscles between tissues from curve concavity and convexity, between JIS and AIS and to identify VDR responsive genes differentiating Juvenile and Adolescent Idiopathic Scoliosis in paravertebral muscles. Methods In a group of 29 patients with JIS and AIS, specimens of bone, cartilage, paravertebral muscles were harvested at the both sides of the curve apex together with peripheral blood samples. Extracted total RNA served as a matrix for VDRs and VDRl mRNA quantification by QRT PCR. Subsequent microarray analysis of paravertebral muscular tissue samples was performed with HG U133A chips (Affymetrix). Quantitative data were compared by a nonparametric Mann Whitney U test. Microarray results were analyzed with GeneSpring 11GX application. Matrix plot of normalized log-intensities visualized the degree of differentiation between muscular tissue transcriptomes of JIS and AIS group. Fold Change Analysis with cutoff of Fold Change ≥2 identified differentially expressed VDR responsive genes in paravertebral muscles of JIS and AIS. Results No significant differences in transcript abundance of VDR isoforms between tissues of the curve concavity and convexity were found. Statistically significant difference between JIS and AIS group in mRNA abundance of VDRl isoform was found in paravertebral muscles of curve concavity. Higher degree of muscular transcriptome differentiation between curve concavity and convexity was visualized in JIS group. In paravertebral muscles Tob2 and MED13 were selected as genes differentially expressed in JIS and AIS group. Conclusions In Idiopathic Scolioses transcriptional activity and alternative splicing of VDR mRNA in osseous, cartilaginous, and paravertebral muscular tissues are tissue specific and equal on both sides of the curve. The number of mRNA copies of VDRl izoform in concave paravertebral muscles might be one of the factors differentiating JIS and AIS. In paravertebral muscles Tob2 and Med13 genes differentiate Adolescent and Juvenile type of Idiopathic Scoliosis. PMID:23259508

Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multi-gene family

PubMed Central

Dean, Caroline; Elzen, Peter van den; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Of the eight nuclear genes in the plant multi-gene family which encodes the small subunit (rbcS) of Petunia (Mitchell) ribulose bisphosphate carboxylase, one rbcS gene accounts for 47% of the total rbcS gene expression in petunia leaf tissue. Expression of each of five other rbcS genes is detected at levels between 2 and 23% of the total rbcS expression in leaf tissue, while expression of the remaining two rbcS genes is not detected. There is considerable variation (500-fold) in the levels of total rbcS mRNA in six organs of petunia (leaves, sepals, petals, stems, roots and stigmas/anthers). One gene, SSU301, showed the highest levels of steady-state mRNA in each of the organs examined. We discuss the differences in the steady-state mRNA levels of the individual rbcS genes in relation to their gene structure, nucleotide sequence and genomic linkage. ImagesFig. 2.Fig. 3. PMID:16453647

MSX-1 gene expression and regulation in embryonic palatal tissue.

PubMed

Nugent, P; Greene, R M

1998-01-01

The palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.

Decoy receptor 3 expression in esophageal squamous cell carcinoma: correlation with tumour invasion and metastasis.

PubMed

Xiong, Gang; Guo, Hong; Ge, Xiaodong; Xu, Xueqing; Yang, Xiaoya; Yang, Kang; Jiang, Yaoguang; Bai, Yun

2011-03-01

Decoy receptor 3 (DcR3) is a soluble receptor, which can bind to and inactivate the apoptosis-inducing ligands. We studied a possible association between DcR3 expression and clinicopathologic features in patients with esophageal squamous cell carcinoma (ESCC). The mRNA expression of DcR3 was examined by RT-PCR in 109 primary ESCC patients. For the 52 pairs of DcR3 positive tissues, the protein expression was determined by immunohistochemistry. There was a strong correlation among DcR3 mRNA expression and tumor invasion (P=0.01) and lymph node metastasis (P=0.036). We also found that there was a correlation between DcR3 overexpression with lymph node metastasis (P=0.014) in 52 pairs of DCR3 mRNA positive tissues. Our finding suggested that the overexpression of DcR3 is significantly related with ESCC clinical staging. DcR3 might be a candidate as a tumor specific biomarker for ESCC.

Cloning of peroxisome proliferators activated receptors in the cobia (Rachycentron canadum) and their expression at different life-cycle stages under cage aquaculture.

PubMed

Tsai, Mei-Ling; Chen, Houng-Yung; Tseng, Mei-Cheuh; Chang, Rey-Chang

2008-12-01

We present the cDNA sequences and tissue mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, beta and gamma isotypes in the cobia (Rachycentron canadum), a warm water pelagic fish that is becoming a fish of choice for offshore cage farming. RT-PCR and real-time PCR showed that PPARalpha mRNA predominated in red muscle, heart and liver whereas PPARbeta was expressed mainly in liver and pyloric caeca. In contrast, PPARgamma transcripts were detected in all of the tissues examined, with the highest level occurring in visceral fat depot. Our 52-wk time-series investigation showed that while the mRNA expression of PPARgamma in the cobia was positively (P < 0.05) related to its body lipid deposition, a negative (P < 0.05) relationship was found between PPARalpha expression in the liver and body lipid deposition. There was a significant increase in body lipid deposition and hepatic PPARgamma expression as the fish grew. The hepatic PPARgamma expression could be a sufficient parameter describing the bodily expression of PPARgamma because of its positive correlation with PPARgamma expressions in all other tissues. These results showed that PPARgamma and alpha played a pivotal role in the control of lipid metabolic and storage functions in the liver, muscle and visceral fat depot of the cobia.

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

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

Feng, Guoxing; Shi, Hui; Li, Jiong

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

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.

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

Over, and Underexpression of Endothelin 1 and TGF-Beta Family Ligands and Receptors in Lung Tissue of Broilers with Pulmonary Hypertension

PubMed Central

Dominguez-Avila, Norma; Ruiz-Castañeda, Gabriel; González-Ramírez, Javier; Fernandez-Jaramillo, Nora; Escoto, Jorge; Sánchez-Muñoz, Fausto; Marquez-Velasco, Ricardo; Bojalil, Rafael; Espinosa-Cervantes, Román; Sánchez, Fausto

2013-01-01

Transforming growth factor beta (TGFβ) is a family of genes that play a key role in mediating tissue remodeling in various forms of acute and chronic lung disease. In order to assess their role on pulmonary hypertension in broilers, we determined mRNA expression of genes of the TGFβ family and endothelin 1 in lung samples from 4-week-old chickens raised either under normal or cold temperature conditions. Both in control and cold-treated groups of broilers, endothelin 1 mRNA expression levels in lungs from ascitic chickens were higher than levels from healthy birds (P < 0.05), whereas levels in animals with cardiac failure were intermediate. Conversely, TGFβ2 and TGFβ3 gene expression in lungs were higher in healthy animals than in ascitic animals in both groups (P < 0.05). TGFβ1, TβRI, and TβRII mRNA gene expression among healthy, ascitic, and chickens with cardiac failure showed no differences (P > 0.05). BAMBI mRNA gene expression was lowest in birds with ascites only in the control group as compared with the values from healthy birds (P < 0.05). PMID:24286074

CFTR expression and organ damage in cystic fibrosis

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

Tizzano, E.; Chitayat, D.; Buchwald, M.

1994-09-01

To assist our understanding of the origin of organ damage caused by cystic fibrosis (CF) disease, we have analyzed the pattern of expression of the CF gene (CFTR). mRNA in situ hybridization analysis was carried out in human fetal, newborn, infant and adult tissues and the abundance of the mRNA was correlated with the known pathology at the various stages of human development. Analysis of the pattern of expression indicates a constitutive level of mRNA in gastrointestinal tissues starting during early development and maintained throughout life. Prenatal respiratory tissues show qualitative and quantitative major differences in comparison to postnatal lungmore » samples. Male reproductive tissues show high levels of expression in the head of the epididymis compared with the rest of the male ducts. Female reproductive tissues show a variable pattern of expression at different stages during fetal development and during puberty probably due to changes in hormonal levels. Gastrointestinal and male reproductive tissues have a consistent pathology at birth, whereas no lung abnormalities have been described in newborns affected by CF. Our results show that there is no exact correlations between organ damage present at birth and the degree of CFTR expression. To explain these observations, we hypothesize that the pathogenesis of organ damage in CF depend on at least three factors: the rate of CFTR-mediated fluid secretion, differences in genotype and environmental factors, such as the amount of macromolecules in the lumen of the ducts. This last element predicts that damage will occur in tissues with high protein loads and low flow rates (e.g. pancreas, epididymis), where the absence of CFTR function leads to obstruction and pathology. Organs that express CFTR but with no significant damage (e.g. prenatal lung, female reproductive tissues), will have a low protein load and a high flow rates.« less

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

PubMed

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

2013-01-01

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

Effect of energy restriction and physical exercise intervention on phenotypic flexibility as examined by transcriptomics analyses of mRNA from adipose tissue and whole body magnetic resonance imaging.

PubMed

Lee, Sindre; Norheim, Frode; Langleite, Torgrim M; Noreng, Hans J; Storås, Trygve H; Afman, Lydia A; Frost, Gary; Bell, Jimmy D; Thomas, E Louise; Kolnes, Kristoffer J; Tangen, Daniel S; Stadheim, Hans K; Gilfillan, Gregor D; Gulseth, Hanne L; Birkeland, Kåre I; Jensen, Jørgen; Drevon, Christian A; Holen, Torgeir

2016-11-01

Overweight and obesity lead to changes in adipose tissue such as inflammation and reduced insulin sensitivity. The aim of this study was to assess how altered energy balance by reduced food intake or enhanced physical activity affect these processes. We studied sedentary subjects with overweight/obesity in two intervention studies, each lasting 12 weeks affecting energy balance either by energy restriction (~20% reduced intake of energy from food) in one group, or by enhanced energy expenditure due to physical exercise (combined endurance- and strength-training) in the other group. We monitored mRNA expression by microarray and mRNA sequencing from adipose tissue biopsies. We also measured several plasma parameters as well as fat distribution with magnetic resonance imaging and spectroscopy. Comparison of microarray and mRNA sequencing showed strong correlations, which were also confirmed using RT-PCR In the energy restricted subjects (body weight reduced by 5% during a 12 weeks intervention), there were clear signs of enhanced lipolysis as monitored by mRNA in adipose tissue as well as plasma concentration of free-fatty acids. This increase was strongly related to increased expression of markers for M1-like macrophages in adipose tissue. In the exercising subjects (glucose infusion rate increased by 29% during a 12-week intervention), there was a marked reduction in the expression of markers of M2-like macrophages and T cells, suggesting that physical exercise was especially important for reducing inflammation in adipose tissue with insignificant reduction in total body weight. Our data indicate that energy restriction and physical exercise affect energy-related pathways as well as inflammatory processes in different ways, probably related to macrophages in adipose tissue. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis.

PubMed

Serce, Nuran Bektas; Boesl, Andreas; Klaman, Irina; von Serényi, Sonja; Noetzel, Erik; Press, Michael F; Dimmler, Arno; Hartmann, Arndt; Sehouli, Jalid; Knuechel, Ruth; Beckmann, Matthias W; Fasching, Peter A; Dahl, Edgar

2012-12-13

Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation.

Analysis of thyroid hormone receptor {beta}A mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action

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

Opitz, Robert; Lutz, Ilka; Nguyen, Ngoc-Ha

2006-04-01

Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors {alpha} (TR{alpha}) and {beta}A (TR{beta}A) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TR{alpha} gene expression whereas a marked up-regulation of TR{beta}A mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TR{beta}A mRNA in head and tail tissue withinmore » 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TR{beta}A mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TR{beta}A gene expression analysis for detection of agonistic and antagonistic effects on T3 action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TR{beta}A expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TR{beta}A mRNA up-regulation. Results of this study suggest that TR{beta}A mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.« less

[Effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction].

PubMed

Tan, Wenqing; Wang, Wei; Zheng, Xuan; Chen, Jiying; Yuan, Xiangning; Zhang, Fangfang; Wang, Shuting; Tao, Lijian

2018-05-28

To investigate the effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction (UUO) and to observe the effect of fluorofenidone on the expressions of collagen type I (Col I), collagen type III (Col III), α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), platelet derived growth factor (PDGF) in the renal tissues of UUO rats.
 Methods: Male Sprague-Dawley (SD) rats were randomly divided into a sham-operated group, a UUO group, and a flurofenidone group (n=5). UUO model was induced by ligating the left ureter in rats. The rats were treated with 125 mg/(kg.d) fluorofenidone by gastric gavage in the fluorofenidone group at 24 h before the operation, and the rats were treated with the identical dose of 0.5% sodium carboxyl methyl cellulose (CMC-Na) in the other 2 groups. The rats were sacrificed at 14 days after UUO. Pathological changes of the renal tissue were observed by HE and Masson staining, the mRNA expressions of Col I, Col III, α-SMA, PDGF and CTGF were detected by real-time PCR, and the protein expressions of Col I, Col III, PDGF and CTGF were detected by immunohistochemical staining.
 Results: The renal interstitial damage index, relative collagen area and mRNA and protein expressions of Col I and Col III in the renal tissues of the rats in the UUO group significantly increased (P<0.05), and fluorofenidone could reduce these indexes (P<0.05). Compared with the sham-operated group, the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF in the renal tissues of the rats in the UUO group were increased, but fluorofenidone could decrease the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF (P<0.05).
 Conclusion: Fluorofenidone (125 mg/kg.d) could attenuate renal interstitial fibrosis through inhibition of fibroblast proliferation, myofibroblastic activation, PDGF and CTGF expression.

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

PubMed

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

2007-06-01

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

Effects of chronic sumatriptan and zolmitriptan treatment on 5-HT receptor expression and function in rats.

PubMed

Reuter, U; Salomone, S; Ickenstein, G W; Waeber, C

2004-05-01

Triptans are commonly used anti-migraine drugs and show agonist action mainly at serotonin 5-HT(1B/1D/1F) receptors. It is not known whether frequent or long-term treatment with these drugs would alter 5-HT receptor function. We investigated the effects of protracted (14-18 days) sumatriptan and zolmitriptan treatment in rats on 5-HT(1) receptor mRNA expression and function in tissues related to migraine pathophysiology. RT-PCR analysis revealed that 5-HT(1B/1D/1F) receptor mRNA was reduced in the trigeminal ganglion after treatment with either triptan (reduction by: sumatriptan 39% and zolmitriptan 61% for 5-HT(1B); 60%vs 41% for 5-HT(1D); 32%vs 68% for 5-HT(1F)). Sumatriptan attenuated 5-HT(1D) receptor mRNA by 49% in the basilar artery, whereas zolmitriptan reduced 5-HT(1B) mRNA in this tissue by 70%. No change in 5-HT(1) receptor mRNA expression was observed in coronary artery and dura mater. Chronic triptan treatment had no effect in two functional assays [sumatriptan mediated inhibition (50 mg/kg, i.p.) of electrically induced plasma protein extravasation in dura mater and 5-nonyloxytryptamine-stimulated [(35)S]guanosine-5'-O-(3-thio)triphosphate binding in substantia nigra]. Furthermore, vasoconstriction to 5-HT in isolated basilar artery was not affected by chronic triptan treatment, while it was slightly reduced in coronary artery. We conclude that, although our treatment protocol altered mRNA receptor expression in several tissues relevant to migraine pathophysiology, it did not attenuate 5-HT(1) receptor-dependent functions in rats.

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

PubMed

Karbowska, Joanna; Kochan, Zdzislaw

2012-03-01

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

Squalene epoxidase, located on chromosome 8q24.1, is upregulated in 8q+ breast cancer and indicates poor clinical outcome in stage I and II disease.

PubMed

Helms, M W; Kemming, D; Pospisil, H; Vogt, U; Buerger, H; Korsching, E; Liedtke, C; Schlotter, C M; Wang, A; Chan, S Y; Brandt, B H

2008-09-02

Gains of chromosomes 7p and 8q are associated with poor prognosis among oestrogen receptor-positive (ER+) stage I/II breast cancer. To identify transcriptional changes associated with this breast cancer subtype, we applied suppression subtractive hybridisation method to analyse differentially expressed genes among six breast tumours with and without chromosomal 7p and 8q gains. Identified mRNAs were validated by real-time RT-PCR in tissue samples obtained from 186 patients with stage I/II breast cancer. Advanced statistical methods were applied to identify associations of mRNA expression with distant metastasis-free survival (DMFS). mRNA expression of the key enzyme of cholesterol biosynthesis, squalene epoxidase (SQLE, chromosomal location 8q24.1), was associated with ER+ 7p+/8q+ breast cancer. Distant metastasis-free survival in stage I/II breast cancer cases was significantly inversely related to SQLE mRNA in multivariate Cox analysis (P<0.001) in two independent patient cohorts of 160 patients each. The clinically favourable group associated with a low SQLE mRNA expression could be further divided by mRNA expression levels of the oestrogen-regulated zinc transporter LIV-1. The data strongly support that SQLE mRNA expression might indicate high-risk ER+ stage I/II breast cancers. Further studies on tumour tissue from standardised treated patients, for example with tamoxifen, may validate the role of SQLE as a novel diagnostic parameter for ER+ early stage breast cancers.

PPARα, PPARβ, and PPARγ expression in prenatal and ...

EPA Pesticide Factsheets

PFOA is developmentally toxic, reducing in utero and neonatal survival, and altering development and growth in mice. PFOA activates PPARα and studies in PPARα knockout mice showed that PPARα signaling is required to produce these effects. This study examines the expression of PPARα, PPARβ, and PPARγ in fetal and postnatal mice. Timed pregnant CD-1 mice were dosed orally from GD1-17 with vehicle or 5 mg PFOA/kg body weight. Tissues were collected on GD14, GD17, PND1, 7, 14, 21, and 28. Each tissue was pooled by litter and divided for preparation of RNA and protein. qPCR and Western blot (WB) data were normalized to internal controls (GAPDH, β-actin) and expressed relative to adult liver. In the developing liver, PPARα mRNA increased with age up to PND14 and then declined. A similar profile was observed for PPARγ with the peak mRNA expression on PND7. PPARβ decreased from GD14 to 17 and then gradually increased to PND14, followed by decline. PFOA exposure decreased mRNA levels of all isoforms relative to controls. For PPARα and γ, the rise in mRNA from GD14 to PND14 appeared to shift to a later age. Hepatic PPAR protein was measured on PND1 and 14. Levels of PPARα increased with age, but PPARβ and PPARγ did not change. PFOA reduced PPARα and increased PPARβ protein expression on PND14, but did not alter PPARγ. In the intestine, PPARα mRNA gradually increased with age and PFOA-exposure had no effect on expression. Intestinal PPARβ mRNA expressio

Maintenance of the Extracellular Matrix in Rat Anterior Pituitary Gland: Identification of Cells Expressing Tissue Inhibitors of Metalloproteinases.

PubMed

Azuma, Morio; Tofrizal, Alimuddin; Maliza, Rita; Batchuluun, Khongorzul; Ramadhani, Dini; Syaidah, Rahimi; Tsukada, Takehiro; Fujiwara, Ken; Kikuchi, Motoshi; Horiguchi, Kotaro; Yashiro, Takashi

2015-12-25

The extracellular matrix (ECM) is important in creating cellular environments in tissues. Recent studies have demonstrated that ECM components are localized in anterior pituitary cells and affect cell activity. Thus, clarifying the mechanism responsible for ECM maintenance would improve understanding of gland function. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases and participate in ECM degradation. In this study, we investigated whether cells expressing TIMPs are present in rat anterior pituitary gland. Reverse transcription polymerase chain reaction was used to analyze expression of the TIMP family (TIMP1-4), and cells producing TIMPs in the gland were identified by using in situ hybridization. Expression of TIMP1, TIMP2, and TIMP3 mRNAs was detected, and the TIMP-expressing cells were located in the gland. The TIMP-expressing cells were also investigated by means of double-staining with in situ hybridization and immunohistochemical techniques. Double-staining revealed that TIMP1 mRNA was expressed in folliculostellate cells. TIMP2 mRNA was detected in folliculostellate cells, prolactin cells, and thyroid-stimulating hormone cells. TIMP3 mRNA was identified in endothelial cells, pericytes, novel desmin-immunopositive perivascular cells, and folliculostellate cells. These findings indicate that TIMP1-, TIMP2-, and TIMP3-expressing cells are present in rat anterior pituitary gland and that they are involved in maintaining ECM components.

Maintenance of the Extracellular Matrix in Rat Anterior Pituitary Gland: Identification of Cells Expressing Tissue Inhibitors of Metalloproteinases

PubMed Central

Azuma, Morio; Tofrizal, Alimuddin; Maliza, Rita; Batchuluun, Khongorzul; Ramadhani, Dini; Syaidah, Rahimi; Tsukada, Takehiro; Fujiwara, Ken; Kikuchi, Motoshi; Horiguchi, Kotaro; Yashiro, Takashi

2015-01-01

The extracellular matrix (ECM) is important in creating cellular environments in tissues. Recent studies have demonstrated that ECM components are localized in anterior pituitary cells and affect cell activity. Thus, clarifying the mechanism responsible for ECM maintenance would improve understanding of gland function. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases and participate in ECM degradation. In this study, we investigated whether cells expressing TIMPs are present in rat anterior pituitary gland. Reverse transcription polymerase chain reaction was used to analyze expression of the TIMP family (TIMP1-4), and cells producing TIMPs in the gland were identified by using in situ hybridization. Expression of TIMP1, TIMP2, and TIMP3 mRNAs was detected, and the TIMP-expressing cells were located in the gland. The TIMP-expressing cells were also investigated by means of double-staining with in situ hybridization and immunohistochemical techniques. Double-staining revealed that TIMP1 mRNA was expressed in folliculostellate cells. TIMP2 mRNA was detected in folliculostellate cells, prolactin cells, and thyroid-stimulating hormone cells. TIMP3 mRNA was identified in endothelial cells, pericytes, novel desmin-immunopositive perivascular cells, and folliculostellate cells. These findings indicate that TIMP1-, TIMP2-, and TIMP3-expressing cells are present in rat anterior pituitary gland and that they are involved in maintaining ECM components. PMID:26855451

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

PubMed

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

2011-04-01

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

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.

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta).

PubMed

Tedeschi, J N; Kennington, W J; Berry, O; Whiting, S; Meekan, M; Mitchell, N J

2015-01-01

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34°C and 36°C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36°C, 3h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cancer testis antigen OY-TES-1 expression and serum immunogenicity in colorectal cancer: its relationship to clinicopathological parameters

PubMed Central

Luo, Bin; Yun, Xiang; Fan, Rong; Lin, Yong-Da; He, Shu-Jia; Zhang, Qing-Mei; Mo, Fa-Rong; Chen, Fang; Xiao, Shao-Wen; Xie, Xiao-Xun

2013-01-01

Cancer testis (CT) antigens are attractive targets for cancer immunotherapy because their expression is restricted in normal germ line tissues but frequently detected in variety of tumors. OY-TES-1 is identified as a member of CT antigens. Current knowledge about OY-TES-1 expression in colorectal cancer (CRC) is solely based on mRNA analysis. None of previous researches has studied OY-TES-1 at protein level. In this study, OY-TES-1 polyclonal antibody was generated. The expression of OY-TES-1 mRNA and protein was detected by RT-PCR and immunohistochemistry in 60 CRC and paired adjacent non-tumor tissues, 24 colorectal adenoma and 3 normal colon tissues, respectively. Sera from 73 CRC patients were also tested for OY-TES-1 antibody by ELISA. Our results showed that the frequency of OY-TES-1 mRNA expression was statistically higher in CRC (73.3%, 44/60) than that in adjacent non-tumor tissue (55.0%, 33/60) and colorectal adenoma (45.8%, 11/24). For the first time, OY-TES-1 protein expression was found in (43.3%, 26/60) of CRC tissues, but absent in any of adjacent non-tumor and colorectal adenoma tissues. No OY-TES-1 expression was found in normal colon by either RT-PCR or immunohistochemistry. Furthermore, OY-TES-1 protein expression was correlated with tumor invasion stage (P=0.004) and histological grade (P=0.040). Anti-OY-TES-1 antibody was detected in (9.6%, 7/73) of CRC patients’ sera but not in 76 healthy donors. This finding demonstrates that OY-TES-1 is frequently expressed in CRC and is able to induce humoral immune response spontaneously in CRC patients, suggesting that it might be a promising immunotherapy target for CRC. PMID:24294369

Progesterone and 17β-estradiol regulate expression of nesfatin-1/NUCB2 in mouse pituitary gland.

PubMed

Chung, Yiwa; Kim, Jinhee; Im, Eunji; Kim, Heejeong; Yang, Hyunwon

2015-01-01

Nesfatin-1 was first shown to be involved in the control of appetite and energy metabolism in the hypothalamus. Many recent reports have shown nesfatin-1 expression in various tissues including the pituitary gland, but its expression and regulation mechanisms in the pituitary gland are unclear. Therefore, first, we investigated the mRNA and protein expression of nesfatin-1 in the pituitary using qRT-PCR and Western blotting, respectively. Expression of NUCB2 mRNA and nesfatin-1 protein was higher in the pituitary gland than in other organs, and nesfatin-1 protein was localized in many cells in the anterior pituitary gland. Next, we investigated whether NUCB2 mRNA expression in the pituitary gland was regulated by sex steroid hormones secreted by the ovary. Mice were ovariectomized and injected with progesterone (P4) and 17β-estradiol (E2). The expression of NUCB2 in the pituitary gland was dramatically decreased after ovariectomy and increased with injection of P4 and E2, respectively. The in vitro experiment to elucidate the direct effect of P4 and E2 on NUCB2 mRNA expression showed NUCB2 mRNA expression was significantly increased with E2 and decreased with P4 alone and P4 plus E2 in cultured pituitary tissue. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the mouse pituitary and was regulated by P4 and E2. These data suggest that reproductive-endocrine regulation through hypothalamus-pituitary-ovary axis may contribute to nesfatin-1/NUCB2 expression in the pituitary gland. Copyright © 2014 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.

Effects of fasting, temperature, and photoperiod on preproghrelin mRNA expression in Chinese perch.

PubMed

Song, Yi; Zhao, Cheng; Liang, Xu-Fang; He, Shan; Tian, Changxu; Cheng, Xiaoyan; Yuan, Xiaochen; Lv, Liyuan; Guo, Wenjie; Xue, Min; Tao, Ya-Xiong

2017-06-01

Preproghrelin, a gut/brain peptide, plays an important role in the regulation of food intake and energy homeostasis in teleost and mammals. In the present study, we obtained the full-length preproghrelin cDNA in Chinese perch. The preproghrelin messenger RNA (mRNA) tissue expression showed that level was much higher in stomach and pituitary than in other tissues. The fasting study showed, after gastric emptying (3-6 h), short-term fasting (6-12 h) increased preproghrelin expression in the stomach. While in the pituitary, fasting reduced preproghrelin expression at 1, 3, 12, and 48 h, presenting state fluctuation of self-adjustment. The temperature study showed that the mRNA expression of preproghrelin was the highest in the brain at 26 °C and highest in the stomach at 32 °C, respectively, with different optimum temperature in these two tissues, reflecting spatiotemporal differences of regulation by central nervous system and peripheral organs. The photoperiod study showed that normal light (11 h of lightness and 13 h of darkness) led to highest preproghrelin expression, both in the brain and in the stomach, than continuous light or continuous dark, proving food intake is adapted to natural photoperiod or normal light in this study. These results all indicated that tissue-specific preproghrelin expression of Chinese perch could be significantly affected by environmental factors. Short-term fasting of 6 h after gastric emptying, 26 °C, and normal light led to higher preproghrelin expression, which indicated potential appetite increase in Chinese perch.

Isolation of Xenopus frizzled-10A and frizzled-10B genomic clones and their expression in adult tissues and embryos.

PubMed

Moriwaki, J; Kajita, E; Kirikoshi, H; Koike, J; Sagara, N; Yasuhiko, Y; Saitoh, T; Hirai, M; Katoh, M; Shiokawa, K

2000-11-19

Frizzled genes, encoding WNT receptors, play key roles in cell fate determination. Here, we isolated two Xenopus frizzled genes (Xfz10A and Xfz10B), probably reflecting pseudotetraploidy in Xenopus. Xfz10A (586 amino acids) and Xfz10B (580 amino acids) both encoded by a single exon, consisted of the N-terminal cysteine-rich domain, seven transmembrane domains, and the C-terminal Ser/Thr-X-Val motif. Xfz10A and Xfz10B were 97.0% identical at the amino acid level, and Xfz10B was 100% identical to previously reported Xfz9, yet Xfz10A was 85.3% and 62.4% identical to FZD10 and FZD9, respectively. Xfz10 mRNA appeared as 3.4 kb in adult tissues and embryos. RT-PCR analyses revealed the expression of more Xfz10A mRNA in stomach, kidney, eye, skeletal muscle, and skin, and more Xfz10B mRNA in heart and ovary, but in embryos, two mRNAs were equally expressed from the blastula stage with their peak expression at the late gastrula stage. The main site of Xfz10 mRNA expression was neural fold at the neurula stage and the dorsal region of midbrain, hindbrain, and spinal cord at the tadpole stage. These results suggest that Xfz10 has important roles in neural tissue formation. Copyright 2000 Academic Press.

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

PubMed

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

1991-04-01

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

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

PubMed

Randhawa, Harsharan; Chikara, Shireen; Gehring, Drew; Yildirim, Tuba; Menon, Jyotsana; Reindl, Katie M

2013-07-01

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

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

PubMed Central

2013-01-01

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

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.

Increase of CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells.

PubMed

Kunzmann, Steffen; Krempl, Christine; Seidenspinner, Silvia; Glaser, Kirsten; Speer, Christian P; Fehrholz, Markus

2018-04-16

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase of CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

Depot-specific Regulation of the Conversion of Cortisone to Cortisol in Human Adipose Tissue

PubMed Central

Lee, Mi-Jeong; Fried, Susan K.; Mundt, Steven S.; Wang, Yanxin; Sullivan, Sean; Stefanni, Alice; Daugherty, Bruce L.; Hermanowski-Vosatka, Anne

2015-01-01

Objective Our main objective was to compare the regulation of cortisol production within omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue. Methods and Procedures Om and Abd sc adipose tissue were obtained at surgery from subjects with a wide range of BMI. Hydroxysteroid dehydrogenase (HSD) activity (3H-cortisone and 3H-cortisol interconversion) and expression were measured before and after organ culture with insulin and/or dexamethasone. Results Type 1 HSD (HSD1) mRNA and reductase activity were mainly expressed within adipocytes and tightly correlated with adipocyte size within both depots. There was no depot difference in HSD1 expression or reductase activity, while cortisol inactivation and HSD2 mRNA expression (expressed in stromal cells) were higher in Om suggesting higher cortisol turnover in this depot. Culture with insulin decreased HSD reductase activity in both depots. Culture with dexamethasone plus insulin compared to insulin alone increased HSD reductase activity only in the Om depot. This depot-specific increase in reductase activity could not be explained by an alteration in HSD1 mRNA or protein, which was paradoxically decreased. However, in Om only, hexose-6-phosphate dehydrogenase (H6PDH) mRNA levels were increased by culture with dexamethasone plus insulin compared to insulin alone, suggesting that higher nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) production within the endoplasmic reticulum (ER) contributed to the higher HSD reductase activity. Discussion We conclude that in the presence of insulin, glucocorticoids cause a depot-specific increase in the activation of cortisone within Om adipose tissue, and that this mechanism may contribute to adipocyte hypertrophy and visceral obesity. PMID:18388900

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

PubMed

Proszkowiec-Weglarz, M; Richards, M P

2009-01-01

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

Prostaglandin H2 synthase-1 and -2 expression in guinea pig gestational tissues during late pregnancy and parturition

PubMed Central

Welsh, Toni; Mitchell, Carolyn M; Walters, William A; Mesiano, Sam; Zakar, Tamas

2005-01-01

Increased intrauterine prostaglandin (PG) production is crucial for the initiation of parturition. To investigate the mechanisms controlling intrauterine PG synthesis, we examined the expression of the key PG biosynthetic isoenzymes, PG-H2 synthase (PTGS)-1 and -2, in the amnion, visceral yolk sac (VYS), placenta and myo-endometrium of pregnant guinea pigs. This animal model was chosen because the hormonal milieu of pregnancy and the role of PGs in the hormonal control of parturition are similar to those in the human. PTGS1 mRNA abundance, measured by real-time RT-PCR, increased in the amnion and the placenta during the last third of gestation. During labour, PTGS1 mRNA levels decreased precipitously in all four tissues. PTGS1 protein abundance, assessed by immunoblotting, increased to high levels in the amnion and the placenta by the end of pregnancy and remained high during labour. PTGS2 mRNA expression was higher in the placenta than in the other tissues, but did not change before and during labour. PTGS2 protein expression decreased in the placenta and remained low in the other tissues during labour. Immunohistochemistry showed pervasive PTGS1 protein expression in the amnion and strong expression in the parietal yolk sac membrane (PYS) covering the placenta. PTGS2 was expressed in the PYS and the endometrium. The PTGS inhibitor piroxicam, administered in doses that inhibited PTGS1 but not PTGS2, significantly prolonged gestation. These data suggest that PGs generated by intrauterine PTGS1 are involved in the timing of birth in guinea pigs. The induction of PTGS1 in the amnion and the PYS is a critical event leading to labour in guinea pigs and models analogous changes in the human gestational tissues before labour. PMID:16239272

Tissue distribution and effects of fasting and obesity on the ghrelin axis in mice.

PubMed

Morash, Michael G; Gagnon, Jeffrey; Nelson, Stephanie; Anini, Younes

2010-08-09

Ghrelin is a 28 amino acid peptide hormone derived from the 117 amino acid proghrelin, following cleavage by proprotein convertase 1 (PC1). In this study, we comprehensively assessed the tissue distribution and the effect of fasting and obesity on preproghrelin, Exon-4D, PC1 and GOAT expression and proghrelin-derived peptide (PGDP) secretion. The stomach was the major source of preproghrelin expression and PDGPs, followed by the small intestine. The remaining peripheral tissues (including the brain and pancreas) contained negligible expression levels. We detected obestatin in all stomach proghrelin cells, however, 22% of proghrelin cells in the small intestine did not express obestatin. There were strain differences in ghrelin secretion in response to fasting between CD1 and C57BL/6 mice. After a 24 hour-fast, CD1 mice had increased plasma levels of total ghrelin and obestatin with no change in preproghrelin mRNA or PGDP tissues levels. C57BL/6 mice showed a different response to a 24 hour-fast having increased proghrelin mRNA expression, stomach acylated ghrelin peptide and no change in plasma obestatin in C57BL/6 mice. In obese mice (ob/ob and diet-induced obesity (DIO)) there was a significant increase in preproghrelin mRNA levels while tissue and plasma PGDP levels were significantly reduced. Fasting did not affect PGDP in obese mice. Obese models displayed differences in GOAT expression, which was elevated in DIO mice, but reduced in ob/ob mice. We did not find co-localization of the leptin receptor in ghrelin expressing stomach cells, ruling out a direct effect of leptin on stomach ghrelin synthesis and secretion. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Role of Krüppel-like factor 4 and heat shock protein 27 in cancer of the larynx

PubMed Central

Karam, Jihad; Fadous-Khalifé, Marie Claude; Tannous, Rita; Fakhreddine, Sally; Massoud, Marcel; Hadchity, Joseph; Aftimos, Georges; Hadchity, Elie

2017-01-01

Late detection and lack of standard treatment strategies in larynx cancer patients result in high levels of mortality and poor prognosis. Prognostic stratification of larynx cancer patients based on molecular prognostic tumor biomarkers may lead to more efficient clinical management. Krüppel-like factor 4 (KLF4) and Heat Shock Protein 27 (HSP27) have an important role in tumorigenesis and are considered promising candidate biomarkers for various types of cancer. However, their role in larynx carcinoma remains to be elucidated. The present study aimed to determine KLF4 and HSP27 expression profiles in laryngeal tumors. The protein and mRNA expression levels of KLF4 and HSP27 were evaluated by immunohistochemical and reverse transcription-polymerase chain reaction analyses in 44 larynx carcinoma samples and 21 normal tissue samples, and then correlated with clinical characteristics. A differential expression of KLF4 and HSP27 was observed between normal and tumor tissues. The protein and mRNA expression levels of KLF4 were significantly decreased in larynx squamous cell carcinoma (LSCC) compared with normal tissue, whereas HSP27 was significantly overexpressed in tumor tissues compared with normal tissues, at the protein and mRNA levels. KLF4 expression decreased gradually with tumor progression whereas HSP27 expression increased. A significant difference was observed between stages I and IV. KLF4 and HSP27 exhibit opposite functions and roles in the carcinogenic process of LSCC. Their role in laryngeal cancer initiation and progression emphasizes their use as potential future targets for prognosis and treatment. KLF4 and HSP27 expression levels may act as potential biomarkers in patients with cancer of the larynx. PMID:29181170

Changes in obestatin gene and GPR39 receptor expression in peripheral tissues of rat models of obesity, type 1 and type 2 diabetes.

PubMed

Kolodziejski, Pawel Antoni; Pruszynska-Oszmalek, Ewa; Sassek, Maciej; Kaczmarek, Przemyslaw; Szczepankiewicz, Dawid; Billert, Maria; Mackowiak, Paweł; Strowski, Mathias Z; Nowak, Krzysztof W

2017-04-01

Obestatin has a role in regulating food intake and energy expenditure, but the roles of obestatin and the GPR39 receptor in obesity and type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively) are not well understood. The aim of the present study was to investigate changes in obestatin and GPR39 in pathophysiological conditions like obesity, T1DM, and T2DM. Using rat models of diet-induced obesity (DIO), T1DM and T2DM (n = 14 per group), obestatin, its precursor protein preproghrelin, and GPR39 expression was investigated in tissues involved in glucose and lipid homeostasis regulation. Furthermore, serum obestatin and ghrelin concentrations were determined. Serum obestatin concentrations were positively correlated with glucagon (r = 0.6456; P < 0.001) and visfatin (r = 0.5560; P < 0.001), and negatively correlated with insulin (r = -0.4362; P < 0.05), adiponectin (r = -0.3998; P < 0.05), and leptin (r = -0.4180; P < 0.05). There were differences in GPR39 and preproghrelin expression in the three animal models. Hepatic GPR39 and preproghrelin mRNA expression was greater in T1DM, T2DM, and obese rats than in lean controls, whereas pancreatic GPR39 mRNA and protein and preproghrelin mRNA expression was decreased in T1DM, T2DM, and DIO rats. Higher GPR39 and preproghrelin protein and mRNA levels were found in adipose tissues of T1DM compared with control. In adipose tissues of T2DM and DIO rats, GPR39 protein levels were lower than in lean or T1DM rats. Preproghrelin mRNA was higher in adipose tissues of T1DM, T2DM, and DIO than lean rats. We hypothesize that changes in obestatin, GPR39, and ghrelin may contribute to metabolic abnormalities in T1DM, T2DM, and obesity. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

l-DOPA Decarboxylase (DDC) Expression Status as a Novel Molecular Tumor Marker for Diagnostic and Prognostic Purposes in Laryngeal Cancer.

