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

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

PubMed

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

1999-01-01

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

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

PubMed

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

2006-03-07

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

[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.

Decreased TIM-3 mRNA expression in peripheral blood mononuclear cells from nephropathy patients.

PubMed

Cai, X Z; Liu, N; Qiao, Y; Du, S Y; Chen, Y; Chen, D; Yu, S; Jiang, Y

2015-06-12

Increasing evidence shows that TIM-1 and TIM-3 in-fluence chronic autoimmune diseases, and their expression levels in immune cells from nephritic patients are still unknown. Real-time transcription-polymerase chain reaction analysis was used to deter-mine expression levels of TIM-1 and TIM-3 mRNA in peripheral blood mononuclear cells (PBMCs) from 36 patients with minimal change glo-merulopathy (MCG), 65 patients with lupus nephritis (LN), 78 patients with IgA nephropathy (IgAN), 55 patients with membranous nephropa-thy (MN), 22 patients with crescentic glomerulonephritis (CGN), 26 patients with anaphylactoid purpura nephritis (APN), and 63 healthy controls. TIM-3 mRNA expression significantly decreased in PBMCs from nephritic patients (LN, P < 0.0001; MCG, P < 0.0001; MN, P = 0.0031; CGN, P = 0.0464; IgAN, P = 0.0002; APN, P = 0.0392) com-pared with healthy controls. In contrast, there was no significant differ-ence in TIM-1 mRNA expression between the patients and the healthy controls. Our results suggest that insufficient expression of TIM-3 mRNA may be involved in the pathogenesis of nephropathy.

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.

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.

In situ hybridization reveals that type I and III collagens are produced by pericytes in the anterior pituitary gland of rats.

PubMed

Fujiwara, Ken; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

2010-12-01

Type I and III collagens widely occur in the rat anterior pituitary gland and are the main components of the extracellular matrix (ECM). Although ECM components possibly play an important role in the function of the anterior pituitary gland, little is known about collagen-producing cells. Type I collagen is a heterotrimer of two α1(I) chains (the product of the col1a1 gene) and one α2(I) chain (the product of the col1a2 gene). Type III collagen is a homotrimer of α1(III) chains (the product of the col3a1 gene). We used in situ hybridization with digoxigenin-labeled cRNA probes to examine the expression of col1a1, col1a2, and col3a1 mRNAs in the pituitary gland of adult rats. mRNA expression for these collagen genes was clearly observed, and cells expressing col1a1, col1a2, and col3a1 mRNA were located around capillaries in the gland. We also investigated the possible double-staining of collagen mRNA and pituitary hormones, S-100 protein (a marker of folliculo-stellate cells), or desmin (a marker of pericytes). Col1a1 and col3a1 mRNA were identified in desmin-immunopositive cells. Thus, only pericytes produce type I and III collagens in the rat anterior pituitary gland.

Protective Effect of Moderate Exercise for BALB/c Mice with Salmonella Typhimurium Infection.

PubMed

Campos-Rodríguez, R; Godínez-Victoria, M; Arciniega-Martínez, I M; Reséndiz-Albor, A A; Reyna-Garfias, H; Cruz-Hernández, T R; Drago-Serrano, M E

2016-01-01

Moderate exercise enhances resistance to pathogen-associated infections. However, its influence on intestinal IgA levels and resistance to Salmonella typhimurium in mice has not been reported. The aim of this study was to assess the impact of moderate exercise on bacterial resistance and the intestinal-IgA response in a murine typhoid model. Sedentary and exercised (under a protocol of moderate swimming) BALB/c mice were orally infected with Salmonella typhimurium and sacrificed on days 7 or 14 post-infection (n=5 per group). Compared with infected sedentary mice, infected exercised animals had i) lower intestinal and systemic bacterial loads; ii) higher total and specific intestinal-IgA levels, iii) a higher percentage of IgA plasma cells in lamina propria; iv) a higher level on day 7 and lower level on day 14 of intestinal α- and J-chain mRNA and plasma corticosterone, v) unchanged mRNA expression of intestinal pIgR, and vi) a higher mRNA expression of liver pIgR, α-chain and J-chain on day 7. Hence, it is likely that an increase in corticosterone levels (stress response) induced by moderate exercise increased intestinal IgA levels by enabling greater liver expression of pIgR mRNA, leading to a rise in IgA transcytosis from the liver to intestine. The overall effect of these changes is an enhanced resistance to infection. © Georg Thieme Verlag KG Stuttgart · New York.

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

PubMed

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

2018-01-08

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

Dynamic Changes of Smooth Muscle and Endothelial Markers in the Early Healing Process of Dacron Vascular Grafts in the Dog, Using RT-PCR.

PubMed

Ishida; Wu; Shi; Fujita; Sauvage; Hammond; Wijelath

2000-03-01

Previous studies of neointima formation on Dacron vascular grafts mainly focused on the late stages using immunohistochemistry staining for von Willebrand factor (vWF) and smooth muscle (SM) alpha-actin. However, it is impossible to use immunohistochemistry to study the early events of neointima formation, because graft samples lack sufficient cellular material. Therefore, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to demonstrate dynamic changes of SM and endothelial markers during the early stages of neointima formation. Preclotted Dacron grafts were implanted in the descending thoracic aorta of 14 mongrel dogs. Specimens were retrieved at 1-4 weeks. Total RNAs were extracted from mid-portion of graft flow surfaces, and RT-PCR for vWF, SM myosin heavy chain (MHC), and SM alpha-actin were performed and expressed as a ratio to the ribosome s17 signal. SM MHC and vWF mRNA expression was low at 1-2 weeks but elevated at 3-4 weeks (P < 0.05). However, SM alpha-actin mRNA levels were expressed consistently throughout the study period. At 3-4 weeks, vWF mRNA expression was inversely correlated to thrombus formation on the graft flow surface. Increased expressions of SM MHC and vWF mRNA corresponded to the formation of neointima and an endothelial layer at the later stages. However, SM alpha-actin mRNA expression did not vary during the healing process. The application of RT-PCR should permit further studies of gene regulation in the early vascular graft healing process in vivo. This model can also be used to study the molecular events that are involved in SM cell differentiation.

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

PubMed Central

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

1999-01-01

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

Association between EML4-ALK fusion gene and thymidylate synthase mRNA expression in non-small cell lung cancer tissues

PubMed Central

XU, CHUN-WEI; WANG, GANG; WANG, WU-LONG; GAO, WEN-BIN; HAN, CHUAN-JUN; GAO, JING-SHAN; ZHANG, LI-YING; LI, YANG; WANG, LIN; ZHANG, YU-PING; TIAN, YU-WANG; QI, DONG-DONG

2015-01-01

This study aimed to investigate the association of the mRNA expression of the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene with that of thymidylate synthase (TYMS) in non-small cell lung cancer (NSCLC) tissues. Quantitative polymerase chain reaction was used to detect the expression of EML4-ALK fusion gene and TYMS mRNA in 257 cases of NSCLC. The positive rate of EML4-ALK fusion gene was 4.28% in the NSCLC tissues (11/257), and was higher in nonsmokers than in smokers (P<0.05); TYMS mRNA expression was detected in 63.42% (163/257) of cases. An association of the EML4-ALK fusion gene with TYMS expression was detected; a low expression level of TYMS mRNA was observed more frequently when the EML4-ALK fusion gene was present than when it was not detected (P<0.05). In conclusion, patients positive for the EML4-ALK fusion gene in NSCLC tissues are likely to have a low expression level of TYMS, and may benefit from the first-line chemotherapy drug pemetrexed. PMID:26136951

Association between EML4-ALK fusion gene and thymidylate synthase mRNA expression in non-small cell lung cancer tissues.

PubMed

Xu, Chun-Wei; Wang, Gang; Wang, Wu-Long; Gao, Wen-Bin; Han, Chuan-Jun; Gao, Jing-Shan; Zhang, Li-Ying; Li, Yang; Wang, Lin; Zhang, Yu-Ping; Tian, Yu-Wang; Qi, Dong-Dong

2015-06-01

This study aimed to investigate the association of the mRNA expression of the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene with that of thymidylate synthase (TYMS) in non-small cell lung cancer (NSCLC) tissues. Quantitative polymerase chain reaction was used to detect the expression of EML4-ALK fusion gene and TYMS mRNA in 257 cases of NSCLC. The positive rate of EML4-ALK fusion gene was 4.28% in the NSCLC tissues (11/257), and was higher in nonsmokers than in smokers (P<0.05); TYMS mRNA expression was detected in 63.42% (163/257) of cases. An association of the EML4-ALK fusion gene with TYMS expression was detected; a low expression level of TYMS mRNA was observed more frequently when the EML4-ALK fusion gene was present than when it was not detected (P<0.05). In conclusion, patients positive for the EML4-ALK fusion gene in NSCLC tissues are likely to have a low expression level of TYMS, and may benefit from the first-line chemotherapy drug pemetrexed.

Upregulation of estrogen receptor subtypes and vitellogenin mRNA in cinnamon clownfish Amphiprion melanopus during the sex change process: profiles on effects of 17beta-estradiol.

PubMed

Kim, Na Na; Jin, Deuk-Hee; Lee, Jehee; Kil, Gyung-Suk; Choi, Cheol Young

2010-10-01

In the present study, we investigated the expression pattern of estrogen receptors (esr) and vitellogenin (vtg) mRNA in the gonads and liver during sex change in cinnamon clownfish by using quantitative polymerase chain reaction. We divided gonadal development during the sex change from male to female into 3 stages (mature male, male at 90days after removing female, and mature female) and investigated esr and vtg mRNA expressions during the sex change. With female, the esr and vtg mRNA expressions increased. In western blot analysis, Esr1 protein was detected only in the ovaries of female cinnamon clownfish. Also, to understand the effect of 17beta-estradiol (E(2)), we investigated the esr and vtg mRNA expression patterns in the gonads and liver, and the changes in plasma E(2) level after E(2) injection. E(2) treatment increased both mRNA expression levels of esr and vtg and plasma E(2) levels. The present study describes the molecular characterization of esr subtypes and the interactions between esr and vtg after E(2) treatment in cinnamon clownfish. 2010 Elsevier Inc. All rights reserved.

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.

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

PubMed

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

2016-06-12

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

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

PubMed

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

2012-08-29

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

THE 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.

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

PubMed

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

2011-07-01

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

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

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

PubMed

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

2005-01-01

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

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

PubMed Central

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

2017-01-01

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

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

PubMed

Fauser, S; Kern, P

1997-04-01

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

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

Prostaglandin E1 reduces the glomerular mRNA expression of monocyte-chemoattractant protein 1 in anti-thymocyte antibody-induced glomerular injury.

PubMed

Jocks, T; Zahner, G; Freudenberg, J; Wolf, G; Thaiss, F; Helmchen, U; Stahl, R A

1996-06-01

To study whether prostaglandins (PG) can regulate the mRNA expression of monocyte-chemoattractant protein 1 (MCP-1) in glomerular immune injury, MCP-1 mRNA levels were evaluated in anti-thymocyte antibody (ATS) -induced glomerular injury by Northern blotting and reverse transcription-polymerase chain reaction. Immune injury was induced in vivo by the intravenous application of ATS to male Wistar rats and in vitro by the perfusion of isolated rat kidneys with ATS and rat serum. In vivo 3 h and 5 days after antibody application, glomerular mRNA expression of MCP-1 was markedly enhanced compared with controls. In the isolated perfused kidney, antibody and complement also induced an increase in MCP-1 expression at 10 min and 60 min after antibody perfusion. When the rats were treated with PGE (250 micrograms, twice daily), the increase in MCP-1 expression was reduced. This was associated with a reduction of intraglomerular recruitment of monocytes/macrophages. In the isolated perfused kidneys, PGE1 (1 mg/L) prevented the antibody- and rat serum-stimulated increase in glomerular MCP-1 mRNA expression. These data demonstrate that PGE1 reduces glomerular MCP-1 mRNA expression in glomerulonephritis and in the isolated perfused rat kidney after induction of immune injury with antibody and complement. The data suggest that prostaglandins might mediate MCP-1 effects in glomerular immune injuries.

Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness.

PubMed

Gil, Fulvio R; Zitouni, Nedjma B; Azoulay, Eric; Maghni, Karim; Lauzon, Anne-Marie

2006-11-01

Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.

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.

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

PubMed Central

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

2000-01-01

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

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

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

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

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

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.

[Study of signal transduction pathway in the expression of inflammatory factors stimulated by lipopolysaccharides from Porphyromonas endodontalis in osteoblasts].

PubMed

Yang, Di; Qiu, Li-hong; Li, Ren; Li, Zi-mu; Li, Chen

2010-04-01

To quantify the interleukin (IL)-1beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides ([PS) extracted from Porphyromonoas endodontalis (P. endodontalis) in osteoblasts, and to relate P. endodontalis LPS to the bone resorptive pathogenesis in the lesions of chronic apical periodontitis. MG63 cells was pretreated with PD98059 or SB203580 for 1 h and then treated with P. endodontolis LPS for 6 h. The expression of IL-1beta mRNA and IL-6 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) technique. The production of IL-1beta mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with PD98059. Both of the production of IL-1beta mRNA and JL-6 mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with SB203580. The synthesis of IL-1beta mRNA stimulated by Pendodontalis LPS in MG63 probably occur via extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen activated protein kinase (MAPK) signal transduction system. The synthesis of IL-6 mRNA stimulated by P.endodontalis LPS in MG63 probahly occur via p38MAPK signal transduction system.

Effect of medium-chain triglycerides on calbindin-D9k expression in the intestine.

PubMed

Devlin, A; Innis, S M; Wall, K; Krisinger, J

1996-05-01

These studies determined the effect of the saturated fat source in infant formula on the expression of calbindin-D9k (CaBP-9k). Piglets were fed from birth to 8 d with milk or formula containing saturated fatty acids as medium-chain triglycerides (MCT), coconut oil, palm oil (Palm 1), or synthesized triglycerides with 16:0 directed to the sn-2 position (Palm 2). Levels of intestinal CaBP-9k mRNA were significantly (P < 0.01) higher in piglets fed formula with MCT than in piglets fed the other formula or milk; and higher in piglets fed the Palm-1 than in piglets fed Palm-2 formula. This is the first evidence that MCT alter piglet intestinal CaBP-9k mRNA.

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

PubMed

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

2015-06-01

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

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

PubMed

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

2008-10-01

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

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.

Effects of hypothermia and cerebral ischemia on cold-inducible RNA-binding protein mRNA expression in rat brain.

PubMed

Liu, Aijun; Zhang, Zhiwen; Li, Anmin; Xue, Jinghui

2010-08-06

CIRP (cold-inducible RNA-binding protein) mRNA is highly expressed in hypothermic conditions in mammalian cells, and the relationship between CIRP and neuroprotection for cerebral ischemia under hypothermia has been focused upon. At present, however, the expression characteristics of CIRP under hypothermia and cerebral ischemia in vivo are not clearly elucidated. In this study, CIRP mRNA expression in various regions of rat brain was examined by reverse transcriptase polymerase chain reaction (RT-PCR). CIRP expression levels were found to be similar in the hippocampus and cortex. Real-time quantitative PCR analysis revealed increasing CIRP mRNA expression in the cortex during the 24-h observation period following treatment with hypothermia or cerebral ischemia, with a greater increase in the hypothermia group. When cerebral ischemia was induced following hypothermia, CIRP mRNA expression in the cortex again showed a significant increasing tendency, but ischemia delayed the appearance of this increase. To reveal the relationship between CIRP and energy metabolism in the rat brain, lactate and pyruvate concentrations in the cortex of the rats treated with hypothermia, ischemia and ischemia after hypothermia were determined by spectrophotometric assay, and levels of phosphofructokinas-1 (PFK-1), the major regulatory enzyme of the glycolytic pathway, in the rat cortex in the three groups was also analyzed by Western blot. Using linear correlation, lactate and pyruvate concentrations, and PFK-1 levels, were each analyzed in the three groups in association with CIRP mRNA expression levels. The analysis did not reveal any correlation between the three metabolic parameters and CIRP mRNA expression induced by hypothermia, suggesting that while playing a role in neuroprotection under hypothermia, CIRP does not affect cerebral energy metabolism. Copyright 2010. Published by Elsevier B.V.

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.

[Houttuynia Cordata induces expression of human beta-defensin-2 mRNA in pulmonary epithelial cells in vitro].

PubMed

Luo, Li; Dong, Bi-rong; Teng, Li-hua

2008-07-01

To explore the effects of Houttuynia Cordata on expression of human beta-defensin-2 (HBD-2) in pulmonary epithelial cells (SPC-A-1) in vitro; and to observe the correlationship between the level of HBD-2 mRNA and the concentrations or treatment times of Houttuynia Cordata. The SPC-A-1 cells were cultured with different concentrations of Houttuynia Cordata in vitro, including 0, 12.5, 25, 50, 100 and 200 microg/ml. And then, the SPC-A-1 cells were cultured with the optimal concentration of Houttuynia Cordata in different lengths of time, including 1, 2, 4, 8, 16 and 24 hours. After the treatment, the mRNA level of HBD-2 in pulmonary epithelial cells was detected by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). After being cultured with Houttuynia Cordata, the expression of HBD-2 mRNA had positive correlation with the stimulus concentrations (rs=0.829, P=0.042) and stimulus time (rs=0.914, P=0.003). The highest expression of HBD-2 mRNA was induced by 100 microg/ml Houttuynia Cordata after 8-hour treatment. In comparison with the normal control group and the interleukin-1beta group, 100 microg/ml Houttuynia Cordata could significantly up-regulate the expression of HBD-2 mRNA in SPC-A-1 cells after 8-hour treatment (P<0.01). Houttuynia Cordata can up-regulate expression of HBD-2 mRNA in SPC-A-1 cells, and the highest expression level of HBD-2 mRNA can be obtained by culture with 100 microg/ml Houttuynia Cordata for 8 hours.

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

PubMed

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

2003-01-01

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

Expression profile and distribution of Efhc1 gene transcript during rodent brain development.

PubMed

Conte, Fábio F; Ribeiro, Patrícia A O; Marchesini, Rafael B; Pascoal, Vinícius D B; Silva, Joelcimar M; Oliveira, Amanda R; Gilioli, Rovílson; Sbragia, Lourenço; Bittencourt, Jackson C; Lopes-Cendes, Iscia

2009-09-01

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

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

PubMed

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

2012-11-15

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

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.

Induction of IgA B cell differentiation of bone marrow-derived B cells by Peyer's patch autoreactive helper T cells.

PubMed

Kihira, T; Kawanishi, H

1995-08-01

The objective of this study was to demonstrate in vitro that bone marrow-derived pro/pre-B cells bearing mu mRNA can switch their Ig heavy-chain isotype to that of alpha mRNA-expressing B cells after contact with Peyer's patches-derived activated autoreactive CD4+ T cells. Bone marrow-derived pro/pre-B cells and activated autoreactive Peyer's patch, mesenteric lymph node, or spleen CD4+ T cells were co-cultured in the presence of recombinant (r) IL-2, rIL-7, and Con A for 3 days. The mixed cultured cells were isolated for preparation of total RNA. Dot/slot hybridization, using murine C mu (pu3741) and C alpha (P alpha J558) Ig heavy-chain cDNA probes, detected C mu and C alpha Ig heavy-chain mRNA transcripts. The magnitude of each mRNA expression was measured demsitometrically. In addition, the secreted class-specific Ig contents from the co-cultured supernatants were measured. The results indicate that activated autoreactive Peyer's patch and mesenteric lymph node CD4+ T cells provide a specific Ig heavy-chain switch from mu to alpha (Peyer's patch CD4+ T cells > mesenteric lymph node CD4+ T cells) in bone marrow-derived pro/pre-B cells and also assist to develop IgA-secreting plasma cells. The alpha heavy-chain switch and IgA production do not occur in the presence of activated autoreactive spleen CD4+ T cells. These results support the view that autoreactive gut Peyer's patch CD4+ T cells, at least, regulate IgA B cell heavy-chain switching and terminal differentiation during gut mucosal B cell development.

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

PubMed

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

2011-03-01

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

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

PubMed

Chen, Yuanyuan; Shi, Xiaobo

2016-11-01

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

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

PubMed

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

2016-06-01

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

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

PubMed

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

2012-12-01

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

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

PubMed Central

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

2015-01-01

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

[Study on the inhibition effect of siRNA on herpes simplex virus type 2 ICP4 gene].

PubMed

Liu, Ji-feng; Guan, Cui-ping; Tang, Xu; Xu, Ai-e

2010-06-01

To explore the inhibition effect of RNA interference on the ICP4 expression and DNA replication of herpes simplex virus type 2 (HSV2). Four pairs of siRNA targeted to HSV2 ICP4 gene and negative control siRNA were synthetized by chemical method, named as siRNA-1, siRNA-2, siRNA-3, siRNA-4 and siRNA-N respecticely. HSV2 HG52 was used to attack Vero cell after transfection overnight. Vero cell and supernatant were collected at 1d, 2d, 3d, 4d and 5d after virus attacking. Flurogenic quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR)was used to detect the expression of HSV2 ICP4 mRNA, flurogenic quantitative polymerase chain reaction(FG-PCR) was used to detect the expression of HSV2 DNA and Western-Blot was used to detect the expression of HSV2 ICP4 protein. All the four pairs of siRNA could significantly inhibit the expression of HSV2 ICP4 mRNA and protein, especially siRNA-2. The above siRNAs could significantly decrease HSV2 DNA copy number,too. siRNAs targeted to HSV2 ICP4 gene could significantly inhibit expression of HSV2 ICP4 mRNA and protein, and decrease HSV2 DNA copy number, suggesting that siRNA can inhibit HSV2 DNA replication through silencing ICP4 gene.

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

PubMed

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

2017-08-02

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

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

PubMed

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

2018-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-05-01

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

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

PubMed

Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

PubMed

Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

2010-09-01

Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point after transfection. In addition, human VEGF gene transfection increased osteoblast cell proliferation after 3 days. These in vitro results suggest that cell-based human VEGF gene therapy is not only effective at causing human VEGF expression, but also enhances endogenous rat VEGF mRNA expression in both fibroblasts and osteoblasts, particularly the rat VEGF164 isoform.

Effects of Coriolus versicolor polysaccharide B on monocyte chemoattractant protein 1 gene expression in rat.

PubMed

Song, Lie-Chang; Chen, Hai-Sheng; Lou, Ning; Song, Chang; Zeng, Jun; Fu, Ting-Huan

2002-06-01

To investigate the effect of Coriolus versicolor polysaccharide B (CVPS-B), a new water-soluble component of polysaccharides from the fungus Coriolus versicolor (Fr) L on monocyte chemoattractant protein-1 (MCP-1) gene expression in rat splenocytes. Expression of MCP-1 mRNA in rat splenocytes was examined by reverse transcription-polymerase chain reaction (RT-PCR) with beta- actin as an internal standard. Sequencing of RT-PCR products was performed to confirm their specificity in MCP-1 gene composition. (1) Without pre-treatment of lipopolysaccharide (LPS), the relative MCP-1 mRNA expression ratios (MCP-1/beta-actin) for the saline control group and for CVPS-B groups in 3 different doses (10, 20, and 30 mg . kg-1 . d-1, ip, for 4 d) were 1.4 +/- 0.3, 1.6 +/- 0.4, 1.7 +/- 0.5, and 1.5 +/- 0.4, respectively (P > 0.05); (2) LPS (10 microg . kg-1, ip) enhanced the expression of MPC-1 mRNA by the ratio of 114 %; (3) pre-treatment with CVPS-B of 4 different doses (5, 10, 30, and 50 mg . kg-1 . d-1, ip, for 4 d) decreased the LPS induced expression of MPC-1 mRNA by the ratios of 51 %, 70 %, 84 %, and 99 %, respectively (n = 6). In a dose-related fashion, CVPS-B inhibited the expression of MCP-1 mRNA induced by LPS in the rat splenocytes, but did not significantly affect the expression of MPC-1 mRNA in the normal rat.

Expression of a serine protease (motopsin PRSS12) mRNA in the mouse brain: in situ hybridization histochemical study.

PubMed

Iijima, N; Tanaka, M; Mitsui, S; Yamamura, Y; Yamaguchi, N; Ibata, Y

1999-03-20

Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons. Copyright 1999 Elsevier Science B.V.

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

PubMed

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

2000-04-01

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

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

PubMed

Zheng, Jun; Takagi, Hiroyasu; Tsutsui, Chihiro; Adachi, Akihito; Sakai, Takafumi

2008-03-01

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

Effect of chronic alcohol consumption on Hepatic SIRT1 and PGC-1{alpha} in rats

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

Lieber, Charles S.; Department of Medicine, Mount Sinai School of Medicine, New York, NY; Leo, Maria A.

2008-05-23

The nuclear genes, NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-{gamma} coactivator1{alpha} (PGC-1{alpha}) are regulators of energy metabolism. Here, we studied the role of alcohol consumption in expression of these sensing molecules. Alcohol significantly reduced hepatic SIRT1 mRNA by 50% and PGC-1{alpha} mRNA by 46% and it significantly inhibited the protein expression of SIRT1 and PGC-1{alpha}, while the transcription factor PPAR-{gamma} remained unchanged. However, when the lipid composition of the alcohol diet was changed by replacing long-chain triglycerides (LCT) with medium chain triglycerides (MCT), SIRT1 and PGC-1{alpha} mRNA were restored to near control levels. This study demonstrates thatmore » alcohol reduces key energy sensing proteins and that replacement of LCT by MCT affects the transcription of these genes. Since there is a pathophysiological link between SIRT1 and PGC-1{alpha} and mitochondrial energy, the implication of the study is that mitochondrial dysfunction due to alcohol abuse can be treated by dietary modifications.« less

Molecular cloning and functional analysis of MRLC2 in Tianfu, Boer, and Chengdu Ma goats.

PubMed

Xu, H G; Xu, G Y; Wan, L; Ma, J

2013-03-15

To determine the molecular basis of heterosis in goats, fluorescence quantitative polymerase chain reaction (PCR) was performed to investigate myosin-regulatory light chain 2 (MRLC2) gene expression in the longissimus dorsi muscle tissues of the Tianfu goat and its parents, the Boer and Chengdu Ma goats. The goat MRLC2 gene was differentially expressed in the crossbreed, and the purebred mRNA were isolated and identified using fluorescence quantitative reverse transcription-PCR (RT-PCR). The complete coding sequence of MRLC2 was obtained using the cDNA method, and the full-length coding sequence consisted of 513 bp encoding 172 amino acids. The EF-hand superfamily domain of the MRLC2 protein is well conserved in caprine and other animals. The deduced amino acid sequence of MRLC2 shared significant identity with MRLC2 from other mammals. Phylogenetic tree analysis revealed that the MRLC2 protein was closely related to MRLC2 in other mammals. Several predicted miRNA target sites were found in the coding sequence of caprine MRLC2 mRNA. Analysis by RT-PCR showed that MRLC2 mRNA was present in the heart, stomach, liver, spleen, lung, small intestine, kidney, leg muscle, abdominal muscle, and longissimus dorsi muscles. In particular, the high expression of MRLC2 mRNA was detected in the longissimus dorsi, leg muscle, abdominal muscle, stomach, and heart, but low levels of expression were also observed in the liver, spleen, lung, small intestine, and kidney. The expression of the MRLC2 gene was upregulated in the longissimus dorsi muscle of Boer and Tianfu goats, and it was moderately upregulated in Chengdu Ma goats.

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

PubMed

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

2018-06-01

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

Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

PubMed Central

Guyon, T; Levasseur, P; Truffault, F; Cottin, C; Gaud, C; Berrih-Aknin, S

1994-01-01

Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss. Images PMID:8040257

Proportions of myosin heavy chain mRNAs, protein isoforms and fiber types in the slow and fast skeletal muscles are maintained after alterations of thyroid status in rats.

PubMed

Soukup, T; Diallo, M

2015-01-01

Recently, we have established that slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of euthyroid (EU) Lewis rats posses the same proportions between their four myosin heavy chain (MyHC) mRNAs, protein isoforms and fiber types as determined by real time RT-PCR, SDS-PAGE and 2-D stereological fiber type analysis, respectively. In the present paper we investigated if these proportions are maintained in adult Lewis rats with hyperthyroid (HT) and hypothyroid (HY) status. Although HT and HY states change MyHC isoform expression, results from all three methods showed that proportion between MyHC mRNA-1, 2a, -2x/d, -2b, protein isoforms MyHC-1, -2a, -2x/d, -2b and to lesser extent also fiber types 1, 2A, 2X/D, 2B were preserved in both SOL and EDL muscles. Furthermore, in the SOL muscle mRNA expression of slow MyHC-1 remained up to three orders higher compared to fast MyHC transcripts, which explains the predominance of MyHC-1 isoform and fiber type 1 even in HT rats. Although HT status led in the SOL to increased expression of MyHC-2a mRNA, MyHC-2a isoform and 2A fibers, it preserved extremely low expression of MyHC-2x and -2b mRNA and protein isoforms, which explains the absence of pure 2X/D and 2B fibers. HY status, on the other hand, almost completely abolished expression of all three fast MyHC mRNAs, MyHC protein isoforms and fast fiber types in the SOL muscle. Our data present evidence that a correlation between mRNA, protein content and fiber type composition found in EU status is also preserved in HT and HY rats.

Impairment of Hepcidin Upregulation by Lipopolysaccharide in the Interleukin-6 Knockout Mouse Brain.

PubMed

Zhang, Fa-Li; Hou, Hui-Min; Yin, Zhi-Nan; Chang, Lan; Li, Fe-Mi; Chen, Y-J; Ke, Ya; Qian, Zhong-Ming

2017-01-01

To find out whether the Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in the expression of hepcidin in the mouse brain in vivo , we investigated the phosphorylation of STAT3, as well as the expression of hepcidin mRNA, ferroportin 1 (Fpn1) and ferritin light chain (Ft-L) proteins in the cortex and hippocampus of LPS-treated wild type (IL-6+/+) and IL-6 knockout (IL-6-/-) mice. We demonstrated that IL-6 knockout could significantly reduce the response of hepcidin mRNA, phospho-STAT3, Fpn1 and Ft-L protein expression to LPS treatment, in both the cortex and hippocampus of mice. Also, Stattic, an inhibitor of STAT3, significantly reduced the expression of phospho-STAT3 and hepcidin mRNA in the cortex and hippocampus of the LPS-treated wild type mice. These findings provide in vivo evidence for the involvement of the IL-6/STAT3 signaling pathway in the expression of hepcidin.

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

PubMed

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

2015-07-01

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

[Effect of qihuang decoction on mRNA expressions of Bcl-2, Bax, and Caspase-3, 9 in intestinal mucosa epithelium of ischemia/reperfusion injured rats].

PubMed

Yu, Qing-Sheng; Yu, Hong-Liang; Pan, Jin-Fang

2011-02-01

To observe the effect of Qihuang Decoction (QHD) on mRNA expression of apoptosis genes Bcl-2, Bax, and signal transduction molecules Caspase-3, 9 in intestinal mucosa epithelium of ischemia/ reperfusion (I/R) injured rats. Forty Wistar rats were randomized equally into 4 groups, the control group, the model group, the glutamine group, and the QHD group. Rats in the latter two groups were gastric infused with glutamine and QHD respectively for 3 days, but saline was infused instead to rats in the control group and model group. After then, except those in the control group intervened only by sham operation, rats were made into I/R injured model by 45 min occlusion of superior mesenteric artery followed by 1 h reperfusion. Immediately after modeling, mRNA expressions of Bcl-2, Bax, Caspase-3, and Caspase-9 in intestinal mucosa epithelium of rats were detected by reverse transcription-polymerase chain reaction (RT-PCR). Compared with the control group, mRNA expressions of Bcl-2, Bax, Caspase-3 and Caspase-9 were higher in the other three groups (P < 0.05). Compared with the model group, Bcl-2 mRNA expression was higher, while the expressions of the other three indices were lower in both the glutamine group and the QHD group (P < 0.05); and comparisons between the glutamine group and the QHD group showed a more depressed Bax mRNA expression (0.281 +/- 0.087 vs 0.350 +/- 0.053) and higher Bcl-2/Bax ratio (1.648 vs 1. 374) in the QHD group. QHD can reduce the I/R injury in the intestinal mucosa epithelium by inhibiting the cell apoptosis. The mechanism may be correlated with increased Bcl-2 mRNA expressions and decreased mRNA expressions of Bax, Caspase-3 and Caspase-9.

Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics

PubMed Central

Velazquez, J R; Lacy, P; Moqbel, R

2000-01-01

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins. PMID:10792507

Sodium lauryl sulphate alters the mRNA expression of lipid-metabolizing enzymes and PPAR signalling in normal human skin in vivo.