PubMed

Patsis, Christos; Glyka, Vasiliki; Yiotakis, Ioannis; Fragoulis, Emmanuel G; Scorilas, Andreas

2012-08-01

l-DOPA decarboxylase (DDC) plays an essential role in the enzymatic synthesis of dopamine and alterations in its gene expression have been reported in several malignancies. Our objective was to analyze DDC messenger RNA (mRNA) and protein expression in laryngeal tissues and to evaluate the clinical implication of this molecule in laryngeal cancer. In this study, total RNA was isolated from 157 tissue samples surgically removed from 100 laryngeal cancer patients. A highly sensitive real-time polymerase chain reaction methodology based on SYBR Green I fluorescent dye was developed for the quantification of DDC mRNA levels. In addition, Western blot analysis was performed for the detection of DDC protein. DDC mRNA expression was revealed to be significantly downregulated in primary laryngeal cancer samples compared with their nonmalignant counterparts (P = .001). A significant negative association was also disclosed between DDC mRNA levels and TNM staging (P = .034). Univariate analysis showed that patients bearing DDC-positive tumors had a significantly decreased risk of death (hazard ratio = 0.23, P = .012) and local recurrence (hazard ratio = 0.32, P =.006), whereas DDC expression retained its favorable prognostic significance in the multivariate analysis. Kaplan-Meier curves further demonstrated that DDC-positive patients experienced longer overall and disease-free survival periods (P = .006 and P = .004, respectively). Moreover, DDC protein was detected in both neoplastic and noncancerous tissues. Therefore, our results suggest that DDC expression status could qualify as a promising biomarker for the future clinical management of laryngeal cancer patients.

l-DOPA Decarboxylase (DDC) Expression Status as a Novel Molecular Tumor Marker for Diagnostic and Prognostic Purposes in Laryngeal Cancer1

PubMed Central

Patsis, Christos; Glyka, Vasiliki; Yiotakis, Ioannis; Fragoulis, Emmanuel G; Scorilas, Andreas

2012-01-01

l-DOPA decarboxylase (DDC) plays an essential role in the enzymatic synthesis of dopamine and alterations in its gene expression have been reported in several malignancies. Our objective was to analyze DDC messenger RNA (mRNA) and protein expression in laryngeal tissues and to evaluate the clinical implication of this molecule in laryngeal cancer. In this study, total RNA was isolated from 157 tissue samples surgically removed from 100 laryngeal cancer patients. A highly sensitive real-time polymerase chain reaction methodology based on SYBR Green I fluorescent dye was developed for the quantification of DDC mRNA levels. In addition, Western blot analysis was performed for the detection of DDC protein. DDC mRNA expression was revealed to be significantly downregulated in primary laryngeal cancer samples compared with their nonmalignant counterparts (P = .001). A significant negative association was also disclosed between DDC mRNA levels and TNM staging (P = .034). Univariate analysis showed that patients bearing DDC-positive tumors had a significantly decreased risk of death (hazard ratio = 0.23, P = .012) and local recurrence (hazard ratio = 0.32, P =.006), whereas DDC expression retained its favorable prognostic significance in the multivariate analysis. Kaplan-Meier curves further demonstrated that DDC-positive patients experienced longer overall and disease-free survival periods (P = .006 and P = .004, respectively). Moreover, DDC protein was detected in both neoplastic and noncancerous tissues. Therefore, our results suggest that DDC expression status could qualify as a promising biomarker for the future clinical management of laryngeal cancer patients. PMID:22937181

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.

Laser-induced thermotherapy (LITT) elevates mRNA expression of connective tissue growth factor (CTGF) associated with reduced tumor growth of liver metastases compared to hepatic resection.

PubMed

Isbert, Christoph; Ritz, Jörg-Peter; Roggan, André; Schuppan, Detlef; Ajubi, Navid; Buhr, Heinz Johannes; Hohenberger, Werner; Germer, Christoph-Thomas

2007-01-01

Proliferation and synthesis of hepatocellular tissue after tissue damage are promoted by specific growth factors such as hepatic tissue growth factor (HGF) and connective growth factor (CTGF). Laser-induced thermotherapy (LITT) for the treatment of liver metastases is deemed to be a parenchyma-saving procedure compared to hepatic resection. The aim of this study was to compare the impact of LITT and hepatic resection on intrahepatic residual tumor tissue and expression levels of mRNA HGF/CTGF within liver and tumor tissue. Two independent adenocarcinomas (CC531) were implanted into 75 WAG rats, one in the right (untreated tumor) and one in the left liver lobe (treated tumor). The left lobe tumor was treated either by LITT or partial hepatectomy. The control tumor was submitted to in-situ hybridization of HGF and CTGF 24-96 hours and 14 days after intervention. Volumes of the untreated tumors prior to intervention were 38+/-8 mm(3) in group I (laser), 39 +/- 7 mm(3) in group II (resection), and 42 +/- 12 mm(3) in group III (control) and did not differ significantly (P > 0.05). Fourteen days after the intervention the mean tumor+/-SEM volume of untreated tumor in group I (laser) [223 +/- 36] was smaller than in group II (resection) [1233.28 +/- 181.52; P < 0.001], and in group III (control) [978.92 +/- 87.57; P < 0.003]. Forty-eight hours after the intervention intrahepatic mRNA expression level of HGF in group II (resection) was almost twofold higher than in group I (laser) [7.2 +/- 1.0 c/mf vs. 3.9 +/- 0.4 c/mf; P<0.01]. Fourteen days after the intervention intrahepatic mRNA expression level of CTGF in group I (laser) was higher than in group II (resection) [13.89 +/- 0.77 c/mf vs. 9.09 +/- 0.78 c/mf; P < 0.003]. LITT leads to a decrease of residual tumor growth in comparison to hepatic resection. Accelerated tumor growth after hepatic resection is associated with higher mRNA level of HGF and reduced tumor growth after LITT with higher mRNA level of CTGF. The increased CTGF-mediated regulation of ECM may cause reduced residual tumor growth after LITT. (c) 2006 Wiley-Liss, Inc.

Aberrant rhythmic expression of cryptochrome2 regulates the radiosensitivity of rat gliomas.

PubMed

Fan, Wang; Caiyan, Li; Ling, Zhu; Jiayun, Zhao

2017-09-29

In this study, we investigated the role of the clock regulatory protein cryptochrome 2 (Cry2) in determining the radiosensitivity of C6 glioma cells in a rat model. We observed that Cry2 mRNA and protein levels showed aberrant rhythmic periodicity of 8 h in glioma tissues, compared to 24 h in normal brain tissue. Cry2 mRNA and protein levels did not respond to irradiation in normal tissues, but both were increased at the ZT4 (low Cry2) and ZT8 (high Cry2) time points in gliomas. Immunohistochemical staining of PCNA and TUNEL assays demonstrated that high Cry2 expression in glioma tissues was associated with increased cell proliferation and decreased apoptosis. Western blot analysis showed that glioma cell fate was independent of p53, but was probably dependent on p73, which was more highly expressed at ZT4 (low Cry2) than at ZT8 (high Cry2). Levels of both p53 and p73 were unaffected by irradiation in normal brain tissues. These findings suggest aberrant rhythmic expression of Cry2 influence on radiosensitivity in rat gliomas.

Human serum amyloid A genes are expressed in monocyte/macrophage cell lines.

PubMed

Urieli-Shoval, S; Meek, R L; Hanson, R H; Eriksen, N; Benditt, E P

1994-09-01

Serum amyloid A (apoSAA) is a family of proteins found, mainly associated with high density lipoproteins, in the blood plasma of mammals and at least one avian species, the Pekin duck. These proteins are present in small amounts under normal circumstances, but their concentration is capable of rising 100- to 1,000-fold in situations involving tissue injury or infection. Like classic acute phase proteins they are produced in the liver; however, expression of one of the apoSAA genes is known to occur in activated macrophages of mice. We examined three human macrophage precursor cell lines (THP-1, U-937, and HL-60), before and after differentiation with phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxy-vitamin D3, for apoSAA messenger (m)-RNA expression and found that: 1) induction of steady-state apoSAA mRNA by lipopolysaccharide, interleukin-1, or interleukin-6 required the presence of the synthetic glucocorticoid dexamethasone; 2) the three known active genes, apoSAA1, apoSAA2, and apoSAA4, were induced in THP-1 cells, whereas the pseudogene apoSAA3 was not; 3) differentiated and undifferentiated THP-1 cells expressed apoSAA mRNA, but U-937 cells expressed apoSAA mRNA (low levels) only after phorbol 12-myristate 13-acetate differentiation and HL-60 cells did not express apoSAA mRNA whether differentiated or not; 4) apoSAA protein was detectable immunologically at a low level in lyophilized medium from induced THP-1 cells. Our findings are compatible with the hypotheses that 1) apoSAA gene expression in human monocytes/macrophages in vivo is differentiation dependent; 2) activated macrophages provide a local source of apoSAA at sites of tissue injury or inflammation; 3) apoSAA is induced in tissue macrophages by local stimuli, under conditions that may not evoke the systemic acute phase response.

Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

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

Jang, Soo Hwa; Choi, Changsun; Hong, Seong-Geun

2009-06-26

Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a rolemore » in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.« less

Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

NASA Technical Reports Server (NTRS)

Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

1998-01-01

Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

Sequence genomic organization and expression of two channel catfish Ictalurus punctatus Ghrelin receptors

USDA-ARS?s Scientific Manuscript database

Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration ...

High expression of 23 kDa protein of augmenter of liver regeneration (ALR) in human hepatocellular carcinoma

PubMed Central

Yu, Hai-Ying; Zhu, Man-Hua; Xiang, Dai-Rong; Li, Jun; Sheng, Ji-Fang

2014-01-01

Background Augmenter of liver regeneration (ALR) is an important polypeptide that participates in the process of liver regeneration. Two forms of ALR proteins are expressed in hepatocytes. Previous data have shown that ALR is essential for cell survival and has potential antimetastatic properties in hepatocellular carcinoma (HCC). Aims The study aimed to evaluate the expression levels of two forms of ALR proteins in HCC and their possible significance in HCC development. Methods Balb/c mouse monoclonal antibody against ALR protein was prepared in order to detect the ALR protein in HCC by Western blotting and immunohistochemistry. ALR mRNA expression levels were measured by real-time polymerase chain reaction in HCC tissues and compared to paracancerous liver tissues in 22 HCC patients. Results ALR mRNA expression in HCC liver tissues (1.51×106 copies/μL) was higher than in paracancerous tissues (1.04×104 copies/μL). ALR protein expression was also enhanced in HCC liver tissues. The enhanced ALR protein was shown to be 23 kDa by Western blotting. Immunohistochemical analysis showed that the 23 kDa ALR protein mainly existed in the hepatocyte cytosol. Conclusion The 23 kDa ALR protein was highly expressed in HCC and may play an important role in hepatocarcinogenesis. PMID:24940072

Expression of Leptin and Visfatin in Gingival Tissues of Chronic Periodontitis With and Without Type 2 Diabetes Mellitus: A Study Using Enzyme-Linked Immunosorbent Assay and Real-Time Polymerase Chain Reaction.

PubMed

Ghallab, Noha A; Amr, Eman M; Shaker, Olfat G

2015-07-01

The aim of this study is to investigate the protein and gene expression of leptin and visfatin in gingival tissue from patients with chronic periodontitis (CP), patients with CP and type 2 diabetes mellitus (T2DM), and healthy individuals. The study includes 50 individuals: 10 healthy individuals, 20 patients with CP, and 20 patients with CP and T2DM. Plaque index, gingival index, probing depth, and clinical attachment loss were measured, and gingival biopsies were obtained. Leptin and visfatin protein expression in gingival tissues was determined using enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was measured via real-time polymerase chain reaction. The highest leptin mRNA and protein expression was observed in the control group and was significantly (P ≤0.05) different from the CP and CP+T2DM groups. Gingival tissues from patients with CP and T2DM had a significant increase in visfatin and a decrease in leptin gene and protein expression (P <0.05) compared with both controls and patients with CP. Expression of leptin and visfatin in the gingival tissues suggests a possible role for these adipokines in the pathogenesis of CP and T2DM.

Identification of the thiamin pyrophosphokinase gene in rainbow trout: Characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development

USGS Publications Warehouse

Yuge, Shinya; Richter, Catherine A.; Wright-Osment, Maureen K.; Nicks, Diane; Saloka, Stephanie K.; Tillitt, Donald E.; Li, Weiming

2012-01-01

Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-11-01

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

Reproductive Experience Alters Prolactin Receptor Expression in Mammary and Hepatic Tissues in Female Rats1

PubMed Central

Bridges, Robert S.; Scanlan, Victoria F.; Lee, Jong-O; Byrnes, Elizabeth M.

2011-01-01

Recent studies have reported that reproductive experience in female rats alters prolactin (PRL) receptor gene expression in the brain as well as neural sensitivity to PRL. Given PRL's actions in nonneural tissues, that is, mammary tissue and liver, it was asked whether reproductive experience may also alter prolactin receptor (Prlr) gene expression in these tissues. Groups of age-matched female rats were generated with varying reproductive histories. Separate groups of primiparous (first lactation) and multiparous (second lactation) had mammary tissue and liver samples collected on Day 3 or 10 of lactation. A fifth group raised one litter to weaning and then resumed estrous cyclicity. This group and a final group of age-matched, virgin controls were killed on diestrus. Tissue was processed by quantitative PCR for expression rates of the long and short forms of Prlr mRNA as well as casein beta mRNA (mammary tissue only). Western blots were performed to quantify receptor protein content. Multiple lactations as well as lactation itself resulted in alterations in Prlr expression. Prlr gene expression in mammary tissue was increased in primiparous mothers compared with that in multiparous dams, whereas in the liver, Prlr expression was reduced during an initial lactation. In contrast, PRLR protein levels declined during lactation in mammary, but not hepatic, tissues. Overall, the results demonstrate that the prolactin receptor system is altered in nonneural tissues as a result of the female's reproductive history. The findings are discussed in the context of milk and bile production and PRL's possible role in breast cancer. PMID:21508351

Reproductive experience alters prolactin receptor expression in mammary and hepatic tissues in female rats.

PubMed

Bridges, Robert S; Scanlan, Victoria F; Lee, Jong-O; Byrnes, Elizabeth M

2011-08-01

Recent studies have reported that reproductive experience in female rats alters prolactin (PRL) receptor gene expression in the brain as well as neural sensitivity to PRL. Given PRL's actions in nonneural tissues, that is, mammary tissue and liver, it was asked whether reproductive experience may also alter prolactin receptor (Prlr) gene expression in these tissues. Groups of age-matched female rats were generated with varying reproductive histories. Separate groups of primiparous (first lactation) and multiparous (second lactation) had mammary tissue and liver samples collected on Day 3 or 10 of lactation. A fifth group raised one litter to weaning and then resumed estrous cyclicity. This group and a final group of age-matched, virgin controls were killed on diestrus. Tissue was processed by quantitative PCR for expression rates of the long and short forms of Prlr mRNA as well as casein beta mRNA (mammary tissue only). Western blots were performed to quantify receptor protein content. Multiple lactations as well as lactation itself resulted in alterations in Prlr expression. Prlr gene expression in mammary tissue was increased in primiparous mothers compared with that in multiparous dams, whereas in the liver, Prlr expression was reduced during an initial lactation. In contrast, PRLR protein levels declined during lactation in mammary, but not hepatic, tissues. Overall, the results demonstrate that the prolactin receptor system is altered in nonneural tissues as a result of the female's reproductive history. The findings are discussed in the context of milk and bile production and PRL's possible role in breast cancer.

The induced RNA-binding protein, HuR, targets 3'-UTR region of IL-6 mRNA and enhances its stabilization in periodontitis.

PubMed

Ouhara, K; Munenaga, S; Kajiya, M; Takeda, K; Matsuda, S; Sato, Y; Hamamoto, Y; Iwata, T; Yamasaki, S; Akutagawa, K; Mizuno, N; Fujita, T; Sugiyama, E; Kurihara, H

2018-06-01

RNA-binding proteins (RBPs) regulate mRNA stability by binding to the 3'-untranslated region (UTR) region of mRNA. Human antigen-R (HuR), one of the RBPs, is involved in the progression of diseases, such as rheumatoid arthritis, diabetes mellitus and some inflammatory diseases. Interleukin (IL)-6 is a major inflammatory cytokine regulated by HuR binding to mRNA. Periodontal disease (PD) is also an inflammatory disease caused by elevations in IL-6 following an infection by periodontopathogenic bacteria. The involvement of HuR in the progression of PD was assessed using in-vitro and in-vivo experiments. Immunohistochemistry of inflamed periodontal tissue showed strong staining of HuR in the epithelium and connective tissue. HuR mRNA and protein level was increased following stimulation with Porphyromonas gingivalis (Pg), one of the periodontopathogenic bacteria, lipopolysacchride (LPS)-derived from Pg (PgLPS) and tumour necrosis factor (TNF)-α in OBA-9, an immortalized human gingival epithelial cell. The luciferase activity of 3'-UTR of IL-6 mRNA was increased by TNF-α, Pg and PgLPS in OBA-9. Luciferase activity was also increased in HuR-over-expressing OBA-9 following a bacterial stimulation. Down-regulation of HuR by siRNA resulted in a decrease in mRNA expression and production of IL-6. In contrast, the over-expression of HuR increased IL-6 mRNA expression and production in OBA-9. The HuR inhibitor, quercetin, suppressed Pg-induced HuR mRNA expression and IL-6 production in OBA-9. An oral inoculation with quercetin also inhibited bone resorption in ligature-induced periodontitis model mice as a result of down-regulation of IL-6. These results show that HuR modulates inflammatory responses by regulating IL-6. © 2018 British Society for Immunology.

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

PubMed

Williams, C M; Coleman, J W

1995-10-01

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

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

PubMed Central

Williams, C M; Coleman, J W

1995-01-01

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

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.

Expression of circadian gens in different rat tissues is sensitive marker of in vivo silver nanoparticles action

NASA Astrophysics Data System (ADS)

Minchenko, D. O.; Yavorovsky, O. P.; Zinchenko, T. O.; Komisarenko, S. V.; Minchenko, O. H.

2012-09-01

Circadian factors PER1, PER2, ARNTL and CLOCK are important molecular components of biological clock system and play a fundamental role in the metabolism at both the behavioral and molecular levels and potentially have great importance for understanding metabolic health and disease, because disturbance the circadian processes lead to developing of different pathology. The antibacterial effect of silver nanoparticles has resulted in their extensive application in health, electronics, home products, and for water disinfection, but little is yet known about their toxicity. These nanoparticles induce blood-brain barrier destruction, astrocyte swelling, cause degeneration of neurons and impair neurodevelopment as well as embryonic development. We studied the expression of genes encoded the key molecular components of circadian clock system in different rat organs after intratracheally instilled silver nanoparticles which quite rapidly translocate from the lungs into the blood stream and accumulate in different tissues. We have shown that silver nanoparticles significantly affect the expression levels of PER1, PER2, ARNTL and CLOCK mRNA in different rat tissues in time-dependent and tissue-specific manner. High level of PER1, ARNTL and CLOCK mRNA expression was observed in the lung on the 1st 3rd and 14th day after treatment of rats with silver nanoparticles. At the same time, the expression level of PER1 mRNA in the brain and liver increases predominantly on the 1st and 14th day but decreases in the testis. Significant increase of the expression level of PER2 and ARNTL mRNA was detected only in the brain of treated by silver nanoparticles rats. Besides that, intratracheally instilled silver nanoparticles significantly reduced the expression levels of CLOCK mRNA in the brain, heart and kidney. No significant changes in the expression level of PER2 mRNA were found in the lung, liver, heart and testis, except kidney where this mRNA expression decreases on the 3rd and 14th day after treatment of rats with silver nanoparticles. It was also shown that expression level of PFKFB4, a key enzyme of glycolysis regulation, gradually reduces in the brain from 1st to 14th day being up to 4 fold less on 14th day after treatment of animals with silver nanoparticles. Thus, the intratracheally instilled silver nanoparticles significantly affect the expression of PER1, PER2, ARNTL, and CLOCK genes which are an important molecular component of circadian clock system. This is because a disruption of the circadian processes leads to a development of various pathologic processes. The results of this study clearly demonstrate that circadian genes could be a sensitive test for detection of silver nanoparticles toxic action and suggest that more caution is needed in biomedical applications of silver nanoparticles as well as higher level of safety in silver nanoparticles production industry.

Differential expression pattern of heat shock protein 70 gene in tissues and heat stress phenotypes in goats during peak heat stress period.

PubMed

Rout, P K; Kaushik, R; Ramachandran, N

2016-07-01

It has been established that the synthesis of heat shock protein 70 (Hsp70) is temperature-dependent. The Hsp70 response is considered as a cellular thermometer in response to heat stress and other stimuli. The variation in Hsp70 gene expression has been positively correlated with thermotolerance in Drosophila melanogaster, Caenorhabditis elegans, rodents and human. Goats have a wide range of ecological adaptability due to their anatomical and physiological characteristics; however, the productivity of the individual declines during thermal stress. The present study was carried out to analyze the expression of heat shock proteins in different tissues and to contrast heat stress phenotypes in response to chronic heat stress. The investigation has been carried out in Jamunapari, Barbari, Jakhrana and Sirohi goats. These breeds differ in size, coat colour and production performance. The heat stress assessment in goats was carried out at a temperature humidity index (THI) ranging from 85.36-89.80 over the period. Phenotyping for heat stress susceptibility was carried out by combining respiration rate (RR) and heart rate (HR). Based on the distribution of RR and HR over the breeds in the population, individual animals were recognized as heat stress-susceptible (HSS) and heat stress-tolerant (HST). Based on their physiological responses, the selected animals were slaughtered for tissue collection during peak heat stress periods. The tissue samples from different organs such as liver, spleen, heart, testis, brain and lungs were collected and stored at -70 °C for future use. Hsp70 concentrations were analyzed from tissue extract with ELISA. mRNA expression levels were evaluated using the SYBR green method. Kidney, liver and heart had 1.5-2.0-fold higher Hsp70 concentrations as compared to other organs in the tissue extracts. Similarly, the gene expression pattern of Hsp70 in different organs indicated that the liver, spleen, brain and kidney exhibited 5.94, 4.96, 5.29 and 2.63-fold higher expression than control. Liver and brain tissues showed the highest gene expression at mRNA levels as compared to kidney, spleen and heart. HST individuals had higher levels of mRNA level expression than HSS individuals in all breeds. The Sirohi breed showed the highest (6.3-fold) mRNA expression levels as compared to the other three breeds, indicating the better heat stress regulation activity in the breed.

Characterisation of 11β-hydroxysteroid dehydrogenase 1 in human orbital adipose tissue: a comparison with subcutaneous and omental fat

PubMed Central

Bujalska, Iwona J; Durrani, Omar M; Abbott, Joseph; Onyimba, Claire U; Khosla, Pamela; Moosavi, Areeb H; Reuser, Tristan T Q; Stewart, Paul M; Tomlinson, Jeremy W; Walker, Elizabeth A; Rauz, Saaeha

2007-01-01

Glucocorticoids (GCs) have a profound effect on adipose biology increasing tissue mass causing central obesity. The pre-receptor regulation of GCs by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) that activates cortisol from cortisone has been postulated as a fundamental mechanism underlying the metabolic syndrome mediating adipocyte hyperplasia and hypertrophy in the omental (OM) depot. Orbital adipose tissue (OF) is the site of intense inflammation and tissue remodelling in several orbital inflammatory disease states. In this study, we describe features of the GC metabolic pathways in normal human OF depot and compare it with subcutaneous (SC) and OM depots. Using an automated histological characterisation technique, OF adipocytes were found to be significantly smaller (parameters: area, maximum diameter and perimeter) than OM and SC adipocytes (P<0·001). Although immunohistochemical analyses demonstrated resident CD68+ cells in all three whole tissue adipose depots, OF CD68 mRNA and protein expression exceeded that of OM and SC (mRNA, P<0·05; protein, P<0·001). In addition, there was higher expression of glucocorticoid receptor (GR)α mRNA in the OF whole tissue depot (P<0·05). Conversely, 11β-HSD1 mRNA together with the markers of late adipocyte differentiation (FABP4 and G3PDH) were significantly lower in OF. Primary cultures of OF preadipocytes demonstrated predominant 11β-HSD1 oxo-reductase activity with minimal dehydrogenase activity. Orbital adipocytes are smaller, less differentiated, and express low levels of 11β-HSD1 but abundant GRα compared with SC and OM. OF harbours a large CD68+ population. These characteristics define an orbital microenvironment that has the potential to respond to sight-threatening orbital inflammatory disease. PMID:17283228

Identification of MSX1 and DCLK1 as mRNA Biomarkers for Colorectal Cancer Detection Through DNA Methylation Information.

PubMed

Sun, Ai-Jun; Gao, Hai-Bo; Liu, Gao; Ge, Heng-Fa; Ke, Zun-Ping; Li, Sen

2017-07-01

Colorectal cancer is the second most deadly malignancy in the United States. However, the currently screening options had their limitation. Novel biomarkers for colorectal cancer detections are necessary to reduce the mortality. The clinical information, mRNA expression levels and DNA methylation information of colorectal cancer were downloaded from TCGA. The patients were separated into training group and testing group based on their platforms for DNA methylation. Beta values of DNA methylation from tumor tissues and normal tissues were utilized to figure out the position that were differentially methylated. The expression levels of mRNA of thirteen genes, whose CpG islands were differentially methylated, were extracted from the RNA-Seq results from TCGA. The probabilities whether the mRNA was differentially expressed between tumor and normal samples were calculated using Student's t-test. Logistic regression and decision tree were built for cancer detection and their performances were evaluated by the area under the curve (AUC). Twenty-four genomic locations were differentially methylated, which could be mapped to eleven genes. Nine out of eleven genes had differentially expressed mRNA levels, which were used to build the model for cancer detection. The final detection models consisting of mRNA expression levels of these nine genes had great performances on both training group and testing group. The model that constructed in this study suggested MSX1 and DCLK1 might be used in colorectal cancer detection or as target of cancer therapies. J. Cell. Physiol. 232: 1879-1884, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish.

PubMed

Kobayashi, Y; Peterson, B C; Waldbieser, G C

2015-04-01

This study tested the hypothesis that increased growth in channel catfish is associated with expression of the genes that code for uncoupling proteins (UCP) 2 and 3, members of the mitochondrial channel proteins involved in nutrient sensing and metabolism. The specific objective was to contrast the levels of UCP2 messenger RNA (mRNA) in fast vs slow growing catfish as well as in fed vs fasted catfish. Two distinct UCP2 transcripts were identified and named UCP2a and UCP2b, respectively. Nucleotide and amino acid sequence of catfish UCP2s were highly similar to UCP2 and other UCPs from other fish and mammals (>75%). Expression of UCP2a mRNA was detectable at very low levels in various metabolically active tissues, whereas the expression of UCP2b mRNA was readily detectable in the muscle and heart. In a 21-wk feeding study, fish that grew faster had a greater percent body fat at the end of the study (P < 0.01). Expression of UCP2b mRNA tended to be lower (P < 0.10) in fast growing fish in the middle of the study although levels were similar at the beginning and the end of the study. In the fed vs fasted study, expression of UCP2b mRNA in muscle was increased (P < 0.05) in fish assigned to 30 d of fasting. Our results suggest that, based on the nucleotide and amino acid sequence similarities and tissue mRNA distribution, catfish UCP2b may be the analog to UCP3. Moreover, our results suggest selection toward growth and associated fat accumulation appears to be independent of muscle UCP2b mRNA expression and UCP2b-mediated mechanisms. Copyright © 2015 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

The expression of genes involved in myometrial contractility changes during ex situ culture of pregnant human uterine smooth muscle tissue.

PubMed

Ilicic, Marina; Butler, Trent; Zakar, Tamas; Paul, Jonathan W

2017-01-01

Ex situ analyses of human myometrial tissue has been used to investigate the regulation of uterine quiescence and transition to a contractile phenotype. Following concerns about the validity of cultured primary cells, we examined whether myometrial tissue undergoes culture-induced changes ex situ that may affect the validity of in vitro models. To determine whether human myometrial tissue undergoes culture-induced changes ex situ in Estrogen receptor 1 (ESR1), Prostaglandin-endoperoxide synthase 2 (PTGS2) and Oxytocin receptor (OXTR) expression. Additionally, to determine whether culture conditions approaching the in vivo environment influence the expression of these key genes. Term non-laboring human myometrial tissues were cultured in the presence of specific treatments, including; serum supplementation, progesterone and estrogen, cAMP, PMA, stretch or NF-κB inhibitors. ESR1, PTGS2 and OXTR mRNA abundance after 48 h culture was determined using quantitative RT-PCR. Myometrial tissue in culture exhibited culture-induced up-regulation of ESR1 and PTGS2 and down-regulation of OXTR mRNA expression. Progesterone prevented culture-induced increase in ESR1 expression. Estrogen further up-regulated PTGS2 expression. Stretch had no direct effect, but blocked the effects of progesterone and estrogen on ESR1 and PTGS2 expression. cAMP had no effect whereas PMA further up-regulated PTGS2 expression and prevented decline of OXTR expression. Human myometrial tissue in culture undergoes culture-induced gene expression changes consistent with transition toward a laboring phenotype. Changes in ESR1, PTGS2 and OXTR expression could not be controlled simultaneously. Until optimal culture conditions are determined, results of in vitro experiments with myometrial tissues should be interpreted with caution.

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

PubMed

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

2017-01-01

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

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

PubMed

Smits, A; Funa, K; Vassbotn, F S; Beausang-Linder, M; af Ekenstam, F; Heldin, C H; Westermark, B; Nistér, M

1992-03-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

Immunolocalization of Leptin Receptor and mRNA Expression of Leptin and Estrogen Receptors as well as Caspases in the Chorioallantoic Membrane (CAM) of the Chicken Embryo.

PubMed

Grzegorzewska, Agnieszka K; Lis, Marcin W; Sechman, Andrzej

The chicken chorioallantoic membrane (CAM) is used as a model in tests of angiogenesis, the biocompatibility of materials as well as tumor invasive potential. To assess the properties of CAM tissue, the localization of leptin receptor in the CAM, and the mRNA expression of two leptin receptor isoforms, estrogen receptors (ERα and ERβ) and caspases (-1 and -3) in the CAM on embryonic days 12 (E12), 15 (E15) and 18 (E18) were investigated. The leptin receptor was immunolocalized in each structure of the CAM (chorionic epithelium, allantoic epithelium, mesodermal layer and the walls of blood vessels) and did not change among analyzed stages of embryonic development (E12, E15 and E18) and between sexes. Expression of mRNA of genes encoding leptin and estrogen receptors as well as caspases was detected in the CAM of female and male chicken embryos at all three analysed stages of development. The relative mRNA expression of the long form of leptin receptor exceeded that of its short isoform. The mRNA expression of ERβ was significantly higher than ERα as well as caspase-3 in comparison with caspase-1. There were no differences in mRNA expression of these genes between sexes and among analyzed developmental days. The results indicate that the CAM is a target tissue for leptin as well as for estrogens and that CAM development is partially regulated by caspase-1 and caspase-3 dependent cell death. These results should be taken into consideration in studies in which the CAM is used as an experimental model.

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

PubMed

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

2017-10-01

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

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

PubMed

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

2017-01-01

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

Dietary supplementation with arginine and glutamic acid enhances key lipogenic gene expression in growing pigs.

PubMed

Hu, C J; Jiang, Q Y; Zhang, T; Yin, Y L; Li, F N; Su, J Y; Wu, G Y; Kong, X F

2017-12-01

Our previous study showed dietary supplementation with Arg and Glu increased intramuscular fat deposition and decreased back fat thickness in pigs, suggesting that the genes involved in lipid metabolism might be regulated differently in muscle and s.c. adipose (SA) tissues. Sixty Duroc × Large White × Landrace pigs with an average initial BW of 77.1 ± 1.3 kg were randomly assigned to 1 of 5 treatment groups (castrated male to female ratio = 1:1). Pigs in the control group were fed a basic diet, and those in experimental groups were fed the basic diet supplemented with 2.05% alanine (isonitrogenous group), 1.00% arginine (Arg group), 1.00% glutamic acid + 1.44% alanine (Glu group), or 1.00% arginine + 1.00% glutamic acid (Arg+Glu group). Fatty acid percentages and mRNA expression levels of the genes involved in lipid metabolism in muscle and SA tissues were examined. The percentages of C14:0 and C16:0 in the SA tissue of Glu group pigs and C14:0 in the longissimus dorsi (LD) muscle of Glu and Arg+Glu groups decreased ( < 0.05) compared to the basic diet group. The Arg+Glu group showed the highest ( < 0.05) hormone-sensitive lipase expression level in SA tissue and higher ( < 0.05) mRNA levels of in the LD muscle than the basic diet and isonitrogenous groups. Additionally, the mRNA level of fatty acid synthase in the Arg+Glu group was more upregulated ( < 0.05) than that of the Arg group. An increase in the mRNA level of in the biceps femoris muscle was also observed in the Arg+Glu group ( < 0.05) compared with the basic diet and isonitrogenous groups. Collectively, these findings suggest that dietary supplementation with Arg and Glu upregulates the expression of genes involved in adipogenesis in muscle tissues and lipolysis in SA tissues.

Expression of three sHSP genes involved in heat pretreatment-induced chilling tolerance in banana fruit.

PubMed

He, Li-hong; Chen, Jian-ye; Kuang, Jian-fei; Lu, Wang-jin

2012-07-01

Banana fruit is highly susceptible to chilling injury. In previous research it was shown that heat pretreatment of banana fruit at 38 °C for 3 days before storage at a chilling temperature of 8 °C for 12 days prevented increases in visible chilling injury index, electrolyte leakage and malondialdehyde content and also decreases in lightness and chroma, indicating that heat pretreatment could effectively alleviate chilling injury of banana fruit. However, little is known about the role of small heat shock proteins (sHSPs) in postharvest chilling tolerance of banana fruit. In the present study, three cytosolic sHSP expression profiles in peel and pulp tissues of banana fruit during heat pretreatment and subsequent chilled storage (8 °C) were investigated in relation to heat pretreatment-induced chilling tolerance. Three full-length cDNAs of cytosolic sHSP genes, including two class I sHSP (CI sHSP) and one class II sHSP (CII sHSP) cDNAs, named Ma-CI sHSP1, Ma-CI sHSP2 and Ma-CII sHSP3 respectively, were isolated and characterised from harvested banana fruit. Accumulation of Ma-CI sHSP1 mRNA transcripts in peel and pulp tissues and Ma-CII sHSP3 mRNA transcripts in peel tissue increased during heat pretreatment. Expression of all three Ma-sHSP genes in peel and pulp tissues was induced during subsequent chilled storage. Furthermore, Ma-CI sHSP1 and Ma-CII sHSP3 mRNA transcripts in pulp tissue and Ma-CI sHSP2 mRNA transcripts in peel and pulp tissues were obviously enhanced by heat pretreatment at days 6 and 9 of subsequent chilled storage. These results suggested that heat pretreatment enhanced the expression of Ma-sHSPs, which might be involved in heat pretreatment-induced chilling tolerance of banana fruit. Copyright © 2012 Society of Chemical Industry.

Developmentally Regulated Expression of the Nerve Growth Factor Receptor Gene in the Periphery and Brain

NASA Astrophysics Data System (ADS)

Buck, C. R.; Martinez, Humberto J.; Black, Ira B.; Chao, Moses V.

1987-05-01

Nerve growth factor (NGF) regulates development and maintenance of function of peripheral sympathetic and sensory neurons. A potential role for the trophic factor in brain has been detected only recently. The ability of a cell to respond to NGF is due, in part, to expression of specific receptors on the cell surface. To study tissue-specific expression of the NGF receptor gene, we have used sensitive cRNA probes for detection of NGF receptor mRNA. Our studies indicate that the receptor gene is selectively and specifically expressed in sympathetic (superior cervical) and sensory (dorsal root) ganglia in the periphery, and by the septum-basal forebrain centrally, in the neonatal rat in vivo. Moreover, examination of tissues from neonatal and adult rats reveals a marked reduction in steady-state NGF receptor mRNA levels in sensory ganglia. In contrast, a 2- to 4-fold increase was observed in the basal forebrain and in the sympathetic ganglia over the same time period. Our observations suggest that NGF receptor mRNA expression is developmentally regulated in specific areas of the nervous system in a differential fashion.

Identification of a differentially-expressed gene in fatty liver of overfeeding geese.

PubMed

Zhao, Ayong; Tang, Huachun; Lu, Sufang; He, Ruiguo

2007-09-01

In response to overfeeding, geese develop fatty liver. To understand the fattening mechanism, mRNA differential display reverse transcription PCR was used to study the gene expression differences between French Landes grey geese and Xupu white geese in conditions of overfeeding and normal feeding. One gene was found to be up-regulated in the fatty liver in both breeds, and it has a 1797 bp cDNA with 83% identity to chicken SELENBP1. The sequence analysis revealed that its open reading frame of 1413 bp encodes a protein of 471 amino acids, which contains a putative conserved domain of 56 kDa selenium binding protein with high homology to its homologues of chicken (95%), rat (86%), mouse (84%), human (86%), monkey (86%), dog (86%), and cattle (86%). The function of this protein has been briefly reviewed based on published information. In tissue expression analysis, the expression of geese SELENBP1 mRNA was found to be higher in liver or kidney than in other tested tissues. The results showed that overfeeding could increase the mRNA expression level of geese SELENBP1.

Artesunate modulates expression of matrix metalloproteinases and their inhibitors as well as collagen-IV to attenuate pulmonary fibrosis in rats.

PubMed

Wang, Y; Huang, G; Mo, B; Wang, C

2016-06-03

The aim of this study was to determine the effect of artesunate on extracellular matrix (ECM) accumulation and the expression of collagen-IV, matrix metalloproteinase (MMP), and tissue inhibitor of matrix metalloproteinase (TIMP) to understand the pharmacological role of artesunate in pulmonary fibrosis. Eighty Sprague-Dawley rats were randomly assigned to four groups that were administered saline alone, bleomycin (BLM) alone, BLM + artesunate, or artesunate alone for 28 days. Lung tissues from 10 rats in each group were used to obtain lung fibroblast (LF) primary cells, and the rest were used to analyze protein expression. The mRNA expression of collagen-IV, MMP-2, MMP-9, TIMP-1, and TIMP-2 in lung fibroblasts was detected by real-time quantitative reverse transcriptase polymerase chain reaction. The protein levels of collagen-IV, MMP-2, MMP-9, TIMP-1, and TIMP-2 protein in lung tissues were analyzed by western blotting. Artesunate treatment alleviated alveolitis and pulmonary fibrosis induced by bleomycin in rats, as indicated by a decreased lung coefficient and improvement of lung tissue morphology. Artesunate treatment also led to decreased collagen-IV protein levels, which might be a result of its downregulated expression and increased MMP-2 and MMP-9 protein and mRNA levels. Increased TIMP-1 and TIMP- 2 protein and mRNA levels were detected after artesunate treatment in lung tissues and primary lung fibroblast cells and may contribute to enhanced activity of MMP-2 and -9. These findings suggested that artesunate attenuates alveolitis and pulmonary fibrosis by regulating expression of collagen-IV, TIMP-1 and 2, as well as MMP-2 and -9, to reduce ECM accumulation.

Class I β-1,3-Glucanase and Chitinase Are Expressed in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence1

PubMed Central

Wu, Chun-Ta; Leubner-Metzger, Gerhard; Meins, Frederick; Bradford, Kent J.

2001-01-01

β-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. β-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. β-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of β-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 μM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both β-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed. PMID:11457981

Differential expression of glucose transporters in normal and pathologic thyroid tissue.

PubMed

Matsuzu, Kenichi; Segade, Fernando; Matsuzu, Utako; Carter, Aaron; Bowden, Donald W; Perrier, Nancy D

2004-10-01

Malignant cells demonstrate increased glucose uptake and utilization. Immunohistochemical studies have suggested that enhanced glucose uptake in cancer cells may be caused by the overexpression of glucose transporters (GLUTs), in most cases GLUT1 and/or GLUT3. The aim of this study was to examine in detail the expression pattern and levels of GLUT genes in normal and pathologic thyroid tissues and to evaluate the clinical significance of GLUT mRNA levels. One hundred fifty-two surgically resected thyroid tissue samples from 103 patients were evaluated. Samples included: normal thyroid tissue (n = 58), benign thyroid disease (n = 61), and thyroid carcinoma (n = 33). Expression of the GLUT1, GLUT2, GLUT3, GLUT4, and GLUT10 genes were examined by reverse transcription-polymerase chain reaction (RT-PCR) and mRNA levels were quantitated by real-time RT-PCR. All thyroid parenchymal cells expressed GLUT1, GLUT3, GLUT4, and GLUT10. GLUT1 showed increased expression in carcinoma cases (p < 0.0001) and also in comparison with paired normal tissue samples from the same patient (p < 0.0001). Other GLUTs were statistically unchanged in pathologic tissues. These results are consistent with the theory that GLUT1 is upregulated during carcinogenesis and may play a major role in enhanced glucose uptake in thyroid cancer cells.