PubMed

Törmä, Hans; Berne, Berit

2009-12-01

Detergents irritate skin and affect skin barrier homeostasis. In this study, healthy skin was exposed to 1% sodium lauryl sulphate (SLS) in water for 24 h. Biopsies were taken 6 h to 8 days post exposure. Lipid patterns were stained in situ and real-time polymerase chain reaction (PCR) was used to examine mRNA expression of enzymes synthesizing barrier lipids, peroxisome proliferator-activated receptors (PPAR) and lipoxygenases. The lipid pattern was disorganized from 6 h to 3 days after SLS exposure. Concomitant changes in mRNA expression included: (i) reduction, followed by induction, of ceramide-generating beta-glucocerebrosidase, (ii) increase on day 1 of two other enzymes for ceramide biosynthesis and (iii) persistent reduction of acetyl-CoA carboxylase-B, a key enzyme in fatty acid synthesis. Surprisingly, the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, was unaltered. Among putative regulators of barrier lipids synthesis, PPARalpha and PPARgamma exhibited reduced mRNA expression, while PPARbeta/delta and LXRbeta were unaltered. Epidermal lipoxygenase-3, which may generate PPARalpha agonists, exhibited reduced expression. In conclusion, SLS induces reorganization of lipids in the stratum corneum, which play a role in detergents' destruction of the barrier. The changes in mRNA expression of enzymes involved in synthesizing barrier lipids are probably important for the restoration of the barrier.

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.

Coordinate tropic hormone regulation of mRNAs for insulin-like growth factor II and the cholesterol side-chain-cleavage enzyme, P450scc [corrected], in human steroidogenic tissues.

PubMed Central

Voutilainen, R; Miller, W L

1987-01-01

Insulin-like growth factors (IGFs) are single-chain polypeptides important for cell proliferation and growth. IGFs are produced in several tissues, suggesting that they function in a paracrine or autocrine fashion as well as functioning as endocrine hormones. We studied the hormonal regulation of IGF-I and IGF-II mRNA in human steroidogenic tissues. In cultured human ovarian granulosa cells, follicle-stimulating hormone, human chorionic gonadotropin, and dibutyryl cAMP increased IGF-II mRNA, but corticotropin [adrenocorticotropic hormone (ACTH)], chorionic somatomammotropin, growth hormone, prolactin, dexamethasone, estradiol, and progesterone had no effect. In cultured human fetal adrenal cells, ACTH and dibutyryl cAMP increased IGF-II mRNA accumulation, but human chorionic gonadotropin and angiotensin II did not. The same five size species of IGF-II mRNA were detected in transfer blots of RNA from granulosa cells and fetal adrenal cells, and all of these increased after hormonal stimuli. Dibutyryl cAMP also increased IGF-II mRNA accumulation in cultured human placental cells. Accumulation of mRNA for the cholesterol side-chain-cleavage monooxygenase [P450scc [corrected]; cholesterol, reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.6] was regulated in parallel with IGF-II mRNA in all these steroidogenic tissues. IGF-I mRNA was not detected in transfer blots of these RNAs, and the minimal amounts detected in dot blots showed no detectable change after any of the hormonal stimuli studied. The data indicate that the IGF-II gene is expressed in human steroidogenic tissues and is regulated by cAMP. These data suggest that IGF-II may act in an autocrine or paracrine fashion to stimulate the adrenal and gonadal growth stimulated by ACTH and gonadotropins, respectively. Images PMID:3031644

Downregulation in GATA4 and Downstream Structural and Contractile Genes in the db/db Mouse Heart

PubMed Central

Broderick, Tom L.; Jankowski, Marek; Wang, Donghao; Danalache, Bogdan A.; Parrott, Cassandra R.; Gutkowska, Jolanta

2012-01-01

Reduced expression of GATA4, a transcriptional factor for structural and cardioprotective genes, has been proposed as a factor contributing to the development of cardiomyopathy. We investigated whether the reduction of cardiac GATA4 expression reported in diabetes alters the expression of downstream genes, namely, atrial natriuretic peptide (ANP), B-type natriuretic, peptide (BNP), and α- and β-myosin heavy chain (MHC). db/db mice, a model of type 2 diabetes, with lean littermates serving as controls, were studied. db/db mice exhibited obesity, hyperglycemia, and reduced protein expression of cardiac GLUT4 and IRAP (insulin-regulated aminopeptidase), the structural protein cosecreted with GLUT4. Hearts from db/db mice had reduced protein expression of GATA4 (~35%) with accompanying reductions in mRNA expression of ANP (~40%), BNP (~85%), and α-MHC mRNA (~50%) whereas expression of β-MHC mRNA was increased by ~60%. Low GATA4 was not explained by an increased ligase or atrogin1 expression. CHIP protein content was modestly downregulated (27%) in db/db mice whereas mRNA and protein expression of the CHIP cochaperone HSP70 was significantly decreased in db/db hearts. Our results indicate that low GATA4 in db/db mouse heart is accompanied by reduced expression of GATA4-regulated cardioprotective and structural genes, which may explain the development of cardiomyopathy in diabetes. PMID:22474596

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

PubMed

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

2016-04-01

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

Influence of cyclic hydrostatic pressure on fibrocartilaginous metaplasia of achilles tendon fibroblasts.

PubMed

Shim, J W; Elder, S H

2006-11-01

The goal of this study was to demonstrate whether cyclically imposed hydrostatic pressure, compressive in nature, could induce fibrocartilaginous metaplasia in a purely tendinous cell source in vitro. The effect of short-duration cyclic hydrostatic pressure on tendon fibroblasts (tenocytes) expanded from rat Achilles tendon was studied. Total RNA was isolated either immediately after loading or 24 h later. The mRNA expression of tendon and cartilage specific markers - Collagen types I and II, Sox9, and Aggrecan was quantified by real-time reverse transcription polymerase chain reaction over multiple biological samples (n=6). For immediately isolated RNA samples, there were statistically significant increases in mRNA expression of Aggrecan and Collagen type II, while Collagen type I significantly decreased. Noticeably, for RNA samples isolated 24 h later, there were further increases in mRNA expression of Aggrecan and Collagen type II, whereas Collagen type I increased roughly three-fold relative to the non-loaded control. These findings support the hypothesis that cyclic hydrostatic pressurization can induce fibrocartilaginous metaplasia in tenocytes by upregulation of cartilaginous gene expression. Also, it was demonstrated that changes in mRNA expression as a result of single 2 h pressurization persist even up to 24 h.

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

PubMed

Yasuda, Yuka; Hashimoto, Ryota; Yamamori, Hidenaga; Ohi, Kazutaka; Fukumoto, Motoyuki; Umeda-Yano, Satomi; Mohri, Ikuko; Ito, Akira; Taniike, Masako; Takeda, Masatoshi

2011-05-26

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

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

PubMed

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

2014-04-01

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

Expression of small leucine-rich proteoglycans in rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Syaidah, Rahimi; Fujiwara, Ken; Tsukada, Takehiro; Ramadhani, Dini; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

2013-01-01

Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

Effects of castration on expression of lipid metabolism genes in the liver of korean cattle.

PubMed

Baik, Myunggi; Nguyen, Trang Hoa; Jeong, Jin Young; Piao, Min Yu; Kang, Hyeok Joong

2015-01-01

Castration induces the accumulation of body fat and deposition of intramuscular fat in Korean cattle, resulting in improved beef quality. However, little is known about the metabolic adaptations in the liver following castration. To understand changes in lipid metabolism following castration, hepatic expression levels of lipid metabolism genes were compared between Korean bulls and steers. Steers had higher (p<0.001) hepatic lipids contents and higher (p<0.01) mRNA levels of lipogenic acetyl-CoA carboxylase. This differential gene expression may, in part, contribute to increased hepatic lipid content following the castration of bulls. However, we found no differences in the hepatic expression levels of genes related to triglyceride synthesis (mitochondrial glycerol-3-phosphate acyltransferase, diacylglycerol O-acyltransferase 1 and 2) and fatty acid (FA) oxidation (carnitine palmitoyltransferase 1A, C-4 to C-12 straight chain acyl-CoA dehydrogenase, very long chain acyl-CoA dehydrogenase) between bulls and steers. No differences in gene expression for very-low-density lipoprotein (VLDL) secretion, including apolipoprotein B mRNA and microsomal triglyceride transfer protein (MTTP) protein, were observed in the liver although MTTP mRNA levels were higher in steers compared to bulls. In conclusion, FA synthesis may contribute to increased hepatic lipid deposition in steers following castration. However, hepatic lipid metabolism, including triglyceride synthesis, FA oxidation, and VLDL secretion, was not significantly altered by castration. Our results suggest that hepatic lipid metabolism does not significantly contribute to increased body fat deposition in steers following castration.

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

PubMed

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

2004-05-01

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

Promoter hypermethylation of the RECK gene is associated with its low expression and poor survival of esophageal squamous cell carcinoma

PubMed Central

Zhu, Jing; Ling, Yang; Xu, Yun; Lu, Mingzhu; Liu, Yongping; Zhang, Changsong

2017-01-01

The present study aimed to investigate the association between the methylation status of the reversion-inducing cysteine-rich protein with kazal motifs (RECK) gene and its mRNA expression levels in patients with esophageal squamous cell carcinoma (ESCC). The methylation status of RECK was analyzed by methylation-specific polymerase chain reaction (PCR), and RECK mRNA expression levels were analyzed by quantitative PCR, in 310 paired ESCC tissues. The mean RECK methylation index (MI) was 0.65 in ESCCs and 0.49 in non-tumor samples. There was a significant association between RECK methylation and the American Joint Committee on Cancer stage and lymph node metastasis in ESCC (P<0.0001; P=0.001). The mRNA expression level of RECK was lower in ESCC tissues (mean-∆Cq=−4.66) compared with non-tumor tissues (mean-∆Cq=−2.79), and decreased RECK mRNA expression levels were associated with lymph node metastasis in ESCC. In addition, RECK mRNA levels were decreased in ESCC patients with hypermethylation of the RECK gene (∆MI >0.16; mean-∆∆Cq=−2.85) compared with those with hypomethylation of the RECK gene (∆MI ≤0.16; mean-∆∆Ct=−0.83), and there was a significant difference in the mRNA expression levels of RECK between those with N0–1 and N2–3 lymph node metastasis (P<0.0001). A significant correlation was observed between RECK mRNA expression levels, the MI of RECK and poor postoperative survival (P=0.0003; P<0.0001). The results of the present study suggested that promoter hypermethylation may be an important factor for loss of RECK mRNA expression and may be an indicator of poor survival in ESCC. PMID:28454343

Rapid switch-off of the human myosin heavy chain IIX gene after heavy load muscle contractions is sustained for at least four days.

PubMed

Andersen, J L; Gruschy-Knudsen, T

2018-02-01

Long-term heavy load contractions decrease the relative amount of the myosin heavy chain (MHC) IIX isoform in human skeletal muscle, but the timing of the down-regulation in the short term is unknown. Untrained subjects performed two resistance bouts, in two consecutive days, with one leg, the other leg serving as a control (age 24±1, n=5). Muscle biopsies were obtained in both legs before, immediately after, and 24, 54, and 96 hours after exercise. Serial cryosection analysis combined immunohistochemistry and ATPase histochemistry with In Situ hybridization to identify the distribution of MHC isoforms and their corresponding transcripts, enabling identification of transitional fibers. Fibers positive solely for MHC IIX mRNA decreased in the exercised leg throughout the study period. At 96 hours post-exercise, no fibers solely expressed MHC IIX mRNA. In contrast, the number of fibers expressing MHC IIA mRNA increased throughout the study period. The percentage of fibers expressing mRNA for MHC I was unchanged in both legs at all time points. Pronounced depletion of glycogen in the MHC IIX fibers of the exercised leg verifies that the type IIX fibers were active during the heavy load contractions. Major mismatch between MHC at the mRNA and protein levels was only found in the fibers of the exercised leg. These data provide unequivocal in situ evidence of an immediate shutdown of the MHC IIX gene after resistance exercise. A further novel finding was that the silencing of the MHC IIX gene is sustained at least 4 days after removal of the stimulus. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

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

PubMed

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

2017-10-19

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

Interleukin 18 messenger RNA and proIL-18 protein expression in chorioamniotic membranes from pregnant women with preterm prelabor rupture of membranes.

PubMed

Polettini, Jossimara; Vieira, Eliane Passarelli; Santos, Mariana Perlati dos; Peraçoli, José Carlos; Witkin, Steven S; da Silva, Márcia Guimarães

2012-04-01

To quantify the expression of IL-18 mRNA and protein in the chorioamniotic membranes of pregnant women with PPROM and correlate expression with histological chorioamnionitis. A case control study that included 42 pregnant women not in labor in the following groups: PPROM (n=28) and controls with intact membranes submitted to selective cesarean section at term (n=14). Expression of IL-18 mRNA in chorioamniotic membranes was determined by real-time polymerase chain reaction, and IL-18 protein expression was measured by western blot. Histopathological analyses and immunolocalization of IL-18 by immunohistochemistry were also performed. Analyses were performed using the Mann-Whitney or Fisher's exact tests and the group effect was considered significant if the adjusted p-values were <0.05 and the magnitude of change was greater than 2-fold for mRNA expression. IL-18 mRNA was present in 100% of samples and no difference in expression was observed between term vs. PPROM membranes (fold-change 0.12; p=0.88). In the PPROM group, no difference was observed in IL-18 mRNA regarding gestational age (fold-change 0.11; p=0.42) or the presence of histological chorioamnionitis (fold-change 0.26; p=0.15). ProIL-18 was present in all samples. IL-18 was immunolocalized to amnion, chorion and decidua cells, with intense immunohistochemical staining at the choriodecidual junction. Chorioamniotic membranes are sources of IL-18 mRNA and proIL-18, and their expression is unrelated to PPROM or histological chorioamnionitis. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of Ghrelin miRNA on Inflammation and Calcium Pathway in Pancreatic Acinar Cells of Acute Pancreatitis.

PubMed

Tang, Xiping; Tang, Guodu; Liang, Zhihai; Qin, Mengbin; Fang, Chunyun; Zhang, Luyi

The study investigated the effects of endogenous targeted inhibition of ghrelin gene on inflammation and calcium pathway in an in vitro pancreatic acinar cell model of acute pancreatitis. Lentiviral expression vector against ghrelin gene was constructed and transfected into AR42J cells. The mRNA and protein expression of each gene were detected by reverse transcription polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. The concentration of intracellular calcium ([Ca]i) was determined by calcium fluorescence mark probe combined with laser scanning confocal microscopy. Compared with the control group, cerulein could upregulate mRNA and protein expression of inflammatory factors, calcium pathway, ghrelin, and [Ca]i. mRNA and protein expression of inflammatory factors increased significantly in cells transfected with ghrelin miRNA compared with the other groups. Intracellular calcium and expression of some calcium pathway proteins decreased significantly in cells transfected with ghrelin miRNA compared with the other groups. Targeted inhibition of ghrelin gene in pancreatic acinar cells of acute pancreatitis can upregulate the expression of the intracellular inflammatory factors and alleviate the intracellular calcium overload.

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.

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

Effects of dietary valine:lysine ratio on the performance, amino acid composition of tissues and mRNA expression of genes involved in branched-chain amino acid metabolism of weaned piglets

PubMed Central

Xu, Ye Tong; Ma, Xiao Kang; Wang, Chun Lin; Yuan, Ming Feng; Piao, Xiang Shu

2018-01-01

Objective The goal of this study was to investigate the effects of dietary standard ileal digestible (SID) valine:lysine ratios on performance, intestinal morphology, amino acids of liver and muscle, plasma indices and mRNA expression of branched-chain amino acid (BCAA) metabolism enzymes. Methods A total of 144 crossbred pigs (Duroc×Landrace×Large White) weaned at 28±4 days of age (8.79±0.02 kg body weight) were randomly allotted to 1 of 4 diets formulated to provide SID valine:lysine ratios of 50%, 60%, 70%, or 80%. Each diet was fed to 6 pens of pigs with 6 pigs per pen (3 gilts and 3 barrows) for 28 days. Results Average daily gain increased quadratically (p<0.05), the villous height of the duodenum, jejunum and ileum increased linearly (p<0.05) as the SID valine:lysine ratio increased. The concentrations of plasma α-keto isovaleric and valine increased linearly (p<0.05), plasma aspartate, asparagine and cysteine decreased (p<0.05) as the SID valine:lysine ratio increased. An increase in SID lysine:valine levels increased mRNA expression levels of mitochondrial BCAA transaminase and branched-chain α-keto acid dehydrogenase in the longissimus dorsi muscle (p<0.05). Conclusion Using a quadratic model, a SID valine:lysine ratio of 68% was shown to maximize the growth of weaned pigs which is slightly higher than the level recommended by the National Research Council [6]. PMID:28728397

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

PubMed

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

2005-09-21

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

Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis

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

Trapnell, B.C.; Chinshyan Chu; Paakko, P.K.

1991-08-01

The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called {Delta}Phe{sup 508}, a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR protein. In the context that the lung is the major site of morbidity and mortality in CF, the authors evaluated airway epithelial cells for CFTR mRNA transcripts in normal individuals, normal-{Delta}Phe{sup 508} heterozygotes, and {Delta}Phe{sup 508} homozygotes to determine if the normal and {Delta}Phe{sup 508} CFTR alleles are expressed in the respiratory epithelium, to what extent they aremore » expressed, and whether there are relative differences in the expression of the normal and abnormal alleles at the mRNA level. Respiratory tract epithelial cells recovered by fiberoptic bronchoscopy with a cytology brush demonstrated CFTR mRNA transcripts with sequences appropriately reflecting the normal and {Delta}Phe{sup 508} CFTR alleles of the various study groups. CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells.« less

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

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 branched-chain volatile fatty acids on lactation performance and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows.

PubMed

Liu, Q; Wang, C; Guo, G; Huo, W J; Zhang, S L; Pei, C X; Zhang, Y L; Wang, H

2018-02-12

Branched-chain volatile fatty acids (BCVFA) supplements could promote lactation performance and milk quality by improving ruminal fermentation and milk fatty acid synthesis. This study was conducted to evaluate the effects of BCVFA supplementation on milk performance, ruminal fermentation, nutrient digestibility and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows. A total of 36 multiparous Chinese Holstein cows averaging 606±4.7 kg of BW, 65±5.2 day in milk (DIM) with daily milk production of 30.6±0.72 kg were assigned to one of four groups blocked by lactation number, milk yield and DIM. The treatments were control, low-BCVFA (LBCVFA), medium-BCVFA (MBCVFA) and high-BCVFA (HBCVFA) with 0, 30, 60 and 90 g BCVFA per cow per day, respectively. Experimental periods were 105 days with 15 days of adaptation and 90 days of data collection. Dry matter (DM) intake tended to increase, but BW changes were similar among treatments. Yields of actual milk, 4% fat corrected milk, milk fat and true protein linearly increased, but feed conversion ratio (FCR) linearly decreased with increasing BCVFA supplementation. Milk fat content linearly increased, but true protein content tended to increase. Contents of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0 and C15:0 fatty acids in milk fat linearly increased, whereas other fatty acids were not affected with increasing BCVFA supplementation. Ruminal pH, ammonia N concentration and propionate molar proportion linearly decreased, but total VFA production and molar proportions of acetate and butyrate linearly increased with increasing BCVFA supplementation. Consequently, acetate to propionate ratios linearly increased. Digestibilities of DM, organic matter, CP, NDF and ADF also linearly increased. In addition, mRNA expressions of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding factor 1 and fatty acid-binding protein 3 linearly increased, mRNA expressions of acetyl-coenzyme A carboxylase-α, fatty acid synthase and stearoyl-CoA desaturase quadratically increased. However, lipoprotein lipase mRNA expression was not affected by treatments. The results indicated that lactation performance and milk fat synthesis increased with BCVFA supplementation by improving ruminal fermentation, nutrient digestibility and mRNA expressions of genes related to milk fat synthesis.

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.

Use of Trypanosoma equiperdum infected rabbits as a source of splenic mRNA; construction of cDNA clones and identification of a rabbit mu heavy chain clone.

PubMed

Bernstein, K E; Pavirani, A; Alexander, C; Jacobsen, F; Fitzmaurice, L; Mage, R

1983-01-01

Rabbits were infected by Trypanosoma equiperdum and the splenic mRNA was isolated. In vitro translation of this RNA and immunoprecipitation with anti-light chain, anti-heavy chain, anti-mu and anti-VH antibodies demonstrated that T. equiperdum infection elicits large quantities of splenic mRNA encoding mu and kappa chains. The mu and gamma heavy chains and the kappa light chains synthesized in the cell-free translation system were specifically immunoprecipitated by antisera to heavy chain VHa and light chain kappa b allotypes. In vitro labeling of spleen cells from trypanosome-infected animals demonstrated that the biosynthetically labeled IgM has a mu chain of higher molecular weight than the mu chain synthesized by in vitro translation, a difference that is largely abolished when cellular glycosylation is blocked with the antibiotic tunicamycin. Enrichment for heavy chain or light chain mRNA was achieved by fractionating mRNA from trypanosome-infected animals on a sucrose gradient. cDNA clones carrying mu heavy chain sequences were produced using a 'one tube' protocol and identified by cross species hybridization and hybridization selection. Infection of rabbits with T. equiperdum followed by sucrose gradient enrichment of splenic mRNA has provided sufficient quantities of mRNA encoding mu heavy chain suitable for cDNA cloning.

Prostate cancer targeting motifs: expression of αν β3, neurotensin receptor 1, prostate specific membrane antigen, and prostate stem cell antigen in human prostate cancer cell lines and xenografts.

PubMed

Taylor, Robert M; Severns, Virginia; Brown, David C; Bisoffi, Marco; Sillerud, Laurel O

2012-04-01

Membrane receptors are frequent targets of cancer therapeutic and imaging agents. However, promising in vitro results often do not translate to in vivo clinical applications. To better understand this obstacle, we measured the expression differences in receptor signatures among several human prostate cancer cell lines and xenografts as a function of tumorigenicity. Messenger RNA and protein expression levels for integrin α(ν) β(3), neurotensin receptor 1 (NTSR1), prostate specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) were measured in LNCaP, C4-2, and PC-3 human prostate cancer cell lines and in murine xenografts using quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and immunohistochemistry. Stable expression patterns were observed for integrin α(ν) and PSMA in all cells and corresponding xenografts. Integrin β(3) mRNA expression was greatly reduced in C4-2 xenografts and greatly elevated in PC-3 xenografts compared with the corresponding cultured cells. NTSR1 mRNA expression was greatly elevated in LNCaP and PC-3 xenografts. PSCA mRNA expression was elevated in C4-2 xenografts when compared with C4-2 cells cultured in vitro. Furthermore, at the protein level, PSCA was re-expressed in all xenografts compared with cells in culture. The regulation of mRNA and protein expression of the cell-surface target proteins α(ν) β(3), NTSR1, PSMA, and PSCA, in prostate cancer cells with different tumorigenic potential, was influenced by factors of the microenvironment, differing between cell cultures and murine xenotransplants. Integrin α(ν) β(3), NTRS1 and PSCA mRNA expression increased with tumorigenic potential, but mRNA expression levels for these proteins do not translate directly to equivalent expression levels of membrane bound protein. Copyright © 2011 Wiley Periodicals, Inc.

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

PubMed

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

2016-01-01

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

Detection of AR-V7 mRNA in whole blood may not predict the effectiveness of novel endocrine drugs for castration-resistant prostate cancer.

PubMed

Takeuchi, Takumi; Okuno, Yumiko; Hattori-Kato, Mami; Zaitsu, Masayoshi; Mikami, Koji

2016-01-01

A splice variant of androgen receptor (AR), AR-V7, lacks in androgen-binding portion and leads to aggressive cancer characteristics. Reverse transcription-polymerase chain reactions (PCRs) and subsequent nested PCRs for the amplification of AR-V7 and prostate-specific antigen (PSA) transcripts were done for whole blood of patients with prostate cancer and male controls. With primary reverse transcription PCRs, AR-V7 and PSA were detected in 4.5% and 4.7% of prostate cancer, respectively. With nested PCRs, AR-V7 messenger RNA (mRNA) was positive in 43.8% of castration-sensitive prostate cancer and 48.1% of castration-resistant prostate cancer (CRPC), while PSA mRNA was positive in 6.3% of castration-sensitive prostate cancer and 18.5% of CRPC. Whole-blood samples of controls showed AR-V7 mRNA expression by nested PCR. Based on multivariate analysis, expression of AR-V7 mRNA in whole blood was not significantly correlated with clinical parameters and PSA mRNA in blood, while univariate analysis showed a correlation between AR-V7 mRNA and metastasis at initial diagnosis. Detection of AR-V7 mRNA did not predict the reduction of serum PSA in patients with CRPC following abiraterone and enzalutamide administration. In conclusion, AR-V7 mRNA expression in normal hematopoietic cells may have annihilated the manifestation of aggressiveness of prostate cancer and the prediction of the effectiveness of abiraterone and enzalutamide by the assessment of AR-V7 mRNA in blood.

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

PubMed

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

2013-04-01

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

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.

Intake of branched-chain amino acids influences the levels of MAFbx mRNA and MuRF-1 total protein in resting and exercising human muscle.

PubMed

Borgenvik, Marcus; Apró, William; Blomstrand, Eva

2012-03-01

Resistance exercise and amino acids are two major factors that influence muscle protein turnover. Here, we examined the effects of resistance exercise and branched-chain amino acids (BCAA), individually and in combination, on the expression of anabolic and catabolic genes in human skeletal muscle. Seven subjects performed two sessions of unilateral leg press exercise with randomized supplementation with BCAA or flavored water. Biopsies were collected from the vastus lateralis muscle of both the resting and exercising legs before and repeatedly after exercise to determine levels of mRNA, protein phosphorylation, and amino acid concentrations. Intake of BCAA reduced (P < 0.05) MAFbx mRNA by 30 and 50% in the resting and exercising legs, respectively. The level of MuRF-1 mRNA was elevated (P < 0.05) in the exercising leg two- and threefold under the placebo and BCAA conditions, respectively, whereas MuRF-1 total protein increased by 20% (P < 0.05) only in the placebo condition. Phosphorylation of p70(S6k) increased to a larger extent (∼2-fold; P < 0.05) in the early recovery period with BCAA supplementation, whereas the expression of genes regulating mTOR activity was not influenced by BCAA. Muscle levels of phenylalanine and tyrosine were reduced (13-17%) throughout recovery (P < 0.05) in the placebo condition and to a greater extent (32-43%; P < 0.05) following BCAA supplementation in both resting and exercising muscle. In conclusion, BCAA ingestion reduced MAFbx mRNA and prevented the exercise-induced increase in MuRF-1 total protein in both resting and exercising leg. Further-more, resistance exercise differently influenced MAFbx and MuRF-1 mRNA expression, suggesting both common and divergent regulation of these two ubiquitin ligases.

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

Periapical cytokine expression in sickle cell disease.

PubMed

Ferreira, Shirlene Barbosa Pimentel; de Brito, Luciana Carla Neves; Oliveira, Michelle Pimenta; Maciel, Kamilla Faria; Martelli Júnior, Hercílio; Vieira, Leda Quercia; Sobrinho, Antônio Paulino Ribeiro

2015-03-01

Sickle cell anemia (SCA) is the most prevalent genetic disease worldwide. Patients with SCA exhibit increased levels of proinflammatory mediators as part of a permanently activated immunoinflammatory status. The aim of this study was to evaluate the mRNA expression levels of the cytokines interferon (IFN-γ), tumor necrosis factor, interleukin (IL-1β, IL-17A, IL-10), receptor activator for nuclear factor kappa B ligand, and the chemokines CCL2/MCP-1 and CCL5 in the periapical interstitial fluid from SCA individuals compared with healthy individuals. Samples were collected from 12 teeth of SCA patients and 12 non-SCA patients with apical periodontitis. In addition, 12 teeth were sampled from the periapical region of healthy patients with vital pulp (control). The expression of cytokine mRNA was detected by using real-time polymerase chain reaction. The expression of mRNA for the Th1-associated cytokines IFN-γ, tumor necrosis factor-α, and IL-1β were significantly higher in SCA individuals than in the control individuals (P < .05). Among Th1-associated cytokines, only IFN-γ was significantly increased in non-SCA compared with control patients (vital pulp). The expression of IL-17A mRNA was significant higher in SCA cases than in control samples (P < .05), whereas the IL-10 mRNA expression was significantly increased in SCA and non-SCA individuals when compared with the control group. Similar levels of receptor activator for nuclear factor kappa B ligand, CCL2, and CCL5 mRNA expression were observed in all samples. However, no significant differences were observed in the expression of cytokine or chemokine mRNA between SCA and non-SCA individuals (P > .05). The results were able to demonstrate that SCA patients presented prone proinflammatory ability, despite the fact that any differences in periapical immune responses between SCA and non-SCA individuals were not observed. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

The expression of 11β-hydroxysteroid dehydrogenase type 1 is increased in experimental periodontitis in rats.

PubMed

Nakata, Takaya; Umeda, Makoto; Masuzaki, Hiroaki; Sawai, Hirofumi

2016-10-03

The involvement of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive glucocorticoids into active glucocorticoids intracellularly, in metabolic diseases and chronic inflammatory diseases has been elucidated. We recently reported that an increase in 11β-HSD1 expression was associated with chronic periodontitis in humans irrespective of obesity. To further clarify the role of 11β-HSD1 in chronic periodontitis, the expression of 11β-HSD1 was investigated in experimental periodontitis model in rats. Experimental periodontitis was induced by silk ligature of left maxillary second molars of 7-week-old male Wistar rats, and periodontal tissues were collected at day 3. The expression of 11β-HSD1, 11β-HSD2, and TNFα mRNA was examined using real time reverse transcription-polymerase chain reaction. The expression of TNFα was used as an indicator of inflammation. Thus, the rats in which the levels of TNFα mRNA were increased in the ligature-induced periodontitis compared with the control were analysed. The findings demonstrated that the expression of 11β-HSD1 mRNA was significantly increased in experimental periodontitis compared with the control. The increase in the levels of 11β-HSD1 mRNA in the ligature-induced periodontitis compared with the control was positively correlated with that of TNFα mRNA. On the other hand, the expression of 11β-HSD2 mRNA, which inactivates glucocorticoids, was slightly decreased in experimental periodontitis. Therefore, the ratio of 11β-HSD1 versus 11β-HSD2 mRNA was significantly higher in experimental periodontitis than in the control. These results suggest that the increased expression of 11β-HSD1, which would result in the increased levels of intracellular glucocorticoids, may play a role in the pathophysiology of experimental periodontitis.

[Effect of Bushen Huoxue Compound on Retinal Müller Cells in High Glucose or AGEs Conditions].

PubMed

Xie, Xue-jun; Song, Ming-xia; Zhang, Mei; Qin, Wei; Wan, Li; Fang, Yang

2015-06-01

To explore the effect of Bushen Huoxue Compound (BHC) on lactate dehydrogenase (LDH) leakage, expressions of vascular endothelial growth factor (VEGF) and VEGF mRNA in retinal Muller cells under high glucose condition or advanced glycosylation end products (AGEs) condition by using serum pharmacological method. The retinal Müller cells of 5-7 days post-natal Sprague Dawley (SD) rats were cultured with modified enzyme-digestion method. Purified retinal Muller cells were cultured in normal conditions, high glucose condition (50 mmol/L) or AGEs (50 mg/L and 100 mg/L) conditions, and BHC-containing serum was added to culture medium. The LDH leakage and VEGF expressions were measured by enzyme-linked immunosorbent assay (ELISA). In addition, the relative expression of VEGF mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR). Compared with the normal control group, expressions of VEGF and VEGF mRNA were significantly increased in the high glucose group, the low dose AGEs group and the high dose AGEs group (all P < 0.01). The LDH leakage was obviously increased in the high dose AGEs group, when compared with the normal control group and the high glucose group (P < 0.01). The LDH leakage, expressions of VEGF and VEGF mRNA were obviously decreased by BHC-containing serum both in high glucose and AGEs conditions (P < 0.05, P < 0.01). BHC-containing serum had no significant effect on the LDH leakage and expressions of VEGF and VEGF mRNA in normal conditions (P > 0.05). AGEs intervention could obviously lower the stability of Müller cell membrane. Up-regulated expressions of VEGF and VEGF mRNA in cultured Müller cells could be induced by AGEs or high glucose. BHC-containing serum could stabilize the stability of Müller cell membrane, inhibit the transcription of VEGF mRNA and decrease the protein expression of VEGF, which might be one of important mechanisms for preventing and treating diabetic retinopathy.

Evodiamine Inhibits Angiotensin II-Induced Rat Cardiomyocyte Hypertrophy.

PubMed

He, Na; Gong, Qi-Hai; Zhang, Feng; Zhang, Jing-Yi; Lin, Shu-Xian; Hou, Hua-Hua; Wu, Qin; Sun, An-Sheng

2018-05-01

To investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms. Cardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca 2+ ] i ) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis. Compared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca 2+ ] i ) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca 2+ ] i concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05). Evo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca 2+ ]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

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.

[Effects of Salvianolate on Myosin Heavy Chain in Cardiomyocytes of Congestive Heart Failure Rats].