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.

Immunolocalization of Collagens (I and III) and Cartilage Oligomeric Matrix Protein in the Normal and Injured Equine Superficial Digital Flexor Tendon

PubMed Central

2013-01-01

This is a descriptive study of tendon pathology with different structural appearances of repair tissue correlated to immunolocalization of cartilage oligomeric matrix protein (COMP) and type I and III collagens and expression of COMP mRNA. The material consists of nine tendons from seven horses (5–25 years old; mean age of 10 years) with clinical tendinopathy and three normal tendons from horses (3, 3, and 13 years old) euthanized for non-orthopedic reasons. The injured tendons displayed different repair-tissue appearances with organized and disorganized fibroblastic regions as well as areas of necrosis. The normal tendons presented distinct immunoreactivity for COMP and expression of COMP mRNA and type I collagen in the normal aligned fiber structures, but no immunolabeling of type III collagen. However, immunoreactivity for type III collagen was present in the endotenon surrounding the fiber bundles, where no expression of COMP could be seen. Immunostaining for type I and III collagens was present in all of the pathologic regions indicating repair tissue. Interestingly, the granulation tissues showed immunostaining for COMP and expression of COMP mRNA, indicating a role for COMP in repair and remodeling of the tendon after fiber degeneration and rupture. The present results suggest that not only type III collagen but also COMP is involved in the repair and remodeling processes of the tendon. PMID:23020676

Differentiation of rat brown adipocytes during late foetal development: role of insulin-like growth factor I.

PubMed Central

Teruel, T; Valverde, A M; Alvarez, A; Benito, M; Lorenzo, M

1995-01-01

Rat brown adipocytes at day 22 of foetal development showed greater size, higher mitochondria content and larger amounts of lipids, as determined by flow cytometry, than 20-day foetal cells. Simultaneously, an inhibition on the percentage of brown adipocytes into S+G2/M phases of the cell cycle was observed between days 20 and 22 of foetal development. The expression of several adipogenesis-related genes, such as fatty acid synthase, malic enzyme, glucose-6-phosphate dehydrogenase and insulin-regulated glucose transporter, increased at the end of foetal life in brown adipose tissue. In addition, the lipogenic enzyme activities and the lipogenic flux increased during late foetal development, resulting in mature brown adipocytes showing a multilocular fat droplet phenotype. Concurrently, brown adipocytes induced the expression of the uncoupling protein (UP) mRNA and UP protein, as visualized by immunofluorescence. The three isoforms of CCAAT enhancer-binding proteins (C/EBPs) were expressed at the mRNA level in brown adipose tissue at day 20. C/EBP alpha decreased and C/EBP beta and delta increased their expression between days 20 and 22 of foetal development, respectively. Brown adipose tissue constitutively expressed insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNAs. Moreover, IGF-IR mRNA content increased between days 20 and 22 in parallel with the occurrence of tissue differentiation. Images Figure 2 Figure 3 Figure 4 PMID:7575409

Genetic background modulates lncRNA-coordinated tissue response to low dose ionizing radiation

DOE PAGES

Tang, Jonathan; Huang, Yurong; Nguyen, David H.; ...

2015-02-04

Long noncoding RNAs (lncRNAs) are emerging as key regulators of diverse cell functions and processes. However, the relevance of lncRNAs in the cell and tissue response to ionizing radiation has not yet been characterized. Here we used microarray profiling to determine lncRNA and mRNA expression in mammary glands of BALB/c and SPRET/EiJ mice after low-dose ionizing radiation (LDIR) exposure. We found that unirradiated mammary tissues of these strains differed significantly in baseline expressions of 290 lncRNAs. LDIR exposure (10 cGy) induced a significant change in the expression of many lncRNAs. The vast majority of lncRNAs identified to be differentially expressed aftermore » LDIR in either BALB/c or SPRET/EiJ had a significantly correlated expression pattern with at least one LDIR responsive mRNA. Functional analysis revealed that the response to LDIR in BALB/c mice is highly dynamic with enrichment for genes involved in tissue injury, inflammatory responses, and mammary gland development at 2, 4, and 8 weeks after LDIR, respectively. Our study demonstrates that genetic background strongly influences the expression of lncRNAs and their response to radiation and that lncRNAs may coordinate the tissue response to LDIR exposure via regulation of coding mRNAs.« less

[Study on the correlation between EGFR-STAT3 signal pathway and laryngeal papilloma].

PubMed

Wang, Xinhua; Sun, Jingwu

2009-09-01

To explore the relationship between the expression of EGFR and STAT3 in human laryngeal papilloma and its biological behavior. Reverse transcription polymerase chain reaction(RT-PCR), immunohistochemical staining and Western blot were used to evaluate the mRNA and protein expression of EGFR and STAT3 (p-STAT3) in 42 laryngeal papilloma tissues and 15 samples of normal laryngeal tissue, and the relationship between the protein expression of them and clinic pathological parameters was also analyzed. The mRNA expression levels of EGFR and STAT3 in laryngeal papilloma tissue were significantly higher than that in normal laryngeal tissue (P < 0.05, P < 0.01). Protein positive expression of EGFR and p-STAT3 were also detected in a significantly greater proportion of laryngeal papilloma than normal laryngeal tissue by immunohistochemistry and western blot (P < 0.01, P < 0.05). There was relationship between EGFR and p-STAT3 overexpression in laryngeal papilloma (P < 0.05). The expression p-STAT3 was correlated with the recurrence and canceration of laryngeal papilloma (P < 0.05). The EGFR-STAT3 signal transduction pathway may be involved in the pathogenesis of laryngeal papilloma,, and the persistent activation of STAT3 gene plays an important role in the recurrence and canceration of laryngeal papilloma.

Genetic background modulates lncRNA-coordinated tissue response to low dose ionizing radiation

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

Tang, Jonathan; Huang, Yurong; Nguyen, David H.

Long noncoding RNAs (lncRNAs) are emerging as key regulators of diverse cell functions and processes. However, the relevance of lncRNAs in the cell and tissue response to ionizing radiation has not yet been characterized. Here we used microarray profiling to determine lncRNA and mRNA expression in mammary glands of BALB/c and SPRET/EiJ mice after low-dose ionizing radiation (LDIR) exposure. We found that unirradiated mammary tissues of these strains differed significantly in baseline expressions of 290 lncRNAs. LDIR exposure (10 cGy) induced a significant change in the expression of many lncRNAs. The vast majority of lncRNAs identified to be differentially expressed aftermore » LDIR in either BALB/c or SPRET/EiJ had a significantly correlated expression pattern with at least one LDIR responsive mRNA. Functional analysis revealed that the response to LDIR in BALB/c mice is highly dynamic with enrichment for genes involved in tissue injury, inflammatory responses, and mammary gland development at 2, 4, and 8 weeks after LDIR, respectively. Our study demonstrates that genetic background strongly influences the expression of lncRNAs and their response to radiation and that lncRNAs may coordinate the tissue response to LDIR exposure via regulation of coding mRNAs.« less

Regucalcin Expression in Bovine Tissues and Its Regulation by Sex Steroid Hormones in Accessory Sex Glands

PubMed Central

Starvaggi Cucuzza, Laura; Divari, Sara; Mulasso, Chiara; Biolatti, Bartolomeo; Cannizzo, Francesca T.

2014-01-01

Regucalcin (RGN) is a mammalian Ca2+-binding protein that plays an important role in intracellular Ca2+ homeostasis. Recently, RGN has been identified as a target gene for sex steroid hormones in the prostate glands and testis of rats and humans, but no studies have focused on RGN expression in bovine tissues. Thus, in the present study, we examined RGN mRNA and protein expression in the different tissues and organs of veal calves and beef cattle. Moreover, we investigated whether RGN expression is controlled through sex steroid hormones in bovine target tissues, namely the bulbo-urethral and prostate glands and the testis. Sex steroid hormones are still illegally used in bovine husbandry to increase muscle mass. The screening of the regulation and function of anabolic sex steroids via modified gene expression levels in various tissues represents a new approach for the detection of illicit drug treatments. Herein, we used quantitative PCR, western blot and immunohistochemistry analyses to demonstrate RGN mRNA and protein expression in bovine tissues. In addition, estrogen administration down-regulated RGN gene expression in the accessory sex glands of veal calves and beef cattle, while androgen treatment reduced RGN gene expression only in the testis. The confirmation of the regulation of RGN gene expression through sex steroid hormones might facilitate the potential detection of hormone abuse in bovine husbandry. Particularly, the specific response in the testis suggests that this tissue is ideal for the detection of illicit androgen administration in veal calves and beef cattle. PMID:25415588

Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

PubMed

Ferder, Ianina; Parborell, Fernanda; Sundblad, Victoria; Chiauzzi, Violeta; Gómez, Karina; Charreau, Eduardo H; Tesone, Marta; Dain, Liliana

2013-04-01

Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

Comparison of Osteogenic and Chondrogenic Differentiation Ability of Buccal Fat Pad Derived Mesenchymal Stem Cells and Gingival Derived Cells.

PubMed

Ghaderi, Hamid; Razmkhah, Mahboobeh; Kiany, Farin; Chenari, Nooshafarin; Haghshenas, Mohammad Reza; Ghaderi, Abbas

2018-06-01

One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells (MSCs) with higher differentiation ability. In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs (BFP-MSCs) with gingival derived cells (GDCs) were compared. BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I (COLL), BGLA and bone morphogenetic protein 2 (BMP2) using qRT-PCR. Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls. Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries.

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium.

PubMed

Shynlova, Oksana; Mitchell, Jennifer A; Tsampalieros, Anne; Langille, B Lowell; Lye, Stephen J

2004-04-01

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

Nesfatin-1-Like Peptide Encoded in Nucleobindin-1 in Goldfish is a Novel Anorexigen Modulated by Sex Steroids, Macronutrients and Daily Rhythm

PubMed Central

Sundarrajan, Lakshminarasimhan; Blanco, Ayelén Melisa; Bertucci, Juan Ignacio; Ramesh, Naresh; Canosa, Luis Fabián; Unniappan, Suraj

2016-01-01

Nesfatin-1 is an 82 amino acid anorexigen encoded in a secreted precursor nucleobindin-2 (NUCB2). NUCB2 was named so due to its high sequence similarity with nucleobindin-1 (NUCB1). It was recently reported that NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP) in mice. Here, we aimed to characterize NLP in fish. RT- qPCR showed NUCB1 expression in both central and peripheral tissues. Western blot analysis and/or fluorescence immunohistochemistry determined NUCB1/NLP in the brain, pituitary, testis, ovary and gut of goldfish. NUCB1 mRNA expression in goldfish pituitary and gut displayed a daily rhythmic pattern of expression. Pituitary NUCB1 mRNA expression was downregulated by estradiol, while testosterone upregulated its expression in female goldfish brain. High carbohydrate and fat suppressed NUCB1 mRNA expression in the brain and gut. Intraperitoneal injection of synthetic rat NLP and goldfish NLP at 10 and 100 ng/g body weight doses caused potent inhibition of food intake in goldfish. NLP injection also downregulated the expression of mRNAs encoding orexigens, preproghrelin and orexin-A, and upregulated anorexigen cocaine and amphetamine regulated transcript mRNA in goldfish brain. Collectively, these results provide the first set of results supporting the anorectic action of NLP, and the regulation of tissue specific expression of goldfish NUCB1. PMID:27329836

Elevated expression of FABP3 and FABP4 cooperatively correlates with poor prognosis in non-small cell lung cancer (NSCLC).

PubMed

Tang, Zhiyuan; Shen, Qin; Xie, Hao; Zhou, Xiaoyu; Li, Jun; Feng, Jian; Liu, Hua; Wang, Wei; Zhang, Shu; Ni, Songshi

2016-07-19

Fatty acid binding proteins (FABPs) are intracellular lipid-binding proteins that are involved in a variety of biological cellular processes, including tumorigenesis. In this study, we explored the expression pattern of FABP3 and FABP4 in non-small cell lung cancer (NSCLC) as well as their roles in prognosis. We determined mRNA expression of FABP3 and FABP4 in matched pairs of cancerous and non-cancerous fresh frozen tissues from 30 NSCLC patients. Tissue microarray immunohistochemical analysis (TMA-IHC) was applied to determine the protein expression of FABP3 and FABP4 in 281 cancerous and 121 matched adjacent non-cancerous tissue samples. Our results showed that both mRNA and protein expression of FABP3 and FABP4 were significantly higher in cancerous tissues when compared to non-cancerous tissues. Furthermore, high expression of FABP3 or FABP4 in NSCLC was significantly associated with advanced tumor node metastasis (TNM) stage and had a negative impact on the overall survival of NSCLC patients. Concurrent high expression of FABP3 and FABP4 was significantly related to TNM stage. In conclusion, our research demonstrated that high FABP3 or FABP4 expression had strong prognostic value for overall survival in NSCLC. Detection of FABP3 and FABP4 cooperatively was helpful to predict the prognosis of NSCLC.

Elevated expression of FABP3 and FABP4 cooperatively correlates with poor prognosis in non-small cell lung cancer (NSCLC)

PubMed Central

Tang, Zhiyuan; Shen, Qin; Xie, Hao; Zhou, Xiaoyu; Li, Jun; Feng, Jian; Liu, Hua; Wang, Wei; Zhang, Shu; Ni, Songshi

2016-01-01

Fatty acid binding proteins (FABPs) are intracellular lipid-binding proteins that are involved in a variety of biological cellular processes, including tumorigenesis. In this study, we explored the expression pattern of FABP3 and FABP4 in non-small cell lung cancer (NSCLC) as well as their roles in prognosis. We determined mRNA expression of FABP3 and FABP4 in matched pairs of cancerous and non-cancerous fresh frozen tissues from 30 NSCLC patients. Tissue microarray immunohistochemical analysis (TMA-IHC) was applied to determine the protein expression of FABP3 and FABP4 in 281 cancerous and 121 matched adjacent non-cancerous tissue samples. Our results showed that both mRNA and protein expression of FABP3 and FABP4 were significantly higher in cancerous tissues when compared to non-cancerous tissues. Furthermore, high expression of FABP3 or FABP4 in NSCLC was significantly associated with advanced tumor node metastasis (TNM) stage and had a negative impact on the overall survival of NSCLC patients. Concurrent high expression of FABP3 and FABP4 was significantly related to TNM stage. In conclusion, our research demonstrated that high FABP3 or FABP4 expression had strong prognostic value for overall survival in NSCLC. Detection of FABP3 and FABP4 cooperatively was helpful to predict the prognosis of NSCLC. PMID:27323829

Genome-Wide DNA Methylation Indicates Silencing of Tumor Suppressor Genes in Uterine Leiomyoma

PubMed Central

Navarro, Antonia; Yin, Ping; Monsivais, Diana; Lin, Simon M.; Du, Pan; Wei, Jian-Jun; Bulun, Serdar E.

2012-01-01

Background Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown. Principal Findings We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels. Conclusions These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women. PMID:22428009

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.

Effects of thalidomide in experimental models of post-endoscopic retrograde cholangiopancreatography pancreatitis.

PubMed

Xiong, Guang-Su; Wu, Shu-Ming; Wang, Zhen-Hua; Mo, Jian-Zhong; Xiao, Shu-Dong

2007-03-01

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in the pathogenesis of acute pancreatitis and related systemic complications. The authors hypothesized that it may also play an important role in the development of pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP). The aim of the study was to evaluate the effectiveness of thalidomide, an immunomodulator that exerts an inhibitory action on TNF-alpha by enhancing mRNA degradation, in reducing post-ERCP pancreatitis in a rat model. A total of 200 mg/kg thalidomide was given intragastric once a day (total 8 days) before the experimental models of post-ERCP pancreatitis were established. After 24 h, histology and edema of pancreas, serum amylase, and TNF-alpha mRNA in the pancreatic tissue were evaluated. Intraductal contrast infusion caused increases in serum amylase, edema, histological grade, and TNF-alpha mRNA of pancreas. The prophylactic use of thalidomide significantly reduced serum amylase, pancreatic edema and the histologic grade of pancreatitis accompanied by a decrease in mRNA expression of TNF-alpha in the pancreatic tissue. Prophylactic intragastric administration of thalidomide provides a protective effect in post-ERCP pancreatitis. The mechanism of the protective effects of thalidomide seems to be the reduction of expression of TNF-alpha mRNA in pancreatic tissue.

Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque

PubMed Central

Hu, Minlu; Zhou, Tian; Pearlman, Andrew P; Paton, Dorothy L; Rohan, Lisa C

2017-01-01

Summary This manuscript summarizes our recent progress in examine the CYP1A1 and CYP1B1 as well as a number of nuclear receptors in the female genital and colorectal tissues of human and pigtailed macaque. Understanding the nuclear receptor mediated regulation of CYP1A1 and 1B1 expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. However, there is a lack of systematic and comparative analysis of the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and clinically relevant animal models. The current study aims to fill this gap. We found CYP1A1, CYP1B1 and a number of nuclear receptors were expressed in the female genital and colorectal tissues of human and macaque. However, the mRNA level and protein localization of these CYP enzymes and NRs depended on the type of tissue examined. Cytochrome P450 (CYP) 1A1 and CYP1B1 activate hormonal and environmental procarcinogens, and are associated with carcinogenesis in female genital and colorectal tissues. Understanding the nuclear receptor (NR) mediated regulation of CYP expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. The study aims to analyze the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and pigtailed macaques. We found that compared to the liver, human CYP1A1 mRNA level in the genital and colorectal tissues was significantly lower, while the CYP1B1 level was significantly higher. CYP1A1 protein was mainly localized in the plasma membrane of the uterine and endocervical epithelial cells. The CYP1B1 protein was concentrated in the nucleus of genital and colorectal tissues. Fourteen NRs in the genital tract and 12 NRs in colorectal tissue were expressed at levels similar to or higher than the liver. The expression and localization of CYP1A1, CYP1B1, and NRs in macaque tissues were usually comparable to those of human tissues. In addition, menopause did not significantly alter the ectocervical mRNA levels of CYP1A1, CYP1B1, or NRs. PMID:29276805

Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque.

PubMed

Hu, Minlu; Zhou, Tian; Pearlman, Andrew P; Paton, Dorothy L; Rohan, Lisa C

2016-01-01

This manuscript summarizes our recent progress in examine the CYP1A1 and CYP1B1 as well as a number of nuclear receptors in the female genital and colorectal tissues of human and pigtailed macaque. Understanding the nuclear receptor mediated regulation of CYP1A1 and 1B1 expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. However, there is a lack of systematic and comparative analysis of the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and clinically relevant animal models. The current study aims to fill this gap. We found CYP1A1, CYP1B1 and a number of nuclear receptors were expressed in the female genital and colorectal tissues of human and macaque. However, the mRNA level and protein localization of these CYP enzymes and NRs depended on the type of tissue examined. Cytochrome P450 (CYP) 1A1 and CYP1B1 activate hormonal and environmental procarcinogens, and are associated with carcinogenesis in female genital and colorectal tissues. Understanding the nuclear receptor (NR) mediated regulation of CYP expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. The study aims to analyze the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and pigtailed macaques. We found that compared to the liver, human CYP1A1 mRNA level in the genital and colorectal tissues was significantly lower, while the CYP1B1 level was significantly higher. CYP1A1 protein was mainly localized in the plasma membrane of the uterine and endocervical epithelial cells. The CYP1B1 protein was concentrated in the nucleus of genital and colorectal tissues. Fourteen NRs in the genital tract and 12 NRs in colorectal tissue were expressed at levels similar to or higher than the liver. The expression and localization of CYP1A1, CYP1B1, and NRs in macaque tissues were usually comparable to those of human tissues. In addition, menopause did not significantly alter the ectocervical mRNA levels of CYP1A1, CYP1B1, or NRs.

Inflammation and Fibrosis in Perirenal Adipose Tissue of Patients With Aldosterone-Producing Adenoma.

PubMed

Wu, Chunyan; Zhang, Huijian; Zhang, Jiajun; Xie, Cuihua; Fan, Cunxia; Zhang, Hongbin; Wu, Peng; Wei, Qiang; Tan, Wanlong; Xu, Lingling; Wang, Ling; Xue, Yaoming; Guan, Meiping

2018-01-01

The prevalence of primary aldosteronism is much higher than previously thought. Recent studies have shown that primary aldosteronism is related to a higher risk of cardiovascular events. However, the underlying mechanism is not yet clear. Here we investigate the characteristics, including inflammation, fibrosis, and adipokine expression, of adipose tissues from different deposits in patients with aldosterone-producing adenoma (APA). Inflammation and fibrosis changes were evaluated in perirenal and subcutaneous adipose tissues obtained from patients with APA (n = 16), normotension (NT; n = 10), and essential hypertension (EH; n = 5) undergoing laparoscopic surgery. We also evaluated the effect of aldosterone in isolated human perirenal adipose tissue stromal vascular fraction (SVF) cells and investigated the effect of aldosterone in mouse 3T3-L1 and brown preadipocytes. Compared with the EH group, significantly higher levels of interleukin-6 (IL-6) and tumor necrosis factor-α messenger RNA (mRNA) and protein were observed in perirenal adipose tissue of patients with APA. Expression of genes related to fibrosis and adipogenesis in perirenal adipose tissue was notably higher in patients with APA than in patients with NT and EH. Aldosterone significantly induced IL-6 and fibrosis gene mRNA expression in differentiated SVF cells. Aldosterone treatment enhanced mRNA expression of genes associated with inflammation and fibrosis and stimulated differentiation of 3T3-L1 and brown preadipocytes. In conclusion, these data indicate that high aldosterone in patients with APA may induce perirenal adipose tissue dysfunction and lead to inflammation and fibrosis, which may be involved in the high risk of cardiovascular events observed in patients with primary aldosteronism. Copyright © 2018 Endocrine Society.

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.

Presence of brown adipocytes in retroperitoneal fat from patients with benign adrenal tumors: relationship with outdoor temperature.

PubMed

Betz, Matthias Johannes; Slawik, Marc; Lidell, Martin E; Osswald, Andrea; Heglind, Mikael; Nilsson, Daniel; Lichtenauer, Urs Daniel; Mauracher, Brigitte; Mussack, Thomas; Beuschlein, Felix; Enerbäck, Sven

2013-10-01

Brown adipose tissue (BAT) is a metabolically highly active organ with increased thermogenic activity in rodents exposed to cold temperature. Recently its presence in the cervical adipose tissue of human adults and its association with a favorable metabolic phenotype have been reported. The objective of the study was to determine the prevalence of retroperitoneal BAT in human adults. This was an observational cohort study. The study was conducted at a tertiary referral hospital. Fifty-seven patients who underwent surgery for benign adrenal tumors were included in this study. Prevalence of retroperitoneal BAT adjacent to the removed adrenal tumor as determined by uncoupling protein 1 (UCP1) protein and mRNA expression was measured. Using protein and mRNA expression analysis, we detected UCP1 protein in 26 of 57 patients (45.6%) as well as high mRNA expression of genes characteristic for brown adipocytes, independent of the adrenal tumor type. The presence of brown adipocytes within the retroperitoneal fat was associated with a significantly lower outdoor temperature during the month prior to surgery. Importantly, UCP1 expression on both mRNA and protein level was inversely correlated to outdoor temperature, whereas body mass index, sex, age, and diabetes status were not. These findings suggest that human retroperitoneal adipose tissue can acquire a BAT phenotype, thereby adapting to environmental challenges. These adaptive processes might provide a valuable therapeutic target in the treatment of obesity and insulin resistance.

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).

Effects of Dietary Selenium Against Lead Toxicity on mRNA Levels of 25 Selenoprotein Genes in the Cartilage Tissue of Broiler Chicken.

PubMed

Gao, H; Liu, C P; Song, S Q; Fu, J

2016-07-01

The interactions between the essential element selenium (Se) and the toxic element lead (Pb) have been reported extensively; however, little is known about the effect of Se on Pb toxicity and the expression pattern of selenoproteins in the cartilage of chicken. To investigate the effects of Se on Pb toxicity and the messenger RNA (mRNA) expressions of selenoproteins in cartilage tissue, an in vitro study was performed on 1-day-old broiler chickens (randomly allocated into four groups) with diet of different concentration of Se and Pb. After 90 days, the meniscus cartilage and sword cartilage tissue were examined for the mRNA levels of 25 selenoprotein genes. The results showed that Se and Pb influenced the expression of selenoprotein genes in the chicken cartilage tissue. In detail, Se could alleviate the downtrend of the expression of Gpx1, Gpx2, Gpx4, Txnrd2, Txnrd3, Dio1, Dio2, Seli, Selu, Sepx1, Selk, Selw, Selo, Selm, Sep15, Sepnn1, Sels, and Selt induced by Pb exposure in the meniscus cartilage. In the sword cartilage, Se alleviated the downtrend of the expression of Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Dio2, Dio3, Seli, Selh, SPS2, Sepx1, Selk, Selw, Selo, Selm, Sep15, Selpb, Sepn1, and Selt induced by Pb exposure. The present study provided some compensated data about the roles of Se against Pb toxicity in the regulation of selenoprotein expression.

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

Extracellular matrix metalloproteinase inducer expression in the baboon endometrium: menstrual cycle and endometriosis

PubMed Central

Braundmeier, A G; Fazleabas, A T; Nowak, R A

2016-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN; BSG) regulates tissue remodeling through matrix metalloproteinases (MMPs). In human and non-human primates, endometrial remodeling is important for menstruation and the pathogenesis of endometriosis. We hypothesized that as in humans, BSG and MMPs are expressed in the endometrium of cycling baboons, and their expression is hormonally regulated by ovarian hormones, but endometriosis disrupts this regulation. BSG expression was evaluated in the baboon endometrium by q-PCR and immunohistochemistry. In the endometrium of control cycling animals, BSG mRNA levels were highest in late secretory stage tissue. BSG protein localized to glandular epithelial cells during the proliferative phase; whereas, secretory stage tissues expressed BSG in glandular and luminal epithelia with weak stromal staining. Several MMPs were differentially expressed throughout the menstrual cycle with the highest levels found during menstruation. In ovariectomized animals, BSG endometrial mRNA levels were highest with treatment of both estrogen and progesterone than that with only estrogen. Estrogen alone resulted in BSG protein localization primarily in the endometrial glandular epithelia, while estrogen and progesterone treatment displayed BSG protein localization in both the glandular and stromal cells. Exogenous hormone treatment resulted in differential expression patterns of all MMPs compared with the control cycling animals. In the eutopic endometrium of endometriotic animals, BSG mRNA levels and protein were elevated early but decreased later in disease progression. Endometriosis elevated the expression of all MMPs except MMP7 compared with the control animals. In baboons, BSG and MMP endometrial expression is regulated by both ovarian hormones, and their expression patterns are dysregulated in endometriotic animals. PMID:20841363

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.

Association between macrophage migration inhibitory factor in the endometrium and estrogen in endometriosis

PubMed Central

ZHANG, XIAO; MU, LIN

2015-01-01

Recent studies have shown that macrophage migration inhibitory factor (MIF) has a possible role in endometriosis-related pain and infertility, yet it has not been explored whether the mRNA level of MIF is altered in endometrial tissues from patients with endometriosis. The aim of the present study was to compare the expression of MIF in endometrial tissues from women with and without endometriosis, and to analyze the association between endometrial MIF expression and 17β-estradiol (E2). The protein and mRNA expression of MIF in the human endometrial tissue was assessed by western blotting and reverse transcription-polymerase chain reaction analysis, respectively. The MIF expression of women with endometriosis was found to be significantly higher than that of the controls. A positive correlation was noted between the serum E2 level and MIF expression. In endometrial cells from women with endometriosis, the level of E2-induced MIF upregulation was significantly higher than that in cells from women without endometriosis. In conclusion, this study demonstrated a significant increase in MIF expression in the endometrial tissues of women with endometriosis and an association between MIF expression and E2 level. MIF expression in endometrial cells from patients with endometriosis showed an increased sensitivity to stimulation by E2. PMID:26622394

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

PubMed

Endo, Daisuke; Park, Min Kyun

2003-12-01

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

Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis

PubMed Central

2012-01-01

Background Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Methods Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. Results SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. Conclusions The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation. PMID:23236990

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

PubMed

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

2007-12-01

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

Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

PubMed Central

Pistoi, S; Roland, J; Babinet, C; Morello, D

1996-01-01

We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation. PMID:8756668

Aberrant Hypermethylation of SALL3 with HPV Involvement Contributes to the Carcinogenesis of Cervical Cancer.

PubMed

Wei, Xing; Zhang, Shaohua; Cao, Di; Zhao, Minyi; Zhang, Qian; Zhao, Juan; Yang, Ting; Pei, Meili; Wang, Li; Li, Yang; Yang, Xiaofeng

2015-01-01

This study aimed to investigate the methylation status of the promoter region of spalt-like transcription factor 3 (SALL3) and the expression of SALL3 in cervical cancer to explore the function of this gene in cervical cancer carcinogenesis. The methylation status of SALL3 was detected by methylation-specific PCR, and SALL3 gene expression was assessed by real-time quantitative PCR in the cervical cancer cell lines, SiHa, HeLa and C33A, as well as in cervical cancer tissue samples (n = 23), matched pericarcinomatous tissue samples (n = 23) and normal cervix tissue samples (n = 17). MTT was used to measure the cell viability and proliferation capacity of SiHa and HeLa cells. The SALL3 promoter was completely methylated in SiHa cells, unmethylated in C33A cells and partially methylated in HeLa cells. After treatment of SiHa and HeLa cells with 5 μM and 10 μM of 5-Azacytidine (5-Aza), respectively, the methylation level of the SALL3 promoter decreased and observed increase in the degree of unmethylation in a dose-dependent manner. Moreover, the relative expression of SALL3 mRNA increased as the concentration of 5-Aza increased in SiHa (p<0.05) and HeLa (p<0.05) cells. This above-mentioned increase in SALL3 mRNA in SiHa cells was more remarkable than that observed in HeLa cells. Cell proliferation capacity also decreased after administration of 5-Aza to SiHa and HeLa cells (p<0.05). Methylation of the SALL3 promoter was observed in 15 of 23 (65.21%) cervical cancer tissue samples, 15 of 23 (65.21%) matched pericarcinomatous tissue samples and 5 of 17 (29.41%) normal cervical tissue samples (p<0.05). SALL3 mRNA expression was significantly lower in cervical cancer and pericarcinomatous tissues compared with normal cervical tissues (p<0.05). In all cervix tissue samples, HPV infection was positively associated with hypermethylation of the promoter region of SALL3 (p<0.05, r = 0.408), and the expression of SALL3 mRNA in HPV-positive tissues was lower than that in HPV-negative tissues (p<0.05). The aberrant hypermethylation of SALL3 together with HPV involvement inactivated its function as a tumor suppressor and contributed to carcinogenesis in cervical cancer.

[Study on effect of cordyceps sinensis on early-stage silicotic pulmonary fibrosis in rabbits].

PubMed

Liu, Qianzhong; Zhang, Wei; Cui, Hongfu; Ying, Yanhong

2014-07-01

To establish a rabbit model of silicotic pulmonary fibrosis and to investigate the effect of cordyceps sinensis in this model. Thirty healthy male white rabbits were randomly divided into control group, silicosis model group, and intervention group. The rabbits in silicosis model group and intervention group received endotracheal perfusion of silicon dioxide suspension (120 mg/kg), and the control group was treated with the same volume of saline. All the rabbits were sacrificed 30 days later. The lung coefficient was calculated by comparing the lung weight and body weight; the right lung tissue was stained with hematoxylin-eosin (HE). The content of hydroxyproline in lung tissue was measured by alkaline hydrolysis. The mRNA levels of transforming growth factor beta 1 (TGF-β₁) and mothers against decapentaplegic homolog 7 (Smad7) in rabbit lung sections were determined by real-time PCR. No abnormalities were observed by HE staining in the lung tissues of control group, while fibrosis and silicotic nodules were discovered in the silicosis model group and intervention group. The lung coefficient and the content of hydroxyproline in lung tissue were significantly higher in the silicosis model group than in the control group and intervention group (P < 0.05 or P < 0.01). Compared with the control group, the silicosis model group and intervention group had significantly increased TGF-β₁ mRNA levels but significantly reduced Smad7 mRNA levels (P < 0.02). Compared with the silicosis model group, the intervention group had a significantly reduced TGF-β₁ mRNA level but a significantly increased Smad7 mRNA level (P < 0.05). Cordyceps sinensis is able to reduce the expression of TGF-β₁ mRNA and increase the expression of Smad7 mRNA in lung tissues of rabbits with silicotic pulmonary fibrosis, and thus postpone the progression of fibrosis.

Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

PubMed

Cathcart, Mary-Clare; Gray, Steven G; Baird, Anne-Marie; Boyle, Elaine; Gately, Kathy; Kay, Elaine; Cummins, Robert; Pidgeon, Graham P; O'Byrne, Kenneth J

2011-11-15

Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. PGIS expression was reduced/absent in human NSCLC protein samples (P < .0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P = .004) and in male patients (P < .05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P < .001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. Copyright © 2011 American Cancer Society.

Expression of Caspase-1 in breast cancer tissues and its effects on cell proliferation, apoptosis and invasion.

PubMed

Sun, Yanxia; Guo, Yingzhen

2018-05-01

The present study aimed to detect the expression of Caspase-1 in the tumor tissues and tumor-adjacent tissues of patients with breast cancer, and to investigate the effects of Caspase-1 on the proliferation, apoptosis and invasion of breast cancer cells. Reverse transcription-quantitative polymerase chain reaction was used to detect Caspase-1 mRNA expression in breast cancer tissues and tumor-adjacent tissues from patients. Additionally, the human breast cancer MDA-MB-231 cell line was treated with the Caspase-1 small molecule inhibitor Ac-YVAD-CMK, following which the changes to Caspase-1 protein expression were detected via western blotting. The MTT method detected the changes to cell proliferation, flow cytometry detected the rate of apoptosis, and a Transwell assay was employed to assess invasion. Caspase-1 mRNA expression was significantly decreased in the breast cancer tissues of patients, compared with in the tumor-adjacent tissues, a difference that was statistically significant (P<0.05). Treatment with the Ac-YVAD-CMK markedly decreased the protein expression of Caspase-1 in MDA-MB-231 cells, and the difference was statistically significant (P<0.05). Following this treatment of Ac-YVAD-CMK cells, the proliferation and invasion abilities markedly increased, while the apoptotic levels significantly decreased (P<0.05). In conclusion, the expression of Caspase-1 is low in breast cancer tissues, which may promote the proliferation and invasion of breast cancer cells and could be closely associated with the occurrence and development of breast cancer.

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

PubMed

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

2017-11-01

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

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

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

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

Cold-Induced Accumulation of hsp90 Transcripts in Brassica napus.

PubMed Central

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

1995-01-01

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

Expression of nestin in embryonic tissues and its effects on clinicopathological characteristics of patients with placenta previa.

PubMed

Qiao, Yan-Yan; Chu, Ping

2018-02-01

In this study, we examined expression of nestin in the spinal cord, lung, kidney, stomach, colon, and intestine tissues at different stages of embryos in patients with placenta previa. Fetuses of 75 patients with placenta previa were assigned to case group and 80 fetuses from healthy pregnant women with normal placenta who voluntarily terminated pregnancy to control group. Clinical data of pregnant women were collected at the time of admission. Blood from elbow vein was collected to determine expression of serum nestin. Tissues from spinal cord, lung, kidney, stomach, colon, and intestine in 3-7 months fetuses of the two groups were extracted. Expression of nestin in tissues was detected by immunohistochemistry, Western blotting and RT-qPCR. The mRNA expression of nestin in the case group was increased. Nestin expression was correlated with the gestational age, age of foetus, and type of placenta previa in patients with placenta previa. Positive nestin expression was detected in the spinal cord, lung, kidney, stomach, intestine, and colon tissues in normal and placenta previa embryo at Stage I. The positive cell density and nestin expression decreased at Stage II, and further decreased at Stage III. The case group had higher nestin mRNA and protein levels throughout human fetal development. Findings of this study suggested that, nestin, as a specific marker of neural precursor cells, was expressed in various tissues of the embryo in patients with placenta previa and nestin expression was lower with increased maturation of the embryo. © 2017 Wiley Periodicals, Inc.

11beta-Hydroxysteroid dehydrogenase Type 1: genetic polymorphisms are associated with Type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle.

PubMed

Nair, S; Lee, Y H; Lindsay, R S; Walker, B R; Tataranni, P A; Bogardus, C; Baier, L J; Permana, P A

2004-06-01

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) modulates tissue-specific glucocorticoid concentrations by generating active cortisol. We have shown that adipose tissue 11beta-HSD1 mRNA levels were associated with adiposity and insulinaemia. Here we conducted further expression and genetic association studies in Pima Indians. The 11beta-HSD1 mRNA concentrations were measured in abdominal subcutaneous adipocytes (n=61) and skeletal muscle tissues (n=64). Single nucleotide polymorphisms in the HSD11B1 gene were genotyped in a larger group of full-blooded Pima Indians. Two representative SNPs (SNP1, n=706; SNP5, n=839) were associated with Type 2 diabetes mellitus (p=0.01), although neither SNP was associated with obesity. Among subjects with normal glucose tolerance, SNP1 (n=127) and SNP5 (n=159) were associated with insulin-mediated glucose uptake rates (p=0.03 and p=0.04), and SNP1 was further associated with fasting, 30-min, and 2-h plasma insulin concentrations (p=0.002, p=0.002 and p=0.03). Adipocyte 11beta-HSD1 mRNA concentrations were correlated positively with adiposity and insulinaemia, and were additionally negatively correlated with insulin-mediated glucose uptake rates; nevertheless, the adipocyte 11beta-HSD1 expression did not correlate with genotypes of the donors. The muscle 11beta-HSD1 mRNA concentrations did not correlate with any anthropometric or metabolic variables. We confirmed that adipocyte 11beta-HSD1 mRNA concentrations were associated with adiposity, and showed that genetic variations in the HSD11B1 gene were associated with Type 2 diabetes mellitus, plasma insulin concentrations and insulin action, independent of obesity. The variable adipose expression might not be a primary consequence of these HSD11B1 SNPs. Therefore, it is possible that the HSD11B1 gene is under tissue-specific regulation, and has tissue-specific consequences.

Expression of Estrogen Receptors in Relation to Hormone Levels and the Nottingham Prognostic Index.

PubMed

Fahlén, Mia; Zhang, Hua; Löfgren, Lars; Masironi, Britt; VON Schoultz, Eva; VON Schoultz, B O; Sahlin, Lena

2016-06-01

Estrogen hormones have a large impact on both normal development and tumorigenesis of the breast. Breast tissue samples from 49 women undergoing surgery were included. The estrogen receptors (ERα and ERβ), ERα36 and G-coupled estrogen receptor-1 (GPER) were determined in benign and malignant breast tissue. The ERα36 and ERα mRNA levels were highest in malignant tumors. Stromal ERβ immunostaining in benign tumors was higher than in the paired normal tissue. GPER expression was lowest in benign tumors. In the malignant tumors, the Nottingham Prognostic Index (NPI) correlated positively with stromal GPER and the serum testosterone level. The serum insulin-like growth factor-1 (IGF-1) level correlated negatively with GPER mRNA and glandular ERα. The expression of ERα36 is stronger in malignant breast tissue. The strong positive correlation between NPI and GPER in malignant breast stroma indicates an important role for GPER in breast cancer prognosis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Longitudinal Profiling of the Tissue-Specific Expression of Genes Related with Insulin Sensitivity in Dairy Cows during Lactation Focusing on Different Fat Depots

PubMed Central

Saremi, Behnam; Winand, Sarah; Friedrichs, Paula; Kinoshita, Asako; Rehage, Jürgen; Dänicke, Sven; Häussler, Susanne; Breves, Gerhard; Mielenz, Manfred; Sauerwein, Helga

2014-01-01

In dairy cows the milk associated energy output in early lactation exceeds the input via voluntary feed intake. To spare glucose for mammary lactose synthesis, peripheral insulin sensitivity (IS) is reduced and fat mobilization is stimulated. For these processes a link between IS and the endocrine functions of adipose tissue (AT) is likely; we thus aimed to characterise the mRNA expression from bovine AT derived proteins and receptors that are related to IS according to the literature in metabolically active tissues plus systemic IS throughout lactation. Conjugated linoleic acids (CLA) reduce milk fat thus decreasing the milk drain of energy and potentially dampening lipolysis, but may also affect IS. Subcutaneous (s.c.) AT and liver from pluriparous cows receiving either control fat or CLA supplement (100 g/day from 1 to 182 days in milk each) were biopsied covering week −3 to 36 relative to parturition. In an additional trial with primiparous cows treated analogously and slaughtered on days in milk 1, 42 or 105, samples from liver, udder, skeletal muscle and 3 visceral and 3 s.c. AT were obtained and assayed for mRNA abundance of adiponectin, its receptors, leptin, leptin receptor, PPARγ, PPARγ2, IL-6, and TNF-α. In pluriparous animals, the mRNA abundance of most of the target genes decreased after parturition in s.c. AT but increased in liver. In primiparous cows, AT depot specific differences were mostly related to retroperitoneal AT; adiponectin receptor 1 and TNF-α were affected predominantly. CLA effects in primiparous cows were largely limited to decreased PPARγ2 mRNA abundance in udder tissue. In pluriparous cows, insulin secretion was increased by CLA resulting in decreased systemic IS but without consistent changes in tissue target mRNA abundance. The temporal gene expression profiles from the adipokines and related receptors support their coactive function in adapting to the needs of lactation. PMID:24465964

Increase in substance P precursor mRNA in noninflamed small-bowel sections in patients with Crohn's disease.