PubMed

Chen, Cheng; Zou, Xiang-gu; Qiu, Shan-dong; Chen, Hui; Chen, Yong-zhong; Lin, Xiu-ming

2015-07-01

To explore the effect of Salvianolate on myosin heavy chain (MHC) in cardiomyocytes of congestive heart failure (CHF) rats. Sixty male SD rats were divided into 6 groups according to random digit table, i.e., the normal control group (NCG), the model group, the Captopril group (CAG), the low dose Salvianolate group (LSG), the high dose Salvianolate group (HSG), the Captopril and high dose Salvianolate group (CSG), 10 in each group. CHF rat model was established with peritoneal injection of adriamycin in all rats except those in the NCG. Equal volume of normal saline was peritoneally injected to rats in the NCG, once per week for 6 successive weeks. Corresponding medication was started from the 5th week of injecting adriamycin. Rats in the CAG were administered with Captopril solution at the daily dose of 10 mg/kg by gastrogavage. Rats in the LSG and the HSG were administered with Salvianolate solution at the daily dose of 24.219 mg/kg and 48.438 mg/kg respectively by gastrogavage. Salvianolate was dissolved in 2 mL 5% glucose solution and administered by peritoneal injection. Rats in the CSG were peritoneally injected with high dose Salvianolate solution and administered with Captopril solution by gastrogavage. Two mL normal saline was peritoneally injected to rats in the model group, once per day for 8 successive weeks. Eight weeks later, the cardiac function and myocardial hypertrophy indices were detected by biological signal collecting and processing system. mRNA expression levels of alpha-MHC and beta-MHC in cardiac muscle were detected by fluorescence quantitative PCR. Expressions of protein kinase C (PKC) in cardiac muscle were detected by Western blot. Compared with the normal control group, heart mass index (HMI) and left ventricular mass index (LVMI) obviously increased in the model group (P < 0.01). Compared with the model group, HMI and LVMI decreased in HSG, CAG, and CSG groups (P < 0.05, P < 0.01). It was more obviously lowered in the CSG group than in the CAG group (P < 0.05). Compared with the NCG, the mRNA expression level of alpha-MHC in cardiac muscle decreased, the mRNA expression level of p-MHC and the expression of PKC in cardiac muscle increased in the model group (P < 0.01). Compared with the model group, the mRNA expression level of alpha-MHC in cardiac muscle was increased, and the mRNA expression level of beta-MHC and the expression of PKC in cardiac muscle were decreased in HSG, CAG, and CSG groups (P < 0.05, P < 0.01). There was statistical difference between the CSG group and the CAG group (P < 0.05). Salvianolate could up-regulate the mRNA expression level of alpha-MHC, and down-regulate the mRNA expression level of beta-MHC in cardiac muscle. Its mechanism might be related to decreasing the expression of PKC.

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

PubMed

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

2014-02-01

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

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

PubMed

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

2017-09-01

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

Effects of Castration on Expression of Lipid Metabolism Genes in the Liver of Korean Cattle

PubMed Central

Baik, Myunggi; Nguyen, Trang Hoa; Jeong, Jin Young; Piao, Min Yu; Kang, Hyeok Joong

2015-01-01

Castration induces the accumulation of body fat and deposition of intramuscular fat in Korean cattle, resulting in improved beef quality. However, little is known about the metabolic adaptations in the liver following castration. To understand changes in lipid metabolism following castration, hepatic expression levels of lipid metabolism genes were compared between Korean bulls and steers. Steers had higher (p<0.001) hepatic lipids contents and higher (p<0.01) mRNA levels of lipogenic acetyl-CoA carboxylase. This differential gene expression may, in part, contribute to increased hepatic lipid content following the castration of bulls. However, we found no differences in the hepatic expression levels of genes related to triglyceride synthesis (mitochondrial glycerol-3-phosphate acyltransferase, diacylglycerol O-acyltransferase 1 and 2) and fatty acid (FA) oxidation (carnitine palmitoyltransferase 1A, C-4 to C-12 straight chain acyl-CoA dehydrogenase, very long chain acyl-CoA dehydrogenase) between bulls and steers. No differences in gene expression for very-low-density lipoprotein (VLDL) secretion, including apolipoprotein B mRNA and microsomal triglyceride transfer protein (MTTP) protein, were observed in the liver although MTTP mRNA levels were higher in steers compared to bulls. In conclusion, FA synthesis may contribute to increased hepatic lipid deposition in steers following castration. However, hepatic lipid metabolism, including triglyceride synthesis, FA oxidation, and VLDL secretion, was not significantly altered by castration. Our results suggest that hepatic lipid metabolism does not significantly contribute to increased body fat deposition in steers following castration. PMID:25557684

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.

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

PubMed Central

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

2010-01-01

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

Metabolic Modulation by Medium-Chain Triglycerides Reduces Oxidative Stress and Ameliorates CD36-Mediated Cardiac Remodeling in Spontaneously Hypertensive Rat in the Initial and Established Stages of Hypertrophy.

PubMed

Saifudeen, Ismael; Subhadra, Lakshmi; Konnottil, Remani; Nair, R Renuka

2017-03-01

Left ventricular hypertrophy (LVH) is characterized by a decrease in oxidation of long-chain fatty acids, possibly mediated by reduced expression of the cell-surface protein cluster of differentiation 36 (CD36). Spontaneously hypertensive rats (SHRs) were therefore supplemented with medium-chain triglycerides (MCT), a substrate that bypasses CD36, based on the assumption that the metabolic modulation will ameliorate ventricular remodeling. The diet of 2-month-old and 6-month-old SHRs was supplemented with 5% MCT (Tricaprylin), for 4 months. Metabolic modulation was assessed by mRNA expression of peroxisome proliferator-activated receptor α and medium-chain acyl-CoA dehydrogenase. Blood pressure was measured noninvasively. LVH was assessed with the use of hypertrophy index, cardiomyocyte cross-sectional area, mRNA expression of B-type natriuretic peptide, cardiac fibrosis, and calcineurin-A levels. Oxidative stress indicators (cardiac malondialdehyde, protein carbonyl, and 3-nitrotyrosine levels), myocardial energy level (ATP, phosphocreatine), and lipid profile were determined. Supplementation of MCT stimulated fatty acid oxidation in animals of both age groups, reduced hypertrophy and oxidative stress along with the maintenance of energy level. Blood pressure, body weight, and lipid profile were unaffected by the treatment. The results indicate that modulation of myocardial fatty acid metabolism by MCT prevents progressive cardiac remodeling in SHRs, possibly by maintenance of energy level and decrease in oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Region-specific expression and hormonal regulation of the first exon variants of rat prolactin receptor mRNA in rat brain and anterior pituitary gland.

PubMed

Nogami, H; Hoshino, R; Ogasawara, K; Miyamoto, S; Hisano, S

2007-08-01

Recent studies have revealed the occurrence of five first exon variants of the rat prolactin receptor mRNA, suggesting that multiple promoters direct prolactin receptor transcription in response to different regulatory factors. In the present study, regional expression of these first exon variants, as well as two prolactin receptor subtypes generated by alternative splicing, was examined in the brains and anterior pituitary glands of female rats. Expression of the long-form was detected in the choroid plexus, hypothalamus, hippocampus, cerebral cortex and anterior pituitary gland, whereas the short form was detected only in the choroid plexus. E1-3 mRNA, a first exon variant, was detected in the choroid plexus, hypothalamus, and anterior pituitary gland, whereas E1-4 was detected only in the choroid plexus. Other variants were not detectable by the polymerase chain reaction protocol employed in this study. Ovariectomy increased the short form in the choroid plexus and the E1-3 expression in the choroid plexus and pituitary gland, but changes in the long-form and E1-4 expression were minimal. Replacement of oestrogens and prolactin suggest that oestrogens down-regulate E1-3 expression in the choroid plexus and pituitary gland, and that the negative effect of oestrogen is mediated by prolactin in the pituitary gland. The present results revealed the region-specific promoter usage in prolactin receptor mRNA transcription, as well as the involvement of oestrogens in the regulation of E1-3 mRNA expression in the brain and pituitary gland.

Neuroleptic Drugs and PACAP Differentially Affect the mRNA Expression of Genes Encoding PAC1/VPAC Type Receptors.

PubMed

Jóźwiak-Bębenista, Marta; Kowalczyk, Edward

2017-04-01

Several lines of evidence suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide playing an important role as a neuromodulator. It has been indicated that PACAP is associated with mental diseases, and that regulation of the PACAPergic signals could be a potential target for the treatment of such psychiatric states as schizophrenia. Recent studies have suggested that action of neuroleptic drugs is mediated not only by dopaminergic and serotonergic neurotransmission, but also via neuropeptides which may act both as neurotransmitters and as neuromodulators. The present study examines whether currently-used neuroleptics influence the action of PACAP receptors, whose expression is altered in a schizophrenic patient. Real-time polymerase chain reaction (PCR) was used to examine the effects of haloperidol, olanzapine and amisulpride on the expression of genes coding PAC1/VPAC type receptors in the T98G glioblastoma cell line, as an example of an in vitro model of glial cells. PAC1 mRNA expression fell after 24-h incubation with haloperidol or olanzapine; however the effect was not maintained after 72 h, and haloperidol even up-regulated PAC1 mRNA expression in a dose-dependent manner. All the examined drugs decreased VPAC2 mRNA expression, especially after 72-h incubation. Haloperidol (typical neuroleptic) was distinctly more potent than atypical neuroleptic drugs (olanzapine and amisulpride). In addition, PACAP increased PAC1 and VPAC2 mRNA expression. In conclusion, our findings suggest PACAP receptors may be involved in the mechanism of typical and atypical neuroleptic drugs.

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.

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.

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

PubMed Central

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

2013-01-01

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

Expression of genes of the cardiac and renal renin-angiotensin systems in preterm piglets: is this system a suitable target for therapeutic intervention?

PubMed

Kim, Eleanor; Eiby, Yvonne; Lumbers, Eugenie; Boyce, Amanda; Gibson, Karen; Lingwood, Barbara

2015-10-01

The newborn circulating, cardiac and renal renin-angiotensin systems (RASs) are essential for blood pressure control, and for cardiac and renal development. If cardiac and renal RASs are immature this may contribute to cardiovascular compromise in preterm infants. This study measured mRNA expression of cardiac and renal RAS components in preterm, glucocorticoid (GC) exposed preterm, and term piglets. Renal and cardiac RAS mRNA levels were measured using real-time polymerase chain reaction (PCR). Genes studied were: (pro)renin receptor, renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). All the genes studied were expressed in the kidney; neither renin nor AT2R mRNA were detected in the heart. There were no gestational changes in (pro)renin receptor, renin, ACE or AT1R mRNA levels. Right ventricular angiotensinogen mRNA levels in females were lower in preterm animals than at term, and GC exposure increased levels in male piglets. Renal angiotensinogen mRNA levels in female term piglets were lower than females from both preterm groups, and lower than male term piglets. Left ventricular ACE2 mRNA expression was lower in GC treated preterm piglets. Renal AT2R mRNA abundance was highest in GC treated preterm piglets, and the AT1R/AT2R ratio was increased at term. Preterm cardiac and renal RAS mRNA levels were similar to term piglets, suggesting that immaturity of these RASs does not contribute to preterm cardiovascular compromise. Since preterm expression of both renal and cardiac angiotensin II-AT1R is similar to term animals, cardiovascular dysfunction in the sick preterm human neonate might be effectively treated by agents acting on their RASs. © The Author(s), 2015.

Ovarian kisspeptin expression is related to age and to monocyte chemoattractant protein-1.

PubMed

Merhi, Zaher; Thornton, Kimberley; Bonney, Elizabeth; Cipolla, Marilyn J; Charron, Maureen J; Buyuk, Erkan

2016-04-01

The objective of this study was to test the hypothesis that ovarian kisspeptin (kiss1) and its receptor (kiss1r) expression are affected by age, obesity, and the age- and obesity-related chemokine monocyte chemoattractant protein-1 (MCP-1). Ovaries from reproductive-aged and older C57BL/6J mice fed normal chow (NC) or high-fat (HF) diet, ovaries from age-matched young MCP-1 knockout and young control mice on NC, and finally, cumulus and mural granulosa cells (GCs) from women who underwent in vitro fertilization (IVF) were collected. Kiss1, kiss1r, anti-Mullerian hormone (AMH), and AMH receptor (AMHR-II) messenger RNA (mRNA) expression levels were quantified using real-time polymerase chain reaction (RT-PCR). In mouse ovaries, kiss1 and kiss1r mRNA levels were significantly higher in old compared to reproductive-aged mice, and diet-induced obesity did not alter kiss1 or kiss1r mRNA levels. Compared to young control mice, young MCP-1 knockout mice had significantly lower ovarian kiss1 mRNA but significantly higher AMH and AMHR-II mRNA levels. In human cumulus GCs, kiss1r mRNA levels were positively correlated with age but not with BMI. There was no expression of kiss1 mRNA in either cumulus or mural GCs. These data suggest a possible age-related physiologic role for the kisspeptinergic system in ovarian physiology. Additionally, the inflammatory MCP-1 may be associated with kiss1 and AMH genes, which are important in ovulation and folliculogenesis, respectively.

Epidermal growth factor receptor expression in gastric tumors and its relationship with the germline polymorphisms - 216 G>T, -191 C>A, (CA) n IVS1, and R521K.

PubMed

Torres-Jasso, J H; Bustos-Carpinteyro, A R; Garcia-Gonzalez, J R; Peregrina-Sandoval, J; Cruz-Ramos, J A; Santiago-Luna, E; Sanchez-Lopez, J Y

2016-01-01

Gastric cancer (GC) is the third worldwide leading cause of cancer-related death affecting both sexes. The aberrant expression of epidermal growth factor receptor (EGFR) gene has been detected in many human epithelial malignancies and linked to advanced disease, more aggressive phenotype, and poor prognosis. To analyze the relation that the expression of EGFR in gastric tumors holds with pathological characteristics and with the germline polymorphisms -216 G>T, -191 C>A, (CA) n IVS1, and R521K. We studied 22 biopsies from gastric tumors obtained by endoscopy. EGFR expression was determined by relative quantification real-time polymerase chain reaction with the glyceraldehyde-3-phosphate dehydrogenase reference gene (as for messenger RNA [mRNA]) and by immunohistochemistry (IHC) (as for protein). EGFR germline polymorphisms were analyzed by sequencing, GeneScan, and restriction fragment length polymorphisms. EGFR mRNA expression was increased (>2-fold) in 13.6% of GC cases, decreased (<0.5-fold) in 68.2%, and normal in 18.2%; overexpression was related to well-differentiated gastric tumors, whereas underexpression was linked to moderate or poorly differentiated gastric tumors (P < 0.001). EGFR protein expression was high (IHC 2+ and 3+) in 29.4% of gastric tumors and was normal or low (score 0 to 1+) in 70.6% cases. EGFR expression, in both mRNA and protein, was not related to any EGFR polymorphism (P > 0.05). Most gastric tumors showed low EGFR expression (mRNA and protein), whereas EGFR overexpression was related to well-differentiated gastric tumors. Furthermore, germinal polymorphisms -216, -191, (CA) n IVS1, and R521K were not related to EGFR expression (mRNA or protein).

Endoplasmic reticulum stress in vasopressin neurons of familial diabetes insipidus model mice: aggregate formation and mRNA poly(A) tail shortening.

PubMed

Arima, Hiroshi; Morishita, Yoshiaki; Hagiwara, Daisuke; Hayashi, Masayuki; Oiso, Yutaka

2014-01-01

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone, which binds to newly synthesized secretory and transmembrane proteins to facilitate protein folding. BiP mRNA is expressed in the arginine vasopressin (AVP) neurons in the supraoptic nucleus of wild-type mice even in basal conditions, and the expression levels increase in response to dehydration. These data suggest that AVP neurons are subjected to ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is caused by mutations in the gene locus of AVP. The mutant proteins could accumulate in the ER and possibly increase ER stress in the AVP neurons. We bred mice possessing a mutation causing FNDI, which manifested progressive polyuria, as do the patients with FNDI. Electron microscopic analyses demonstrated that aggregates accumulated in the ER of AVP neurons in FNDI mice. Despite polyuria, which could potentially induce dehydration, AVP mRNA expression was decreased in the supraoptic nucleus, and the AVP mRNA poly(A) tail length was shortened in FNDI mice compared with wild-type mice. Incubation of hypothalamic explants of wild-type mice with ER stressors caused shortening of the poly(A) tail length of AVP mRNA, accompanied by decreases in the expression. These data revealed a mechanism by which ER stress decreases poly(A) tail length of AVP mRNA, and this reduces the load of unfolded proteins that form the aggregates in ER of the AVP neurons in FNDI mice.

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

PubMed

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

2017-08-01

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

Aberrant expression of erythropoietin in uterine leiomyoma: implications in tumor growth.

PubMed

Asano, Ryoko; Asai-Sato, Mikiko; Miyagi, Yohei; Mizushima, Taichi; Koyama-Sato, Makiko; Nagashima, Yoji; Taguri, Masataka; Sakakibara, Hideya; Hirahara, Fumiki; Miyagi, Etsuko

2015-08-01

Myomatous erythrocytosis syndrome is a rare complication of uterine leiomyoma caused by erythropoietin (EPO) that is produced by tumor cells. We assessed the EPO expression in leiomyomas and investigated the effects of EPO on the tumor growth. Tissue samples were collected from 114 patients with uterine leiomyomas who underwent myomectomy or hysterectomy in Yokohama City University Hospital. From 17 patients, the corresponding normal myometrium was also collected. All samples were analyzed for EPO messenger RNA (mRNA) expression by real-time reverse transcription-polymerase chain reaction. EPO protein expression was determined by an enzyme-linked immunosorbent assay. The relationships between EPO expression and clinicopathological features were retrospectively analyzed using the patients' charts. Blood vessel density and maturity were assessed using hematoxylin-eosin staining and CD34 immunohistochemistry. EPO mRNA expression was detected in 108 of 114, or 95%, of the leiomyomas. The mean EPO mRNA expression in the leiomyoma was higher than the corresponding normal myometrium (3836 ± 4122 vs 1455 ± 2141; P = .025 by Wilcoxon rank test). The EPO mRNA expression in the leiomyomas varied extensively among samples, ranging from undetectable levels to 18-fold above the mean EPO mRNA of normal myometrium. EPO protein production was observed concomitant with mRNA expression. A positive correlation of leiomyoma size and EPO mRNA expression was shown by Spearman rank correlation coefficient (ρ = 0.294; P = .001), suggesting the involvement of EPO in leiomyoma growth. The blood vessel maturity was also significantly increased in EPO-producing leiomyomas (high vessel maturity in high vs low EPO group: 67% vs 20%; P = .013 by Fisher exact test). This report demonstrates that EPO is produced in most of conventional leiomyomas and supports a model in which EPO accelerates tumor growth, possibly by inducing vessel maturity. Our study suggests one possible mechanism by which some uterine leiomyomas reach a large size, and the understanding of EPO expression patterns in these tumors may be useful for management of the patients with leiomyomas. Copyright © 2015 Elsevier Inc. All rights reserved.

Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading

NASA Technical Reports Server (NTRS)

Carlson, C. J.; Booth, F. W.; Gordon, S. E.

1999-01-01

Transgenic mice lacking a functional myostatin (MSTN) gene demonstrate greater skeletal muscle mass resulting from muscle fiber hypertrophy and hyperplasia (McPherron, A. C., A. M. Lawler, and S. -J. Lee. Nature 387: 83-90, 1997). Therefore, we hypothesized that, in normal mice, MSTN may act as a negative regulator of muscle mass. Specifically, we hypothesized that the predominately slow (type I) soleus muscle, which demonstrates greater atrophy than the fast (type II) gastrocnemius-plantaris complex (Gast/PLT), would show more elevation in MSTN mRNA abundance during hindlimb unloading (HU). Surprisingly, MSTN mRNA was not detectable in weight-bearing or HU soleus muscle, which atrophied 42% by the 7th day of HU in female ICR mice. In contrast, MSTN mRNA was present in weight-bearing Gast/PLT muscle and was significantly elevated (67%) at 1 day but not at 3 or 7 days of HU. However, the Gast/PLT muscle had only atrophied 17% by the 7th day of HU. Because the soleus is composed only of type I and IIa fibers, whereas the Gast/PLT expresses type IId/x and IIb in addition to type I and IIa, it was necessary to perform a more careful analysis of the relationship between MSTN mRNA levels and myosin heavy-chain (MHC) isoform expression (as a marker of fiber type). A significant correlation (r = 0.725, P < 0. 0005) was noted between the percentage of MHC isoform IIb expression and MSTN mRNA abundance in several muscles of the mouse hindlimb. These results indicate that MSTN expression is not strongly associated with muscle atrophy induced by HU; however, it is strongly associated with MHC isoform IIb expression in normal muscle.

Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268.

PubMed

Kimoto, Sohei; Bazmi, H Holly; Lewis, David A

2014-09-01

Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

[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.

Comparison of the expression of neurotransmitter and muscular genesis markers in the postnatal male mouse masseter and trigeminal ganglion during development.

PubMed

Kamata, Hiroaki; Karibe, Hiroyuki; Sato, Iwao

2018-06-01

Calcitonin gene-related peptide (CGRP) is released by motor neurons and affects skeletal muscle fiber and transient receptor potential cation channel subfamily V member 1 (TRPV1), an important marker of pain modulation. However, the expression of CGRP and TRPV1 in the trigeminal ganglion (TG) during changes and in feeding patterns has not been described. We used real-time reverse transcription polymerase chain reaction and in situ hybridization to investigate the mRNA expression levels of CGRP and TRPV1 in the TG. The expression of myosin heavy-chain (MyHC) isoforms was also investigated in the masseter muscle (MM) during the transition from sucking to mastication, an important functional trigger for muscle. The mRNA and protein levels of CGRP increased in the MM and TG from postnatal day 10 (P10) to P20 in male mice. The protein levels of TRPV1 were almost constant in the TG from P10 to P20, in contrast to increases in the MM. The mRNA abundance of TRPV1 in the TG and MM was increased from P10 to P20. The localization of an antisense probe was used to count CGRP cell numbers and found to differentiate the ophthalmic, maxillary, and mandibular nerve divisions of the TG. In particular, the number of CGRP + cells per 10,000 μm 2 in the maxillary and mandibular divisions of the TG gradually changed from P10 to P20. The expression of CGRP and TRPV1 in the TG and MM and the patterns of expression of different MyHC isoforms were affected by changes in feeding during male mouse development. © 2017 Wiley Periodicals, Inc.

Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction.

PubMed

Pinelli, E; van der Kaaij, S Y; Slappendel, R; Fragio, C; Ruitenberg, E J; Bernadina, W; Rutten, V P

1999-08-02

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

Growth hormone and Pit-1 expression in bovine fetal lymphoid cells.

PubMed

Chen, H T; Schuler, L A; Schultz, R D

1997-11-01

Bovine fetal lymphoid cells were examined for growth hormone (GH) and the transcription factor Pit-1/GHF-1 mRNA. GH and Pit-1/GHF-1 transcripts were detected in thymocytes and splenocytes from fetuses at 60, 90, 120, and 270 d of gestation using reverse transcription-polymerase chain reaction (RT-PCR). Northern analysis indicated that the lymphoid GH mRNA was approximately 350 nucleotides larger than in the pituitary. RT-PCR analysis demonstrated that the coding regions as well as 3' untranslated region of the lymphocyte GH and pituitary transcripts were the same. Analysis of the 5'-untranslated region of the lymphocyte GH mRNA showed that transcription began upstream from the start site in the pituitary gland, suggesting differences in regulation in these tissues. Fetal thymocytes and splenocytes expressed Pit-1/GHF-1 mRNA; however, they contained only the 2.5-kb transcript. The GH and Pit-1/GHF-1 mRNA in fetal lymphoid cells supports the hypothesis that lymphocyte-derived GH may function as an autocrine and/or paracrine factor during the development and maturation of the bovine fetal immune system.

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

Increased cardiac alpha-myosin heavy chain in left atria and decreased myocardial insulin-like growth factor (Igf-I) expression accompany low heart rate in hibernating grizzly bears.

PubMed

Barrows, N D; Nelson, O L; Robbins, C T; Rourke, B C

2011-01-01

Grizzly bears (Ursus arctos horribilis) tolerate extended periods of extremely low heart rate during hibernation without developing congestive heart failure or cardiac chamber dilation. Left ventricular atrophy and decreased left ventricular compliance have been reported in this species during hibernation. We evaluated the myocardial response to significantly reduced heart rate during hibernation by measuring relative myosin heavy-chain (MyHC) isoform expression and expression of a set of genes important to muscle plasticity and mass regulation in the left atria and left ventricles of active and hibernating bears. We supplemented these data with measurements of systolic and diastolic function via echocardiography in unanesthetized grizzly bears. Atrial strain imaging revealed decreased atrial contractility, decreased expansion/reservoir function (increased atrial stiffness), and decreased passive-filling function (increased ventricular stiffness) in hibernating bears. Relative MyHC-α protein expression increased significantly in the atrium during hibernation. The left ventricle expressed 100% MyHC-β protein in both groups. Insulin-like growth factor (IGF-I) mRNA expression was reduced by ∼50% in both chambers during hibernation, consistent with the ventricular atrophy observed in these bears. Interestingly, mRNA expression of the atrophy-related ubiquitin ligases Muscle Atrophy F-box (MAFBx) and Muscle Ring Finger 1 did not increase, nor did expression of myostatin or hypoxia-inducible factor 1α (HIF-1α). We report atrium-specific decreases of 40% and 50%, respectively, in MAFBx and creatine kinase mRNA expression during hibernation. Decreased creatine kinase expression is consistent with lowered energy requirements and could relate to reduced atrial emptying function during hibernation. Taken together with our hemodynamic assessment, these data suggest a potential downregulation of atrial chamber function during hibernation to prevent fatigue and dilation due to excessive work against an optimally filled ventricle, a response unpredicted by the Frank-Starling mechanism.

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.

Enrofloxacin and Probiotic Lactobacilli Influence PepT1 and LEAP-2 mRNA Expression in Poultry.

PubMed

Pavlova, Ivelina; Milanova, Aneliya; Danova, Svetla; Fink-Gremmels, Johanna

2016-12-01

Expression of peptide transporter 1 (PepT1) and liver-expressed antimicrobial peptide 2 (LEAP-2) in chickens can be influenced by food deprivation, pathological conditions and drug administration. Effect of three putative probiotic Lactobacillus strains and enrofloxacin on the expression of PepT1 and LEAP-2 mRNA was investigated in Ross 308 chickens. One-day-old chicks (n = 24) were allocated to following groups: control (without treatment); group treated with probiotics via feed; group treated with a combination of probiotics and enrofloxacin; and a group given enrofloxacin only. The drug was administered at a dose of 10 mg kg -1 , via drinking water for 5 days. Samples from liver, duodenum and jejunum were collected 126 h after the start of the treatment. Expression levels of PepT1 and LEAP-2 were determined by real-time polymerase chain reaction and were statistically evaluated by Mann-Whitney test. Enrofloxacin administered alone or in combination with probiotics provoked a statistically significant up-regulation of PepT1 mRNA levels in the measured organ sites. These changes can be attributed to a tendency of improvement in utilization of dietary peptide and in body weight gain. LEAP-2 mRNA expression levels did not change significantly in enrofloxacin-treated chickens in comparison with control group.

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

NASA Astrophysics Data System (ADS)

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

2012-07-01

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

[Up regulation of phenylacetate to glioma homeobox gene expression].

PubMed

Tian, Yu; Yang, Chaohua; Zhao, Conghai

2002-03-01

Even though phenylacetate (PA) bas been shown to inhibit the growth and induce differentiation in rat C6 glioma cell line, its mechanisms are still poorly understood. This study is aimed to identify which Hox gene is related to glioma and to observe the change in expression on mRNA level as treated by phenylasetate. Twenty-two kinds of Hox gene were divided into 3 groups according to their primer sequence. Semiquantitative reverse transcription- polymerase chain reaction (RT-PCR) was used to investigate the mRNA expression of Hox gene groups and some Hox gene in rat C6 glioma cell line following differentiation induced by PA. The level of Hox gene expression was expressed as ratio expression rate (RER) of Hox gene/beta-actin according to computer image analysis and the difference between C6 cells and PA treated C6 cells was analyzed by student t-test. It was found that Hox genes matching to primers P2 were mildly expressed in C6 cells and the expression of HoxB2 mRNA was significantly up-regulated in PA treated C6 cells (P < 0.001). The weak expression of HoxB2 may be involved in glioma origin and the mechanisms of PA action are correlated with transcription process in the glioma cells.

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

PubMed

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

2010-10-01

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

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

PubMed

Nishiura, T; Abe, K

1999-01-01

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

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

PubMed Central

2011-01-01

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

Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss.

PubMed

Hokari, Ryota; Kitagawa, Noritake; Watanabe, Chikako; Komoto, Shunsuke; Kurihara, Chie; Okada, Yoshikiyo; Kawaguchi, Atsushi; Nagao, Shigeaki; Hibi, Toshifumi; Miura, Soichiro

2008-07-01

Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE-1 are specifically expressed in the endothelium of the lymphatic systems. VEGF-C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia. Biopsy samples were obtained from duodenal biopsies in patients with intestinal lymphangiectasia complicated with protein-losing from white spot lesions in which lymphangiectasia was histologically confirmed. Immunohistochemical analysis for VEGFR3 and LYVE-1 was performed. mRNA expression of VEGF-C, VEGF-D, VEGFR3, and transcription factors was determined by the quantitative reverse transcription-polymerase chain reaction method. In the control mucosa, VEGFR3 was weakly expressed on the central lymphatic vessels in the lamina propria and LYVE-1 was expressed mainly on the lymphatic vessels in the submucosa. In intestinal lymphangiectasia, VEGFR3 and LYVE-1 expression levels were increased on the mucosal surface corresponding to widely dilated lymphatic vessels, while they were decreased in the deeper mucosa. mRNA expression study showed a significant increase in the expression level of VEGFR3 in lymphangiectasia, but the expression of VEGF-C and -D mRNA was significantly suppressed compared with that in controls despite the presence of lymphangiectasia. The mRNA expression levels of FOXC2 and SOX18 were also decreased, whereas Prox 1 was not altered. There is an altered expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa in these patients.

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.

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.

Effect of feeding diets of varying fatty acid composition on apolipoprotein expression in newborn swine.

PubMed

Wang, H; Hunter, F; Black, D D

1998-10-01

The purpose of this study was to determine the effect of chronic (1 wk) feeding of dietary triacylglycerol (TG) of varying fatty acid composition on small intestinal and hepatic apolipoprotein expression, as well as serum lipid and apolipoprotein concentrations, in newborn swine. Two-day-old female swine were fed one of three diets by gavage with the following lipid composition: medium-chain TG (MCT; MCT oil), intermediate-chain saturated TG (ICST; coconut oil), and long-chain polyunsaturated TG (LCPUT; safflower oil) at 753 kJ . kg-1 . day-1 with 51% of energy from fat. After 1 wk, serum lipids and apolipoprotein concentrations were measured, and jejunal apolipoprotein B (apo B) and apo A-I mass and apo B, apo A-I, apo A-IV, and apo C-III synthesis were measured. Liver was processed for determination of apo B and apo A-I mass and apo B, apo A-I, apo C-III, and beta-actin mRNA abundance by slot blot hybridization. Compared with the MCT and LCPUT groups, the ICST group had higher total serum cholesterol, TG, high-density lipoprotein (HDL)-cholesterol, and apo A-I concentrations. There were no differences among the three groups for intestinal apolipoprotein mass or synthesis. In liver, apo A-I mass was highest in the ICST group. Liver apo A-I and apo C-III mRNA abundance was highest in the ICST group. Among all three groups, hepatic apo A-I mass correlated significantly with plasma HDL-cholesterol concentrations, and serum TG concentrations correlated with hepatic apo C-III mRNA abundance. In conclusion, we found that in the newborn piglet, chronic feeding of ICST increases serum total cholesterol, TG, HDL-cholesterol, and apo A-I concentrations and hepatic expression of apo A-I and apo C-III mRNA, compared with feeding of MCT or LCPUT. We speculate that increased hepatic apo A-I expression may contribute to the higher serum HDL and apo A-I concentrations in the ICST animals. Increased hepatic expression of apo C-III with ICST feeding may contribute to the higher serum TG concentrations by apo C-III-mediated inhibition of the catabolism of triacylglycerol-rich lipoproteins.

Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

DTIC Science & Technology

2007-05-08

deoxynucleotide triphosphates, from Sigma. Sequences for glyceraldehyde-3-phosphate dehydrogenase ( G3PDH ), IL-8,and TNF-a were amplified with primer...This was accomplished by normalizing all samples to the mRNA for the moderately expressed housekeeping function glyceraldehyde-3 -phosphate...without and with isolation of cells before reverse transcription and PCR. G3PDH mRNA target amplifies at 983 base pairs. The 630 base pair band is the

Omega-3 fatty acid deficiency selectively up-regulates delta6-desaturase expression and activity indices in rat liver: prevention by normalization of omega-3 fatty acid status.