PubMed

Michalski, Christoph W; Autschbach, Frank; Selvaggi, Federico; Shi, Xin; Di Mola, Fabio Francesco; Roggo, Antoine; Müller, Michael W; Di Sebastiano, Pierluigi; Büchler, Markus W; Giese, Thomas; Friess, Helmut

2007-04-01

Neuropeptides, such as substance P (SP), are mediators of neurogenic inflammation and play an important role in inflammatory disorders. To further investigate the role of the SP pathway in inflammatory bowel disease (IBD), we analyzed the following in normal intestinal tissue specimens and in tissue specimens from patients with Crohn's disease (CD) and ulcerative colitis (UC): neurokinin receptor-1 (NK-1R); its isoforms (NK-1R-L and NK-1R-S); its ligand SP, encoded by preprotachykinin-A (PPT-A); and the SP-degradation enzyme, neutral endopeptidase (NEP). Real-time quantitative reverse transcription-polymerase chain reaction was used to simultaneously determine the expression of NK-1R-L, NK-1R-S, and PPT-A. Protein levels of NK-1R and NEP were determined by immunoblot analysis. In noninflamed small-bowel tissue samples of CD patients, PPT-A mRNA expression was significantly increased, whereas there was no difference between inflamed or noninflamed UC and normal intestinal tissue samples. Examining subgroups of diverse intestinal segments from CD and UC samples with various levels of inflammation revealed no differences in NK-1R-L and NK-1R-S mRNA expression, whereas there was a tendency toward overall lower NK-1R-S mRNA copy numbers. Immunoblot analysis showed upregulation of NK-1R protein levels in cases of IBD, with more pronounced enhancement in cases of CD than in UC. For NEP, there were no differences in protein levels in normal, CD, and UC intestinal tissues. These observations suggest a contribution of SP and its receptor, NK-1R, in the local inflammatory reaction in IBD and particularly in ileal CD. Moreover, significant upregulation of PPT-A mRNA in the noninflamed ileum of these patients suggests an influence of inflamed intestines on their healthy counterparts.

Similar Properties of Chondrocytes from Osteoarthritis Joints and Mesenchymal Stem Cells from Healthy Donors for Tissue Engineering of Articular Cartilage

PubMed Central

Fernandes, Amilton M.; Herlofsen, Sarah R.; Karlsen, Tommy A.; Küchler, Axel M.; Fløisand, Yngvar; Brinchmann, Jan E.

2013-01-01

Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA) may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM) derived mesenchymal stem cells (MSCs) from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin), ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage. PMID:23671648

Status of epigenetic chromatin modification enzymes and esophageal squamous cell carcinoma risk in northeast Indian population.

PubMed

Singh, Virendra; Singh, Laishram C; Singh, Avninder P; Sharma, Jagannath; Borthakur, Bibhuti B; Debnath, Arundhati; Rai, Avdhesh K; Phukan, Rup K; Mahanta, Jagadish; Kataki, Amal C; Kapur, Sujala; Saxena, Sunita

2015-01-01

Esophageal cancer incidence is reported in high frequency in northeast India. The etiology is different from other population at India due to wide variations in dietary habits or nutritional factors, tobacco/betel quid chewing and alcohol habits. Since DNA methylation, histone modification and miRNA-mediated epigenetic processes alter the gene expression, the involvement of these processes might be useful to find out epigenetic markers of esophageal cancer risk in northeast Indian population. The present investigation was aimed to carryout differential expression profiling of chromatin modification enzymes in tumor and normal tissue collected from esophageal squamous cell carcinoma (ESCC) patients. Differential mRNA expression profiling and their validation was done by quantitative real time PCR and tissue microarray respectively. Univariate and multiple logistic regression analysis were used to analyze the epidemiological data. mRNA expression data was analyzed by Student t-test. Fisher exact test was used for tissue microarray data analysis. Higher expression of enzymes regulating methylation (DOT1L and PRMT1) and acetylation (KAT7, KAT8, KAT2A and KAT6A) of histone was found associated with ESCC risk. Tissue microarray done in independent cohort of 75 patients revealed higher nuclear protein expression of KAT8 and PRMT1 in tumor similar to mRNA expression. Expression status of PRMT1 and KAT8 was found declined as we move from low grade to high grade tumor. Betel nut chewing, alcohol drinking and dried fish intake were significantly associated with increased risk of esophageal cancer among the study subject. Study suggests the association of PRMT1 and KAT8 with esophageal cancer risk and its involvement in the transition process of low to high grade tumor formation. The study exposes the differential status of chromatin modification enzymes between tumor and normal tissue and points out that relaxed state of chromatin facilitates more transcriptionally active genome in esophageal carcinogenesis.

In situ Localization of the Human Multidrug‐resistance Gene mRNA Using Thymine‐Thymine Dimerized Single‐stranded cDNA

PubMed Central

Koji, Takehiko; Ueda, Kazumitsu; Pastan, Ira; Gottesman, Michael M.; Nakane, Paul K.; Mori, Shigeo

1990-01-01

In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T‐T dimerized single‐stranded DNA (300–400 bases) so that they could penetrate well into the cytoplasm. The hybridized T‐T DNA was detected immunohisto‐chemically using rabbit anti‐T‐T DNA antibody (Ab) and peroxidase‐labeled goat anti‐rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug‐resistant cell lines K562/ADM, CEM/VLB, 2780ad, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene. PMID:1977730

Messenger RNA Delivery for Tissue Engineering and Regenerative Medicine Applications.

PubMed

Patel, Siddharth; Athirasala, Avathamsa; Menezes, Paula P; Ashwanikumar, N; Zou, Ting; Sahay, Gaurav; Bertassoni, Luiz E

2018-06-07

The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed (IVT) mRNA technology with chemical modifications have led to development of methods that control spatiotemporal gene expression. Additionally, there is a current thrust toward the development of safe, integration-free approaches to gene therapy for translational purposes. In this review, we describe strategies of synthetic IVT mRNA modifications and nonviral technologies for intracellular delivery. We provide insights into the current tissue engineering approaches that use a hydrogel scaffold with genetic material. Furthermore, we discuss the transformative potential of novel mRNA formulations that when embedded in hydrogels can trigger controlled genetic manipulation to regenerate tissues and organs in vitro and in vivo. The role of mRNA delivery in vascularization, cytoprotection, and Cas9-mediated xenotransplantation is additionally highlighted. Harmonizing mRNA delivery vehicle interactions with polymeric scaffolds can be used to present genetic cues that lead to precise command over cellular reprogramming, differentiation, and secretome activity of stem cells-an ultimate goal for tissue engineering.

[Nuclear factor-kappaB mRNA and protein expression in stomach tissue of rats with gastric ulcer recurrence and effect of jianwei yuyang granule on its expression].

PubMed

Ling, Jiang-Hong; Li, Jia-Bang; Shen, Ding-Zhu; Zhou, Bing

2006-03-01

To observe the inflammatory reaction, nuclear factor-kappaB (NF-kappaB) mRNA and protein expression in stomach tissue of rats with gastric ulcer recurrence and the effect of Jianwei Yuyang granule (JYG) on them. Gastric ulcer and its recurrent lesion were successively induced by acetic acid and interliukin1-beta (IL-1beta), and the model rats were divided into the sham operation group, the model group, the omeprazole (correction of omepraxole) group and the JYG group to observe the state of chronic inflammatory cell, neutrophil count, NF-kappaBmRNA and protein expression in stomach tissue. On the 16th and 92th day after administration, the increase of chronic inflammatory cell, neutrophil, NF-kappaBmRNA and protein expression in the model group was more significant than those in the sham operated group (P < 0.01), while that was lower in the JYG group than in the model group (P < 0.05, P <0.01), but with no remarkable difference to the omepraxole group. In addition, the mRNA and protein expression of NF-kappaB were correlated closely with the count of chronic inflammatory cell and neutrophil respectively (P < 0.01). NF-kappaB may play an important role in regulating inflammatory reaction during the healing and recurrence processes of gastric ulcer induced by acetic acid. JYG may suppress inflammatory reaction by inhibiting the activation and expression of NF-kappaB in stomach tissue, which may be one of the mechanisms of JYG in preventing the recurrence of gastric ulcer.

Expression of peroxisome proliferator-activated receptor alpha messenger ribonucleic acid and protein in human and rat testis.

PubMed

Schultz, R; Yan, W; Toppari, J; Völkl, A; Gustafsson, J A; Pelto-Huikko, M

1999-07-01

Peroxisome proliferator-activated receptor a (PPARalpha), a member of the steroid hormone receptor superfamily, has been linked to lipid homeostasis and tumorigenesis in tissues with high expression of receptor protein. On the other hand, the role of PPARalpha in tissues with a lower expression is not well known. Here we demonstrate the localization of PPARalpha messenger RNA (mRNA) and protein in developing and adult rat testis. Additionally, we demonstrate the expression of PPARalpha protein in adult human testis. Our experiments with Northern analysis, in situ hybridization and immunocytochemistry reveal a complex distribution of PPARalpha in tubular and interstitial cells of both adult and developing rat testis. The overall expression is rather low but may be modified by exogenous or endogenous stimuli. An up-regulation of PPARalpha mRNA could be observed after stimulation with FSH. In the developing rat testis, a clear expression of PPARalpha mRNA was present from the first days after birth. Additionally, PPARalpha mRNA and protein increased toward adulthood. In adult human testis PPARalpha immunoreactivity (IR) was present in interstitial Leydig cells and tubular cells. In the seminiferous epithelium of adult human testis the expression of PPARalpha-IR could be seen in meiotic spermatocytes, spermatids and myoid peritubular cells. The findings of our study suggest that PPARalpha may be involved in the regulation of growth and differentiation of tubular and interstitial cells in rat and human testis.

[Effects of electromagnetic pulses on apoptosis and TGF-β3 expression of mouse testis tissue].

PubMed

Luo, Yaning; Ding, Guirong; Chen, Yongbin; Xu, Shenglong; Wang, Xiaowu

2014-04-01

To investigate the effects of electromagnetic pulses (EMP) on the apoptosis and transforming growth factor beta 3 (TGF-β3) expression of mouse testis tissue. Thirty-two male BALB/c mice were randomly and equally divided into one control group and three EMP treated groups, which were whole-body exposed to EMP at 200 kV/m with 100, 200, and 400 pulses, respectively. The control group received no treatment. The pathological changes and cell apoptosis in testis tissue were analyzed by TUNEL assay. The mRNA expression of TGF-β3 in testis tissue was determined by RT-PCR, and the protein expression of TGF-β3 was determined by immunohistochemistry and Western blot. No obvious pathological changes were found in testis tissue after EMP exposure at 200 kV/m with 100 and 200 pulses. However, after EMP exposure with 400 pulses, degeneration and shedding of testis tissue, accompanied by significant increase in apoptosis rate (P < 0.05), was observed. The RT-PCR, immunohistochemistry, and Western blot showed that the expression of TGF-β3 mRNA and protein increased significantly after EMP exposure with 400 pulses as compared with that of the control group (P < 0.05). EMP exposure at 200 kV/m with 400 pulses increases the incidence of apoptosis and expression of TGF-β3 in mouse testis tissue, which is potentially one of the mechanisms by which EMP increases blood-testis barrier permeability in mice.

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

PubMed

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

2018-01-02

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

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.

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.

Developmental expression of VGF mRNA in the prenatal and postnatal rat.

PubMed

Snyder, S E; Pintar, J E; Salton, S R

1998-04-27

VGF is a developmentally regulated, secretory peptide precursor that is expressed by neurons and neuroendocrine cells and that has its transcription and secretion induced rapidly by neurotrophins and by depolarization. To gain insight into the possible functions and regulation of VGF in vivo, we have characterized the distribution of VGF mRNA in the developing rat nervous system. VGF expression was first detectable at embryonic day 11.5 in the primordia of cranial, sympathetic, and dorsal root ganglia, and its distribution expanded throughout development to include significant expression throughout the brain, spinal cord, and retina of the adult rat. The earliest expression of VGF, therefore, appeared in the peripheral nervous system as developing neurons settled in their designated ganglia. In many regions of the brain, VGF mRNA levels were found to be highest during periods when axonal outgrowth and synaptogenesis predominate. Areas of the central nervous system that contain predominantly dividing cells never displayed any VGF mRNA expression, nor did the vast majority of nonneural tissues.

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

PubMed

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

2018-06-21

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

Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

PubMed

Pinsonneault, Julia K; Frater, John T; Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

2017-01-01

Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits

PubMed Central

Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

2017-01-01

Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3’UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12–33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders. PMID:28617822

Cinnamon extract regulates plasma levels of adipose-derived factors and expression of multiple genes related to carbohydrate metabolism and lipogenesis in adipose tissue of fructose-fed rats.

PubMed

Qin, B; Polansky, M M; Anderson, R A

2010-03-01

We reported earlier that dietary cinnamon extract (CE) improves systemic insulin sensitivity and dyslipidemia by enhancing insulin signaling. In the present study, we have examined the effects of CE on several biomarkers including plasma levels of adipose-derived adipokines, and the potential molecular mechanisms of CE in epididymal adipose tissue (EAT). In Wistar rats fed a high-fructose diet (HFD) to induce insulin resistance, supplementation with a CE (Cinnulin PF, 50 mg/kg daily) for 8 weeks reduced blood glucose, plasma insulin, triglycerides, total cholesterol, chylomicron-apoB48, VLDL-apoB100, and soluble CD36. CE also inhibited plasma retinol binding protein 4 (RBP4) and fatty acid binding protein 4 (FABP4) levels. CE-induced increases in plasma adiponectin were not significant. CE did not affect food intake, bodyweight, and EAT weight. In EAT, there were increases in the insulin receptor ( IR) and IR substrate 2 ( IRS2) mRNA, but CE-induced increases in mRNA expression of IRS1, phosphoinositide-3-kinase, AKT1, glucose transporters 1 and 4 , and glycogen synthase 1 expression and decreased trends in mRNA expression of glycogen synthase kinase 3beta were not statistically significant. CE also enhanced the mRNA levels of ADIPOQ, and inhibited sterol regulatory element binding protein-1c mRNA levels. mRNA and protein levels of fatty acid synthase and FABP4 were inhibited by CE and RBP4, and CD36 protein levels were also decreased by CE. These results suggest that CE effectively ameliorates circulating levels of adipokines partially mediated via regulation of the expression of multiple genes involved in insulin sensitivity and lipogenesis in the EAT.

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

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.

Desmoplakin II expression is not restricted to stratified epithelia.

PubMed

Angst, B D; Nilles, L A; Green, K J

1990-10-01

Desmosomes are major intercellular junctions found in association with intermediate filaments in epithelial, cardiac and arachnoidal tissue. Desmoplakins I and II (DPI and II) are highly related proteins localized in the innermost part of the desmosomal plaque and are candidates for linking intermediate filaments (IF) to the desmosomal complex. While investigators agree that DPI is present in all epithelia, they disagree on the distribution of DPII. Some have reported DPII to be restricted to stratified tissue and have furthermore suggested that the expression of DPII may be linked to stratification. We have compared the expression of DPI and II at the mRNA and protein levels in cell lines derived from simple, transitional and stratified epithelia. Northern blot analysis revealed DPI and II mRNA to be present in all cell lines as well as simple and stratified epithelial tissues. However, DPII mRNA could not be detected in cardiac muscle tissue. Immunoblotting and immunoprecipitation demonstrated the presence of DPI and II in all cell lines at the whole-cell protein level as well as in association with cytoskeletal fractions. Immunofluorescence staining was used to correlate the biochemical findings with the localization of DPI and II. While most cell lines exhibited typical intercellular and in many cases cytoplasmic DP staining, T24 cells exhibited predominantly diffuse and dotty cytoplasmic staining. In addition, we investigated whether changes in DPI and II expression occurred following calcium-induced cell contact formation and stratification in the human pharyngeal cell line, FaDu. No significant changes in mRNA or whole-cell protein levels were observed during a period of 5 days following the calcium switch. However, immunoblotting revealed a significant increase in DPI and II levels in the insoluble protein pool during desmosome formation. These observations indicated a possible recruitment of soluble DPI/II into an insoluble pool after induction of desmosome assembly by the calcium switch, consistent with earlier reports for MDCK cells. In summary, our results suggest that the expression of DPII is not strictly linked to stratification or differentiation; however, the apparent absence of DPII mRNA from cardiac muscle suggests it may not be a constituent of all desmosomes.

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

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.

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

PubMed

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

2018-04-01

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

[Effects of bushen yinao tablet on physiology and cerebral gene expression in senescence-accelerated mice].

PubMed

Zhang, Chong; Wang, Jin-gang; Yang, Ting

2006-06-01

To study the effects of Bushen Yin' ao Tablet (BSYNT) on physiology and cerebral gene expression in senescence-accelerated mice (SAM). The change of cerebral tissues mRNA expression in SAM was analyzed and compared by messenger ribonucleic acids reverse transcription differential display polymerase chain reaction (mRNA DDRT-PCR) between the medicated group and the control group. BSYNT could increase the level of hemoglobin (Hb) and amount of erythrocyte (RBC) of blood deficiency mice, improve the spatial learning and memory function and the escape response by conditional stimulus. In this study, 14 differential display bands had been discerned, and three of them had been sequenced. The sequence of the three fragments was similar to fatty acid binding protein 7, ubiquinol-cytochrome C reductase complex (7. 2 kD) and 60S ribosomal protein L21 respectively. And the homogeneity was 97% , 100% , and 99% , respectively. BSYNT has effect on the physiological changing of mice, and its effect on cerebral tissues mRNA expression maybe play an important role in anti-aging on the molecular level.

Expression of Selenoprotein Genes Is Affected by Obesity of Pigs Fed a High-Fat Diet123

PubMed Central

Zhao, Hua; Li, Ke; Tang, Jia-Yong; Zhou, Ji-Chang; Wang, Kang-Ning; Xia, Xin-Jie; Lei, Xin Gen

2015-01-01

Background: Relations of the 25 mammalian selenoprotein genes with obesity and the associated inflammation remain unclear. Objective: This study explored impacts of high-fat diet-induced obesity on inflammation and expressions of selenoprotein and obesity-related genes in 10 tissues of pigs. Methods: Plasma and 10 tissues were collected from pigs (n = 10) fed a corn-soy–based control diet or that diet containing 3–7% lard from weanling to finishing (180 d). Plasma concentrations (n = 8) of cytokines and thyroid hormones and tissue mRNA abundance (n = 4) of 25 selenoprotein genes and 16 obesity-related genes were compared between the pigs fed the control and high-fat diets. Stepwise regression was applied to analyze correlations among all these measures, including the previously reported body physical and plasma biochemical variables. Results: The high-fat diet elevated (P < 0.05) plasma concentrations of tumor necrosis factor α, interleukin-6, leptin, and leptin receptor by 29–42% and affected (P < 0.05–0.1) tissue mRNA levels of the selenoprotein and obesity-related genes in 3 patterns. Specifically, the high-fat diet up-regulated 12 selenoprotein genes in 6 tissues, down-regulated 13 selenoprotein genes in 7 tissues, and exerted no effect on 5 genes in any tissue. Body weights and plasma triglyceride concentrations of pigs showed the strongest regressions to tissue mRNA abundances of selenoprotein and obesity-related genes. Among the selenoprotein genes, selenoprotein V and I were ranked as the strongest independent variables for the regression of phenotypic and plasma measures. Meanwhile, agouti signaling protein, adiponectin, and resistin genes represented the strongest independent variables of the obesity-related genes for the regression of tissue selenoprotein mRNA. Conclusions: The high-fat diet induced inflammation in pigs and affected their gene expression of selenoproteins associated with thioredoxin and oxidoreductase systems, local tissue thyroid hormone activity, endoplasmic reticulum protein degradation, and phosphorylation of lipids. This porcine model may be used to study interactive mechanisms between excess fat intake and selenoprotein function. PMID:25972525

Expression of Selenoprotein Genes Is Affected by Obesity of Pigs Fed a High-Fat Diet.

PubMed

Zhao, Hua; Li, Ke; Tang, Jia-Yong; Zhou, Ji-Chang; Wang, Kang-Ning; Xia, Xin-Jie; Lei, Xin Gen

2015-07-01

Relations of the 25 mammalian selenoprotein genes with obesity and the associated inflammation remain unclear. This study explored impacts of high-fat diet-induced obesity on inflammation and expressions of selenoprotein and obesity-related genes in 10 tissues of pigs. Plasma and 10 tissues were collected from pigs (n = 10) fed a corn-soy-based control diet or that diet containing 3-7% lard from weanling to finishing (180 d). Plasma concentrations (n = 8) of cytokines and thyroid hormones and tissue mRNA abundance (n = 4) of 25 selenoprotein genes and 16 obesity-related genes were compared between the pigs fed the control and high-fat diets. Stepwise regression was applied to analyze correlations among all these measures, including the previously reported body physical and plasma biochemical variables. The high-fat diet elevated (P < 0.05) plasma concentrations of tumor necrosis factor α, interleukin-6, leptin, and leptin receptor by 29-42% and affected (P < 0.05-0.1) tissue mRNA levels of the selenoprotein and obesity-related genes in 3 patterns. Specifically, the high-fat diet up-regulated 12 selenoprotein genes in 6 tissues, down-regulated 13 selenoprotein genes in 7 tissues, and exerted no effect on 5 genes in any tissue. Body weights and plasma triglyceride concentrations of pigs showed the strongest regressions to tissue mRNA abundances of selenoprotein and obesity-related genes. Among the selenoprotein genes, selenoprotein V and I were ranked as the strongest independent variables for the regression of phenotypic and plasma measures. Meanwhile, agouti signaling protein, adiponectin, and resistin genes represented the strongest independent variables of the obesity-related genes for the regression of tissue selenoprotein mRNA. The high-fat diet induced inflammation in pigs and affected their gene expression of selenoproteins associated with thioredoxin and oxidoreductase systems, local tissue thyroid hormone activity, endoplasmic reticulum protein degradation, and phosphorylation of lipids. This porcine model may be used to study interactive mechanisms between excess fat intake and selenoprotein function. © 2015 American Society for Nutrition.

[Effects of heat shock protein 70-2 gene polymorphisms on the transcription of HSP 70-2 mRNA and the translation of HSP 70 protein in lung cancer].

PubMed

Lu, Hao-quan; Wang, Yu-zhen; Sun, Peng-hui; Liang, Shou-pei; Li, Jie; Wang, Xiao-long; Xu, Dong; Yao, Wu; Wu, Yi-ming; Zhou, Fang

2012-05-01

This study aimed to investigate the effects of gene polymorphism of heat shock protein 70-2 (HSP 70-2) 1267A/G on the mRNA level HSP 70-2 mRNA and the protein level HSP 70 in human lung cancer. Forty six lung cancer patients diagnosed histopathologically between February and August 2008 from a hospital in zhengzhou were enrolled as the subjects in this study. Gene polymorphism of HSP 70-2 1276A/G in 46 patients with lung cancer was detected by PCR-RFLP. The mRNA levels of HSP 70-2 mRNA and the protein levels of HSP 70 in lung tissue and para-cancerous tissues of these subjects were determined by RT-PCR and Western blotting respectively. The expression levels of HSP 70-2 mRNA (1.02 ± 0.30) and HSP 70 protein (0.44 ± 0.12) in the lung cancer tissues was significantly higher than that in para-cancerous tissues (0.19 ± 0.04, 0.12 ± 0.02). The relative levels of HSP 70-2 mRNA in the subjects with AA genotype (1.32 ± 0.22) were significantly higher than the patients with AG genotype or GG genotype (0.95 ± 0.17, 0.70 ± 0.16) at the site of 1267 (A/G) (P < 0.01); however, the relative protein levels of HSP 70 were 0.47 ± 0.13 (AA genotype), 0.42 ± 0.11 (AG genotype), 0.45 ± 0.11 (GG genotype), respectively, which showed no statistically significant difference (P > 0.05). The polymorphism of HSP 70-2 1267 (A/G) is highly associated with the transcription level of HSP 70-2 mRNA, but not with the expression level of HSP 70 protein.

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

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

PubMed

Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

1995-02-10

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Changes in the expression of α-tocopherol-related genes in liver and mammary gland biopsy specimens of peripartum dairy cows.

PubMed

Haga, S; Miyaji, M; Nakano, M; Ishizaki, H; Matsuyama, H; Katoh, K; Roh, S G

2018-03-28

Blood α-tocopherol (α-Toc) concentrations decline gradually throughout the prepartum period, reaching the nadir after calving in dairy cows. The 6 α-Toc-related molecules [α-Toc transfer protein (TTPA); afamin; scavenger receptor class B, Type I; ATP-binding cassette transporter A1; tocopherol-associated protein (SEC14L2); and cytochrome P450 family 4, subfamily F, polypeptide 2 (CYP4F2)] are expressed in liver and other peripheral tissues. These molecules could regulate α-Toc transport, blood concentrations, and metabolism of α-Toc. Therefore, the aim of this study was to evaluate the changes in the expression of α-Toc-related genes in liver and mammary gland tissues of dairy cows around calving, which have remained elusive until now. In experiment (Exp.) 1, 28 multiparous Holstein cows were used (from -5 to 6 wk relative to parturition) to monitor the changes in dietary α-Toc intake, blood concentrations of α-Toc, and lipoproteins; in Exp. 2, 7 peripartum Holstein cows were used (from -4 to 4 wk relative to parturition) for liver tissue biopsy; and in Exp. 3, 10 peripartum Holstein cows were used (from -8 to 6 wk relative to parturition) to carry out the mammary gland tissue biopsy and milk sampling. In Exp. 1, the serum α-Toc concentrations declined gradually with decreasing amount of α-Toc intake and plasma high-density lipoprotein concentrations toward calving time. However, in the early lactation period after calving, serum α-Toc concentrations remained at a lower concentration despite the recovery of α-Toc intake and plasma high-density lipoprotein concentrations. In Exp. 2, just after calving, the TTPA, SEC14L2, afamin, and albumin mRNA expression levels in the liver were temporarily downregulated, and the hepatic mRNA levels of endoplasmic reticulum stress-induced unfolded protein response markers and acute-phase response marker increased at calving. In Exp. 3, the concentrations of α-Toc in colostrum were greater than those in precolostrum (samples were collected at wk -1 relative to parturition) and mature milk. The expression of TTPA, SEC14L2, and CYP4F2 mRNA in bovine mammary gland tissue was detected. However, TTPA and SEC14L2 mRNA expressions showed the opposite trends: the expression levels of TTPA mRNA peaked whereas SEC14L2 mRNA reached a nadir at calving. These results indicate that the expression of α-Toc-related genes involved in specific α-Toc transfer and metabolism in the liver and mammary gland are altered during calving. Moreover, these changes might be associated with the maintenance of lower serum α-Toc concentrations after calving. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Local expression of vaginal Th1 and Th2 cytokines in murine vaginal candidiasis under different immunity conditions.

PubMed

Chen, Shanjuan; Li, Shaohua; Wu, Yan; Liu, Zhixiang; Li, Jiawen

2008-08-01

To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1 (IL-2)/Th2 (IL-4, IL-10, TGF-beta1) cytokines in murine vaginal tissues was determined by RT-PCR. The cykotine in local tissues was increased to different extent under normal immune condition. IL-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-beta1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10, and TGF-beta1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans (C. albicans), and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.

Peroxisome proliferator-activated receptor (PPAR) isoforms are differentially expressed in peri-implantation porcine conceptuses.

PubMed

Blitek, Agnieszka; Szymanska, Magdalena

2017-10-01

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are critical regulators of glucose homeostasis and lipid metabolism, and affect cell proliferation and differentiation. In the current study, we examined (1) the profiles of PPARA, PPARD, and PPARG mRNA expression and DNA binding activity in porcine conceptuses collected on Days 10-11 (spherical and tubular conceptuses), 11-12 (filamentous conceptuses), 13-14, and 15-16 (elongated conceptuses) of pregnancy, (2) the presence of PPARA, PPARD, and PPARG proteins in Days 10, 12, and 15 conceptuses. Moreover, we analyzed the abundance of retinoid X receptor (RXR; PPARs heterodimer partner) transcripts as well as the correlation between PPARs mRNA expression and the expression of genes important for and/or associated with elongation of porcine conceptuses: aromatase (CYP19A1), prostaglandin endoperoxide synthase 2 (PTGS2), glucose transporter 1 (SLC2A1), and interleukin 1B (IL1B). PPARA mRNA expression in conceptuses did not change during Days 10-14 of gestation, but was greater on Days 15-16 compared to Days 10-11 (P < 0.05). A considerable increase in PPARD and PPARG mRNA expression was observed in filamentous conceptuses from Days 11-12 compared to spherical and tubular conceptuses from Days 10-11 (P < 0.01), followed by a decrease on Days 13-14 and 15-16 (P < 0.05). PPARA, PPARD, and PPARG proteins were present in conceptus tissue demonstrating nuclear localization clearly visible on Days 12 and 15 of pregnancy. DNA binding activity of the PPARD isoform was greater in filamentous conceptuses from Days 11-12 than in spherical and tubular conceptuses from Days 10-11 (P < 0.01). Moreover, concentrations of active PPARD and PPARG proteins in nuclear fractions of conceptus tissue were greater on Days 11-12 compared to Days 13-14 and 15-16 of pregnancy (P < 0.05). RXRA, RXRD, and RXRG mRNA expression in conceptuses increased on Days 11-12 compared to Days 10-11 (P < 0.05). PPARD and PPARG mRNA expression showed strong positive correlations with PTGS2 mRNA expression (P < 0.0001). Additionally, PPARD gene expression correlated with SLC2A1 and IL1B mRNA expression (P < 0.01). Collectively, these results indicate that among all three PPARs expressed in peri-implantation porcine conceptuses, PPARD and PPARG may be involved in conceptus elongation before implantation. Copyright © 2017 Elsevier Inc. All rights reserved.

Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

PubMed Central

Günthner, Roman; Kumar, Vankayala Ramaiah Santhosh; Lorenz, Georg; Anders, Hans-Joachim; Lech, Maciej

2013-01-01

The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring. PMID:24009023

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

NASA Technical Reports Server (NTRS)

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

2001-01-01

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

Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma.

PubMed

Feng, Ling; Wang, Ru; Lian, Meng; Ma, Hongzhi; He, Ning; Liu, Honggang; Wang, Haizhou; Fang, Jugao

2016-01-01

Long non-coding RNA (lncRNA) plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC) are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

Effects of RNAi-mediated MUC4 gene silencing on the proliferation and migration of human pancreatic carcinoma BxPC-3 cells.

PubMed

Li, Yong; Wu, Changqiang; Chen, Tianwu; Zhang, Juanjuan; Liu, Gang; Pu, Yu; Zhu, Jiang; Shen, Chengyi; Zhang, Yang; Zeng, Nanlin; Zhang, Xiaoming

2016-12-01

It was previously demonstrated that mucin 4 (MUC4) is not expressed in normal pancreatic tissues or in chronic pancreatitis tissue but is highly expressed in pancreatic cancer (PC) tissue. Effective MUC4 gene knockdown in PC may contribute to the elucidation of pancreatic tumor development and metastasis, and may be valuable in new therapeutic approaches. Thus to confirm this, in the present study, the BxPC-3 cell line was transfected with eight pairs of shRNA lentiviral vectors for MUC4. The qPCR results showed that expression of MUC4 mRNA in the BxPC-3 cells was significantly decreased at 96 h after transfection. One of these shRNA lentiviral vectors (shRNA‑A141) had showed the strongest suppressive effect on MUC4 mRNA expression and was used for MUC4 knockdown in BxPC-3 cells. After stable transfection, BxPC-3 cells showed a significantly lower expression of MUC4 mRNA and MUC4 protein, and were suppressed on cell growth and migration. In vivo, lower tumor growth rates and tumor volume were observed in the tumors derived from the MUC4-knockdown cells, whereas the transplanted tumors derived from the control group cells, grew rapidly. Thus, inhibition of MUC4 expression may be an effective means for mitigating metastasis and invasion of PC.

Orosomucoid expression profiles in liver, adipose tissues and serum of lean and obese domestic pigs, Göttingen minipigs and Ossabaw minipigs.

PubMed

Rødgaard, Tina; Stagsted, Jan; Christoffersen, Berit Ø; Cirera, Susanna; Moesgaard, Sophia G; Sturek, Michael; Alloosh, Mouhamad; Heegaard, Peter M H

2013-02-15

The acute phase protein orosomucoid (ORM) has anti-inflammatory and immunomodulatory effects, and may play an important role in the maintenance of metabolic homeostasis in obesity-induced low-grade inflammation. Even though the pig is a widely used model for obesity related metabolic symptoms, the expression of ORM has not yet been characterized in such pig models. The objective of this study was to investigate the expression of ORM1 mRNA in liver, visceral adipose tissue, subcutaneous adipose tissue (SAT) from the abdomen or retroperitoneal abdominal adipose tissue (RPAT) and SAT from the neck, as well as the serum concentration of ORM protein in three porcine obesity models; the domestic pig, Göttingen minipigs and Ossabaw minipigs. No changes in ORM1 mRNA expression were observed in obese pigs compared to lean pigs in the four types of tissues. However, obese Ossabaw minipigs, but none of the other breeds, showed significantly elevated ORM serum concentrations compared to their lean counterparts. Studies in humans have shown that the expression of ORM was unchanged in adipose tissue depots in obese humans with an increased serum concentration of ORM. Thus in this respect, obese Ossabaw minipigs behave more similarly to obese humans than the other two pig breeds investigated. Copyright © 2012 Elsevier B.V. All rights reserved.

Tim-3 Up-regulation in Patients with Gastric Cancer and Peptic Ulcer Disease

PubMed Central

Naghavi-Alhosseini, Mahdieh; Tehrani, Mohsen; Ajami, Abolghasem; Rafiei, Alireza; Taghvaei, Tarang; Vahedi-Larijani, Laleh; Hossein-Nataj, Hadi; Asgarian-Omran, Hossein

2017-01-01

Background: T-cell immunoglobulin and mucin domain protein-3 (Tim-3), an inhibitory immunoregulatory receptor, has been recently implicated in tumor biology and tumor-associated immune suppression. In the present study, expression of Tim-3 was evaluated in gastric cancer (GC) and peptic ulcer disease (PUD) at both mRNA and protein levels. Methods: A total of 133 gastric tissue biopsies, comprising 43 from GC cases, 48 from PUD and 42 from non-ulcer dyspepsia (NUD) serving as controls were collected. Additionally, non-neoplastic adjacent tissue biopsies were also obtained from 6 patients with GC. Infection with Helicobacter pylori was determined by the rapid urease test for all participants and H&E staining was conducted for GC and PUD patients. Tim-3 relative mRNA expression was determined by SYBR Green based Real-Time PCR using β-actin as a reference gene. Tim-3 protein expression was also studied by immunohistochemistry in 7 GC, 7 PUD and 10 NUD tissue samples. Results: Tim-3 was expressed at higher levels in GC (p=0.030) and PUD (p=0.022) cases compared to he NUD group. Among paired samples obtained from gastric cancer patients, tumor tissues showed elevated Tim-3 expression (p=0.019) in comparison with adjacent non-neoplastic biopsies. Tim-3 mRNA findings were supported by detection of more Tim-3 protein in cancerous (p=0.002) and ulcerative (p=0.01) tissues than in controls. Tim-3 was similarly expressed in H. pylori positive and negative cases. Conclusion: Higher Tim-3 expression in patients with gastric cancer and peptic ulcer implies that it might be involved in immune regulation and establishment of these gastrointestinal diseases. Targeted immunotherapy by blocking of inhibitory receptors like Tim-3 could be a promising approach for gastric cancer treatment. PMID:28441784

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

PubMed Central

Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

1992-01-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1372158

Preparation of a nano emodin transfersome and study on its anti-obesity mechanism in adipose tissue of diet-induced obese rats

PubMed Central

2014-01-01

Objective To describe the preparation of nano emodin transfersome (NET) and investigate its effect on mRNA expression of adipose triglyceride lipase (ATGL) and G0/G1 switch gene 2 (G0S2) in adipose tissue of diet-induced obese rats. Methods NET was prepared by film-ultrasonic dispersion method. The effects of emodin components at different ratios on encapsulation efficiency were investigated.The NET envelopment rate was determined by ultraviolet spectrophotometry. The particle size and Zeta potential of NET were evaluated by Zetasizer analyzer. Sixty male SD rats were assigned to groups randomly. After 8-week treatment, body weight, wet weight of visceral fat and the percentage of body fat (PBF) were measured. Fasting blood glucose and serum lipid levels were determined. The adipose tissue section was HE stained, and the cellular diameter and quantity of adipocytes were evaluated by light microscopy. The mRNA expression of ATGL and G0S2 from the peri-renal fat tissue was assayed by RT-PCR. Results The appropriate formulation was deoxycholic acid sodium salt vs. phospholipids 1:8, cholesterol vs. phospholipids 1:3, vitamin Evs. phospholipids 1:20, and emodin vs. phospholipid 1:6. Zeta potential was −15.11 mV, and the particle size was 292.2 nm. The mean encapsulation efficiency was (69.35 ± 0.25)%. Compared with the obese model group, body weight, wet weight of visceral fat, PBF and mRNA expression of G0S2 from peri-renal fat tissue were decreased significantly after NET treatment (all P < 0.05), while high-density lipoprotein cholesterol (HDL-C), the diameter of adipocytes and mRNA expression of ATGL from peri-renal fat tissue were increased significantly (all P < 0.05). Conclusion The preparation method is simple and reasonable. NET with negative electricity was small and uniform in particle size, with high encapsulation efficiency and stability. NET could reduce body weight and adipocyte size, and this effect was associated with the up-regulation of ATGL, down-regulation of G0S2 expression in the adipose tissue, and improved insulin sensitivity. PMID:24641917

The Impact of Small RNA Interference Against Homer1 on Rats with Type 2 Diabetes and ERK Phosphorylation.

PubMed

Lu, Jun; Gan, Jihong; Fu, Guoqiang; Ding, Lu; Zheng, Qiangsun

2015-12-01

The objective of the study is to evaluate Homer1 expression in rats with Type 2 diabetes mellitus (T2DM) and investigate the mechanism by which Homer1 influences the pathogenesis of diabetes through study on rat model with decreased Homer1 expression. Rat model of T2DM was constructed and blood insulin concentration was measured. Homer1 mRNA and protein expressions in rat pancreatic tissue were determined using RT-PCR as well as Western blotting. Homer1 expression in human monocytic THP-1 cells was interfered using short hairpin RNA, and its effect on phosphorylation of extracellular signal-regulated kinase (ERK) was assessed. Fasting glucose concentration in rat model of T2DM was significantly higher than that of normal rats (13.1 ± 2.4 vs 5.1 ± 1.1 mmol/L), and fasting blood insulin concentration of diabetic group was significantly lower than that of normal group (13.6 ± 1.9 18.3 ± 2.2 mIU/L) (P < 0.05). Homer1 mRNA and protein expressions in pancreatic tissue of rats with T2DM were significantly higher than those of normal rats (P < 0.05). Level of ERK phosphorylation in pancreatic tissue of rats with T2DM was significantly higher than that of normal rats. Homer1 mRNA level in rat pancreatic tissue of T2DM was positively correlated with the area of pancreatic islets (r = 0.526, P = 0.014). Homer1 mRNA level was significantly inhibited in high-glucose and high-fat stimulated human monotypic THP-1 cells with interfered Homer1. Compared with controls, P-ERK phosphorylation was significantly decreased in THP-1 cells with interfered Homer1 (P < 0.05). Homer1 can promote the progression of T2DM, which may be achieved through affecting ERK phosphorylation.

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

[Roles of Y box-binding protein 1 in SK-BR-3 breast cancer proliferation].

PubMed

Shi, Jianhong; Lü, Xinrui; Wang, Bing; Daudan, Lin; Yanan, Wang; Yuhui, Bu; Zhenfeng, Ma

2014-09-30

To explore the roles of Y box-binding protein 1 (YB-1) in breast cancer cell proliferation. Twenty cases of surgical removal of breast cancer tissue (diagnosed with invasive ductal carcinoma, stage II, by postoperative paraffin pathology) and normal breast tissues adjacent to carcinoma were collected during June 2013 to August 2013.Quantitative real-time PCR (qRT-PCR) was performed to detect the YB1 mRNA levels. Cultured mammary epithelial cells (HBL-100) and breast cancer cells (MCF7, MDA-MB-231 & SK-BR-3 cells) were harvested and qRT-PCR was performed to detect the YB1 mRNA levels.SK-BR-3 cells were stimulated with various concentrations of PDGF-BB and YB1 expression levels were detected by qRT-PCR. Down-regulation or over-expression of YB1 by si-YB1 or Ad-GFP-YB1 was detected in SK-BR-3 cells. And MTS cell proliferation assay kit was used to detect cell proliferation. YB1 mRNA levels were significantly higher in breast cancer tissues and MDA-MB-231 and SK-BR-3 breast cancer cell lines than that in adjacent normal breast tissues and HBL-100 mammary epithelial cells respectively (P < 0.05).YB1 expression levels increased in PDGF-BB stimulated SK-BR-3 cells in a dose-dependent manner. A down-regulation of endogenous YB1 decreases and an over-expression of exogenous YB1 promotes the proliferation activity in SK-BR-3 cells.

Rebamipide inhibits indomethacin-induced small intestinal injury: possible involvement of intestinal microbiota modulation by upregulation of α-defensin 5.

PubMed

Tanigawa, Tetsuya; Watanabe, Toshio; Otani, Koji; Nadatani, Yuji; Ohkawa, Fumikazu; Sogawa, Mitsue; Yamagami, Hirokazu; Shiba, Masatsugu; Watanabe, Kenji; Tominaga, Kazunari; Fujiwara, Yasuhiro; Takeuchi, Koji; Arakawa, Tetsuo

2013-03-15

Enterobacteria play important roles in the pathophysiology of small intestinal injuries induced by nonsteroidal anti-inflammatory drugs (NSAIDs). We investigated the effects of rebamipide, a gastrointestinal mucoprotective drug, on indomethacin-induced small intestinal injuries, intestinal microbiota, and expression levels of α-defensin 5, which is a Paneth cell-specific antimicrobial peptide and is important for the regulation of intestinal microbiota. Indomethacin (10mg/kg) was orally administered to mice after oral administration of rebamipide (100 or 300 mg/kg) or vehicle for 1 week, and the small intestinal injuries were assessed. After oral administration of rebamipide, the small intestinal contents were subjected to terminal restriction fragment length polymorphism (T-RFLP) analysis to assess the intestinal microbiota composition. Further, the expression levels of mRNA and protein for α-defensin 5 in the ileal tissue were determined by real-time reverse transcription-polymerase chain reaction and western blotting analysis, respectively. Rebamipide inhibited indomethacin-induced small intestinal injuries and T-RFLP analysis showed that rebamipide increased the percentage of Lactobacillales and decreased the percentage of Bacteroides and Clostridium than that in vehicle-treated controls. The mice that were treated with rebamipide showed an increase in α-defensin 5 mRNA expression and protein levels in the ileal tissue compared to vehicle-treated control mice. Indomethacin reduced expression of α-defensin 5 mRNA in ileal tissue, while rebamipide reversed expression of α-defensin 5 mRNA. In conclusion, our study results suggest that rebamipide inhibits indomethacin-induced small intestinal injuries, possibly by modulating microbiota in the small intestine by upregulation of α-defensin 5. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed

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

2012-03-01

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

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

PubMed

Finke, J; Fritzen, R; Ternes, P; Lange, W; Dölken, G

1993-03-01

Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.

New candidate tumor-suppressor gene KLF6 and its splice variant KLF6 SV2 counterbalancing expression in primary hepatocarcinoma.

PubMed

Zhenzhen, Zhou; De'an, Tian; Limin, Xia; Wei, Yan; Min, Luo

2012-01-01

This study aimed to detect the expression of newly discovered zinc finger transcriptional factor KLF6 and its splice variant KLF6 SV2 in primary hepatocarcinoma (PHC) tissues and hepatoma cell strains, and to evaluate their clinicopathologic relationship with PHC. Wild-type KLF6 and KLF6 SV2 mRNA expression was determined by RTPCR in 27 cases of PHC tissues and cell strains of HepG2, SMMC7721 and LO2. Western blotting and immunohistochemical staining were adopted to detect KLF6 protein expression. Positive area ratio of wild-type KLF6 protein expression and its relationship with clinicopathological parameters of PHC was analyzed. Wild-type KLF6 expression in PHC tissues was lower than that in paracancerous tissues. In contrast, KLF6 SV2 mRNA expression was higher in PHC tissues and hepatoma cell strains (p<0.05). Positive area ratio of wild-type KLF6 protein expression was positively correlated with cellular differentiation degree of PHC (p<0.01), but negatively correlated not only with liver cirrhosis, tumor size and extrahepatic metastases (p<0.01), but also with portal vein thrombus and the number of lymph nodes with metastasis (p<0.05). Wild-type KLF6 deletion and inactivation was involved in the growth, cell differentiation and other physiological processes of PHC. The upregulation of KLF6 splice variant might counterbalance the wildtype KLF6 and contribute to the occurrence and development of PHC.

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

PubMed

Williams, Kellie J; Picou, Alicia A; Kish, Sharon L; Giraldo, Angelica M; Godke, Robert A; Bondioli, Kenneth R

2008-01-01

Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright 2008 S. Karger AG, Basel.

Lamina propria macrophage phenotypes in relation to Escherichia coli in Crohn's disease.

PubMed

Elliott, Timothy R; Rayment, Neil B; Hudspith, Barry N; Hands, Rebecca E; Taylor, Kirstin; Parkes, Gareth C; Prescott, Natalie J; Petrovska, Liljana; Hermon-Taylor, John; Brostoff, Jonathan; Boussioutas, Alex; Mathew, Christopher G; Bustin, Stephen A; Sanderson, Jeremy D

2015-07-03

Abnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn's disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage. Mucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR. E. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls. In CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

Upregulation of innate antiviral restricting factor expression in the cord blood and decidual tissue of HIV-infected mothers.

PubMed

Pereira, Nátalli Zanete; Cardoso, Elaine Cristina; Oliveira, Luanda Mara da Silva; de Lima, Josenilson Feitosa; Branco, Anna Cláudia Calvielli Castelo; Ruocco, Rosa Maria de Souza Aveiro; Zugaib, Marcelo; de Oliveira Filho, João Bosco; Duarte, Alberto José da Silva; Sato, Maria Notomi

2013-01-01

Programs for the prevention of mother-to-child transmission of HIV have reduced the transmission rate of perinatal HIV infection and have thereby increased the number of HIV-exposed uninfected (HEU) infants. Natural immunity to HIV-1 infection in both mothers and newborns needs to be further explored. In this study, we compared the expression of antiviral restricting factors in HIV-infected pregnant mothers treated with antiretroviral therapy (ART) in pregnancy (n=23) and in cord blood (CB) (n=16), placental tissues (n=10-13) and colostrum (n=5-6) samples and compared them to expression in samples from uninfected (UN) pregnant mothers (n=21). Mononuclear cells (MNCs) were prepared from maternal and CB samples following deliveries by cesarean section. Maternal (decidua) and fetal (chorionic villus) placental tissues were obtained, and colostrum was collected 24 h after delivery. The mRNA and protein expression levels of antiviral factors were then evaluated. We observed a significant increase in the mRNA expression levels of antiviral factors in MNCs from HIV-infected mothers and CB, including the apolipoprotein B mRNA-editing enzyme 3G (A3G), A3F, tripartite motif family-5α (TRIM-5α), TRIM-22, myxovirus resistance protein A (MxA), stimulator of interferon (IFN) genes (STING) and IFN-β, compared with the levels detected in uninfected (UN) mother-CB pairs. Moreover, A3G transcript and protein levels and α-defensin transcript levels were decreased in the decidua of HIV-infected mothers. Decreased TRIM-5α protein levels in the villi and increased STING mRNA expression in both placental tissues were also observed in HIV-infected mothers compared with uninfected (UN) mothers. Additionally, colostrum cells from infected mothers showed increased tetherin and IFN-β mRNA levels and CXCL9 protein levels. The data presented here indicate that antiviral restricting factor expression can be induced in utero in HIV-infected mothers. Future studies are warranted to determine whether this upregulation of antiviral factors during the perinatal period has a protective effect against HIV-1 infection.

Upregulation of Innate Antiviral Restricting Factor Expression in the Cord Blood and Decidual Tissue of HIV-Infected Mothers

PubMed Central

Pereira, Nátalli Zanete; Cardoso, Elaine Cristina; Oliveira, Luanda Mara da Silva; de Lima, Josenilson Feitosa; Branco, Anna Cláudia Calvielli Castelo; Ruocco, Rosa Maria de Souza Aveiro; Zugaib, Marcelo; de Oliveira Filho, João Bosco; Duarte, Alberto José da Silva; Sato, Maria Notomi

2013-01-01

Programs for the prevention of mother-to-child transmission of HIV have reduced the transmission rate of perinatal HIV infection and have thereby increased the number of HIV-exposed uninfected (HEU) infants. Natural immunity to HIV-1 infection in both mothers and newborns needs to be further explored. In this study, we compared the expression of antiviral restricting factors in HIV-infected pregnant mothers treated with antiretroviral therapy (ART) in pregnancy (n=23) and in cord blood (CB) (n=16), placental tissues (n=10-13) and colostrum (n=5-6) samples and compared them to expression in samples from uninfected (UN) pregnant mothers (n=21). Mononuclear cells (MNCs) were prepared from maternal and CB samples following deliveries by cesarean section. Maternal (decidua) and fetal (chorionic villus) placental tissues were obtained, and colostrum was collected 24 h after delivery. The mRNA and protein expression levels of antiviral factors were then evaluated. We observed a significant increase in the mRNA expression levels of antiviral factors in MNCs from HIV-infected mothers and CB, including the apolipoprotein B mRNA-editing enzyme 3G (A3G), A3F, tripartite motif family-5α (TRIM-5α), TRIM-22, myxovirus resistance protein A (MxA), stimulator of interferon (IFN) genes (STING) and IFN-β, compared with the levels detected in uninfected (UN) mother-CB pairs. Moreover, A3G transcript and protein levels and α-defensin transcript levels were decreased in the decidua of HIV-infected mothers. Decreased TRIM-5α protein levels in the villi and increased STING mRNA expression in both placental tissues were also observed in HIV-infected mothers compared with uninfected (UN) mothers. Additionally, colostrum cells from infected mothers showed increased tetherin and IFN-β mRNA levels and CXCL9 protein levels. The data presented here indicate that antiviral restricting factor expression can be induced in utero in HIV-infected mothers. Future studies are warranted to determine whether this upregulation of antiviral factors during the perinatal period has a protective effect against HIV-1 infection. PMID:24367701

MUC19 expression in human ocular surface and lacrimal gland and its alteration in Sjögren syndrome patients.

PubMed

Yu, D F; Chen, Y; Han, J M; Zhang, H; Chen, X P; Zou, W J; Liang, L Y; Xu, C C; Liu, Z G

2008-02-01

This study investigated the expression of MUC19, a newly discovered gel-forming mucin gene, in normal human lacrimal functional unit components and its alteration in Sjögren syndrome patients. Real-time PCR and immunohistochemistry were performed to determine the expression of MUC19 and MUC5AC in human cornea, conjunctiva, and lacrimal gland tissues. Conjunctival impression cytology specimens were collected from normal control subjects and Sjögren syndrome patients for Real-time PCR, PAS staining, and immunohistochemistry assays. In addition, conjunctiva biopsy specimens from both groups were examined for the expression differences of MUC19 and MUC5AC at both mRNA and protein level. The MUC19 mRNA was found to be present in cornea, conjunctiva and lacrimal gland tissues. The immunohistochemical staining of mucins showed that MUC19 was expressed in epithelial cells from corneal, conjunctival, and lacrimal gland tissues. In contrast, MUC5AC mRNA was only present in conjunctiva and lacrimal gland tissues, but not in cornea. Immunostaining demonstrates the co-staining of MUC19 and MUC5AC in conjunctival goblet cells. Consistent with the significant decrease of mucous secretion, both MUC19 and MUC5AC were decreased in conjunctiva of Sjögren syndrome patients compared to normal subjects. Considering the contribution of gel-forming mucins to the homeostasis of the ocular surface, the decreased expression of MUC19 and MUC5AC in Sjögren syndrome patients suggested that these mucins may be involved in the disruption of the ocular surface homeostasis in this disease.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation.

PubMed

Mishra, B; Kizaki, K; Koshi, K; Ushizawa, K; Takahashi, T; Hosoe, M; Sato, T; Ito, A; Hashizume, K

2012-02-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta. Copyright © 2012 Elsevier Inc. All rights reserved.

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

PubMed Central

2014-01-01

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

[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.

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

Comparison of Dorsocervical With Abdominal Subcutaneous Adipose Tissue in Patients With and Without Antiretroviral Therapy–Associated Lipodystrophy

PubMed Central

Sevastianova, Ksenia; Sutinen, Jussi; Greco, Dario; Sievers, Meline; Salmenkivi, Kaisa; Perttilä, Julia; Olkkonen, Vesa M.; Wågsäter, Dick; Lidell, Martin E.; Enerbäck, Sven; Eriksson, Per; Walker, Ulrich A.; Auvinen, Petri; Ristola, Matti; Yki-Järvinen, Hannele

2011-01-01

OBJECTIVE Combination antiretroviral therapy (cART) is associated with lipodystrophy, i.e., loss of subcutaneous adipose tissue in the abdomen, limbs, and face and its accumulation intra-abdominally. No fat is lost dorsocervically and it can even accumulate in this region (buffalo hump). It is unknown how preserved dorsocervical fat differs from abdominal subcutaneous fat in HIV-1–infected cART-treated patients with (cART+LD+) and without (cART+LD−) lipodystrophy. RESEARCH DESIGN AND METHODS We used histology, microarray, PCR, and magnetic resonance imaging to compare dorsocervical and abdominal subcutaneous adipose tissue in cART+LD+ (n = 21) and cART+LD− (n = 11). RESULTS Albeit dorsocervical adipose tissue in cART+LD+ seems spared from lipoatrophy, its mitochondrial DNA (mtDNA; copies/cell) content was significantly lower (by 62%) than that of the corresponding tissue in cART+LD−. Expression of CD68 mRNA, a marker of macrophages, and numerous inflammatory genes in microarray were significantly lower in dorsocervical versus abdominal subcutaneous adipose tissue. Genes with the greatest difference in expression between the two depots were those involved in regulation of transcription and regionalization (homeobox genes), irrespective of lipodystrophy status. There was negligible mRNA expression of uncoupling protein 1, a gene characteristic of brown adipose tissue, in either depot. CONCLUSIONS Because mtDNA is depleted even in the nonatrophic dorsocervical adipose tissue, it is unlikely that the cause of lipoatrophy is loss of mtDNA. Dorsocervical adipose tissue is less inflamed than lipoatrophic adipose tissue. It does not resemble brown adipose tissue. The greatest difference in gene expression between dorsocervical and abdominal subcutaneous adipose tissue is in expression of homeobox genes. PMID:21602514

[Tissue collagenase MMP-14 and endogenous regulators of its activity in the corpus uteri in squamous cell carcinoma of the cervix].

PubMed

Timoshenko, O S; Gureeva, T A; Kugaevskaya, E V; Zavalishina, L E; Andreeva, Yu Yu; Solovyeva, N I

to investigate the expression of the membrane-bound matrix metalloproteinase MT1-MMP (MMP-14), its tissue inhibitor TIMP-2, and the proMMP-14 activator furin in the corpus uteri from the vaginal wall to the bottom of the uterine cavity in squamous cell carcinoma of the cervix (SCCC). Hysterectomy material was examined in patients with SCCC. Reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and enzyme assays were used. In SCCC, higher levels of MMP-14 expression were established in tumor cells, as evidenced by IHC (+3) and RT-PCR. IHC showed that the expression of MMP-14 was absent or insignificant in the normal uterine endometrial and myometrial tissues. However, that of MMP-14 mRNA was also found in the normal tissues to the bottom of the uterine cavity. Furin activity in the tumor was much higher than that in normal tissues. IHC indicated that TIMP-2 expression was low or absent in both the tumor and normal tissues. The expression of TIMP-2 mRNA was sufficiently obvious in both the tumor and normal tissues to the bottom of the uterine cavity. In SCCC, MMP-14 expression was substantially increased in tumors. The expression of MMP-14 and regulators of its activity is aimed at enhancing the tumor destructive (invasive) potential in the pericellular space and can occur (be induced) in the morphologically normal uterine tissue apparently with involvement of signaling through the epithelial-mesenchymal interaction. Data are important for understanding the role of MMP-14 in the development of a multistage process of carcinogenesis and may have prognostic value and an impact on therapeutic strategy for the patient.

Decidual activin: its role in the apoptotic process and its regulation by prolactin.

PubMed

Tessier, Christian; Prigent-Tessier, Anne; Bao, Lei; Telleria, Carlos M; Ferguson-Gottschall, Susan; Gibori, Gil B; Gu, Yan; Bowen-Shauver, Jennifer M; Horseman, Nelson D; Gibori, Geula

2003-05-01

Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.

DUSP5 functions as a feedback regulator of TNFα-induced ERK1/2 dephosphorylation and inflammatory gene expression in adipocytes.

PubMed

Habibian, Justine S; Jefic, Mitra; Bagchi, Rushita A; Lane, Robert H; McKnight, Robert A; McKinsey, Timothy A; Morrison, Ron F; Ferguson, Bradley S

2017-10-10

Adipose tissue inflammation is a central pathological element that regulates obesity-mediated insulin resistance and type II diabetes. Evidence demonstrates that extracellular signal-regulated kinase (ERK 1/2) activation (i.e. phosphorylation) links tumor necrosis factor α (TNFα) to pro-inflammatory gene expression in the nucleus. Dual specificity phosphatases (DUSPs) inactivate ERK 1/2 through dephosphorylation and can thus inhibit inflammatory gene expression. We report that DUSP5, an ERK1/2 phosphatase, was induced in epididymal white adipose tissue (WAT) in response to diet-induced obesity. Moreover, DUSP5 mRNA expression increased during obesity development concomitant to increases in TNFα expression. Consistent with in vivo findings, DUSP5 mRNA expression increased in adipocytes in response to TNFα, parallel with ERK1/2 dephosphorylation. Genetic loss of DUSP5 exacerbated TNFα-mediated ERK 1/2 signaling in 3T3-L1 adipocytes and in adipose tissue of mice. Furthermore, inhibition of ERK 1/2 and c-Jun N terminal kinase (JNK) signaling attenuated TNFα-induced DUSP5 expression. These data suggest that DUSP5 functions in the feedback inhibition of ERK1/2 signaling in response to TNFα, which resulted in increased inflammatory gene expression. Thus, DUSP5 potentially acts as an endogenous regulator of adipose tissue inflammation; although its role in obesity-mediated inflammation and insulin signaling remains unclear.

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

PubMed

Hu, Kunpeng; Wang, Jiani; Yao, Zhicheng; Liu, Bo; Lin, Yuan; Liu, Lei; Xu, Lihua

2014-05-01

The molecular mechanisms of the development and progression of hepatocellular carcinoma (HCC) are poorly understood. The main objective of this study was to analyze the expression of Enabled [mammalian Ena (Mena)] protein and its clinical significance in human HCC. The Mena expression was examined at mRNA and protein levels by real-time quantitative polymerase chain reaction and Western blotting analysis in ten paired HCC tissues and the adjacent normal tissues. The expression of Mena protein in 81 specimens of HCC tissues was determined by immunohistochemistry. Associations of Mena expression with the clinicopathological features were analyzed, and prognosis of HCC patients was evaluated. The result shows the expression of Mena mRNA and protein was higher in HCC than in the adjacent normal tissues in ten paired samples. Mena was mainly accumulated in the cytoplasm of tumor cells and over-expressed in 40.74% (33/81) patients by immunohistochemical staining. Over-expression of Mena was significantly associated with poor cellular differentiation (P = 0.025), advanced tumor stage (P = 0.003) and worse disease-free survival (DFS, P < 0.001). In addition, Mena is an independent prognostic factor for DFS in multivariate analysis (HR 2.309, 95% CI 1.104-4.828; P = 0.026). Mena is up-regulated in HCC and associated with tumor differentiation and clinical stage. Mena may be an independent prognostic marker for DFS of HCC patients.

Expression of cyclooxygenase-1 and cyclooxygenase-2, syndecan-1 and connective tissue growth factor in benign and malignant breast tissue from premenopausal women.

PubMed

Fahlén, M; Zhang, H; Löfgren, L; Masironi, B; von Schoultz, E; von Schoultz, B; Sahlin, L

2017-05-01

Stromal factors have been identified as important for tumorigenesis and metastases of breast cancer. From 49 premenopausal women, samples were collected from benign or malignant tumors and the seemingly normal tissue adjacent to the tumor. The factors studied, with real-time polymerase chain reaction (PCR) and immunohistochemistry, were cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), syndecan-1 (S-1) and connective tissue growth factor (CTGF). COX-1 and S-1 mRNA levels were higher in the malignant tumors than in normal and benign tissues. The COX-2 mRNA level was lower in the malignant tumor than in the normal tissue, while CTGF mRNA did not differ between the groups. COX-1 immunostaining was higher in stroma from malignant tumors than in benign tissues, whereas COX-2 immunostaining was higher in the malignant tissue. Glandular S-1 immunostaining was lower in malignant tumors compared to benign and normal tissues, and the opposite was found in stroma. Conclusively, mRNA levels of COX-1 and COX-2 were oppositely regulated, with COX-1 being increased in the malignant tumor while COX-2 was decreased. S-1 protein localization switched from glandular to stromal cells in malignant tissues. Thus, these markers are, in premenopausal women, localized and regulated differently in normal/benign breast tissue as compared to the malignant tumor.

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth.

PubMed

Yom Din, S; Hurvitz, A; Goldberg, D; Jackson, K; Levavi-Sivan, B; Degani, G

2008-03-01

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

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.

Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

PubMed Central

Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

2015-01-01

The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. PMID:25834424

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

PubMed

Förander, P; Brené, S; Strömberg, I

2000-02-28

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

Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.

PubMed

Josephson, Anna; Trifunovski, Alexandra; Widmer, Hans Ruedi; Widenfalk, Johan; Olson, Lars; Spenger, Christian

2002-11-18

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. Copyright 2002 Wiley-Liss, Inc.

Molecular signaling in intervertebral disk development.

PubMed

DiPaola, Christian P; Farmer, James C; Manova, Katia; Niswander, Lee A

2005-09-01

The purpose of this investigation is to identify and study the expression pattern of pertinent molecular factors involved in the differentiation of the intervertebral disk (IVD). It is likely that hedgehog genes and the BMP inhibitors are key factors involved in spinal joint formation. Radioactive in situ hybridization with mRNA probes for pax-1, SHH, IHH and Noggin gene was performed on mouse embryo and adult tissue. Immunohistochemistry was performed to localize hedgehog receptor, "patched" (ptc). From 14.5 dpc until birth pax-1 mRNA was expressed in the developing anulus fibrosus (AF). During the same developmental period Noggin mRNA is highly expressed throughout the spine, in the developing AF, while ptc protein and SHH mRNA were expressed in the developing nucleus pulposus (NP). IHH mRNA was expressed by condensing chondrocytes of the vertebral bodies and later becomes confined to the vertebral endplate. We show for the first time that pax-1 is expressed in the adult intervertebral disk. Ptc expression in the NP is an indicator of hedgehog protein signaling in the developing IVD. The expression pattern of the BMP inhibitor Noggin appears to be important for the normal formation of the IVD and may prove to play a role in its segmental pattern formation.

Relative mRNA expression of prostate-derived E-twenty-six factor and E-twenty-six variant 4 transcription factors, and of uridine phosphorylase-1 and thymidine phosphorylase enzymes, in benign and malignant prostatic tissue

PubMed Central

CAVAZZOLA, LUCIANE ROSTIROLA; CARVALHAL, GUSTAVO FRANCO; DEVES, CANDIDA; RENCK, DAIANA; ALMEIDA, RICARDO; SANTOS, DIóGENES SANTIAGO

2015-01-01

Prostate cancer is the most frequent urological tumor, and the second most common cancer diagnosed in men. Incidence and mortality are variable and appear to depend on behavioral factors and genetic predisposition. The prostate-derived E-twenty-six factor (PDEF) and E-twenty-six variant 4 (ETV4) transcription factors, and the thymidine phosphorylase (TP) and uridine phosphorylase-1 (UP-1) enzymes, are reported to be components of the pathways leading to tumorigenesis and/or metastasis in a number of tumors. The present study aimed to analyze the mRNA expression levels of these proteins in prostatic cancerous and benign tissue, and their association with clinical and pathological variables. Using quantitative reverse transcription polymerase chain reaction, the mRNA expression levels of PDEF, ETV4, TP and UP-1 were studied in 52 tissue samples (31 of benign prostatic hyperplasia and 21 of prostate adenocarcinomas) obtained from patients treated by transurethral resection of the prostate or by radical prostatectomy. Relative expression was assessed using the ∆-CT method. Data was analyzed using Spearman's tests for correlation. P<0.05 was considered to indicate a statistically significant difference. The results revealed that PDEF, ETV4, UP-1 and TP were expressed in 85.7, 90.5, 95.2 and 100% of the prostate cancer samples, and in 90.3, 96.8, 90.3 and 96.8% of the benign samples, respectively. PDEF and ETV4 exhibited a significantly higher relative expression level in the tumor samples compared with their benign counterparts. The relative expression of TP and UP-1 did not differ significantly between benign and cancerous prostate tissues. The relative expression of TP was moderately and significantly correlated with the expression of ETV4 in the benign tissues. The relative expression of UP-1 was significantly lower in T3 compared with T1 and T2 cancers. These findings indicate that PDEF, ETV4, TP and UP-1 are typically expressed in benign and malignant prostatic tissues. Further studies are necessary to define the role of these proteins as therapeutic targets in prostate cancer. PMID:26137165

LRIG1, a 3p tumor suppressor, represses EGFR signaling and is a novel epigenetic silenced gene in colorectal cancer

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

Kou, Changhua, E-mail: chkoukou@hotmail.com; Zhou, Tian; Han, Xilin

2015-08-21

Downregulation of LRIG1 was found in many types of cancer. However, data concerning the possible mechanism of LRIG1 reduction in cancers were not reported yet. To analyze the regulation and function of LRIG1 in colorectal cancer (CRC), 6 cell lines, 46 paired tissues from primary CRC cases were employed in this study. In CRC cell lines, under-expression of LRIG1 was correlated with promoter region hypermethylation, and restoration of LRIG1 was induced by 5-Aza-2'-deoxyazacytidine treatment. Subsequently, we ectopically expressed LRIG1 in LRIG1 low-expressing HCT-116 cells and suppressed LRIG1 in LRIG1 high-expressing LoVo cells. We found that over-expression of LRIG1 inhibits cellmore » proliferation and colony formation and tumor growth, while knockdown of LRIG1 promotes cell proliferation and colony formation. Decreased and increased EGFR/AKT signaling pathway may partially explain the lower and higher rates of proliferation in CRC cells transfected with LRIG1 cDNA or shRNA. In clinical samples, we compared the methylation, mRNA and protein expression of LRIG1 in samples of CRC tissues. A significant increase in LRIG1 methylation was identified in CRC specimens compared to adjacent normal tissues and that it was negatively correlated with its mRNA and protein expression. In conclusion, LRIG1 is frequently methylated in human CRC and consequent mRNA and protein downregulation may contribute to tumor growth by activating EGFR/AKT signaling. - Highlights: • Promoter methylation of LRIG1 occurred in colorectal cancer cells and tumors. • Restoration of LRIG1 inhibits tumor growth in vitro and in vivo. • Overexpression or knockdown of LRIG1 regulates EGFR/AKT and downstream apoptosis. • Methylation of LRIG1 correlates with its mRNA and protein downregulation. • LRIG1 was firstly identified as an epigenetic target in cancer.« less

Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis.

PubMed

Dai, W; Pan, H; Hassanain, H; Gupta, S L; Murphy, M J

1994-03-01

Using a combination of polymerase chain reaction and conventional cDNA library screening approaches, we have cloned and characterized a putative receptor tyrosine kinase termed tif. The extracellular domain of tif has an immunoglobulin-like loop and a fibronectin type III structure. The intracellular domain contains a tyrosine kinase domain. Compared with ryk, a ubiquitously expressed receptor tyrosine kinase, tif expression is tissue-specific with human ovary and testis containing the highest amount of tif mRNA. Many other tested human tissues such as heart, liver, pancreas and thymus do not contain detectable levels of tif mRNA. The molecular cloning and characterization of tif cDNA will facilitate the identification of a potential ligand(s) for the putative receptor and the study of its biological role.

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

PubMed Central

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

2016-01-01

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

Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

PubMed Central

VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

2014-01-01

The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

[Effect of acute intra-peritoneal infection on leptin expression levels in peripheral blood and vital organs of rats].

PubMed

Lin, Ji; Yan, Guang-Tao; Wang, Lu-Huan

2008-02-01

To explore the effect of acute intra-peritoneal infection on leptin expression levels in peripheral blood and vital organs, and find out the role leptin plays in acute inflammation. A cecal ligation and perforation model of rats was established, setting groups of sham-operation, intralipid injection, injury, estradiol injection and insulin injection. A rat leptin radioimmunoassay was used to check serum leptin concentrations at 12 h after the injury, and RT-PCR was also used to detect leptin mRNA expressions in adipose tissue, lung and liver. Compared with serum leptin level of sham-operation group after injury, that of all the other four groups showed no significant difference, while the level of intralipid group was significantly higher than that of injury group and estradiol group. Compared with leptin mRNA expression level of sham-operation group after injury, that of the other four groups had different changes. Leptin mRNA expression of intralipid group was significantly increased in adipose tissue but decreased in lung and liver. Leptin expression levels may be affected by the changes of energy metabolism and neuroendocrine function after injury, which suggests a possible protective role for leptin in the recovery of body homeostasis.

Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice

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

Jambaldorj, Jamiyansuren; Advanced Molecular Epidemiology Research Institute, Yamagata University Faculty of Medicine, Yamagata 990-9585; Central Scientific Research Laboratory, Institute of Medical Sciences, Ulaanbaatar

2012-08-24

Highlights: Black-Right-Pointing-Pointer We identified the mouse homologue of neuron-specific TAF1 (N-Taf1). Black-Right-Pointing-Pointer Taf1 mRNA was expressed in most tissues and cell lines. Black-Right-Pointing-Pointer N-Taf1 mRNA was expressed in the brain and Neuroblastoma N2a cell lines. Black-Right-Pointing-Pointer Taf1 and N-Taf1 showed different expression profile in development stage and aging. -- Abstract: TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II-mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1),more » which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.« less

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

PubMed Central

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

2011-01-01

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

Insight into mechanism of oxidative DNA damage in angiomyolipomas from TSC patients

PubMed Central

Habib, Samy L

2009-01-01

Background The tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors, both angiomyolipomas and renal cell carcinomas. Loss of heterozygosity at the 8-oxoG-DNA glycosylase (OGG1) allele is found in human kidney clear cell carcinoma identifying loss of OGG1 function as a possible contributor to tumorigenesis in the kidney. Tuberin regulates OGG1 through the transcription factor NF-YA in cultured cells. The purpose of this study is to determine the effect of tuberin-deficiency on OGG1 protein and mRNA levels as well as on 8-oxodG levels in kidney tumors from patients with TSC. In addition we evaluated the phophorylation level of downstream targets of mTOR, phospho-S70K, in kidney tumor tissue from TSC patients. Results Kidney angiomyolipoma tissue from TSC patients expresses significant levels of phopho-tuberin and low levels of tuberin compared to control kidney tissue. The increase in tuberin phosphorylation and the decrease tuberin expression are associated with decrease in OGG1 protein and mRNA levels in tumor samples compared to normal kidney samples. The decrease OGG1 expression is also associated with significant decrease in the transcription factor, NF-YA, expression in tumor samples compared to normal tissues. In addition, the levels of 8-oxodG are 4-fold higher in tumors compared to control samples. The significant increase of phospho-tuberin expression is associated with increase phosphorylation of S6K in tumor samples compared to controls. Cyclin D1 expression is also 3-fold higher in increase in the tumor tissues compared to normal kidney tissues. Conclusion These data indicate that tuberin deficiency in angiomyolipoma enhances mTOR activation by phosphorylation of S6K and downregulation of protein and mRNA expression of OGG1 resulted in accumulation of oxidized DNA in patients with TSC. These data suggest that tuberin and OGG1 are important proteins in the pathogenesis of angiomyolipoma in TSC patients. PMID:19265534

MicroRNA-9 promotes the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.

PubMed

Liu, D-Z; Chang, B; Li, X-D; Zhang, Q-H; Zou, Y-H

2017-09-01

The objective of the study was to investigate the role of microRNA-9 (miR-9) targeting forkhead box O1 (FOXO1) in the proliferation, migration, and invasion of breast cancer cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expressions of miR-9 and FOXO1 mRNA in breast cancer tissues, normal breast tissues, breast cancer cell lines, and normal breast epithelial cells. After the up-regulation of miR-9 expression, qRT-PCR and Western blotting were used to determine the expression of FOXO1. The luciferase reporter gene assay was used to validate the target gene. The CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay were used to investigate the changes in the proliferation, migration, and invasion of breast cancer cells, respectively. MicroRNA-9 expression was significantly up-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). FOXO1 mRNA and protein expressions were substantially down-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). There can be a negative correlation between miR-9 and FOXO1 mRNA in breast cancer. Luciferase reporter gene assay indicated that miR-9 can down-regulate FOXO1 expression at a post-transcriptional level through binding specifically to FOXO1 3'UTR. The results of CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay revealed that the inhibition of miR-9 can suppress MCF7 cell proliferation, migration, and invasion. Additionally, the expression of miR-9 increased significantly whilst that of FOXO1 decreased substantially as the disease progressed (P < 0.05). Our study provides evidence that miR-9 can promote the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.

Aberrant Expression of PIWIL1 and PIWIL2 and Their Clinical Significance in Ductal Breast Carcinoma.

PubMed

Litwin, Monika; Szczepańska-Buda, Anna; Michałowska, Dagmara; Grzegrzółka, Jędrzej; Piotrowska, Aleksandra; Gomułkiewicz, Agnieszka; Wojnar, Andrzej; Dzięgiel, Piotr; Witkiewicz, Wojciech

2018-04-01

P-Element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) are involved in epigenetic regulation of gene expression in germline cells. Aberrant expression of piRNA and PIWI proteins have been identified in various types of tumour cells. The aim of this study was to evaluate the expression profiles of PIWI-like protein-1, -2 (PIWIL1 and PIWIL2), their immunohistochemical (IHC) characteristics in ductal breast cancer, and determine their correlation with clinicopathological parameters of this type of cancer. Material for IHC studies comprised of 101 invasive ductal carcinoma (IDC) cases and 31 mastopathy tissues. Frozen fragments of paired tissue specimens (tumour and adjacent non-malignant breast tissue) taken from 55 patients with IDC and 18 samples of mastopathy were used for molecular studies using real-time polymerase chain reaction (RT-PCR). A statistically significantly higher level of PIWIL1 and PIWIL2 was found in IDC compared to mastopathy samples (p≤0.0001). Increased expression of PIWIL1 was correlated with increased PIWIL2 expression in breast cancer tissue. Surprisingly, PIWIL1 mRNA was detected only in cancer and mastopathy, but was not found in most normal breast tissues, although it is noteworthy that the PIWIL2 mRNA level was statistically significantly lower in mastopathy and IDC samples compared to normal breast tissues. Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Identification and tissue distribution of mRNAs encoding salmon-type calcitonins-IV and -V in the rainbow trout.

PubMed

Hidaka, Yoshie; Suzuki, Masakazu

2004-06-01

Four types of calcitonin are produced in salmonid fish, although their functional diversity is almost unknown. To explore the significance of these isoforms, we have characterized salmon-type calcitonin (sCT) mRNAs in the rainbow trout (Oncorhynchus mykiss), and examined their tissue distribution. In addition to the previously isolated sCT-I cDNAs, two new forms of sCT cDNA were cloned from the ultimobranchial gland, and one of them (sCT-IV cDNA) was predicted to encode an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-IV, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. The sCT-IV precursor was 78% identical with the rainbow trout sCT-I precursors. The other cloned cDNA encoded a precursor for a novel CT, sCT-V. The sCT-V peptide was different from sCT-IV by only one amino acid residue: Val at position 8 in the latter was replaced by Met. The sCT-V precursor had 80 and 90% identity with the sCT-I and -IV precursors respectively. No cDNA clones were obtained for sCTs-II or -III.Tissue distribution of sCT-I, -IV and -V mRNAs was examined by RT-PCR and specific cleavage with restriction enzymes. An amplified fragment from sCT-I mRNA was detected not only in the ultimobranchial gland, but also in the gills, testis and ovary. RT-PCR analysis coupled to restriction digestion further revealed that sCT-IV mRNA was expressed in both the testis and the ultimobranchial gland. The expression sites of sCT-IV mRNA were localized to the Leydig cells of the testis and to the parenchymal cells of the ultimobranchial gland, by in situ hybridization histochemistry. Although the amino acid sequence of sCT-V peptide was nearly the same as that of sCT-IV, the sCT-V gene showed a much wider pattern of expression: the band amplified by RT-PCR was detected in all the tissues examined except the kidney, gills and blood cells. The sCT-V mRNA was shown to be localized in the parenchymal cells of the ultimobranchial gland, but not in other tissues at the cellular level, suggesting very low expression of sCT-V mRNA in those tissues. Our results show different patterns of tissue expression of three types of sCT genes in the rainbow trout, suggesting that sCTs-I, -IV and -V might differ in their local actions.

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples

PubMed Central

Zubakov, Dmitry; Hanekamp, Eline; Kokshoorn, Mieke; van IJcken, Wilfred

2007-01-01

Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice. Electronic supplementary material The online version of this article (doi:10.1007/s00414-007-0182-6) contains supplementary material, which is available to authorized users. PMID:17579879

Cathepsin B expression in colorectal cancer in a Middle East population: Potential value as a tumor biomarker for late disease stages

PubMed Central

Abdulla, Maha-Hamadien; Valli-Mohammed, Mansoor-Ali; Al-Khayal, Khayal; Shkieh, Abdulmalik Al; Zubaidi, Ahmad; Ahmad, Rehan; Al-Saleh, Khalid; Al-Obeed, Omar; McKerrow, James

2017-01-01

Cathepsin B (CTSB), is a cysteine protease belonging to the cathepsin (Clan CA) family. The diagnostic and prognostic significance of increased CTSB in the serum of cancer patients have been evaluated for some tumor types. CTSB serum and protein levels have also been reported previously in colorectal cancer (CRC) with contradictory results. The aim of the present study was to investigate CTSB expression in CRC patients and the association of CTSB expression with various tumor stages in a Middle East population. Serum CTSB levels were evaluated in 70 patients and 20 healthy control subjects using enzyme-linked immunosorbant assay (ELISA) technique. CTSB expression was determined in 100 pairs of CRC tumor and adjacent normal colonic tissue using quantitative PCR for mRNA levels. Detection of CTSB protein expression in tissues was carried out using both immunohistochemistry and western blotting techniques. ELISA analysis showed that in sera obtained from CRC patients, the CTSB concentration was significantly higher in late stage patients with lymph node metastases when compared to early stage patients with values of 2.9 and 0.33 ng/ml, respectively (P=0.001). The majority of tumors studied had detectable CTSB protein expression with significant increased positive staining in tumors cells when compared with matched normal colon subjects (P=0.006). The mRNA expression in early stage CRC compared to late stage CRC was 0.04±0.01 and 0.07±0.02, respectively. Increased mRNA expression was more frequently observed in the advanced cancer stages with lymph node metastases when compared with the control (P=0.002). Mann-Whitney test and paired t-test were used to compare serum CTSB and mRNA levels in early and late tumor stage. A subset of four paired tissue extracts were analyzed by western blotting. The result confirmed a consistent increase in the CTSB protein expression level in tumor tissues compared with that noted in the adjacent normal mucosal cells. These findings indicate that CTSB may be an important prognostic biomarker for late stage CRC and cases with lymph node metastases in the Middle Eastern population. Monitoring serum CTSB in CRC patients may predict and/or diagnose cases with lymph node metastases. PMID:28440429

[Changes and role evaluation of TNF-α and IL-1β in lung tissues of ARDS mice].

PubMed

Liang, Jianing; Zhou, Qianqian; Zhang, Tianxiang; Wang, Xiaosu; Song, Liqiang

2017-02-01

Objective To study the expression levels of TNF-α and IL-1β in the lung tissues of acute respiratory distress syndrome (ARDS) mice and their relationships with the severity of lung injury in the mice. Methods A mouse model of ARDS was induced by lipopolysaccharide (LPS). The morphological changes of lung tissue was observed by HE staining, and the lung injury score was calculated. Quantitative real-time PCR was employed to detect the mRNA expression levels of TNF-α and IL-1β in lung tissues and ELISA was performed to test the protein levels of TNF-α and IL-1β in bronchoalveolar lavage fluid (BALF). Results Compared with the control group, the alveolar and interstitial tissue structure of ARDS model mice was impaired and filled with inflammatory cells. The lung injury score of ARDS model mice reached the peak at the third day. The mRNA levels of TNF-α and IL-1β in lung tissues of ARDS mice significantly increased, and respectively peaked at 30 minutes and 6 hours after LPS instillation. Simultaneously, the levels of TNF-α and IL-1β in BALF of ARDS mice significantly increased, and the tendency was consistent with mRNA levels in lung tissues. Conclusion LPS-induced lung injury and the expression levels of TNF-α and IL-1β in ARDS mice showed a similar "hump-like" increase over time. The high values of inflammatory mediators appeared before the peak of lung injury, which indicated that these inflammatory cytokines played an important role in the development of ARDS-caused inflammatory injury.

A common carcinogen benzo[a]pyrene causes p53 overexpression in mouse cervix via DNA damage.

PubMed

Gao, Meili; Li, Yongfei; Sun, Ying; Long, Jiangang; Kong, Yu; Yang, Shuiyun; Wang, Yili

2011-09-18

Benzo[a]pyrene (BaP) is cytotoxic and/or genotoxic to lung, stomach and skin tissue in the body. However, the effect of BaP on cervical tissue remains unclear. The present study detected DNA damage and the expression of the p53 gene in BaP-induced cervical tissue in female mice. Animals were intraperitoneally injected and orally gavaged with BaP at the doses of 2.5, 5, and 10mg/kg twice a week for 14 weeks. The single-cell gel electrophoresis (SCGE) assay was used to detect the DNA damage. Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to detect the expression of p53 protein and p53 mRNA, respectively. The results showed that BaP induced a significant and dose-dependent increase of the number of cells with DNA damaged and the tail length as well as Comet tail moment in cervical tissue. The expression level of p53 protein and mRNA was increased. The results demonstrate that BaP may show toxic effect on the cervix by increasing DNA damage and the expression of the p53 gene. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of limb ischemic preconditioning on myocardial apoptosis-related proteins in ischemia-reperfusion injury

PubMed Central

GAO, JIANZHI; ZHAO, LINJING; WANG, YONGLING; TENG, QINGLEI; LIANG, LIDONG; ZHANG, JINYING

2013-01-01

The aim of this study was to investigate the effect of limb ischemic preconditioning (LIPC) on myocardial apoptosis in myocardial ischemia-reperfusion injury (MIRI), as well as the regulation of caspase-3 and the B cell lymphoma 2 (Bcl-2) gene in LIPC. A total of 50 rats were divided randomly into 5 groups (n=10). Four rats in each group were drawn out for detection of apoptosis. The sham, MIRI and LIPC groups underwent surgery without additional treatment. In the LY294002 group, LY294002 preconditioning was administered 15 min before reperfusion. In the LY294002+LIPC group, following LIPC, LY294002 was administered 15 min before reperfusion. The relative expression of myocardial Bcl-2 and caspase-3 mRNA and the apoptotic index for each group were determined by reverse transcription-polymerase chain reaction (RT-PCR) and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), respectively. The ultrastructure of the cardiac muscle tissues was observed by election microscopy. Compared with the sham group, the expression of caspase-3 mRNA in the MIRI group significantly increased (P<0.05) and the expression of Bcl-2 mRNA clearly decreased. Compared with the MIRI group, LIPC reduced the expression of caspase-3 and increased the expression of Bcl-2 mRNA (P<0.05). There were no significant differences between the LY294002+LIPC group and the MIRI group. Compared with the sham group, the apoptotic index of myocardial cells in the MIRI group significantly increased (P<0.05). Compared with the MIRI group, LIPC significantly decreased the apoptotic index of myocardial cells (P<0.05) and LY294002 increased the apoptotic index of myocardial cells. Compared with the LIPC group, LY294002+LIPC significantly increased the apoptotic index of myocardial cells (P<0.05). There were no significant differences between the LY294002+LIPC and MIRI groups. In conclusion, LIPC increased the expression of Bcl-2 and decreased caspase-3 mRNA and apoptosis in myocardial tissue following MIRI. Therefore, LIPC plays a protective role in myocardial tissue. PMID:23737869

Lactococcus lactis and Lactobacillus salivarius differently modulate early immunological response of Wistar rats co-administered with Listeria monocytogenes.

PubMed

Lukic, J; Jancic, I; Mirkovic, N; Bufan, B; Djokic, J; Milenkovic, M; Begovic, J; Strahinic, I; Lozo, J

2017-10-13

In the light of the increasing resistance of bacterial pathogens to antibiotics, one of the main global strategies in applied science is development of alternative treatments, which would be safe both for the host and from the environmental perspective. Accordingly, the aim of this study was to test whether two lactic acid bacteria (LAB) strains, Lactococcus lactis BGBU1-4 and Lactobacillus salivarius BGHO1, could be applied as safe supplements for Listeria infection. Two major research objectives were set: to compare the effects of BGBU1-4 and BGHO1 on early immune response in gut tissue of Wistar rats co-administered with Listeria monocytogenes ATCC19111 and next, to test how this applies to their usage as therapeutics in acute ATCC19111 infection. Intestinal villi (IV), Peyer's patches (PP) and mesenteric lymph nodes (MLN) were used for the analysis. The results showed that BGHO1 increased the mRNA expression of innate immune markers CD14, interleukin (IL)-1β and tumour necrosis factor (TNF)-α in PP and IV, and, in parallel, caused a decrease of listeriolysin O (LLO) mRNA expression in same tissues. In MLN of BGHO1 treated rats, LLO expression was increased, along with an increase of the expression of OX-62 mRNA and CD69, pointing to the activation of adaptive immunity. On the other hand, in BGBU1-4 treated rats, there was no reduction of LLO mRNA expression and no induction of innate immunity markers in intestinal tissue. Additionally, CD14 and IL-1β, as well as LLO, but not OX-62 mRNA and CD69 expression, were elevated in MLN of BGBU1-4 treated rats. However, when applied therapeutically, both, BGBU1-4 and BGHO1, lowered Listeria count in spleens of infected rats. Our results not only reveal the potential of LAB to ameliorate Listeria infections, but suggest different immunological effects of two different LAB strains, both of which could be effective in Listeria elimination.

Altered gene expression in tree shrew retina and retinal pigment epithelium produced by short periods of minus-lens wear.

PubMed

He, Li; Frost, Michael R; Siegwart, John T; Norton, Thomas T

2018-03-01

Hyperopic refractive error is detected by retinal neurons, which generate GO signals through a direct emmetropization signaling cascade: retinal pigment epithelium (RPE) into choroid and then into sclera, thereby increasing axial elongation. To examine signaling early in this cascade, we measured gene expression in the retina and RPE after short exposure to hyperopia produced by minus-lens wear. Gene expression in each tissue was compared with gene expression in combined retina + RPE. Starting 24 days after normal eye opening, three groups of juvenile tree shrews (n = 7 each) wore a monocular -5 D lens. The untreated fellow eye served as a control. The "6h" group wore the lens for 6 h; the "24h" group wore the lens for 24 h; each group provided separate retina and RPE tissues. Group "24hC" wore the lens for 24 h and provided combined retina + RPE tissue. Quantitative PCR was used to measure the relative differences (treated eye vs. control eye) in mRNA levels for 66 candidate genes. In the retina after 6 h, mRNA levels for seven genes were significantly regulated: EGR1 and FOS (early intermediate genes) were down-regulated in the treated eyes. Genes with secreted protein products, BMP2 and CTGF, were down-regulated, whilst FGF10, IL18, and SST were up-regulated. After 24 h the pattern changed; only one of the seven genes still showed differential expression; BMP2 was still down-regulated. Two new genes with secreted protein products, IGF2 and VIP, were up-regulated. In the RPE, consistent with its role in receiving, processing, and transmitting GO signaling, differential expression was found for genes whose protein products are at the cell surface, intracellular, in the nucleus, and are secreted. After 6 h, mRNA levels for 17 genes were down-regulated in the treated eyes, whilst four genes (GJA1, IGF2R, LRP2, and IL18) were up-regulated. After 24 h the pattern was similar; mRNA levels for 14 of the same genes were still down-regulated; only LRP2 remained up-regulated. mRNA levels for six genes no longer showed differential expression, whilst nine genes, not differentially expressed at 6 h, now showed differential expression. In the combined retina + RPE after 24 h, mRNA levels for only seven genes were differentially regulated despite the differential expression of many genes in the RPE. Four genes showed the same expression in combined tissue as in retina alone, including up-regulation of VIP despite significant VIP down-regulation in RPE. Thus, hyperopia-induced GO signaling, as measured by differential gene expression, differs in the retina and the RPE. Retinal gene expression changed between 6 h and 24 h of treatment, suggesting evolution of the retinal response. Gene expression in the RPE was similar at both time points, suggesting sustained signaling. The combined retina + RPE does not accurately represent gene expression in either retina or, especially, RPE. When gene expression signatures were compared with those in choroid and sclera, GO signaling, as encoded by differential gene expression, differs in each compartment of the direct emmetropization signaling cascade. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

The expression and prognostic value of protein tyrosine kinase 6 in early-stage cervical squamous cell cancer.

PubMed

Wang, Xiao-Jing; Xiong, Ying; Ma, Ze-Biao; Xia, Jian-Chuan; Li, Yan-Fang

2016-06-16

Protein tyrosine kinase 6 (PTK6) is overexpressed in many epithelial tumors and predicts poor prognosis. However, PTK6 expression status and its role in cervical squamous cell cancer are unknown. This study aimed to investigate the expression level and clinical significance of PTK6 in early-stage cervical squamous cell cancer. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting analysis were performed to detect PTK6 mRNA and protein expression levels in 10 freshly frozen, early-stage cervical squamous cell cancer specimens and adjacent non-tumorous cervical tissues. The expression of PTK6 was detected using immunohistochemical staining in 150 formalin-fixed, paraffin-embedded, early-stage cervical squamous cell cancer sections and 10 normal cervical tissue sections. The mRNA and protein levels of PTK6 in cancer tissues were higher than those in adjacent non-tumorous cervical tissues. Immunohistochemical analysis showed that PTK6 was not expressed in normal cervical tissues but was overexpressed in the cytoplasm of cervical squamous cell cancer cells. The level of PTK6 expression was significantly associated with tumor grade (P = 0.020). The 5-year overall survival rate of patients with high PTK6 expression was lower than that of patients with low PTK6 expression (81.3% vs. 96.2%, P = 0.008). Multivariate Cox regression analysis showed that the expression level of PTK6 in cervical squamous cell cancer was an independent prognostic factor for patient survival (hazard ratio = 5.999, 95% confidence interval 1.622-22.191, P < 0.05). PTK6 is overexpressed in cervical squamous cell cancer. Increased PTK6 expression is associated with reduced 5-year overall survival. PTK6 expression is an independent prognostic predictor for cervical cancer.

Extended specificity studies of mRNA assays used to infer human organ tissues and body fluids.

PubMed

van den Berge, Margreet; Sijen, Titia

2017-12-01

Messenger RNA (mRNA) profiling is a technique increasingly applied for the forensic identification of body fluids and skin. More recently, an mRNA-based organ typing assay was developed which allows for the inference of brain, lung, liver, skeletal muscle, heart, kidney, and skin tissue. When applying this organ typing system in forensic casework for the presence of animal, rather than human, tissue is an alternative scenario to be proposed, for instance that bullets carry cell material from a hunting event. Even though mRNA profiling systems are commonly in silico designed to be primate specific, physical testing against other animal species is generally limited. In this study, human specificity of the organ tissue inferring system was assessed against organ tissue RNAs of various animals. Results confirm human specificity of the system, especially when utilizing interpretation rules considering multiple markers per cell type. Besides, we cross-tested our organ and body fluid mRNA assays against the target types covered by the other assay. Marker expression in the nontarget organ tissues and body fluids was observed to a limited extent, which emphasizes the importance of involving the case-specific context of the forensic samples in deciding which mRNA profiling assay to use and when for interpreting results. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The association of SNPs in Hsp90β gene 5' flanking region with thermo tolerance traits and tissue mRNA expression in two chicken breeds.

PubMed

Chen, Zhuo-Yu; Gan, Jian-Kang; Xiao, Xiong; Jiang, Li-Yan; Zhang, Xi-Quan; Luo, Qing-Bin

2013-09-01

Thermo stress induces heat shock proteins (HSPs) expression and HSP90 family is one of them that has been reported to involve in cellular protection against heat stress. But whether there is any association of genetic variation in the Hsp90β gene in chicken with thermo tolerance is still unknown. Direct sequencing was used to detect possible SNPs in Hsp90β gene 5' flanking region in 3 chicken breeds (n = 663). Six mutations, among which 2 SNPs were chosen and genotypes were analyzed with PCR-RFLP method, were found in Hsp90β gene in these 3 chicken breeds. Association analysis indicated that SNP of C.-141G>A in the 5' flanking region of the Hsp90β gene in chicken had some effect on thermo tolerance traits, which may be a potential molecular marker of thermo tolerance, and the genotype GG was the thermo tolerance genotype. Hsp90β gene mRNA expression in different tissues detected by quantitative real-time PCR assay were demonstrated to be tissue dependent, implying that different tissues have distinct sensibilities to thermo stress. Besides, it was shown time specific and varieties differences. The expression of Hsp90β mRNA in Lingshan chickens in some tissues including heart, liver, brain and spleen were significantly higher or lower than that of White Recessive Rock (WRR). In this study, we presume that these mutations could be used in marker assisted selection for anti-heat stress chickens in our breeding program, and WRR were vulnerable to tropical thermo stress whereas Lingshan chickens were well adapted.

Analysis of the association of the expression of KiSS-1 in colorectal cancer tissues with the pathology and prognosis.

PubMed

Huo, Xinkai; Zhang, Lei; Li, Tao

2018-03-01

Colorectal cancer is a common malignant tumor of the digestive tract with high morbidity and mortality rates. The aim of the present study was to examine the expression level of KiSS-1 in tumor tissues of patients with colorectal cancer, and to explore the relationship with the clinicopathology and prognosis of patients with colorectal cancer. Frozen tumor tissue and corresponding cancer-adjacent normal tissue specimens were selected from 56 patients with colorectal cancer who were treated in the Department of Surgery of our hospital from May 2009 to December 2011. The expression levels of KiSS-1 messenger ribonucleic acid (mRNA) in tumor tissues and cancer-adjacent normal tissues were detected by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The expression levels of KiSS-1 proteins in colorectal cancer tissues and cancer-adjacent normal tissues were further detected by immunohistochemistry. In addition, the association of the expression level of KiSS-1 proteins in tissues of colorectal cancer patients with pathological parameters and the prognosis of patients with colorectal cancer was analyzed combined with clinical data. The RT-qPCR results showed that the expression of KiSS-1 mRNA in colorectal cancer tissues was significantly lower than that in cancer-adjacent normal tissues (P<0.05). Immunohistochemistry results indicated that the positive expression rate of KiSS-1 proteins in colorectal cancer tissues (26.79%) was significantly lower than that in cancer-adjacent normal tissues (80.36%). The low expression of KiSS-1 in colorectal cancer tissues was associated with the degree of differentiation, invasion and metastasis, as well as clinical staging. The 5-year overall survival rate of patients with colorectal cancer was 55.36% (31/56). The univariate survival analysis showed that patients with lowly expressed KiSS-1 had worse prognosis. The low expression of KiSS-1 is closely associated with the occurrence and development of colorectal cancer, especially to the degree of differentiation, invasion and metastasis, as well as clinical staging. Thus, the expression of KiSS-1 in colorectal cancer tissues can be used as a reference for the prognosis of colorectal cancer, and KiSS-1 is a potential new target for the treatment of colorectal cancer.

Expression of adrenomedullin 2/intermedin in human adrenal tumors and attached non-neoplastic adrenal tissues.

PubMed

Morimoto, Ryo; Satoh, Fumitoshi; Murakami, Osamu; Hirose, Takuo; Totsune, Kazuhito; Imai, Yutaka; Arai, Yoichi; Suzuki, Takashi; Sasano, Hironobu; Ito, Sadayoshi; Takahashi, Kazuhiro

2008-07-01

Adrenomedullin 2/intermedin (AM2/IMD) is a new member of calcitonin/calcitonin gene-related peptide family. AM is expressed in various tumors including adrenocortical tumors and modulates tumor growth. The AM2/IMD expression has not been studied, however, in adrenal tumors. The expression of AM2/IMD and AM was therefore studied in human adrenal tumors and attached non-neoplastic adrenal tissues by immunocytochemistry (ICC). Immunoreactive (IR)-AM2/IMD was measured by RIA. Furthermore, the expression of AM2/IMD and its receptor components, calcitonin receptor-like receptor (CRLR), and receptor activity-modifying proteins (RAMPs) 1, 2, and 3 mRNA in these tissues was studied by reverse transcription PCR (RT-PCR). ICC showed that AM2/IMD and AM immunoreactivities were localized in adrenocortical tumors and pheochromocytomas. AM2/IMD and AM immunoreactivities were detected in medulla of attached non-neoplastic tissues, while the degree of immunoreactivity for AM2/IMD and AM in cortices of attached adrenals was relatively weak or undetectable. RIA detected IR-AM2/IMD in adrenal tumors (0.414+/-0.12 to 0.786+/-0.27 pmol/g wet weight, mean+/-S.E.M.) and attached adrenal tissues (0.397+/-0.052 pmol/g wet weight). Reverse-phase high-performance liquid chromatography showed one broad peak eluted in the similar position to synthetic AM2/IMD with several minor peaks. RT-PCR showed expression of AM2/IMD, CRLR, and RAMP1, RAMP2, and RAMP3 mRNA in tissues of adrenal tumors and attached adrenal glands. In conclusion, AM2/IMD is expressed in human adrenal tumors and attached non-neoplastic adrenal tissues and may play (patho-)physiological roles in normal and neoplastic adrenals as an autocrine/paracrine regulator.

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

PubMed

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

2003-07-18

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

The growth hormone-releasing hormone (GHRH) antagonist JV-1-36 inhibits proliferation and survival of human ectopic endometriotic stromal cells (ESCs) and the T HESC cell line.

PubMed

Annunziata, Marta; Grande, Cristina; Scarlatti, Francesca; Deltetto, Francesco; Delpiano, Elena; Camanni, Marco; Ghigo, Ezio; Granata, Riccarda

2010-08-01

To determine the effect of the GHRH antagonist JV-1-36 on proliferation and survival of primary ectopic human endometriotic stromal cells (ESCs) and the T HESC cell line. Prospective laboratory study. University hospital. 22 women with endometriosis (aged 34.8+/-5.7 years) undergoing therapeutic laparoscopy. Eutopic (n=10) and ectopic (n=22) endometrial tissues were collected from women who underwent therapeutic laparoscopic surgery for endometriosis (stage III/IV). Expression of GHRH, GHRH receptor (GHRH-R) and GHRH-R splice variant (SV) 1 mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR). The ESC proliferation was assessed by 5-bromo-2-deoxyuridine incorporation, cell survival by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Trypan blue assay. The T HESC survival was evaluated by MTT, cyclic adenosine monophosphate (cAMP) levels by ELISA, extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation by Western blot, and insulin-like growth factor (IGF)-2 mRNA by real-time PCR. The ESCs and T HESCs, but not normal endometrial tissues, expressed GHRH-R mRNA; SV1 mRNA was determined in normal endometrial tissues, ESCs, and T HESCs; GHRH mRNAwas found in T HESCs; JV-1-36 inhibited ESC proliferation and ESC and T HESC survival. In T HESCs, JV-1-36 reduced cAMP production and ERK1/2 phosphorylation but had no effect on IGF-2 mRNA expression. The GHRH antagonist JV-1-36 inhibits endometriotic cell proliferation and survival, suggesting that GHRH antagonist may represent promising tools for treatment of endometriosis. Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Serum vaspin levels and vaspin mRNA expression in subcutaneous adipose tissue in women with gestational diabetes mellitus.

PubMed

Mm, Wei Qian; Fan, Jianxia; Khor, Shuzin; Song, Mengfan; Hong, Wei; Dai, Xiaobei

2014-11-01

To compare serum vaspin level and mRNA and protein levels of vaspin in adipose tissue in women with gestational diabetes mellitus (GDM) and normal glucose tolerance (NGR), along with the correlation between serum vaspin level with fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR) and birth-weight. Thirty-seven women with GDM and 36 with NGR were enrolled. Total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting plasma glucose (FPG), FINS and vaspin levels were measured. The mRNA and protein levels were detected using RT-PCR and Western blot. Pearson correlation analysis (PCA) was performed to reveal the correlation between serum vaspin level and FINS, HOMA-IR. Spearman correlation analysis (SCA) was conducted to examine the association between serum vaspin level and birth-weight. HDL-C level in GDM was lower than NGR group (P<0.05), and there were no statistical differences in TC, TG, LDL-C, FPG, FINS and HOMA-IR between the two groups. Serum vaspin level, mRNA and protein expression levels of vaspin in GDM were higher than NGR group (P<0.05). Serum vaspin level was not significantly correlated with FINS and HOMA-IR, but had a positive correlation with birth-weight (P=0.023). Serum vaspin level cannot serve as an independent predictor of IR. The increased serum vaspin level and increased vaspin mRNA and protein expression in adipose tissues in GDM women indicate that vaspin may be involved in the pathogenesis of GDM, but its exact mechanism needs further study. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

mRNA expression of CDH3, IGF2BP3, and BIRC5 in biliary brush cytology specimens is a useful adjunctive tool of cytology for the diagnosis of malignant biliary stricture

PubMed Central

Kim, Tae Ho; Chang, Jae Hyuck; Lee, Hee Jin; Kim, Jean A; Lim, Yeon Soo; Kim, Chang Whan; Han, Sok Won

2016-01-01

Abstract Although advances have been made in diagnostic tools, the distinction between malignant and benign biliary strictures still remains challenging. Intraductal brush cytology is a convenient and safe method that is used for the diagnosis of biliary stricture, but, low sensitivity limits its usefulness. This study aimed to demonstrate the usefulness of mRNA expression levels of target genes in brush cytology specimens combined with cytology for the diagnosis of malignant biliary stricture. Immunohistochemistry for cadherin 3 (CDH3), p53, insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), and baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) was performed in 4 benign and 4 malignant bile duct tissues. Through endoscopic or interventional radiologic procedures, brush cytology specimens were prospectively obtained in 21 and 35 paitents with biliary strictures. In the brush cytology specimens, the mRNA expressions levels of 5 genes were determined by real-time polymerase chain reaction. Immunohistochemistry for CDH3, p53, IGF2BP3, HOXB7, and BIRC5 all showed positive staining in malignant tissues in contrast to benign tissues, which were negative. In the brush cytology specimens, the mRNA expression levels of CDH3, IGF2BP3, HOXB7, and BIRC5 were significantly higher in cases of malignant biliary stricture compared with cases of benign stricture (P = 0.006, P < 0.001, P < 0.001, and P = 0.001). The receiver-operating characteristic curves of these 4 mRNAs demonstrated that mRNA expression levels are useful for the prediction of malignant biliary stricture (P = 0.006, P < 0.001, P < 0.001, and P = 0.002). The sensitivity and specificity, respectively, for malignant biliary stricture were 57.1% and 100% for cytology, 57.1% and 64.3% for CDH3, 76.2% and 100% for IGF2BP3, 71.4% and 57.1% for HOXB7, and 76.2% and 64.3% for BIRC5. When cytology was combined with the mRNA levels of CDH3, IGF2BP3, or BIRC5, the sensitivity for malignant biliary stricture improved to 90.5%. The measurement of the mRNA expression levels of CDH3, IGF2BP3, and BIRC5 by real-time polymerase chain reaction combined with cytology was useful for the differentiation of malignant and benign biliary strictures in brush cytology specimens. PMID:27399126

Effects of various cryoprotectants on the quality of frozen-thawed immature bovine (Qinchuan cattle) calf testicular tissue.

PubMed

Zhang, X-G; Li, H; Hu, J-H

2017-11-01

To investigate the effects of different concentrations of various cryoprotectants (CPs) on the cell viability as well as expression of spermatogenesis-related genes, such as CREM, Stra8 and HSP70-2 in frozen-thawed bovine calf testicular tissue, immature bovine (Qinchuan cattle) calf testicular tissue was collected and cryopreserved in the cryomedia containing different concentrations (5%, 10%, 15% and 20%) of the following three CPs: glycerol, ethylene glycol (EG) and dimethyl sulphoxide (DMSO) respectively. After 1 month cryopreservation in liquid nitrogen, cell viability was evaluated using Trypan blue exclusion under a bright-field microscope. The mRNA expression of the three genes was also evaluated using qRT-PCR. The results indicated that different concentrations of glycerol, EG and DMSO in cryomedia during cryopreservation could protect bovine calf testicular tissue in various ways to avoid freezing or cryopreservation-induced expression changes in spermatogenesis-related genes. The highest cell viability and the three spermatogenesis-related genes (CREM, Stra8 and HSP70-2) expression level came from the cryomedia containing glycerol, EG and DMSO at 10% concentration respectively (p < .05). Meanwhile, compared with the other CPs, the frozen-thawed bovine calf testicular tissue treated with 10% DMSO exhibited the highest cell viability and mRNA expression level of the spermatogenesis-related genes (CREM, Stra8 and HSP70-2). © 2017 Blackwell Verlag GmbH.

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.

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.

Advanced Glycation End-Products Induce Connective Tissue Growth Factor-Mediated Renal Fibrosis Predominantly through Transforming Growth Factor β-Independent Pathway

PubMed Central

Zhou, Guihua; Li, Cai; Cai, Lu

2004-01-01

Advanced glycation end-products (AGEs) play a critical role in diabetic nephropathy by stimulating extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF) is a potent inducer of ECM synthesis and increases in the diabetic kidneys. To determine the critical role of CTGF in AGE-induced ECM accumulation leading to diabetic nephropathy, rats were given AGEs by intravenous injection for 6 weeks. AGE treatment induced a significant renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) accumulation in glomeruli, and a mild renal dysfunction, as shown by increases in urinary volume and protein content. AGE treatment also caused significant increases in renal CTGF and transforming growth factor (TGF)-β1 mRNA and protein expression. Direct exposure of rat mesangial cells to AGEs in vitro significantly induced increases in fibronectin and Col IV production, which could be completely prevented by pretreatment with anti-CTGF antibody. AGE treatment also significantly increased both TGF-β1 and CTGF mRNA expression; however, inhibition of TGF-β1 mRNA expression by shRNA or neutralization of TGF-β1 protein by anti-TGF-β1 antibody did not significantly prevent AGE-increased expression of CTGF mRNA and protein. These results suggest that AGE-induced CTGF expression, predominantly through a TGF-β1-independent pathway, plays a critical role in renal ECM accumulation leading to diabetic nephropathy. PMID:15579446

Sex-dependent effects of antenatal glucocorticoids on insulin sensitivity in adult sheep: role of the adipose tissue renin angiotensin system

PubMed Central

Massmann, G. Angela; Zhang, Jie; Seong, Won Joon; Kim, Minhyoung

2017-01-01

Exposure to glucocorticoids in utero is associated with changes in organ function and structure in the adult. The aims of this study were to characterize the effects of antenatal exposure to glucocorticoids on glucose handling and the role of adipose tissue. Pregnant sheep received betamethasone (Beta, 0.17 mg/kg) or vehicle 24 h apart at 80 days of gestation and allowed to deliver at term. At 9 mo, male and female offspring were fed at either 100% of nutritional allowance (lean) or ad libitum for 3 mo (obese). At 1 yr, they were chronically instrumented under general anesthesia. Glucose tolerance was evaluated using a bolus of glucose (0.25 g/kg). Adipose tissue was harvested after death to determine mRNA expression levels of angiotensinogen (AGT), angiotensin-converting enzyme (ACE) 1, ACE2, and peroxisome proliferator-activated receptor γ (PPAR-γ). Data are expressed as means ± SE and analyzed by ANOVA. Sex, obesity, and Beta exposure had significant effects on glucose tolerance and mRNA expression. Beta impaired glucose tolerance in lean females but not males. Superimposed obesity worsened the impairment in females and unmasked the defect in males. Beta increased ACE1 mRNA in females and males and AGT in females only (P < 0.05 by three-way ANOVA). Obesity increased AGT in females but had no effect on ACE1 in either males or females. PPAR-γ mRNA exhibited a significant sex (F = 42.8; P < 0.01) and obesity (F = 6.9; P < 0.05) effect and was significantly higher in males (P < 0.01 by three-way ANOVA). We conclude that adipose tissue may play an important role in the sexually dimorphic response to antenatal glucocorticoids. PMID:28356296

Downregulation of SASH1 correlates with poor prognosis in cervical cancer.

PubMed

Xie, J; Zhang, W; Zhang, J; Lv, Q-Y; Luan, Y-F

2017-10-01

The aim of this study was to analyze the association of SASH1 expression with clinicopathological features and prognosis in patients suffering cervical cancer. The expressions of SASH1 mRNA and protein in cervical cancer tissues and matched normal cervical tissues were detected by Real-time PCR and Immunohistochemistry. Based on the above findings, the association among SASH1 expression and clinicopathological features was analyzed. Overall survival was evaluated using the Kaplan-Meier method. The variables were used in univariate and multivariate analysis by the Cox proportional hazards model. The results demonstrated that both SASH1 mRNA and proteins were downregulated in cervical cancer tissues compared with those in matched normal tissues (both p < 0.05). Also, decreased SASH1 expression in cervical cancer was found to be significantly associated with high FIGO Stage (p = 0.001), lymph nodes metastasis (p = 0.003) and differentiation (p = 0.018). Furthermore, Kaplan-Meier analysis demonstrated that low SASH1 expression level was associated with poorer overall survival (p < 0.01). Univariate and multivariate analyses indicated that status of SASH1 was an independent prognostic factor for patients with cervical cancer. These findings suggested that SASH1 can be useful as a new prognostic marker and therapeutic target in cervical cancer patients.

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

PubMed Central

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

2017-01-01

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

Increased expression of tumor necrosis factor-α is associated with advanced colorectal cancer stages.

PubMed

Al Obeed, Omar A; Alkhayal, Khayal A; Al Sheikh, Abdulmalik; Zubaidi, Ahmad M; Vaali-Mohammed, Mansoor-Ali; Boushey, Robin; Mckerrow, James H; Abdulla, Maha-Hamadien

2014-12-28

To detect the expression of tumor necrosis factor-α (TNF-α) in colorectal cancer (CRC) cells among Saudi patients, and correlate its expression with clinical stages of cancer. Archival tissue specimens were collected from 30 patients with CRC who had undergone surgical intervention at King Khalid University Hospital. Patient demographic information, including age and gender, tumor sites, and histological type of CRC, was recorded. To measure TNF-α mRNA expression in CRC, total RNA was extracted from tumor formalin-fixed, paraffin-embedded, and adjacent normal tissues. Reverse transcription and reverse transcription polymerase chain reaction were performed. Colorectal tissue microarrays were constructed to investigate the protein expression of TNF-α by immunohistochemistry. The relative expression of TNF-α mRNA in colorectal cancer was significantly higher than that seen in adjacent normal colorectal tissue. High TNF-α gene expression was associated with Stage III and IV neoplasms when compared with earlier tumor stages (P = 0.004). Eighty-three percent of patients (25/30) showed strong TNF-α positive staining, while only 10% (n = 3/30) of patients showed weak staining, and 7% (n = 2/30) were negative. We showed the presence of elevated TNF-α gene expression in cancer cells, which strongly correlated with advanced stages of tumor. High levels of TNF-α expression could be an independent diagnostic indicator of colorectal cancer, and targeting TNF-α might be a promising prognostic tool by assessment of the clinical stages of CRC.

Increased expression of tumor necrosis factor-α is associated with advanced colorectal cancer stages

PubMed Central

Al Obeed, Omar A; Alkhayal, Khayal A; Al Sheikh, Abdulmalik; Zubaidi, Ahmad M; Vaali-Mohammed, Mansoor-Ali; Boushey, Robin; Mckerrow, James H; Abdulla, Maha-Hamadien

2014-01-01

AIM: To detect the expression of tumor necrosis factor-α (TNF-α) in colorectal cancer (CRC) cells among Saudi patients, and correlate its expression with clinical stages of cancer. METHODS: Archival tissue specimens were collected from 30 patients with CRC who had undergone surgical intervention at King Khalid University Hospital. Patient demographic information, including age and gender, tumor sites, and histological type of CRC, was recorded. To measure TNF-α mRNA expression in CRC, total RNA was extracted from tumor formalin-fixed, paraffin-embedded, and adjacent normal tissues. Reverse transcription and reverse transcription polymerase chain reaction were performed. Colorectal tissue microarrays were constructed to investigate the protein expression of TNF-α by immunohistochemistry. RESULTS: The relative expression of TNF-α mRNA in colorectal cancer was significantly higher than that seen in adjacent normal colorectal tissue. High TNF-α gene expression was associated with Stage III and IV neoplasms when compared with earlier tumor stages (P = 0.004). Eighty-three percent of patients (25/30) showed strong TNF-α positive staining, while only 10% (n = 3/30) of patients showed weak staining, and 7% (n = 2/30) were negative. We showed the presence of elevated TNF-α gene expression in cancer cells, which strongly correlated with advanced stages of tumor. CONCLUSION: High levels of TNF-α expression could be an independent diagnostic indicator of colorectal cancer, and targeting TNF-α might be a promising prognostic tool by assessment of the clinical stages of CRC. PMID:25561807

Tissue specific expression of the retinoic acid receptor-beta 2: regulation by short open reading frames in the 5'-noncoding region

PubMed Central

1994-01-01

The 40-S subunit of eukaryotic ribosomes binds to the capped 5'-end of mRNA and scans for the first AUG in a favorable sequence context to initiate translation. Most eukaryotic mRNAs therefore have a short 5'- untranslated region (5'-UTR) and no AUGs upstream of the translational start site; features that seem to assure efficient translation. However, approximately 5-10% of all eukaryotic mRNAs, particularly those encoding for regulatory proteins, have complex leader sequences that seem to compromise translational initiation. The retinoic-acid- receptor-beta 2 (RAR beta 2) mRNA is such a transcript with a long (461 nucleotides) 5'-UTR that contains five, partially overlapping, upstream open reading frames (uORFs) that precede the major ORF. We have begun to investigate the function of this complex 5'-UTR in transgenic mice, by introducing mutations in the start/stop codons of the uORFs in RAR beta 2-lacZ reporter constructs. When we compared the expression patterns of mutant and wild-type constructs we found that these mutations affected expression of the downstream RAR beta 2-ORF, resulting in an altered regulation of RAR beta 2-lacZ expression in heart and brain. Other tissues were unaffected. RNA analysis of adult tissues demonstrated that the uORFs act at the level of translation; adult brains and hearts of transgenic mice carrying a construct with either the wild-type or a mutant UTR, had the same levels of mRNA, but only the mutant produced protein. Our study outlines an unexpected role for uORFs: control of tissue-specific and developmentally regulated gene expression. PMID:7962071

Diminished androgen and estrogen receptors and aromatase levels in hypogonadal diabetic men: reversal with testosterone.

PubMed

Ghanim, Husam; Dhindsa, Sandeep; Abuaysheh, Sanaa; Batra, Manav; Kuhadiya, Nitesh D; Makdissi, Antoine; Chaudhuri, Ajay; Dandona, Paresh

2018-03-01

One-third of males with type 2 diabetes (T2DM) have hypogonadism, characterized by low total and free testosterone concentrations. We hypothesized that this condition is associated with a compensatory increase in the expression of androgen receptors (AR) and that testosterone replacement reverses these changes. We also measured estrogen receptor and aromatase expression. This is a randomized double-blind placebo-controlled trial. Thirty-two hypogonadal and 32 eugonadal men with T2DM were recruited. Hypogonadal men were randomized to receive intramuscular testosterone or saline every 2 weeks for 22 weeks. We measured AR, ERα and aromatase expression in peripheral blood mononuclear cells (MNC), adipose tissue and skeletal muscle in hypogonadal and eugonadal males with T2DM at baseline and after 22 weeks of treatment in those with hypogonadism. The mRNA expression of AR, ERα (ESR1) and aromatase in adipose tissue from hypogonadal men was significantly lower as compared to eugonadal men, and it increased significantly to levels comparable to those in eugonadal patients with T2DM following testosterone treatment. AR mRNA expression was also significantly lower in MNC from hypogonadal patients compared to eugonadal T2DM patients. Testosterone administration in hypogonadal patients also restored AR mRNA and nuclear extract protein levels from MNC to that in eugonadal patients. In the skeletal muscle, AR mRNA and protein expression are lower in men with hypogonadism. Testosterone treatment restored AR expression levels to that comparable to levels in eugonadal men. We conclude that, contrary to our hypothesis, the expression of AR, ERα and aromatase is significantly diminished in hypogonadal men as compared to eugonadal men with type 2 diabetes. Following testosterone replacement, there is a reversal of these deficits. © 2018 European Society of Endocrinology.

Selenium-dependent pre- and posttranscriptional mechanisms are responsible for sexual dimorphic expression of selenoproteins in murine tissues.

PubMed

Riese, Cornelia; Michaelis, Marten; Mentrup, Birgit; Götz, Franziska; Köhrle, Josef; Schweizer, Ulrich; Schomburg, Lutz

2006-12-01

Important enzymes for thyroid hormone metabolism, antioxidative defense, and intracellular redox control contain selenocysteine (Sec) in their active centers. Expression of these selenoproteins is tightly controlled, and a sex-specific phenotype is observed on disturbance of selenium (Se) transport in mice. Therefore, we analyzed Se concentrations and expression levels of several selenoproteins including type I iodothyronine deiodinase (Dio1) and glutathione peroxidase (GPx) isozymes in male and female mice. On regular lab chow, serum Se levels were comparable, but serum GPx3 activity was higher in females than males (1.3-fold). Selenoprotein P (SePP) mRNA levels were higher in livers (1.3-fold) and lower in kidneys (to 31%) in female compared with male mice. Orchidectomy alleviated the sex-specific differences in SePP mRNA amounts, indicating modulatory effects of androgens on SePP expression. Female mice expressed higher levels of Dio1 mRNA in kidney (2.6-fold) and liver (1.4-fold) in comparison with male mice. This sexual dimorphic expression of Dio1 mRNA was paralleled by increased Dio1 activity in female kidney (1.8-fold) but not in liver in which males expressed higher Dio1 activity (2.8-fold). Interestingly, Se deficiency decreased Dio1 activity more effectively in males than females, and resulting hepatic enzyme levels were then comparable between the sexes. At the same time, the sex-specific difference of Dio1 activity widened in kidney. Orchidectomy or estradiol treatment of ovariectomized females impacted stronger on renal than hepatic Dio1 expression. Thus, we conclude that Se-dependent posttranscriptional mechanisms are operational that affect either translational efficiency or Dio1 stability in a sex- and tissue-specific manner.

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.

Tissue-specific selection of stable reference genes for real-time PCR normalization in an obese rat model.

PubMed

Cabiati, Manuela; Raucci, Serena; Caselli, Chiara; Guzzardi, Maria Angela; D'Amico, Andrea; Prescimone, Tommaso; Giannessi, Daniela; Del Ry, Silvia

2012-06-01

Obesity is a complex pathology with interacting and confounding causes due to the environment, hormonal signaling patterns, and genetic predisposition. At present, the Zucker rat is an eligible genetic model for research on obesity and metabolic syndrome, allowing scrutiny of gene expression profiles. Real-time PCR is the benchmark method for measuring mRNA expressions, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. In the Zucker rat model, no specific reference genes have been identified in myocardium, kidney, and lung, the main organs involved in this syndrome. The aim of this study was to select among ten candidates (Actb, Gapdh, Polr2a, Ywhag, Rpl13a, Sdha, Ppia, Tbp, Hprt1 and Tfrc) a set of reference genes that can be used for the normalization of mRNA expression data obtained by real-time PCR in obese and lean Zucker rats both at fasting and during acute hyperglycemia. The most stable genes in the heart were Sdha, Tbp, and Hprt1; in kidney, Tbp, Actb, and Gapdh were chosen, while Actb, Ywhag, and Sdha were selected as the most stably expressed set for pulmonary tissue. The normalization strategy was used to analyze mRNA expression of tumor necrosis factor α, the main inflammatory mediator in obesity, whose variations were more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance of having three stably expressed reference gene sets for use in the cardiac, renal, and pulmonary tissues of an experimental model of obese and hyperglycemic Zucker rats.

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.

Low-level laser therapy (LLLT) attenuates RhoA mRNA expression in the rat bronchi smooth muscle exposed to tumor necrosis factor-alpha.

PubMed

de Lima, Flávia Mafra; Bjordal, Jan M; Albertini, Regiane; Santos, Fábio V; Aimbire, Flavio

2010-09-01

Low-level laser therapy (LLLT) has been found to produce anti-inflammatory effects in a variety of disorders. Bronchial smooth muscle (BSM) hyperreactivity is associated with increased Ca+2 sensitivity and increased RhoA mRNA expression. In the current study, we investigated if LLLT could reduce BSM contraction force and RhoA mRNA expression in tumor necrosis factor-alpha (TNF-alpha)-induced BSM hyperreactivity. In the study, 112 male Wistar rats were divided randomly into 16 groups, and BSM was harvested and suspended in TNF-alpha baths for 6 and 24 h, respectively. Irradiation with LLLT was performed with a wavelength of 660 nm for 42 s with a dose of 1.3 J/cm2. This LLLT dose was administered once in the 6-h group and twice in the 24-h group. LLLT significantly decreased contraction force in BSM at 6 h (TNF-alpha + LLLT: 11.65+/-1.10 g/100 mg of tissue) (F=3115) and at 24 h (TNF-alpha+ LLLT: 14.15+/-1.1 g/100 mg of tissue) (F=3245, p<0.05) after TNF-alpha, respectively, when compared to vehicle-bathed groups (control). LLLT also significantly decreased the expression of RhoA mRNA in BSM segments at 6 h (1.22+/-0.20) (F=2820, p<0.05) and 24 h (2.13+/-0.20) (F=3324, p<0.05) when compared to BSM segments incubated with TNF-alpha without LLLT irradiation. We conclude that LLLT administered with this protocol, reduces RhoA mRNA expression and BSM contraction force in TNF-alpha-induced BSM hyperreactivity.

Neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) in winter skate (Raja ocellata): cDNA cloning, tissue distribution and mRNA expression responses to fasting.

PubMed

MacDonald, Erin; Volkoff, Hélène

2009-04-01

cDNAs encoding for neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) were cloned in an elasmobranch fish, the winter skate. mRNA tissue distribution was examined for the three peptides as well as the effects of two weeks of fasting on their expression. Skate NPY, CART and CCK sequences display similarities with sequences for teleost fish but in general the degree of identity is relatively low (50%). All three peptides are present in brain and in several peripheral tissues, including gut and gonads. Within the brain, the three peptides are expressed in the hypothalamus, telencephalon, optic tectum and cerebellum. Two weeks of fasting induced an increase in telencephalon NPY and an increase in CCK in the gut but had no effects on hypothalamic NPY, CART and CCK, or on telencephalon CART. Our results provide basis for further investigation into the regulation of feeding in winter skate.

Amphiphysin I but not dynamin I nor synaptojanin mRNA expression increased after repeated methamphetamine administration in the rat cerebrum and cerebellum.

PubMed

Hamamura, Mitsuko; Okouchi, Jiro; Ozawa, Hidetoshi; Kimuro, Yoshihiko; Iwaki, Akiko; Fukumaki, Yasuyuki

2013-07-01

Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3' region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3' UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca²⁺ and Mg²⁺ devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.

Tripartite motif-containing 29 (TRIM29) is a novel marker for lymph node metastasis in gastric cancer.

PubMed

Kosaka, Yoshimasa; Inoue, Hiroshi; Ohmachi, Takahiro; Yokoe, Takeshi; Matsumoto, Toshifumi; Mimori, Koshi; Tanaka, Fumiaki; Watanabe, Masahiko; Mori, Masaki

2007-09-01

Tripartite motif-containing 29 (TRIM29) belongs to the TRIM protein family, which has unique structural characteristics, including multiple zinc finger motifs and a leucine zipper motif. TRIM29, also known as ataxia telangiectasia group D complementing gene, possesses radiosensitivity suppressor functions. Although TRIM29 has been reported to be underexpressed in prostate and breast cancer, its expression in gastrointestinal cancer has not been studied. By use of real-time reverse transcriptase-polymerase chain reaction, we analyzed TRIM29 mRNA expression status with respect to various clinicopathological parameters in 124 patients with gastric cancer. An immunohistochemical study was also conducted. The expression of TRIM29 was far higher in gastric cancer tumor tissue. Increased TRIM29 mRNA expression was markedly associated with such parameters as histological grade, large tumor size, extent of tumor invasion, and lymph node metastasis. In the TRIM29 high-expression group, it was an independent predictor for lymph node metastasis. Furthermore, patients with high TRIM29 mRNA expression showed a far poorer survival rate than those with low TRIM29 mRNA expression. TRIM29 expression may serve as a good marker of lymph node metastasis in gastric cancer.

Genetic Polymorphism and Expression of CXCR4 in Breast Cancer

PubMed Central

Ariza, Carolina Batista; de Oliveira, Carlos Eduardo Coral; Losi Guembarovski, Roberta; Banin Hirata, Bruna Karina; Vitiello, Glauco Akelinghton Freire; Campos, Clodoaldo Zago; Watanabe, Maria Angelica Ehara

2015-01-01

CXCR4 genetic polymorphisms, as well as their expression level, have been associated with cancer development and prognosis. The present study aimed to investigate the influence of CXCR4 rs2228014 polymorphism on its mRNA and protein expression in breast cancer samples. It was observed that patients presented higher CXCR4 mRNA relative expression (5.7-fold) than normal mammary gland, but this expression was not correlated with patients clinicopathological features (nuclear grade, nodal status, ER status, PR status, p53 staining, Ki67 index, and HER-2 status). Moreover, CXCR4 mRNA relative expression also did not differ regarding the presence or absence of T allele (p = 0.301). In the immunohistochemical assay, no difference was observed for CXCR4 cytoplasmic protein staining in relation to different genotypes (p = 0.757); however, high cytoplasmic CXCR4 staining was verified in invasive breast carcinoma (p < 0.01). All in all, the results from present study indicated that rs2228014 genetic variant does not alter CXCR4 mRNA or protein expression. However, this receptor was more expressed in tumor compared to normal tissue, in both RNA and protein levels, suggesting its promising applicability in the general context of mammary carcinogenesis. PMID:26576337

The Expression of Type-1 and Type-2 Nitric Oxide Synthase in Selected Tissues of the Gastrointestinal Tract during Mixed Mycotoxicosis

PubMed Central

Gajęcka, Magdalena; Stopa, Ewa; Tarasiuk, Michał; Zielonka, Łukasz; Gajęcki, Maciej

2013-01-01

The aim of the study was to verify the hypothesis that intoxication with low doses of mycotoxins leads to changes in the mRNA expression levels of nitric oxide synthase-1 and nitric oxide synthase-2 genes in tissues of the gastrointestinal tract and the liver. The experiment involved four groups of immature gilts (with body weight of up to 25 kg) which were orally administered zearalenone in a daily dose of 40 μg/kg BW (group Z, n = 18), deoxynivalenol at 12 μg/kg BW (group D, n = 18), zearalenone and deoxynivalenol (group M, n = 18) or placebo (group C, n = 21) over a period of 42 days. The lowest mRNA expression levels of nitric oxide synthase-1 and nitric oxide synthase-2 genes were noted in the sixth week of the study, in particular in group M. Our results suggest that the presence of low mycotoxin doses in feed slows down the mRNA expression of both nitric oxide synthase isomers, which probably lowers the concentrations of nitric oxide, a common precursor of inflammation. PMID:24284830

Decreased hepatocyte membrane potential differences and GABAA-beta3 expression in human hepatocellular carcinoma.

PubMed

Minuk, Gerald Y; Zhang, Manna; Gong, Yuewen; Minuk, Leonard; Dienes, Hans; Pettigrew, Norman; Kew, Michael; Lipschitz, Jeremy; Sun, Dongfeng

2007-03-01

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and gamma-aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (-19.8+/-1.3 versus -25.9+/-3.2 mV, respectively [P<0.05]), and GABAA-beta3 expression was down-regulated (GABAA-beta3 mRNA and protein expression in HCC; 5,693+/-1,385 and 0.29+/-0.11 versus 11,046+/-4,979 copies/100 mg RNA and 0.62+/-0.16 optical density in adjacent tumor tissues, respectively [P=0.002 and P<0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down-regulation, human malignant hepatocytes deficient in GABAA-beta3 receptor expression (Huh-7 cells) were transfected with GABAA-beta3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n=16-17 group). Tumors developed after a mean (+/-SD) of 51+/-6 days (range: 41-60 days) in 7/16 (44%) mice injected with vector-transfected cells and 70+/-12 days (range: 59-86 days) in 4/17 (24%) mice injected with GABAA-beta3 cDNA-transfected cells (P<0.005). The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA-beta3 receptor expression is down-regulated in human HCC, and (3) restoration of GABAA-beta3 receptor expression results in attenuated in vivo tumor growth in nude mice.

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

PubMed

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

2016-06-23

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

Effects of early overnutrition on the renal response to Ang II and expression of RAAS components in rat renal tissue.

PubMed

Granado, M; Amor, S; Fernández, N; Carreño-Tarragona, G; Iglesias-Cruz, M C; Martín-Carro, B; Monge, L; García-Villalón, A L

2017-10-01

The aim of this study was to analyze the effects of early overnutrition (EON) on the expression of the renin angiotensin aldosterone system (RAAS) components in renal cortex, renal arteries and renal perivascular adipose tissue (PVAT), as well as the vascular response of renal arteries to Angiotensin II (Ang II). On birth day litters were adjusted to twelve (L12-control) or three (L3-overfed) pups per mother. Half of the animals were sacrificed at weaning (21 days old) and the other half at 5 months of age. Ang II-induced vasoconstriction of renal artery segments increased in young overfed rats and decreased in adult overfed rats. EON decreased the gene expression of angiotensinogen (Agt), Ang II receptors AT1 and AT2 and eNOS in renal arteries of young rats, while it increased the mRNA levels of AT-2 and ET-1 in adult rats. In renal PVAT EON up-regulated the gene expression of COX-2 and TNF-α in young rats and the mRNA levels of renin receptor both in young and in adult rats. On the contrary, Ang II receptors mRNA levels were downregulated at both ages. Renal cortex of overfed rats showed increased gene expression of Agt in adult rats and of AT1 in young rats. However the mRNA levels of AT1 were decreased in the renal cortex of overfed adult rats. EON is associated with alterations in the vascular response of renal arteries to Ang II and changes in the gene expression of RAAS components in renal tissue. Copyright © 2017 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. 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.

Hepatic ontogeny and tissue distribution of mRNAs of epigenetic modifiers in mice using RNA-sequencing

PubMed Central

Lu, Hong; Cui, Julia; Gunewardena, Sumedha; Yoo, Byunggil; Zhong, Xiao-bo; Klaassen, Curtis

2012-01-01

Developmental regulation of gene expression is controlled by distinct epigenetic signatures catalyzed by various epigenetic modifiers. Little is known about the ontogeny and tissue distribution of these epigenetic modifiers. In the present study, we used a novel approach of RNA-sequencing to elucidate hepatic ontogeny and tissue distribution of mRNA expression of 142 epigenetic modifiers, including enzymes involved in DNA methylation/demethylation, histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation and chromosome remodeling factors in male C57BL/6 mice. Livers from male C57BL/6 mice were collected at 12 ages from prenatal to adulthood. Many of these epigenetic modifiers were expressed at much higher levels in perinatal livers than adult livers, such as Dnmt1, Dnmt3a, Dnmt3b, Apobec3, Kat1, Ncoa4, Setd8, Ash2l, Dot1l, Cbx1, Cbx3, Cbx5, Cbx6, Ezh2, Suz12, Eed, Suv39h1, Suv420h2, Dek, Hdac1, Hdac2, Hdac7, Kdm2b, Kdm5c, Kdm7, Prmt1–5, Prmt7, Smarca4, Smarcb1, Chd4 and Ino80e. In contrast, hepatic mRNA expression of a few epigenetic modifiers increased during postnatal liver development, such as Smarca2, Kdm1b, Cbx7 and Chd3. In adult mice (60 d of age), most epigenetic modifiers were expressed at moderately (1–3-fold) higher levels in kidney and/or small intestine than liver. In conclusion, this study, for the first time, unveils developmental changes in mRNA abundance of all major known epigenetic modifiers in mouse liver. These data suggest that ontogenic changes in mRNA expression of epigenetic modifiers may play important roles in determining the addition and/or removal of corresponding epigenetic signatures during liver development. PMID:22772165

Hepatic ontogeny and tissue distribution of mRNAs of epigenetic modifiers in mice using RNA-sequencing.

PubMed

Lu, Hong; Cui, Julia Yue; Gunewardena, Sumedha; Yoo, Byunggil; Zhong, Xiao-bo; Klaassen, Curtis D

2012-08-01

Developmental regulation of gene expression is controlled by distinct epigenetic signatures catalyzed by various epigenetic modifiers. Little is known about the ontogeny and tissue distribution of these epigenetic modifiers. In the present study, we used a novel approach of RNA-sequencing to elucidate hepatic ontogeny and tissue distribution of mRNA expression of 142 epigenetic modifiers, including enzymes involved in DNA methylation/demethylation, histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation and chromosome remodeling factors in male C57BL/6 mice. Livers from male C57BL/6 mice were collected at 12 ages from prenatal to adulthood. Many of these epigenetic modifiers were expressed at much higher levels in perinatal livers than adult livers, such as Dnmt1, Dnmt3a, Dnmt3b, Apobec3, Kat1, Ncoa4, Setd8, Ash2l, Dot1l, Cbx1, Cbx3, Cbx5, Cbx6, Ezh2, Suz12, Eed, Suv39h1, Suv420h2, Dek, Hdac1, Hdac2, Hdac7, Kdm2b, Kdm5c, Kdm7, Prmt1-5, Prmt7, Smarca4, Smarcb1, Chd4 and Ino80e. In contrast, hepatic mRNA expression of a few epigenetic modifiers increased during postnatal liver development, such as Smarca2, Kdm1b, Cbx7 and Chd3. In adult mice (60 d of age), most epigenetic modifiers were expressed at moderately (1-3-fold) higher levels in kidney and/or small intestine than liver. In conclusion, this study, for the first time, unveils developmental changes in mRNA abundance of all major known epigenetic modifiers in mouse liver. These data suggest that ontogenic changes in mRNA expression of epigenetic modifiers may play important roles in determining the addition and/or removal of corresponding epigenetic signatures during liver development.

Expression of Toll-like receptors, interleukin 8, macrophage migration inhibitory factor, and osteopontin in tissues from pigs challenged with Salmonella enterica serovar Typhimurium or serovar Choleraesuis.

PubMed

Burkey, T E; Skjolaas, K A; Dritz, S S; Minton, J E

2007-02-15

Two serovars of Salmonella enterica, namely serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for the vast majority of clinical cases of swine salmonellosis worldwide. These serovars are thought to be transmitted among pigs in production settings mainly through fecal-oral routes. Yet, few studies have evaluated effects of these serovars on expression of innate immune targets when presented to pigs via repeated oral dosing in an attempt to model transmission in production settings. Thus, a primary objective of the current experiments was to evaluate expression of Toll-like receptors (TLR) and selected chemoattractive mediators (interleukin 8, IL8; macrophage migration inhibitory factor, MIF; osteopontin, OPN) in tissues from pigs exposed to ST or SC that had been transformed with kanamycin resistance and green (STG) or red (SCR) fluorescent protein to facilitate isolation from pen fecal samples. In vitro studies confirmed that STG and SCR largely (though not completely) retained their ability to upregulate IL8 and CC chemokine ligand 20 (CCL20) in cultured swine jejunal epithelial cells. Transformed bacteria were then fed to pigs in an in vivo study to determine tissue specific effects on mRNA relative expression. Pigs were fed cookie dough inoculated with bacteria on days 0, 3, 7, and 10 with 10(8)CFU STG (n=8) or SCR (n=8), while control (CTL) pigs (n=8) received dough without bacteria. Animals were sacrificed 14 days from the initial bacterial challenge and samples of tonsil, jejunum, ileum, colon, mesenteric lymph node (MLN), spleen, and liver were removed for subsequent RNA isolation. Expression of mRNA in tissues was determined using real-time quantitative PCR and expressed relative to 18S rRNA. Within CTL pigs, when expressed relative to the content in liver, mRNA for all targets demonstrated substantial tissue effects (P<0.001 for all TLR; MIF, and OPN; P<0.05 for IL8). Feeding STG and SCR resulted in significant (P

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

PubMed

Wojciechowska, A; Mlynarczuk, J; Kotwica, J

2017-01-15

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

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.

Extracellular matrix metalloproteinase inducer (EMMPRIN) remodels the extracellular matrix through enhancing matrix metalloproteinases (MMPs) and inhibiting tissue inhibitors of MMPs expression in HPV-positive cervical cancer cells.

PubMed

Xu, Q; Cao, X; Pan, J; Ye, Y; Xie, Y; Ohara, N; Ji, H

2015-01-01

PUPOSE OF INVESTIGATION: To study the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), and tissue inhibitors of MMP (TIMPs) in uterine cervical cancer cell lines in vitro. EMMPRIN, MMPs, and TIMPs expression were assessed by Western blot and real-time RT-PCR from cervical carcinoma SiHa, HeLa, and C33-A cells. EMMPRIN recombinant significantly increased MMP-2, MMP-9 protein and mRNA expression in SiHa and Hela cells, but not in C33-A cells by Western blot analysis and real-time RT-PCR. EMMPRIN recombinant significantly inhibited TIMP-1 protein and mRNA levels in SiHa and Hela cells, but not in C33-A cells. There was no difference on the TIMP-2 expression in those cells with the treatment of EMMPRIN recombinant. EMMPRIN RNAi decreased MMP-2 and MMP-9 and increased TIMP-1 expression in SiHa and HeLa cells, but not in C33-A cells. There was no change on the expression of TIMP-2 mRNA levels in SiHa, HeLa and C33-A cells transfected with siEMMPRIN. EMMPRIN may induce MMP-2 and MMP-9, and downregulate TIMP-1 in HPV-positive cervical cancer cells in vitro.

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

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

Li Jinmei; Wang Xuefeng; Xi Zhiqin

2006-10-06

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

Evaluation of elevated dietary corn fiber from corn germ meal in growing female pigs.

PubMed

Weber, T E; Trabue, S L; Ziemer, C J; Kerr, B J

2010-01-01

To evaluate the effects of dietary hemicellulose from corn on growth and metabolic measures, female pigs (n = 48; initial BW 30.8 kg) were fed diets containing 0 to 38.6% solvent-extracted corn germ meal for 28 d. Increasing the hemicellulose level had no impact on ADG or ADFI, but resulted in a quadratic response (P < 0.03) on G:F. To investigate physiological changes that occur with increased dietary hemicellulose, blood, colon contents, and tissue samples from the liver and intestine were obtained from a subset (n = 16; 8 pigs/treatment) of pigs fed the least and greatest hemicellulose levels. The abundance of phospho-adenosine monophosphate-activated protein kinase (AMPK) and the mitochondrial respiratory protein, cytochrome C oxidase II (COXII) were determined in liver, jejunum, ileum, and colon by Western blotting. The mRNA expression levels of AMPKalpha1, AMPKalpha2, PPAR coactivator 1alpha (PGC1-alpha), PPARgamma2, and sirtuin 1 (Sirt1) were determined in liver and intestinal tissues. When compared with pigs fed the control diet, pigs fed the high hemicellulose diet had increased (P < 0.02) plasma triglycerides, but there was no difference in plasma cholesterol, glucose, or insulin. Absolute and relative liver weights were decreased (P < 0.03) in pigs consuming the high hemicellulose diet. The high-fiber diet led to a tendency (P < 0.12) for decreased liver triglyceride content. In pigs fed the high hemicellulose diet, ileal mucosal alkaline phosphatase activity was increased (P < 0.08) and sucrase activity tended (P < 0.12) to be increased. The high hemicellulose diet had no effect on phospho-AMPK, AMPK mRNA, or colonic VFA, but in pigs consuming the high fiber diet there was a greater (P < 0.05) abundance of COXII in colon tissue. The expression of PGC1-alpha, PPARgamma, or Sirt1 mRNA was not altered by dietary fiber in liver, jejunum, or ileum tissue. In colon tissue from pigs fed the high fiber diet there was an increase (P < 0.09) in Sirt1 mRNA and a trend (P < 0.12) toward increased of PGC1-alpha mRNA. These data suggest that alterations in metabolism involved in adaptation to a diet high in hemicellulose are associated with increased colonic Sirt1 mRNA and COXII expression, indicating an increased propensity for oxidative metabolism by the intestine.

RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site.

PubMed

Cheng, Feng; Pan, Ying; Lu, Yi-Min; Zhu, Lei; Chen, Shuzheng

2017-01-01

RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated ( p = 0.04). Patients with higher Dnd1 expression level had longer overall survival ( p = 0.0014) by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3'UTR, the stability of Bim-5'UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3'UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3'UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

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.

Small and intermediate conductance Ca(2+)-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum.

PubMed

Chen, Mao Xiang; Gorman, Shelby A; Benson, Bill; Singh, Kuljit; Hieble, J Paul; Michel, Martin C; Tate, Simon N; Trezise, Derek J

2004-06-01

The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.

[Abnormal expression of genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer].

PubMed

Kuznetsova, E S; Zinovieva, O L; Oparina, N Yu; Prokofjeva, M M; Spirin, P V; Favorskaya, I A; Zborovskaya, I B; Lisitsyn, N A; Prassolov, V S; Mashkova, T D

2016-01-01

Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.

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

PubMed

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

2015-09-01

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

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.

Stomatin-like protein 2 is overexpressed in epithelial ovarian cancer and predicts poor patient survival.

PubMed

Sun, Fei; Ding, Wen; He, Jie-Hua; Wang, Xiao-Jing; Ma, Ze-Biao; Li, Yan-Fang

2015-10-20

Stomatin-like protein 2 (SLP-2, also known as STOML2) is a stomatin homologue of uncertain function. SLP-2 overexpression has been suggested to be associated with cancer progression, resulting in adverse clinical outcomes in patients. Our study aim to investigate SLP-2 expression in epithelial ovarian cancer cells and its correlation with patient survival. SLP-2 mRNA and protein expression levels were analysed in five epithelial ovarian cancer cell lines and normal ovarian epithelial cells using real-time PCR and western blotting analysis. SLP-2 expression was investigated in eight matched-pair samples of epithelial ovarian cancer and adjacent noncancerous tissues from the same patients. Using immunohistochemistry, we examined the protein expression of paraffin-embedded specimens from 140 patients with epithelial ovarian cancer, 20 cases with borderline ovarian tumours, 20 cases with benign ovarian tumours, and 20 cases with normal ovarian tissues. Statistical analyses were applied to evaluate the clinicopathological significance of SLP-2 expression. SLP-2 mRNA and protein expression levels were significantly up-regulated in epithelial ovarian cancer cell lines and cancer tissues compared with normal ovarian epithelial cells and adjacent noncancerous ovarian tissues. Immunohistochemistry analysis revealed that the relative overexpression of SLP-2 was detected in 73.6 % (103/140) of the epithelial ovarian cancer specimens, 45.0 % (9/20) of the borderline ovarian specimens, 30.0 % (6/20) of the benign ovarian specimens and none of the normal ovarian specimens. SLP-2 protein expression in epithelial ovarian cancer was significantly correlated with the tumour stage (P < 0.001). Epithelial ovarian cancer patients with higher SLP-2 protein expression levels had shorter progress free survival and overall survival times compared to patients with lower SLP-2 protein expression levels. Multivariate analyses showed that SLP-2 expression levels were an independent prognostic factor for survival in epithelial ovarian cancer patients. SLP-2 mRNA and proteins were overexpressed in epithelial ovarian cancer tissues. SLP-2 protein overexpression was associated with advanced stage disease. Patients with higher SLP-2 protein expression had shorter progress free survival and poor overall survival times. Thus, SLP-2 protein expression was an independent prognostic factor for patients with epithelial ovarian cancer.

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

PubMed

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

2017-02-28

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

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

PubMed

Marchetto, G S; Henry, H L

1997-02-01

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

Histological change and heat shock protein 70 expression in different tissues of Japanese flounder Paralichthys olivaceus in response to elevated temperature

NASA Astrophysics Data System (ADS)

Liu, Yifan; Ma, Daoyuan; Xiao, Zhizhong; Xu, Shihong; Wang, Yanfeng; Wang, Yufu; Xiao, Yongshuang; Song, Zongcheng; Teng, Zhaojun; Liu, Qinghua; Li, Jun

2015-01-01

High temperature influences the homeostasis of fish. We investigated the effects of elevated temperature on tissues of Japanese flounder ( Paralichthys olivaceus) by analyzing the histology and heat shock protein 70 ( hsp70) expression of fish reared in warm conditions. In this study, temperature was increased at 1±0.5°C/day starting at 24±0.5°C, and was kept at that temperature for 5 days before the next rise. After raising temperature at the rate up to 32±0.5°C, tissue samples from midgut, spleen, stomach, liver, muscle, gill, heart, trunk kidney and brain were collected for histological analysis and mRNA assay. Almost all the tissues showed changes in morphological structure and hsp70 level at 32±0.5°C. Histological assessment of the tissues indicated that the gill had the most serious damage, including highly severe epithelial lifting and edema, curved tips and hyperemia at the ending of the lamellars, desquamation and necrosis. The next most severe damage was found in liver and kidney. The hsp70 levels in all the tissues first increased and then decreased. The gut, stomach, muscle, heart, and brain had the highest expressions in 6 h, whereas the spleen, liver, gill and kidney had the highest expressions in 2 h. Therefore, tissues with the most significant lesions (especially gill and liver) responded much earlier (2 h) in hsp70 expression than other tissues, and these tissues demonstrated the most marked histological disruption and elevated mRNA levels, making them ideal candidates for further studies on the thermal physiology of this species.

Neurotrophins and tonsillar hypertrophy in children with obstructive sleep apnea.

PubMed

Goldbart, Aviv D; Mager, Edward; Veling, Maria C; Goldman, Julie L; Kheirandish-Gozal, Leila; Serpero, Laura D; Piedimonte, Giovanni; Gozal, David

2007-10-01

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p<0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p<0.01), and substance P concentrations in OSA patients were higher than in RI (p<0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA.

Neurotrophins and Tonsillar Hypertrophy in Children With Obstructive Sleep Apnea

PubMed Central

GOLDBART, AVIV D.; MAGER, EDWARD; VELING, MARIA C.; GOLDMAN, JULIE L.; KHEIRANDISH-GOZAL, LEILA; SERPERO, LAURA D.; PIEDIMONTE, GIOVANNI; GOZAL, DAVID

2013-01-01

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p < 0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p < 0.01), and substance P concentrations in OSA patients were higher than in RI (p < 0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA. PMID:17667845

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

PubMed Central

2015-01-01

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

Integrated MicroRNA and mRNA Signatures Associated with Survival in Triple Negative Breast Cancer

PubMed Central

Lovat, Francesca; Carasi, Stefania; Pulvirenti, Alfredo; Ferro, Alfredo; Alder, Hansjuerg; He, Gang; Vecchione, Andrea; Croce, Carlo M.; Shapiro, Charles L.; Huebner, Kay

2013-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways. Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis. PMID:23405235

Integrated microRNA and mRNA signatures associated with survival in triple negative breast cancer.

PubMed

Cascione, Luciano; Gasparini, Pierluigi; Lovat, Francesca; Carasi, Stefania; Pulvirenti, Alfredo; Ferro, Alfredo; Alder, Hansjuerg; He, Gang; Vecchione, Andrea; Croce, Carlo M; Shapiro, Charles L; Huebner, Kay

2013-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways.Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis.

[Expressions of heat shock protein (HSP) family HSP 60, 70 and 90alpha in colorectal cancer tissues and their correlations to pathohistological characteristics].

PubMed

Zhang, Wen-Li; Gao, Xue-Qin; Han, Jin-Xiang; Wang, Guo-Qiang; Yue, Long-Tao

2009-06-01

Colorectal cancer is the third common malignant tumor in the world. Heat shock protein (HSP) family has been reported to play an important role in carcinogenesis of various cancers. However, little is known about expressions of HSP60,HSP70 and HSP90alpha in colorectal cancer. This study was to investigate expressions of HSP 60, 70 and 90alpha, and analyzed their correlations to pathohistologic characteristics in colorectal cancer. Colorectal cancer tissues and adjacent normal tissues 2 cm away from the tumor focus were collected from 49 patients. Expressions of HSP60, HSP70 and HSP90alpha mRNA were detected by RT-PCR. The protein expressions of HSP60, HSP70 and HSP90alpha were determined by immunohistochemistry and western blot. The mRNA and protein levels of HSP60, HSP70 and HSP90alpha, as well as their positive rates were significantly increased in tumor tissues compared with those in para-cancerous tissues. The overexpression rates of HSP60, HSP70 and HSP90alpha were also significantly higher in the colorectal cancer tissues than those in the corresponding para-cancerous tissues. The positive and overexpression rates of HSP60, HSP70 and HSP90alpha in well, moderately and poorly differentiated colorectal cancer were not significantly different. HSP60, HSP70 and HSP90alpha may play important roles in the pathogenesis of colorectal cancer, although they are not correlated with the pathological grading.

Identification of the collagen type 1 alpha 1 gene (COL1A1) as a candidate survival-related factor associated with hepatocellular carcinoma

PubMed Central

2014-01-01

Background Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death especially among Asian and African populations. It is urgent that we identify carcinogenesis-related genes to establish an innovative treatment strategy for this disease. Methods Triple-combination array analysis was performed using one pair each of HCC and noncancerous liver samples from a 68-year-old woman. This analysis consists of expression array, single nucleotide polymorphism array and methylation array. The gene encoding collagen type 1 alpha 1 (COL1A1) was identified and verified using HCC cell lines and 48 tissues from patients with primary HCC. Results Expression array revealed that COL1A1 gene expression was markedly decreased in tumor tissues (log2 ratio –1.1). The single nucleotide polymorphism array showed no chromosomal deletion in the locus of COL1A1. Importantly, the methylation value in the tumor tissue was higher (0.557) than that of the adjacent liver tissue (0.008). We verified that expression of this gene was suppressed by promoter methylation. Reactivation of COL1A1 expression by 5-aza-2′-deoxycytidine treatment was seen in HCC cell lines, and sequence analysis identified methylated CpG sites in the COL1A1 promoter region. Among 48 pairs of surgical specimens, 13 (27.1%) showed decreased COL1A1 mRNA expression in tumor sites. Among these 13 cases, 10 had promoter methylation at the tumor site. The log-rank test indicated that mRNA down-regulated tumors were significantly correlated with a poor overall survival rate (P = 0.013). Conclusions Triple-combination array analysis successfully identified COL1A1 as a candidate survival-related gene in HCCs. Epigenetic down-regulation of COL1A1 mRNA expression might have a role as a prognostic biomarker of HCC. PMID:24552139

[Tissue localization and expression difference of endogenous beta-glucosidase in digestive system of Musca domestica third instar larvae].

PubMed

Hu, Rong; Zhang, Shu; Wu, Jian-Wei; Guo, Guo; Fu, Ping

2013-08-01

To study the tissue localization and expression difference of endogenous beta-glucosidase in digestive system of Musca domestica third instar larvae. The digestive system of the 3rd instar larvae of Musca domestic was taken for the below tests. Tissue localization of endogenous beta-glucosidase mRNA was identified by in situ hybridization. Cellulase was localized by immunohistochemistry. The enzymatic activity of beta-glucosidase was measured by 3, 5-dinitrosalicylic acid(DNS) assay. The relative mRNA expression levels of M. domestica beta-glucosidase gene in these organs were determined by RT-PCR. Beta-glucosidase mRNA, with in situ hybridization, was shown in the epithelial cells of midgut, salivary glands and foregut of the larvae. The immunohistochemical analysis on larvae tissues revealed that cellulase was produced and secreted by the epithelial cells of the midgut, salivary glands and foregut. beta-glucosidase activity in salivary glands, foregut, midgut, and hindgut was (0.80 +/- 0.06), (0.38 +/- 0.02), (1.20 +/- 0.05) and (0.26 +/- 0.02) IU/mg, respectively. There was significant difference in beta-glucosidase activity among these digestive organs (P < 0.05). The activity level of beta-glucosidase was highest in midgut [(45.45 +/- 1.27)%], and lowest in hindgut [(9.85 +/- 0.88)%]. However, beta-glucosidase gene were only expressed in the salivary gland, foregut and midgut. Significant differences in gene expression level of beta-glucosidase was found among these organs (P < 0.05). The relative expression quantity of beta-glucosidase gene in midgut and salivary glands were 5 and 3 times higher than that in foregut. The endogenous beta-glucosidase gene is expressed in the foregut, midgut and salivary glands. The midgut and salivary glands of Musca domestica 3rd instar larvae are the primary organs of this enzyme secretion.

[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.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-01

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

Response of genes involved in lipid metabolism in rat epididymal white adipose tissue to different fasting conditions after long-term fructose consumption.

PubMed

Li, Jin-Xiu; Ke, Da-Zhi; Yao, Ling; Wang, Shang; Ma, Peng; Liu, Li; Zuo, Guo-Wei; Jiang, Li-Rong; Wang, Jian-Wei

2017-03-04

There has been much concern regarding the dietary fructose contributes to the development of metabolic syndrome. High-fructose diet changes the expression of genes involved in lipid metabolism. Levels of a number of hepatic lipogenic enzymes are increased by a high-carbohydrate diet in fasted-refed model rats/mice. Both the white adipose tissue (WAT) and the liver play a key role in the maintenance of nutrient homeostasis. Here, the aim of this study was to analyze the expression of key genes related to lipid metabolism in epididymal WAT (eWAT) in response to different fasting condition after long-term chronic fructose consumption. Rats were fed standard chow supplemented with 10% w/v fructose solution for 5 weeks, and killed after chow-fasting and fructose withdrawal (fasting) or chow-fasting and continued fructose (fructose alone) for 14 h. Blood parameters and the expression of genes involved in fatty acid synthesis (ChREBP, SREBP-1c, FAS, SCD1), triglyceride biosynthesis (DGAT-1, DGAT-2) and lipid mobilization (ATGL, HSL) in eWAT were analyzed. In addition, mRNA levels of PPAR-γ, CD36 and LPL were also detected. As expected, fructose alone increased the mRNA expression of FAS, SCD1, and correspondingly decreased ATGL and HSL mRNA levels. However, ChREBP, DGAT-2, ATGL and HSL mRNA levels restored near to normal while FAS and SCD1 tend to basic level under fasting condition. The mRNA expression of SREBP-1c, PPAR-γ and LPL did not changed at any situations but CD36 mRNA decreased remarkably in fructose alone group. In conclusion, these findings demonstrate that genes involved in lipid metabolism in rat eWAT are varied in response to different fasting conditions after long-term fructose consumption. Copyright © 2017. Published by Elsevier Inc.

Porcine calbindin-D9k gene: expression in endometrium, myometrium, and placenta in the absence of a functional estrogen response element in intron A.

PubMed

Krisinger, J; Jeung, E B; Simmen, R C; Leung, P C

1995-01-01

The expression of Calbindin-D9k (CaBP-9k) in the pig uterus and placenta was measured by Northern blot analysis and reverse transcription polymerase chain reaction (PCR), respectively. Progesterone (P4) administration to ovariectomized pigs decreased CaBP-9k mRNA levels. Expression of endometrial CaBP-9k mRNA was high on pregnancy Days 10-12 and below the detection limit on Days 15 and 18. On Day 60, expression could be detected at low levels. In myometrium and placenta, CaBP-9k mRNA expression was not detectable by Northern analysis using total RNA. Reverse-transcribed RNA from both tissues demonstrated the presence of CaBP-9k transcripts by means of PCR. The partial CaBP-9k gene was amplified by PCR and cloned to determine the sequence of intron A. In contrast to the rat CaBP-9k gene, the pig gene does not contain a functional estrogen response element (ERE) within this region. A similar ERE-like sequence located at the identical location was examined by gel retardation analysis and failed to bind the estradiol receptor. A similar disruption of this ERE-like sequence has been described in the human CaBP-9k gene, which is not expressed at any level in placenta, myometrium, or endometrium. It is concluded that the pig CaBP-9k gene is regulated in these reproductive tissues in a manner distinct from that in rat and human tissues. The regulation is probably due to a regulatory region outside of intron A, which in the rat gene contains the key cis element for uterine expression of the CaBP-9k gene.

Bexarotene, a Selective RXRα Agonist, Reverses Hypotension Associated with Inflammation and Tissue Injury in a Rat Model of Septic Shock.

PubMed

Tunctan, Bahar; Kucukkavruk, Sefika P; Temiz-Resitoglu, Meryem; Guden, Demet S; Sari, Ayse N; Sahan-Firat, Seyhan

2018-02-01

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that can activate or inhibit the expression of many target genes by forming a heterodimer complex with the retinoid X receptor (RXR). The aim of this study was to investigate effects of bexarotene, a selective RXRα agonist, on the changes in renal, cardiac, hepatic, and pulmonary expression/activity of inducible nitric oxide synthase (iNOS) and cytochrome P450 (CYP) 4F6 in relation to PPARα/β/γ-RXRα heterodimer formation in a rat model of septic shock. Rats were injected with dimethyl sulfoxide or bexarotene 1 h after administration of saline or lipopolysaccharide (LPS). Mean arterial pressure (MAP) and heart rate (HR) were recorded from rats, which had received either saline or LPS before and after 1, 2, 3, and 4 h. Serum iNOS, LTB 4 , myeloperoxidase (MPO), and lactate dehydrogenase (LDH) levels as well as tissue iNOS and CYP4F6 mRNA expression in addition to PPARα/β/γ and RXRα proteins were measured. LPS-induced decrease in MAP and increase in HR were associated with a decrease in PPARα/β/γ-RXRα heterodimer formation and CYP4F6 mRNA expression. LPS also caused an increase in systemic iNOS, LTB 4 , MPO, and LDH levels as well as iNOS mRNA expression. Bexarotene at 0.1 mg/kg (i.p.) prevented the LPS-induced changes, except tachycardia. The results suggest that increased formation of PPARα/β/γ-RXRα heterodimers and CYP4F6 expression/activity in addition to decreased iNOS expression contributes to the beneficial effect of bexarotene to prevent the hypotension associated with inflammation and tissue injury during rat endotoxemia.

Inhibitory effects of blockage of intermediate conductance Ca(2+)-activated K (+) channels on proliferation of hepatocellular carcinoma cells.

PubMed

Yang, Xiao-wei; Liu, Jin-wen; Zhang, Ru-chao; Yin, Qian; Shen, Wen-zhuang; Yi, Ji-lin

2013-02-01

The roles of intermediate conductance Ca(2+)-activated K(+) channel (IKCa1) in the pathogenesis of hepatocellular carcinoma (HCC) were investigated. Immunohistochemistry and Western blotting were used to detect the expression of IKCa1 protein in 50 HCC and 20 para-carcinoma tissue samples. Real-time PCR was used to detect the transcription level of IKCa1 mRNA in 13 HCC and 11 para-carcinoma tissue samples. The MTT assay was used to measure the function of IKCa1 in human HCC cell line HepG2 in vitro. TRAM-34, a specific blocker of IKCa1, was used to intervene with the function of IKCa1. As compared with para-carcinoma tissue, an over-expression of IKCa1 protein was detected in HCC tissue samples (P<0.05). The mRNA expression level of IKCa1 in HCC tissues was 2.17 times higher than that in para-carcinoma tissues. The proliferation of HepG2 cells was suppressed by TRAM-34 (0.5, 1.0, 2.0 and 4.0 μmol/L) in vitro (P<0.05). Our results suggested that IKCa1 may play a role in the proliferation of human HCC, and IKCa1 blockers may represent a potential therapeutic strategy for HCC.

Garlic Influences Gene Expression In Vivo and In Vitro.

PubMed

Charron, Craig S; Dawson, Harry D; Novotny, Janet A

2016-02-01

There is a large body of preclinical research aimed at understanding the roles of garlic and garlic-derived preparations in the promotion of human health. Most of this research has targeted the possible functions of garlic in maintaining cardiovascular health and in preventing and treating cancer. A wide range of outcome variables has been used to investigate the bioactivity of garlic, ranging from direct measures of health status such as cholesterol concentrations, blood pressure, and changes in tumor size and number, to molecular and biochemical measures such as mRNA gene expression, protein concentration, enzyme activity, and histone acetylation status. Determination of how garlic influences mRNA gene expression has proven to be a valuable approach to elucidating the mechanisms of garlic bioactivity. Preclinical studies investigating the health benefits of garlic far outnumber human studies and have made frequent use of mRNA gene expression measurement. There is an immediate need to understand mRNA gene expression in humans as well. Although safety and ethical constraints limit the types of available human tissue, peripheral whole blood is readily accessible, and measuring mRNA gene expression in whole blood may provide a unique window to understanding how garlic intake affects human health. © 2016 American Society for Nutrition.

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

PubMed

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

2016-11-01

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

Sodium/iodide symporter: a key transport system in thyroid cancer cell metabolism.

PubMed

Filetti, S; Bidart, J M; Arturi, F; Caillou, B; Russo, D; Schlumberger, M

1999-11-01

The recent cloning of the gene encoding the sodium/iodide symporter (NIS) has enabled better characterization of the molecular mechanisms underlying iodide transport, thus opening the way to clarifying its role in thyroid diseases. Several studies, at both the mRNA and the protein expression levels, have demonstrated that TSH, the primary regulator of iodide uptake, upregulates NIS gene expression and NIS protein abundance, both in vitro and in vivo. However, other factors, including iodide, retinoic acid, transforming growth factor-beta, interleukin-1alpha and tumour necrosis factor alpha, may participate in the regulation of NIS expression. Investigation of NIS mRNA expression in different thyroid tissues has revealed increased levels of expression in Graves' disease and toxic adenomas, whereas a reduction or loss of NIS transcript was detected in differentiated thyroid carcinomas, despite the expression of other specific thyroid markers. NIS mRNA was also detected in non-thyroid tissues able to concentrate radioiodine, including salivary glands, stomach, thymus and breast. The production of specific antibodies against the NIS has facilitated study of the expression of the symporter protein. Despite of the presence of high levels of human (h)NIS mRNA, normal thyroid glands exhibit a heterogeneous expression of NIS protein, limited to the basolateral membrane of the thyrocytes. By immunohistochemistry, staining of hNIS protein was stronger in Graves' and toxic adenomas and reduced in thyroid carcinomas. Measurement of iodide uptake by thyroid cancer cells is the cornerstone of the follow-up and treatment of patients with thyroid cancer. However, radioiodide uptake is found only in about 67% of patients with persistent or recurrent disease. Several studies have demonstrated a decrease in or a loss of NIS expression in primary human thyroid carcinomas, and immunohistochemical studies have confirmed this considerably decreased expression of the NIS protein in thyroid cancer tissues, suggesting that the low expression of NIS may represent an early abnormality in the pathway of thyroid cell transformation, rather than being a consequence of cancer progression. The relationship between radioiodine uptake and NIS expression by thyroid cancer cells require further study. New strategies, based on manipulation of NIS expression, to obtain NIS gene reactivation or for use as NIS gene therapy in the treatment of radiosensitive cancer, are also being investigated.

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.

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.

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

PubMed

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

2012-08-01

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

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.

A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

PubMed

Abraham, S; Solomon, W B

2000-09-19

We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin structure (ARC) are identical to portions of the deduced open reading frame of TIG-1 mRNA.

Increased Expression of Cathelicidin by Direct Activation of Protease-Activated Receptor 2: Possible Implications on the Pathogenesis of Rosacea

PubMed Central

Kim, Ji Young; Kim, Yoon Jee; Lim, Beom Jin; Sohn, Hyo Jung; Shin, Dongyun

2014-01-01

Purpose Recent findings of increased cathelicidin protein and its proteolytic fragments in rosacea suggest a pathogenic role for cathelicidin in this disease. The relationship between cathelicidin and protease-activated receptor 2 (PAR-2) is therefore of interest, as PAR-2, expressed principally in keratinocytes, regulates pro-inflammatory cytokine expression in the skin. The purpose of this study was to determine the relationship between expression of PAR-2 and cathelicidin in rosacea and to test the effect of direct PAR-2 activation on cathelicidin expression in keratinocytes. Materials and Methods Samples from 40 patients with clinicopathologic diagnosis of rosacea and facial skin tissue samples from 20 patients with no specific findings or milium without inflammation were retrieved. Intensities of immunohistochemical staining for PAR-2 and cathelicidin were compared between normal and rosacea-affected skin tissues. Additionally, correlations between PAR-2 and cathelicidin staining intensities within rosacea patients were analyzed. In cultured keratinocytes, changes in PAR-2, cathelicidin, and vascular endothelial growth factor (VEGF) mRNA and protein were analyzed after treatment with PAR-2 activating peptide (AP). Results Cathelicidin expression was significantly higher in rosacea skin tissues than in normal tissues (p<0.001), while PAR-2 expression was not significantly higher in rosacea tissues than in normal skin tissues. A positive correlation between PAR-2 and cathelicidin within rosacea samples was observed (R=0.330, p=0.037). After treatment of PAR-2 AP, both mRNA and protein levels for PAR-2, cathelicidin, and VEGF significantly increased in cultured keratinocytes, compared with PAR-2 control peptide treatment. Conclusion PAR-2 may participate in the pathogenesis of rosacea through activation of cathelicidin LL-37, a mediator of innate immune responses in the skin. PMID:25323904

Expression and localization of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) in human pancreas and pancreatic adenocarcinoma.

PubMed

Morales, Angélica; Vilchis, Felipe; Chávez, Bertha; Chan, Carlos; Robles-Díaz, Guillermo; Díaz-Sánchez, Vicente

2007-10-01

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) was recently identified as the first tissue-specific angiogenic molecule. EG-VEGF (the gene product of PROK-1) appears to be expressed exclusively in steroid-producing organs such as the ovary, testis, adrenals and placenta. Since the human pancreatic cells retain steroidogenic activity, in the present study we ascertained whether this angiogenic factor is expressed in normal pancreas and pancreatic adenocarcinoma. Tissue samples from normal males (n=5), normal females (n=5) and from surgically resected adenocarcinomas (n=2) were processed for RT-PCR and immunohistochemical studies. Results from semi-quantitative analysis by RT-PCR suggest a distinct expression level for EG-VEGF in the different tissue samples. The relative amount of EG-VEGF mRNA in pancreas was more abundant in female adenocarcinoma (0.89) followed by male adenocarcinoma (0.71), than normal female (0.64) and normal male (0.38). The expression of mRNA for EG-VEGF in normal tissue was significantly higher in females than in males. All samples examined showed specific immunostaining for EG-VEGF. In male preparations, the positive labeling was localized predominantly within the pancreatic islets while in female preparations the main staining was detected towards the exocrine portion. Specific immunolabeling was also observed in endothelial cells of pancreatic blood vessels. Our data provide evidence that the human pancreas expresses the EG-VEGF, a highly specific mitogen which regulates proliferation and differentiation of the vascular endothelium. The significance of this finding could be interpreted as either, EG-VEGF is not exclusive of endocrine organs, or the pancreas should be considered as a functional steroidogenic tissue. The extent of the expression of EG-VEGF appears to have a dimorphic pattern in normal and tumoral pancreatic tissue.

Cbl downregulation increases RBP4 expression in adipocytes of female mice

PubMed Central

Ameen, Gulizar Issa

2018-01-01

Obesity leads to adipose tissue dysfunction, insulin resistance and diabetes. Adipose tissue produces adipokines that contribute to regulate insulin sensitivity. In turn, insulin stimulates the production and release of some adipokines. Casitas-b-lymphoma proteins (c-Cbl, Cbl-b and Cbl3) are intracellular adaptor signalling proteins that are rapidly phosphorylated by activation of tyrosine kinase receptors. c-Cbl is rapidly phosphorylated by insulin in adipocytes. Here, we tested the hypothesis that Cbl signalling regulates adipokine expression in adipose tissue. We determined the adipokine profile of WAT of Cbl−/− and Cbl+/+ mice in the C57BL6 background. Female Cbl−/− mice exhibited altered expression of adiponectin, leptin and RBP4 in visceral adipose tissue, while no significant changes were seen in male mice. TNFα and IL6 levels were unaffected by Cbl depletion. RBP4 expression was unchanged in liver. Adipose tissue of Cbl−/− animals showed increased basal activation of extracellular regulated kinases (ERK1/2) compared to Cbl+/+. c-Cbl knockdown in 3T3L1 adipocytes also increased basal ERK phosphorylation and RBP4 expression. Inhibition of ERK1/2 phosphorylation in Cbl-depleted 3T3L1 adipocytes or in adipose tissue explants of Cbl−/− mice reduced RBP4 mRNA. 17β-Estradiol increased RBP4 mRNA in adipocytes. Cbl depletion did not change ER expression but increased phosphorylation of ERα at S118, a target site for ERK1/2. ERK1/2 inhibition reduced phosphoER and RBP4 levels. These findings suggest that Cbl contributes to regulate RBP4 expression in adipose of female mice through ERK1/2-mediated activation of ERα. Since Cbl signalling is compromised in diabetes, these data highlight a novel mechanism that upregulates RBP4 locally. PMID:29114012

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

PubMed

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

2003-07-01

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

Brown adipose tissue (BAT) specific vaspin expression is increased after obesogenic diets and cold exposure and linked to acute changes in DNA-methylation.

PubMed

Weiner, Juliane; Rohde, Kerstin; Krause, Kerstin; Zieger, Konstanze; Klöting, Nora; Kralisch, Susan; Kovacs, Peter; Stumvoll, Michael; Blüher, Matthias; Böttcher, Yvonne; Heiker, John T

2017-06-01

Several studies have demonstrated anti-diabetic and anti-obesogenic properties of visceral adipose tissue-derived serine protease inhibitor (vaspin) and so evoked its potential use for treatment of obesity-related diseases. The aim of the study was to unravel physiological regulators of vaspin expression and secretion with a particular focus on its role in brown adipose tissue (BAT) biology. We analyzed the effects of obesogenic diets and cold exposure on vaspin expression in liver and white and brown adipose tissue (AT) and plasma levels. Vaspin expression was analyzed in isolated white and brown adipocytes during adipogenesis and in response to adrenergic stimuli. DNA-methylation within the vaspin promoter was analyzed to investigate acute epigenetic changes after cold-exposure in BAT. Our results demonstrate a strong induction of vaspin mRNA and protein expression specifically in BAT of both cold-exposed and high-fat (HF) or high-sugar (HS) fed mice. While obesogenic diets also upregulated hepatic vaspin mRNA levels, cold exposure tended to increase vaspin gene expression of inguinal white adipose tissue (iWAT) depots. Concomitantly, vaspin plasma levels were decreased upon obesogenic or thermogenic triggers. Vaspin expression was increased during adipogenesis but unaffected by sympathetic activation in brown adipocytes. Analysis of vaspin promoter methylation in AT revealed lowest methylation levels in BAT, which were acutely reduced after cold exposure. Our data demonstrate a novel BAT-specific regulation of vaspin gene expression upon physiological stimuli in vivo with acute epigenetic changes that may contribute to cold-induced expression in BAT. We conclude that these findings indicate functional relevance and potentially beneficial effects of vaspin in BAT function.

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

PubMed Central

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

2013-01-01

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

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

PubMed

Joo, Jin Woo; Kim, Hyun-Soo; Do, Sung-Im; Sung, Ji-Youn

2018-05-01

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma.

PubMed

Zhu, Yi; Zhang, Jing-jing; Zhu, Rong; Zhu, Yan; Liang, Wen-biao; Gao, Wen-tao; Yu, Jun-bo; Xu, Ze-kuan; Miao, Yi

2011-12-01

The MUC4 gene could have a key role in the progression of pancreatic cancer, but the quantitative measurement of its expression in clinical tissue samples remains a challenge. The correlations between MUC4 promoter methylation status in vivo and either pancreatic cancer progression or MUC4 mRNA expression need to be demonstrated. We used the techniques of quantitative real-time PCR and DNA methylation-specific PCR combined microdissection to precisely detect MUC4 expression and promoter methylation status in 116 microdissected foci from 57 patients with pancreatic ductal adenocarcinoma. Both mRNA expression and hypomethylation frequency increased from normal to precancerous lesions to pancreatic cancer. Multivariate Cox regression analysis showed that high-level MUC4 expression (P = 0.008) and tumor-node-metastasis staging (P = 0.038) were significant independent risk factors for predicting the prognosis of 57 patients. The MUC4 mRNA expression was not significantly correlated with promoter methylation status in 30 foci of pancreatic ductal adenocarcinoma. These results suggest that high mRNA expression and hypomethylation of the MUC4 gene could be involved in carcinogenesis and in the malignant development of pancreatic ductal adenocarcinoma. The MUC4 mRNA expression may become a new prognostic marker for pancreatic cancer. Microdissection-based quantitative real-time PCR and methylation-specific PCR contribute to the quantitative detection of MUC4 expression in clinical samples and reflect the epigenetic regulatory mechanisms of MUC4 in vivo.

B7-H4 expression is correlated with tumor progression and clinical outcome in urothelial cell carcinoma.

PubMed

Fan, Min; Zhuang, Qianfeng; Chen, Yiming; Ding, Tao; Yao, Hongwei; Chen, Lujun; He, Xiaozhou; Xu, Xianlin

2014-01-01

To investigate the mRNA and protein levels of B7-H4, a B7 family molecule, in human urothelial cell carcinoma (UCC), to analyze the relationship between B7-H4 protein expression level and pathological stage of UCC, and to examine the potential of B7-H4 as a prognostic factor in UCC. mRNA and protein levels of B7-H4 were measured in pairs of tumor tissues and matched adjacent nontumor tissue obtained from patients with UCC by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical staining, respectively. Association of the protein level of B7-H4 with pathological tumor stage and the overall survival of UCC patients were also analyzed. B7-H4 mRNA and protein level were significantly higher in UCC tumor tissues compared with adjacent nontumor tissues as assessed by qRT-PCR and immunohistochemical staining, respectively. Higher B7-H4 protein levels were observed in patients with more advanced pathological stage of UCC and were also associated with decreased overall survival of patients with UCC. The findings from this study indicate that B7-H4 has the potential to be an independent prognostic indicator for UCC.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development.

PubMed

Rhen, Turk; Jangula, Adam; Schroeder, Anthony; Woodward-Bosh, Rikki

2009-05-01

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-alpha and PdgfR-beta in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-alpha mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-beta mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-alpha and PdgfR-beta in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development

PubMed Central

Rhen, Turk; Jangula, Adam; Schroeder, Anthony; Woodward-Bosh, Rikki

2009-01-01

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-α and PdgfR-β in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-α mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-β mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-α and PdgfR-β in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle. PMID:19523392

Further insights into the characterization of equine adipose tissue-derived mesenchymal stem cells.

PubMed

Raabe, Oksana; Shell, Katja; Würtz, Antonia; Reich, Christine Maria; Wenisch, Sabine; Arnhold, Stefan

2011-08-01

Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic lineages and peroxisome proliferator activated receptor (Pparγ2) mRNA was positive in adipogenic lineages. ADSCs show stem cell and surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs). The availability of an easily accessible and reproducible cell source may greatly facilitate the development of stem cell based tissue engineering and therapies for regenerative equine medicine.

The Accumulation of Versican in the Nodules of Benign Prostatic Hyperplasia

PubMed Central

True, Lawrence D.; Hawley, Sarah; Norwood, Thomas H.; Braun, Kathleen R.; Evanko, Stephen P.; Chan, Christina K.; LeBaron, Richard C.; Wight, Thomas N.

2014-01-01

Background Proteoglycans, a complex group of extracellular matrix (ECM) molecules, are elevated in benign prostatic hyperplasia (BPH). Versican is a stromal proteoglycan present in prostate tissue. Versican expression is elevated in tissues with increased proliferation. Based on these observations, we determined the extent and distribution of versican expression in prostates with BPH. Methods The involvement of versican in BPH nodules was compared with levels in non-nodular transition (TZ) and peripheral zone (PZ) tissues from 18 human prostate glands using immunohistochemistry, Northern blots and/or QRTPCR to localize versican and quantify versican mRNA transcript levels, and Western blots to assess gene product levels. Results Increased versican immunoreactivity was observed in the stroma of BPH nodules. Higher steady state levels of versican variants V0, V1, and V3 mRNA transcript and gene product were detected in the nodular tissues than in the non-nodular TZ or PZ parenchyma. Conclusions These results suggest that versican may play a role in nodule formation in BPH. PMID:18819099

Concerted elevation of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) activity through independent stimulation of mRNA expression of DGAT1 and DGAT2 by carbohydrate and insulin.

PubMed

Meegalla, Rupalie L; Billheimer, Jeffrey T; Cheng, Dong

2002-11-01

Glucose and insulin are anabolic signals which upregulate the transcriptions of a series of lipogenic enzymes to convert excess carbohydrate into triglycerides for efficient energy storage. These enzymes include ATP-citrate lyase (ACL), acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (G3PA). Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is important to synthesize fatty acids into triglycerides. Two DGATs from different gene families have recently been identified. In the current study, we report that glucose preferentially enhances DGAT1 mRNA expression, whereas insulin specifically increases the level of DGAT2 mRNA. Treatment of adipocytes with glucose and insulin together results in higher DGAT activity in the membrane than cells treated with either of the agents alone, indicating that glucose and insulin have additive effect on DGAT activation. In mice treated with fast/refeeding protocol, DGAT2 mRNA decreased upon fasting and was replenished upon refeeding in adipose tissue and liver. This pattern of change was not observed for DGAT1. Inasmuch as DGAT1 mRNA is less abundant in liver, we suggest that DGAT1 is more involved in fat absorption in the intestine and in basal level triglyceride synthesis in adipose tissue where it is more highly expressed. In contrast, DGAT2 is more likely to play important roles in assembly of de novo synthesized fatty acids into VLDL particles in the liver.

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

PubMed

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

2008-08-01

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

Expression of genes involved in carbohydrate-lipid metabolism in muscle and fat tissues in the initial stage of adult-age obesity in fed and fasted mice.

PubMed

Bazhan, Nadezhda M; Baklanov, Alexandr V; Piskunova, Julia V; Kazantseva, Antonina J; Makarova, Elena N

2017-10-01

C57Bl mice exhibit impaired glucose metabolism by the late adult age under standard living conditions. The aim of this study was to evaluate white adipose tissue (WAT), brown adipose tissue (BAT), and skeletal muscle expression of genes involved in carbohydrate-lipid metabolism at postpubertal stages preceding the late adult age in C57Bl mice. Muscle mRNA levels of uncoupling protein 3 ( Ucp3 ) and carnitine palmitoyltransferase 1 ( Cpt1 ) (indicators of FFA oxidation), WAT mRNA levels of hormone-sensitive lipase ( Lipe ) and lipoprotein lipase ( Lpl ) (indicators of lipolysis and lipogenesis), muscle and WAT mRNA levels of the type 4 glucose transporter Slc2a4 (indicators of insulin-dependent glucose uptake), and BAT mRNA levels of uncoupling protein 1 ( Ucp1 ) (indicator of thermogenesis) were measured in fed and 16 h-fasted mice in three age groups: 10-week-old (young), 15-week-old (early adult), and 30-week-old (late adult). Weight gain from young to early adult age was not accompanied by changes in WAT and BAT indexes and biochemical blood parameters. Weight gain from early to late adult age was accompanied by increased WAT and BAT indexes and decreased glucose tolerance. Muscle Ucp3 and Cpt1 mRNA levels and WAT Lipe and Slc2a4 mRNA levels increased from young to early adult age and then sharply decreased by the late adult age. Moreover, BAT Ucp1 mRNA level decreased in the late adult age. Fasting failed to increase muscle Cpt1 mRNA levels in late adult mice. These transcriptional changes could contribute to impaired glucose metabolism and the onset of obesity in late adult mice during normal development. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Protein Kinase A Regulatory Subunits in Human Adipose Tissue

PubMed Central

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

2009-01-01

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

Public data mining plus domestic experimental study defined involvement of the old-yet-uncharacterized gene matrix-remodeling associated 7 (MXRA7) in physiopathology of the eye.

PubMed

Jia, Changkai; Zhang, Feng; Zhu, Ying; Qi, Xia; Wang, Yiqiang

2017-10-20

Matrix-remodeling associated 7 (MXRA7) gene was first reported in 2002 and named so for its co-expression with several genes known to relate with matrix-remodeling. However, not any studies had been intentionally performed to characterize this gene. We started defining the functions of MXRA7 by integrating bioinformatics analysis and experimental study. Data mining of MXRA7 expression in BioGPS, Gene Expression Omnibus and EurExpress platforms highlighted high level expression of Mxra7 in murine ocular tissues. Real-time PCR was employed to measure Mxra7 mRNA in tissues of adult C57BL/6 mice and demonstrated that Mxra7 was preferentially expressed at higher level in retina, corneas and lens than in other tissues. Then the inflammatory corneal neovascularization (CorNV) model and fungal corneal infections were induced in Balb/c mice, and mRNA levels of Mxra7 as well as several matrix-remodeling related genes (Mmp3, Mmp13, Ecm1, Timp1) were monitored with RT-PCR. The results demonstrated a time-dependent Mxra7 under-expression pattern (U-shape curve along timeline), while all other matrix-remodeling related genes manifested an opposite changes pattern (dome-shape curve). When limited data from BioGPS concerning human MXRA7 gene expression in human tissues were looked at, it was found that ocular tissue was also the one expressing highest level of MXRA7. To conclude, integrative assay of MXRA7 gene expression in public databank as well as domestic animal models revealed a selective high expression MXRA7 in murine and human ocular tissues, and its change patterns in two corneal disease models implied that MXRA7 might play a role in pathological processes or diseases involving injury, neovascularization and would healing. Copyright © 2017 Elsevier B.V. All rights reserved.

Clinicopathological significance of SLP-2 overexpression in human gallbladder cancer.

PubMed

Wang, Wei-Xin; Lin, Qing-Feng; Shen, Dong; Liu, Shao-Ping; Mao, Wei-Dong; Ma, Gui; Qi, Wei-Dong

2014-01-01

Several studies have indicated that overexpression of stomatin-like protein 2 (SLP-2) has been identified in several types of cancer. However, its role and clinical relevance in gallbladder cancer (GBC) is unknown. The purpose of this study was to reveal the prognostic significance of SLP-2 in GBC. The SLP-2 expression was examined at mRNA and protein levels by real-time quantitative polymerase chain reaction (qRT-PCR), and immunohistochemistry in GBC tissues and adjacent noncancerous tissues. Statistical analyses were applied to test the associations between SLP-2 expression, clinicopathologic factors, and prognosis. Immunohistochemistry and qRT-PCR showed that the protein and mRNA expression levels of SLP-2 were both significantly higher in GBC tissues than in adjacent noncancerous tissues. In addition, immunohistochemistry analysis showed that SLP-2 expression was significantly correlated with histological grade (P <0.001), pathologic T stage (P = 0.019), clinical stage (P = 0.001), and lymph node metastasis (P = 0.026). The Kaplan-Meier survival curves indicated that patients with high expression of SLP-2 had shorter overall survival than those with low expression (P <0.001). Meanwhile, the Cox multivariate analysis indicated that high expressions of SLP-2 were an independent prognostic factor for patients with GBC. These data showed that SLP-2 may play an important role in human GBC tumorigenesis, and SLP-2 might serve as a novel prognostic marker in human GBC.

MicroRNA-300 targets hypoxia inducible factor-3 alpha to inhibit tumorigenesis of human non-small cell lung cancer.

PubMed

Zhang, Y; Guo, Y; Yang, C; Zhang, S; Zhu, X; Cao, L; Nie, W; Yu, H

2017-01-01

Non-small cell lung cancer (NSCLC) is one of the most deadly human cancers. MicroRNA-300 acts as both tumor promoter and suppressor in different types of cancer. Here, we try to identify the function of microRNA-300 in human NSCLC. We compared MicroRNA-300 levels between tumor tissues versus paired adjacent non-tumor lung tissues from NSCLC patients, and in NSCLC versus normal lung cell lines. Effects of microRNA-300 on cell proliferation, invasion and migration were examined in vitro, and on tumor growth in vivo using a xenograft mouse model. Potential mRNA targets of microRNA-300 were predicted and underlying mechanism was explored. MicroRNA-300 expression was lower in both NSCLC tissues and cell lines. Overexpression of microRNA-300 inhibited proliferation, invasion and migration of NSCLC cells in vitro, and tumor growth in vivo. MicroRNA-300 could directly bind to the 3'-UTR of hypoxia inducible factor-3 alpha (HIF3α) mRNA, and inhibit both its mRNA and protein expressions. Restoring HIF3α expression could rescue the inhibitory effects of microRNA-300 on tumorigenesis of NSCLC both in vitro and in vivo. MicroRNA-300 is a tumor suppressor microRNA in NSCLC by downregulating HIF3α expression. Both microRNA-300 and HIF3α may serve as potential therapeutic targets in NSCLC treatment.

Expression and effect of Calpain9 gene genetic polymorphism on slaughter indicators and intramuscular fat content in chickens.

PubMed

Cui, H X; Wang, S L; Guo, L P; Liu, L; Liu, R R; Li, Q H; Zheng, M Q; Zhao, G P; Wen, J

2018-06-26

Calpain 9 (CAPN9) is expressed in the stomach and small intestine. CAPN9 has regulatory roles in hypertension, heart disease, gastric mucosal defense, and kidney disease. The involvement of CAPN9 has not been reported in the development of chickens. CAPN9 mRNA was found in adipose and muscle tissue in this study. Two linkage single nucleotide polymorphisms (SNP; G7518A and C7542G) in intron 4 were screened from 160 birds of the D2 chicken line. The 2 mutation sites were associated with carcass weight, evisceration weight, abdominal fat weight (AFW), abdominal fat percentage (AFP), and breast muscle percentage (all P < 0.05). Intramuscular fat (IMF) content was not significantly different in the 3 genotypes. But, the AA(7518)/GG(7542) genotype had the highest IMF content, highest breast muscle weight, and lower AFW and AFP. Moreover, the mRNA level of CAPN9 in abdominal fat tissue was significantly different (P < 0.05 or P < 0.01) between any 2 genotypes, consistent with AFW and AFP. In summary, the expression of CAPN9 in adipose and breast muscle tissue is reported for the first time. CAPN9 affected production performance of chickens. As a marker, the linkage G7518A and C7542G polymorphisms in intron 4 of CAPN9 could affect the production traits by regulating mRNA expression. The findings concerning the marker enrich the theoretical foundation for molecular breeding of high-quality broilers.

Inactivation of bone morphogenetic protein 2 may predict clinical outcome and poor overall survival for renal cell carcinoma through epigenetic pathways

PubMed Central

Mitsui, Yozo; Hirata, Hiroshi; Arichi, Naoko; Hiraki, Miho; Yasumoto, Hiroaki; Chang, Inik; Fukuhara, Shinichiro; Yamamura, Soichiro; Shahryari, Varahram; Deng, Guoren; Saini, Sharanjot; Majid, Shahana; Dahiya, Rajvir; Tanaka, Yuichiro; Shiina, Hiroaki

2015-01-01

We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21WAF1/CIP1 and p27KIP1 expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2′-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC. PMID:25797254

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

PubMed Central

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

2012-01-01

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

Increased progesterone receptor expression in uterine leiomyoma: correlation with age, number of leiomyomas, and clinical symptoms.

PubMed

Tsigkou, Anastasia; Reis, Fernando M; Lee, Meng H; Jiang, Bingjie; Tosti, Claudia; Centini, Gabriele; Shen, Fang-Rong; Chen, You-Guo; Petraglia, Felice

2015-07-01

To investigate the possible correlation between progesterone receptor (PR) expression in uterine leiomyoma or adjacent myometrium and patient's age, size/number of leiomyomas, or clinical symptoms such as dysmenorrhea, acyclic pelvic pain, or menstrual and intermenstrual uterine bleeding. Cross-sectional study. Referral center. Sixty-two Chinese women undergoing elective hysterectomy for uterine leiomyomata. None. Evaluation of PR-total and PR-B mRNA with real-time polymerase chain reaction; PR-A and PR-B proteins quantified by Western blot in leiomyoma tissue and myometrium; symptoms rated by the patients using visual analog scores. The PR-B mRNA and PR-A and PR-B proteins were more concentrated in leiomyomas than in matched myometrium. A direct correlation between PR-B mRNA levels in leiomyoma and age (r = 0.347) and number of tumors (r = 0.295) was found. Conversely, there was an inverse correlation between PR-B mRNA levels in leiomyoma and dysmenorrhea (r = -0.260) and intermenstrual bleeding (r = -0.266). Multiple regression analysis indicated that age (β = 0.363) and the number of myomas (β = 0.296) were independently associated with PR-B mRNA levels in leiomyoma tissue. The levels of PR-B mRNA in leiomyoma tissue are directly associated with the number of tumors and inversely correlated with the intensity of intermenstrual bleeding and dysmenorrhea, suggesting that PR signaling may favor leiomyoma growth while attenuating clinical symptoms. This duality should be taken into account in the clinical management of patients with symptomatic uterine leiomyoma. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua.

PubMed

Kolkova, Z; Noskova, V; Ehinger, A; Hansson, S; Casslén, B

2010-10-01

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERβ mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

Altered JS-2 expression in colorectal cancers and its clinical pathological relevance.

PubMed

Lam, Alfred King-Yin; Gopalan, Vinod; Nassiri, Mohammad Reza; Kasim, Kais; Dissanayake, Jayampathy; Tang, Johnny Chuek-On; Smith, Robert Anthony

2011-10-01

JS-2 is a novel gene located at 5p15.2 and originally detected in primary oesophageal cancer. There is no study on the role of JS-2 in colorectal cancer. The aim of this study is to determine the gene copy number and expression of JS-2 in a large cohort of patients with colorectal tumours and correlate these to the clinicopathological features of the cancer patients. We evaluated the DNA copy number and mRNA expression of JS-2 in 176 colorectal tissues (116 adenocarcinomas, 30 adenomas and 30 non-neoplastic tissues) using real-time polymerase chain reaction. JS-2 expression was also evaluated in two colorectal cancer cell lines and a benign colorectal cell line. JS-2 amplification was noted in 35% of the colorectal adenocarcinomas. Significant differences in relative expression levels for JS-2 mRNA between different colorectal tissues were noted (p = 0.05). Distal colorectal adenocarcinoma had significantly higher copy number than proximal adenocarcinoma (p = 0.005). The relative expression level of JS-2 was different between colonic and rectal adenocarcinoma (p = 0.007). Mucinous adenocarcinoma showed higher JS-2 expression than non-mucinous adenocarcinoma (p = 0.02). Early T-stage cancers appear to have higher JS-2 copy number and lower expression of JS-2 mRNA than later stage cancers (p = 0.001 and 0.03 respectively). Colorectal cancer cell lines showed lower expression of JS-2 than the benign colorectal cell line. JS-2 copy number change and expression were shown for the first time to be altered in the carcinogenesis of colorectal cancer. In addition, genetic alteration of JS-2 was found to be related to location, pathological subtypes and staging of colorectal cancer. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Long-term culture of bovine nucleus pulposus explants in a native environment.

PubMed

van Dijk, Bart G M; Potier, Esther; Ito, Keita

2013-04-01

Chronic low back pain is a disease with tremendous financial and social implications, and it is often caused by intervertebral disc degeneration. Regenerative therapies for disc repair are promising treatments, but they need to be tested in physiological models. To develop a physiological in vitro explant model that incorporates the native environment of the intervertebral disc, for example, hypoxia, low glucose, and high tissue osmolarity. Bovine nucleus pulposus (NP) explants were cultured for 42 days in conditions mimicking the native physiological environment. Two different approaches were used to balance the swelling pressure of the NP: raised medium osmolarity or an artificial annulus. Bovine NP explants were either cultured in media with osmolarity balanced at isotonic and hypertonic levels compared with the native tissue or cultured inside a fiber jacket used as an artificial annulus. Oxygen and glucose levels were set at either standard (21% O2 and 4.5 g/L glucose) or physiological (5% O2 and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents and fixed charge density), tissue histology, cell viability, and cellular behavior with messenger RNA (mRNA) expression. Both the hypertonic culture and the artificial annulus approach maintained the tissue matrix composition for 42 days. At Day 3, mRNA expressions of aggrecan, collagen Type I, and collagen Type II in both hypertonic and artificial annulus cultures were not different from Day 0; however, at Day 42, the artificial annulus preserved the mRNA expression closer to Day 0. Gene expressions of matrix metalloprotease 13, tissue inhibitor of matrix metalloprotease 1, and tissue inhibitor of matrix metalloprotease 2 were downregulated under physiological O2 and glucose levels, whereas the other parameters analyzed were not affected. Although the hypertonic culture and the artificial annulus approach are both promising models to test regenerative therapies, the artificial annulus was better able to maintain a cellular behavior closer to the native tissue in longer term cultures. Copyright © 2013 Elsevier Inc. All rights reserved.

FTO regulates the chemo-radiotherapy resistance of cervical squamous cell carcinoma (CSCC) by targeting β-catenin through mRNA demethylation.

PubMed

Zhou, Shun; Bai, Zhou-Lan; Xia, Di; Zhao, Zhi-Jun; Zhao, Ren; Wang, Yan-Yang; Zhe, Hong

2018-05-01

The role of N 6 -methyladenosine (m 6 A) demethylase fat mass and obesity-associated protein (FTO) in the regulation of chemo-radiotherapy resistance remains largely unknown. Here, we show that the mRNA level of FTO is elevated in cervical squamous cell carcinoma (CSCC) tissues when compared with respective adjacent normal tissues. FTO enhances the chemo-radiotherapy resistance both in vitro and in vivo through regulating expression of β-catenin by reducing m 6 A levels in its mRNA transcripts and in turn increases excision repair cross-complementation group 1 (ERCC1) activity. Clinically, the prognostic value of FTO for overall survival is found to be dependent on β-catenin expression in human CSCC samples. Taken together, these findings uncover a critical function for FTO and its substrate m 6 A in the regulation of chemo-radiotherapy resistance, which may bear potential clinical implications for CSCC treatment. © 2018 Wiley Periodicals, Inc.

SASH1 regulates proliferation, apoptosis, and invasion of osteosarcoma cell.

PubMed

Meng, Qingbing; Zheng, Minqian; Liu, Hongbing; Song, Changzhi; Zhang, Wensheng; Yan, Juan; Qin, Ling; Liu, Xiaolan

2013-01-01

SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. The SASH1 protein possesses both the SH3 and SAM domains, indicating that it may play an important role in intracellular signal transduction. Reduced expression of SASH1 is closely related to tumor growth, invasion, metastasis, and poor prognosis. However, the biological role of SASH1 remains unknown in osteosarcoma. To unravel the function of SASH1, we explored the expression of SASH1 in osteosarcoma tissues and its correlation to the clinical pathology of osteosarcoma and analyzed the relationship between SASH1 expression and cell cycle, apoptosis and invasion of osteosarcoma MG-63 cells, using the flow cytometry analysis and transwell invasion chamber experiments. Furthermore, the effect of SASH1 on the expression of cyclin D1, caspase-3, matrix metalloproteinase (MMP)-9 were observed by western blot. Our results showed that the expression rate of SASH1 mRNA in osteosarcoma tissues was significantly lower than that in normal bone tissue (p = 0.000), that the expression rate of SASH1 mRNA in the carcinoma tissues from patients with lung metastasis was significantly lower than that from patients without lung metastasis (p = 0.041), and that the expression rate of SASH1 mRNA also decreased with increasing Enneking stage (p = 0.032). However, the mRNA expression of SASH1 in osteosarcoma was independent of the patient's gender, age, and tumor size (p = 0.983, 0.343, 0.517, respectively). The SASH1 protein displayed a down-regulation in osteosarcoma tissues compared to normal bone tissue (p = 0.000), displayed a down-regulation in osteosarcoma tissues from patients with lung metastasis compared to from patients without lung metastasis (p = 0.000), and displayed a gradual decrease with increasing Enneking stage (p = 0.000). In addition, the MG-63 cells from pcDNA3.1-SASH1 group exhibited significantly reduced cell viability, proliferation, and invasive ability compared to the empty vector group and blank control group (p = 0.023, 0.001, respectively), and there was no difference between the empty vector group and blank control group. The pcDNA3.1-SASH1 group displayed significantly more apoptotic cells than the empty vector group and blank control group (p = 0.004). The expression of cyclin D1, MMP-9 displayed a down-regulation in MG-63 cells from pcDNA3.1-SASH1 group compared to the empty vector group and blank control group (p = 0.000, 0.001, respectively) and the expression levels of caspase-3 displayed an up-regulation in MG-63 cells from pcDNA3.1-SASH1 group compared to the empty vector group and blank control group (p = 0.000). Taken together, these data indicated that the overexpression of SASH1 might be associated with the inhibition of growth, proliferation, and invasion of MG-63 cells and the promotion of apoptosis of MG-63 cells.

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

PubMed

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

2015-08-07

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