PubMed

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

2011-09-01

This study investigated the effects of perinatal dietary omega-3 (n-3) fatty acid depletion and subsequent repletion on the expression of genes that regulate long-chain (LC) polyunsaturated fatty acid biosynthesis in rat liver and brain. It was hypothesized that chronic n-3 fatty acid deficiency would increase liver Fads1 and Fads2 messenger RNA (mRNA) expression/activity and that n-3 fatty acid repletion would normalize this response. Adult rats fed the n-3-free diet during perinatal development exhibited significantly lower erythrocyte, liver, and frontal cortex LCn-3 fatty acid composition and reciprocal elevations in LC omega-6 (n-6) fatty acid composition compared with controls (CONs) and repleted rats. Liver Fads2, but not Fads1, Elovl2, or Elovl5, mRNA expression was significantly greater in n-3-deficient (DEF) rats compared with CONs and was partially normalized in repleted rats. The liver 18:3n-6/18:2n-6 ratio, an index of delta6-desturase activity, was significantly greater in DEF rats compared with CON and repleted rats and was positively correlated with Fads2 mRNA expression among all rats. The liver 18:3n-6/18:2n-6 ratio, but not Fads2 mRNA expression, was also positively correlated with erythrocyte and frontal cortex LCn-6 fatty acid compositions. Neither Fads1 or Fads2 mRNA expression was altered in brain cortex of DEF rats. These results confirm previous findings that liver, but not brain, delta6-desaturase expression and activity indices are negatively regulated by dietary n-3 fatty acids. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-01-01

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

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.

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

PubMed Central

2010-01-01

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

Signal Transduction in T Cell Activation and Tolerance

DTIC Science & Technology

1993-01-01

chains and ’ chains may transduce different signals in intact T cells. These studies demonstrate that while c- deficient and c-containing TCR complexes...three independently derived pairs of CD45- and CD45+ murine T cell lymphomas, the CD45- expressing cells were consistently deficient in...D.B., Larsen, A. and Wilson, C.B. (1986) Reduced interferon-gamma mRNA levels in human neonates: Evidence for an intrinsic T cell deficiency yi 114

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

PubMed

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

2016-04-01

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

Fatty acid profile of maternal and fetal erythrocytes and placental expression of fatty acid transport proteins in normal and intrauterine growth restriction pregnancies.

PubMed

Assumpção, Renata P; Mucci, Daniela B; Fonseca, Fernanda C P; Marcondes, Henrique; Sardinha, Fátima L C; Citelli, Marta; Tavares do Carmo, Maria G

2017-10-01

Long-chain polyunsaturated fatty acids (LC-PUFA), mainly docosahexaenoic (DHA) and arachidonic acids (AA), are critical for adequate fetal growth and development. We investigated mRNA expression of proteins involved in hydrolysis, uptake and/or transport of fatty acids in placenta of fifteen full term normal pregnancies and eleven pregnancies complicated by intrauterine growth restriction (IUGR) with normal umbilical blood flows. The mRNA expression of LPL, FATPs (-1, -2 and -4) and FABPs (-1 and -3) was increased in IUGR placentas, however, tissue profile of LC-PUFA was not different between groups. Erythrocytes from both mothers and fetuses of the IUGR group showed lower concentrations of AA and DHA and inferior DHA/ALA ratio compared to normal pregnancies (P < 0.05). We hypothesize that reduced circulating levels of AA and DHA could up-regulate mRNA expression of placental fatty acids transporters, as a compensatory mechanism, however this failed to sustain normal LC-PUFA supply to the fetus in IUGR. Copyright © 2017 Elsevier Ltd. All rights reserved.

[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.

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

NASA Technical Reports Server (NTRS)

Cubano, L. A.; Lewis, M. L.

2001-01-01

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat).

PubMed

Cubano, L A; Lewis, M L

2001-05-01

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

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.

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

Effects of ultraviolet B irradiation, proinflammatory cytokines and raised extracellular calcium concentration on the expression of ATP2A2 and ATP2C1.

PubMed

Mayuzumi, N; Ikeda, S; Kawada, H; Fan, P S; Ogawa, H

2005-04-01

Darier disease (DD) and Hailey-Hailey disease (HHD) are autosomal dominantly inherited skin disorders that histologically share the characteristics of suprabasal separation and acantholysis of epidermal keratinocytes. Various mutations in the DD gene (ATP2A2) and the HHD gene (ATP2C1) (respectively encoding the calcium pumps of the sarco/endoplasmic reticulum and the Golgi apparatus) have recently been described in multiple families with DD and HHD. Mutations in ATP2A2 or ATP2C1 have been suggested as causing the conditions via the mechanism of haploinsufficiency. Ultraviolet (UV) B irradiation is thought to be an aggravating factor in both diseases. To examine the effects of various stimuli on ATP2A2 and ATP2C1 mRNA expression, and to examine the role of calcium pumps during keratinocyte differentiation. The effects of UVB irradiation, of UVB-inducible inflammatory cytokines produced by keratinocytes and of high-calcium medium (1.8 mmol L(-1) as opposed to 0.08 mmol L(-1) Ca2+) on ATP2A2 and ATP2C1 mRNA expression were quantified in cultured normal human keratinocytes using reverse transcription-polymerase chain reaction. Expression of ATP2A2 and ATP2C1 mRNA was suppressed immediately after exposure to UVB irradiation, and modulation of mRNA expression was achieved in keratinocytes cultured with proinflammatory cytokines. The mRNA expression of both genes was increased significantly after the shift to high extracellular Ca2+ concentration. The results suggest that modulation of ATP2A2 and ATP2C1 mRNA expression by UV or cytokines might contribute to the clinical presentations unique to DD and HHD, and that the controlled expression of these genes plays an important role in keratinocyte homeostasis, function and differentiation.

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.

[Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of interleukin-34 in mouse osteoblasts].

PubMed

Yu, Ya-Qiong; Guo, Jia-Jie; Qiu, Li-Hong; Li, Xiao-Lin; Yang, Di; Guo, Yan

2017-02-01

To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (P.e) on the expression of interleukin-34 (IL-34) mRNA in MC3T3-E1 cells and the role of p38MAPK, ERK1/2, NF-κB and SIRT1 in the process. MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-24 h). The expression of IL-34 mRNA was detected by real-time reverse transcription-polymerase chain reaction (real time RT-PCR). MC3T3-E1 cells were pretreated with inhibitor of NF-κB(BAY 11-7082),inhibitor of p38MAPK (SB203580), inhibitor of ERK1/2 (PD98059), agonist of sirtuin1 (SIRT1) [resveratrol (RES)] and inhibitor of SIRT1 (EX-527) for 1 h, and then were treated with 20 mg/L P.e-LPS. The expression of IL-34 mRNA was detected by real time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. The level of IL-34 mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L)，which indicated that P.e-LPS induced osteoblasts to express IL-34 mRNA in a dose-dependent manner. Maximal induction of IL-34 mRNA expression was observed in MC3T3-E1 cells treated with 20 mg/L P.e-LPS for 24 h.At 48 h, the expression of IL-34 mRNA decreased gradually. The mRNA of IL-34 decreased significantly after pretreatment with 10 μmol/L BAY-117082, SB203580 and PD98059 for 1 h. P.e-LPS-induced IL-34 upregulation was attenuated by pretreatment with RES, but increased by EX-527. These results suggest that P.e-LPS may mediate IL-34 mRNA expression in MC3T3-E1 cells. This process is dependent, at least in part, on p38MAPK, ERK1/2, NF-κB and SIRT1 signaling pathways.

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.

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.

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

PubMed

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

2018-01-01

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

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells.

PubMed

Chen, C; Yang, R L

2013-08-01

MP [4-(3',3'-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27(KIP1) protein and p21(CIP1) mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21(CIP1), p16(INK4a) and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

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.

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls

PubMed Central

Fassah, Dilla Mareistia; Jeong, Jin Young

2018-01-01

Objective This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Methods Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Results Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Conclusion Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat deposition. PMID:29502393

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls.

PubMed

Fassah, Dilla Mareistia; Jeong, Jin Young; Baik, Myunggi

2018-04-01

This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat deposition.

Effect of β-glucan on MUC4 and MUC5B expression in human airway epithelial cells.

PubMed

Kim, Yong-Dae; Bae, Chang Hoon; Song, Si-Youn; Choi, Yoon Seok

2015-08-01

β-Glucan is found in the cell walls of fungi, bacteria, and some plant tissues, and is detected by the innate immune system. Furthermore, this recognition is known to worsen respiratory symptoms in patients with allergic and inflammatory airway diseases. However, the means by which β-glucan affects the secretion of major mucins by human airway epithelial cells has not been elucidated. Therefore, in this study, the effect and signaling pathway of β-glucan on mucins MUC4 and MUC5B were investigated in human airway epithelial cells. In NCI-H292 cells and human normal nasal epithelial cells, the effect and signaling pathway of β-glucan on MUC4 and MUC5B expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA (siRNA). β-Glucan increased MUC4 and MUC5B expression and activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). SB203580 (a p38 MAPK inhibitor) and pyrrolidine dithiocarbamate (PDTC; a NF-κB inhibitor) inhibited β-glucan-induced MUC4 and MUC5B expression. In addition, siRNA knockdown of p38 MAPK blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of NF-κB. Furthermore, Toll-like receptor 4 (TLR4) mRNA expression was increased by β-glucan, and siRNA knockdown of TLR4 blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of p38 MAPK and NF-κB. These results demonstrate that in human airway epithelial cells β-glucan induces MUC4 and MUC5B expression via the TLR4-p38 MAPK-NF-κB signaling pathway. © 2015 ARS-AAOA, LLC.

Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes.

PubMed

Nagasaki, Hiroshi; Kondo, Takaaki; Fuchigami, Masahiro; Hashimoto, Hiroyuki; Sugimura, Yoshihisa; Ozaki, Nobuaki; Arima, Hiroshi; Ota, Akira; Oiso, Yutaka; Hamada, Yoji

2012-02-17

In this study we aimed to identify the physiological roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, co-culture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

Two assays for measuring fibrosis: reverse transcriptase-polymerase chain reaction of collagen alpha(1) (III) mRNA is an early predictor of subsequent collagen deposition while a novel serum N-terminal procollagen (III) propeptide assay reflects manifest fibrosis in carbon tetrachloride-treated rats.

PubMed

Kauschke, S G; Knorr, A; Heke, M; Kohlmeyer, J; Schauer, M; Theiss, G; Waehler, R; Burchardt, E R

1999-11-15

Using a novel quantitative reverse transcriptase-polymerase chain reaction assay, we have determined the amount of specific mRNA for procollagen alpha(1) (III) (PIIIP) in the carbon tetrachloride (CCl(4)) model of liver fibrosis in rats. After a single week of CCl(4) application, the amount of PIIIP mRNA was increased approximately 10 times over the untreated control group and continued to increase to approximately 30 times after 7 weeks of intoxication. In this model substantial fibrosis was demonstrated by computer-aided morphometry after 5 to 7 weeks of treatment. Using recombinant murine N-terminal procollagen alpha(1) (III) propeptide (PIIINP), a novel sensitive immunoassay for the measurement of circulating PIIINP in rodent sera was established. An increase in PIIINP serum levels was observed after 5 to 7 weeks of CCl(4) intoxication. Our results suggest PIIIP gene expression is an early marker of tissue fibrosis. Early PIIIP gene expression is correlated with the extent of the subsequent fibrosis. PIIIP mRNA levels increase much earlier than conventional histological examination or PIIINP levels. PIIINP measurements with our new serum assay, on the other hand, are a good noninvasive marker of manifest fibrosis but are a poor marker of fibrogenesis. Copyright 1999 Academic Press.

Association between toll-like receptors expression and major depressive disorder.

PubMed

Hung, Yi-Yung; Kang, Hong-Yo; Huang, Kai-Wei; Huang, Tiao-Lai

2014-12-15

Accumulating evidences suggest that Toll-like receptors (TLRs) were involved in the pathophysiology of major depressive disorder. TLR4 was thought to be associated with major depressive disorder in animal model, but the others were still unknown. In order to examine TLR1-9 mRNA expression levels in peripheral blood and their relationships with the psychopathology of major depressive disorder, 30 patients with major depressive disorder were compared with 29 healthy controls. The 17-item Hamilton Depression Rating Scale (HAMD-17) was used to assess the severity of major depression. The mRNA expression levels of TLRs were examined in parallel with a housekeeping gene using real-time polymerase chain reaction (RT-PCR). Analysis of covariance with age and body mass index adjustment revealed a significantly higher expression of TLR3, 4, 5 and 7 mRNA but lower expression of TLR1 and 6 in patients with major depressive disorder as compared with healthy controls. Multiple linear regression analysis revealed that TLR4 was an independent risk factor relating to severity of major depression. These findings suggest that TLRs, especially TLR4, may be involved in the psychopathology of major depression. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

PubMed Central

Long, E O; Gross, N; Wake, C T; Mach, J P; Carrel, S; Accolla, R; Mach, B

1982-01-01

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6821356

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum.

PubMed

Guerra, D M; Giometti, I C; Price, C A; Andrade, P B; Castilho, A C; Machado, M F; Ripamonte, P; Papa, P C; Buratini, J

2008-01-01

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

NASA Technical Reports Server (NTRS)

Wu, Liu-Lai; Song, Il; Karuppiah, Nadarajah; Kaufman, Peter B.

1993-01-01

An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

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.

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

PubMed Central

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

2015-01-01

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

Effects of pharmacological treatments on hippocampal NCAM1 and ERK2 expression in epileptic rats with cognitive dysfunction

PubMed Central

Kong, Qingxia; Min, Xia; Sun, Ran; Gao, Jianying; Liang, Ruqing; Li, Lei; Chu, Xu

2016-01-01

The present study aimed to investigate the effects of various pharmacological agents on the hippocampal expression of neural cell adhesion molecule 1 (NCAM1) and extracellular signal-regulated kinase 2 (ERK2) in epileptic rats with cognitive dysfunction. The experiments were conducted using 120 Wistar rats: 20 controls and 100 with pilocarpine-induced status epilepticus (SE). The SE rats were randomly assigned to 5 groups (n=20/group) that received daily treatments for 1 month with one of the following: (i) saline (no effect on epilepsy); (ii) carbamazepine (an anticonvulsant); (iii) oxcarbazepine (an anticonvulsant); (iv) aniracetam (a nootropic); or (v) donepezil (an acetylcholinesterase inhibitor). Spatial learning and memory were assessed using a Morris Water Maze (MWM). Hippocampal tissue was assessed for NCAM1 and ERK2 messenger RNA (mRNA) expression by reverse transcription polymerase chain reaction, and protein expression by immunochemistry. The results revealed that SE rats had significantly poorer MWM performances compared with controls (P<0.01). Performance in SE rats was improved with donepezil treatment (P<0.01), but declined with carbamazepine (P<0.01). Compared with controls, saline-treated SE rats exhibited increased hippocampal NCAM1 mRNA expression (P<0.01). Among SE rats, NCAM1 mRNA expression was highest in those treated with donepezil, followed by aniracetam-, saline-, oxcarbazepine- and carbamazepine-treated rats. Compared to controls, saline-treated SE rats exhibited decreased hippocampal ERK2 mRNA expression (P<0.01). Among SE rats, ERK2 mRNA expression was highest in those treated with donepezil, followed by aniracetam, saline, oxcarbazepine and carbamazepine. NCAM1 and ERK2 protein expression levels were parallel to those of the mRNA. In saline-treated SE rats, hippocampal ERK2 expression was decreased and NCAM1 expression was increased; thus, these two molecules may be involved in the impairment of spatial memory. Carbamazepine augmented this impairment, whereas donepezil was found to ameliorate the dysfunction associated with epilepsy. In conclusion, ERK2 and NCAM1 have significant roles in impairment of spatial memory in SE rats. Carbamazepine may increase this impairment, while donepezil may decrease this impairment. PMID:27588125

[Expression and clinical significance of KNSL4 in breast cancer].

PubMed

Feng, Yu-Mei; Wan, Yan-Fang; Li, Xiao-Qing; Cao, Xu-Chen; Li, Xi

2006-06-01

Previous screening of breast cancer metastasis-related genes found that the mRNA level of kinesin-like 4 (KNSL4) gene is down-regulated in metastatic lymph nodes as compared with the paired primary breast cancer. This study was to clarify the correlations of KNSL4 mRNA expression to metastasis and prognosis of breast cancer, and explore the correlation of KNSL4 expression to c-erbB-2 expression to explore potential mechanisms of promoting metastasis by KNSL4. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the mRNA level of KNSL4 in 108 specimens of primary breast cancer. The correlations of KNSL4 mRNA level to metastasis and prognosis of the 108 cases were analyzed. Immunohistochemistry was used to assess c-erbB-2 protien expression in 76 out of the 108 cases, and the correlation of KNSL4 expression to c-erbB-2 expression was analyzed. The mRNA level of KNSL4 was significantly lower in the cases at stages iii-iv than in the cases at stages iii-iv (P<0.001), significantly lower in the cases with more than 3 metastatic lymph nodes than in the cases with 0-3 metastatic positive lymph nodes (P<0.01), slightly lower in the cases with negative estrogen receptor or prognesterone receptor than in the cases with positive receptors (P>0.05), lower in the 6 cases with distant metastasis than in the rest cases without distant metastasis within 24 month follow up, lower in the 3 cases with bilateral breast cancer than in other cases with unilateral breast cancer, and significantly lower in c-erbB-2-positive group than in c-erB-2-negative group (P<0.01). The down-regulation of KNSL4 mRNA level is correlated to prognosis of primary breast cancer. It may enhance metastatic ability of breast cancer cells through promoting c-erbB-2 transcription and translation.

Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

USDA-ARS?s Scientific Manuscript database

NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

Modulation expression of tumor necrosis factor α in the radiation-induced lung injury by glycyrrhizic acid.

PubMed

Refahi, Soheila; Pourissa, Masoud; Zirak, Mohammad Reza; Hadadi, GholamHassan

2015-01-01

To evaluate the ability of glycyrrhizic acid (GLA) to reduce the tumor necrosis factor α (TNF-α), release on messenger ribonucleic acid (mRNA) and protein production in the lungs using GLA in response to irradiation were studied. The animals were divided into four groups: No treatment (NT group), GLA treatment only (GLA group), irradiation only (XRT group), and GLA treatment plus irradiation (GLA/XRT group). Rats were killed at different time points. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of TNF-α in the lungs (compared with non-irradiated lungs). An enzyme-linked immunosorbant assay (ELISA) assay was used to measure the TNF-α protein level. The TNF-α mRNA expression in the lungs of the XRT rats was clearly higher at all-time points compared to the NT rats. The TNF-α mRNA expression in the lungs of the GLA/XRT rats was lower at all-time points compared to the XRT rats. Release of the TNF-α on protein level in the lungs of the XRT rats increased at all-time points compared to the NT rats. In contrast to the XRT rats, the lungs of the GLA/XRT rats revealed a reduction on TNF-α protein level at 6 h after irradiation. This study has clearly showed the immediate down-regulation of the TNF-α mRNA and protein production in the lungs using GLA in response to irradiation.

Modulation expression of tumor necrosis factor α in the radiation-induced lung injury by glycyrrhizic acid

PubMed Central

Refahi, Soheila; Pourissa, Masoud; Zirak, Mohammad Reza; Hadadi, GholamHassan

2015-01-01

To evaluate the ability of glycyrrhizic acid (GLA) to reduce the tumor necrosis factor α (TNF-α), release on messenger ribonucleic acid (mRNA) and protein production in the lungs using GLA in response to irradiation were studied. The animals were divided into four groups: No treatment (NT group), GLA treatment only (GLA group), irradiation only (XRT group), and GLA treatment plus irradiation (GLA/XRT group). Rats were killed at different time points. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of TNF-α in the lungs (compared with non-irradiated lungs). An enzyme-linked immunosorbant assay (ELISA) assay was used to measure the TNF-α protein level. The TNF-α mRNA expression in the lungs of the XRT rats was clearly higher at all-time points compared to the NT rats. The TNF-α mRNA expression in the lungs of the GLA/XRT rats was lower at all-time points compared to the XRT rats. Release of the TNF-α on protein level in the lungs of the XRT rats increased at all-time points compared to the NT rats. In contrast to the XRT rats, the lungs of the GLA/XRT rats revealed a reduction on TNF-α protein level at 6 h after irradiation. This study has clearly showed the immediate down-regulation of the TNF-α mRNA and protein production in the lungs using GLA in response to irradiation. PMID:26170556

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

PubMed Central

2012-01-01

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

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

PubMed

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

2015-06-01

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

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

PubMed Central

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

2015-01-01

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

Ramipril restores PPARβ/δ and PPARγ expressions and reduces cardiac NADPH oxidase but fails to restore cardiac function and accompanied myosin heavy chain ratio shift in severe anthracycline-induced cardiomyopathy in rat.

PubMed

Cernecka, Hana; Doka, Gabriel; Srankova, Jasna; Pivackova, Lenka; Malikova, Eva; Galkova, Kristina; Kyselovic, Jan; Krenek, Peter; Klimas, Jan

2016-11-15

We hypothesized that peroxisome proliferator-activated receptors (PPARs) might be involved in a complex protective action of ACE inhibitors (ACEi) in anthracyclines-induced cardiomyopathy. For purpose of study, we compared effects of ramipril on cardiac dysfunction, cardiac failure markers and PPAR isoforms in moderate and severe chronic daunorubicin-induced cardiomyopathy. Male Wistar rats were administered with a single intravenous injection of daunorubicin: 5mg/kg (moderate cardiomyopathy), or 15mg/kg (severe cardiomyopathy) or co-administered with daunorubicin and ramipril (1mg/kg/d, orally) or vehicle for 8 weeks. Left ventricular function was measured invasively under anesthesia. Cardiac mRNA levels of heart failure markers (ANP, Myh6, Myh7, Myh7b) and PPARs (alpha, beta/delta and gama) were measured by qRT-PCR. Protein expression of NADPH subunit (gp91phox) was measured by Western blot. Moderate cardiomyopathy exhibited only minor cardiac dysfunction what was corrected by ramipril. In severe cardiomyopathy, hemodynamic dysfunction remained unaltered upon ramipril although it decreased the significantly up-regulated cardiac ANP mRNA expression. Simultaneously, while high-dose daunorubicin significantly decreased PPARbeta/delta and PPARgama mRNA, ramipril normalized these abnormalities. Similarly, ramipril reduced altered levels of oxidative stress-related gp91phox. On the other hand, ramipril was unable to correct both the significantly decreased relative abundance of Myh6 and increased Myh7 mRNA levels, respectively. In conclusion, ramipril had a protective effect on cardiac function exclusively in moderate chronic daunorubicin-induced cardiomyopathy. Although it normalized abnormal PPARs expression and exerted also additional protective effects also in severe cardiomyopathy, it was insufficient to influence impaired cardiac function probably because of a shift in myosin heavy chain isoform content. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of palmitoyl-CoA chain elongation by clofibric acid in the liver of Zucker fa/fa rats.

PubMed

Toyama, Tomoaki; Kudo, Naomi; Mitsumoto, Atsushi; Kawashima, Yoichi

2005-05-01

The regulation of palmitoyl-CoA chain elongation (PCE) by clofibric acid [2-(4-chlorophenoxy)-2-methylpropionic acid] was investigated in comparison with stearoyl-CoA desaturase (SCD) in the liver of obese Zucker fa/fa rats. The proportion of oleic acid in the hepatic lipids of Zucker obese rats is 2.7 times higher than that of lean littermates. The activities of PCE and SCD in the liver of Zucker obese rats were markedly higher than in lean rats, and the hepatic uptake of 2-deoxyglucose (2-DG) was also higher in Zucker obese rats compared with lean rats. The increased activities of SCD and PCE in Zucker obese rats were due to the enhanced expression of mRNA of both SCD1 and rat FA elongase 2 (rELO2), but not SCD2 or rELO1. The proportion of oleic acid in the liver was significantly increased by the administration of clofibric acid to Zucker obese rats, and the hepatic PCE activity and rELO2 mRNA expression, but not the SCD activity or SCD1 mRNA expression, were increased in response to clofibric acid treatment. By contrast, the activities of both PCE and SCD and the mRNA expression of SCD1 and rELO2 in the liver were increased by the treatment of Zucker lean rats with clofibric acid. Multiple regression analysis, which was performed to determine the relationships involving PCE activity, SCD activity, and the proportion of oleic acid, revealed that the three parameters were significantly correlated and that the standardized partial regression coefficient of PCE was higher than that of SCD. These results indicate that oleic acid is synthesized by the concerted action of PCE and SCD and that PCE plays a crucial role in the formation of oleic acid when Zucker fa/fa rats are given clofibric acid.

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

PubMed

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

2017-04-01

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

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

PubMed

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

2016-08-01

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

Stimulation of synthesis and release of brain-derived neurotropic factor from intestinal smooth muscle cells by substance P and pituitary adenylate cyclase-activating peptide.

PubMed

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

2015-08-01

Brain-derived neurotrophic factor (BDNF) is a neurotrophin present in the intestine where it participates in survival and growth of enteric neurons, augmentation of enteric circuits, and stimulation of intestinal peristalsis and propulsion. Previous studies largely focused on the role of neural and mucosal BDNF. The expression and release of BDNF from intestinal smooth muscle and the interaction with enteric neuropeptides has not been studied in gut. The expression and secretion of BDNF from smooth muscle cultured from the rabbit intestinal longitudinal muscle layer in response to substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP) was measured by western blot and enzyme-linked immunosorbent assay. BDNF mRNA was measured by reverse-transcription polymerase chain reaction. The expression of BNDF protein and mRNA was greater in smooth muscle cells (SMCs) from the longitudinal muscle than from circular muscle layer. PACAP and SP increased the expression of BDNF protein and mRNA in cultured longitudinal SMCs. PACAP and SP also stimulated the secretion of BDNF from cultured longitudinal SMCs. Chelation of intracellular calcium with BAPTA (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) prevented SP-induced increase in BDNF mRNA and protein expression and SP-induced secretion of BDNF. Neuropeptides known to be present in enteric neurons innervating the longitudinal layer increase the expression of BDNF mRNA and protein in SMCs and stimulate the release of BDNF. Considering the ability of BDNF to enhance smooth muscle contraction, this autocrine loop may partially explain the characteristic hypercontractility of longitudinal muscle in inflammatory bowel disease. © 2015 John Wiley & Sons Ltd.

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

PubMed Central

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

2009-01-01

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

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

PubMed

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

2009-01-01

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

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

PubMed

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

2012-04-01

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

Cellular antioxidant effects of atorvastatin in vitro and in vivo.

PubMed

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

2002-02-01

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

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

PubMed

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

2002-10-15

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

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

PubMed

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

2010-01-01

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

Molecular cloning, characterization, and expression analysis of an ecdysone receptor homolog in Teleogryllus emma (Orthoptera: Gryllidae).

PubMed

He, Hui; Xi, Gengsi; Lu, Xiao

2015-01-01

Ecdysteroids are steroid hormones that play important roles in the regulation of Arthropoda animal growth development, larvae ecdysis, and reproduction. The effect of ecdysteroids is mediated by ecdysteroid receptor (EcR). The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) that are ligand-dependent transcription factors. Ecdysone receptor is present only in invertebrates and plays a critical role in regulating the expression of a series of genes during development and reproduction. Here, we isolated and characterized cDNA of the cricket Teleopgryllus emma (Ohmachi & Matsuura) (Orthoptera: Gryllidae) and studied mRNA expression pattern using real time-polymerase chain reaction. The full-length cDNA of T. emma EcR, termed TeEcR, is 2,558 bp and contains a 5'-untranslated region of 555 bp and a 3'-untranslated region of 407 bp. The open reading frame of TeEcR encodes deduced 531-amino acid peptides with a predicted molecular mass of 60.7 kDa. The amino acid sequence of T. emma EcR was similar to that of known EcR especially in the ligand-binding domain of insect EcR. Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare TeEcR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeEcR mRNA is differentially expressed during T. emma development, with the highest expression level in late-instar larvae of the body and lowest in third instar. The levels of TeEcR transcripts also vary among gonads development, and levels in ovaries were higher than in testes at every developmental stage. These results suggest that TeEcR may have potential significance to regulate the morphological structure and gonad development of T. emma, due to its expression in different developmental periods. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Molecular Cloning, Characterization, and Expression Analysis of an Ecdysone Receptor Homolog in Teleogryllus emma (Orthoptera: Gryllidae)

PubMed Central

He, Hui; Xi, Gengsi; Lu, Xiao

2015-01-01

Ecdysteroids are steroid hormones that play important roles in the regulation of Arthropoda animal growth development, larvae ecdysis, and reproduction. The effect of ecdysteroids is mediated by ecdysteroid receptor (EcR). The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) that are ligand-dependent transcription factors. Ecdysone receptor is present only in invertebrates and plays a critical role in regulating the expression of a series of genes during development and reproduction. Here, we isolated and characterized cDNA of the cricket Teleopgryllus emma (Ohmachi & Matsuura) (Orthoptera: Gryllidae) and studied mRNA expression pattern using real time-polymerase chain reaction. The full-length cDNA of T. emma EcR, termed TeEcR, is 2,558 bp and contains a 5′-untranslated region of 555 bp and a 3′-untranslated region of 407 bp. The open reading frame of TeEcR encodes deduced 531-amino acid peptides with a predicted molecular mass of 60.7 kDa. The amino acid sequence of T. emma EcR was similar to that of known EcR especially in the ligand-binding domain of insect EcR. Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare TeEcR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeEcR mRNA is differentially expressed during T. emma development, with the highest expression level in late-instar larvae of the body and lowest in third instar. The levels of TeEcR transcripts also vary among gonads development, and levels in ovaries were higher than in testes at every developmental stage. These results suggest that TeEcR may have potential significance to regulate the morphological structure and gonad development of T. emma, due to its expression in different developmental periods. PMID:25797799

Expression and clinical significance of glucose transporter 1 mRNA in bronchial brushing liquid-based cytology specimens from patients with and without lung cancer.

PubMed

Lv, M; Fan, Y-B; Zhao, Y-J; Wang, T-Y; Wu, G-P

2012-04-01

The aim of this study was to evaluate the diagnostic utility of glucose transporter 1 (GLUT1) mRNA expression in bronchial brushing specimens from patients with lung cancer. GLUT1 mRNA levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) in SurePath(TM) liquid-based cytology bronchial brushing specimens from patients with lung cancer (n=76) and benign lung disease (n=154). Compared with patients with benign disease and compared with cytology, GLUT1 mRNA was found significantly more frequently in patients with all carcinomas, squamous cell carcinomas, adenocarcinomas and small cell carcinomas, as well as central, peripheral and diffuse carcinomas (P<0.01). Minor differences were noted in GLUT1 mRNA and cytology results between histological types and tumour location but were not statistically significant. The diagnostic performance of RT-PCR analysis of GLUT1 mRNA was significantly higher than cytology in terms of sensitivity (97.4 ± 3.6% versus 65.8 ± 10.7) and negative predictive value (98.6 ± 1.9%, versus 85.6 ± 5.1%) but specificity (90.9 ± 4.5%) and positive predictive value (84.1 ± 7.6%) were lower than cytology (100%). Using liquid-based cytology, RT-PCR can be performed on bronchial brushing specimens to detect GLUT1 mRNA expression, and may be a useful adjunct to cytology diagnosis. It was more sensitive than cytology but its lower specificity should be taken into account. © 2011 Blackwell Publishing Ltd.

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

PubMed Central

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

2010-01-01

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

Loss of Thrombomodulin in Placental Dysfunction in Preeclampsia.

PubMed

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

2016-04-01

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

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.

[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.

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

PubMed Central

2010-01-01

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

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

PubMed

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

2010-10-31

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

Conjunctival mucin mRNA expression in contact lens wear.

PubMed

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

2009-09-01

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

TC-99m MIBI SPECT imaging in patients with lung carcinoma: is it a functional probe of multidrug resistance genes?

PubMed

Ak, Ilknur; Gülbaş, Zafer; Ocak, Suna; Kaya, Eser; Alataş, Füsun; Vardareli, Erkan; Metintaş, Muzaffer

2007-01-01

Multidrug-resistance (MDR) phenotype concerns altered membrane transport that results in lower cell concentrations of cytotoxic drug in many cancer types, including lung cancer, and is related to the overexpression of a variety of proteins that act as adenosine triphosphate-dependent extrusion pumps. Tc-99m Sestamibi (MIBI) is a transport substrate for P-glycoprotein (Pgp) pump. In this study, we assessed the uptake and clearance of technetium-99m-2-hexakis 2-methoxyisobutylisonitrile (Tc-99m MIBI) from the tumor and its correlation with messenger RNA (mRNA) levels of Pgp, MDR-associated protein (MRP1), and lung resistance protein (LRP) in lung carcinoma. This study was carried out on 19 patients (mean age, 60.1 +/- 2.07 years) with advanced-stage lung carcinoma. The tumor samples obtained by bronchoscopy were assessed to estimate the levels of Pgp, MRP1, and LRP expression on mRNA level by quantitative real-time reverse-transcription polymerase chain reaction. Tc-99m MIBI chest imaging was performed 15 and 180 minutes after injection of 740 MBq Tc-99m MIBI. The early (T/Be) and delayed (T/Bd) Tc-99m MIBI uptakes and washout rate (WR) of Tc-99m MIBI from the tumor were measured. No correlation was found between the T/Be Tc-99m MIBI uptake of tumors (T/Be) and the levels of Pgp mRNA, MRP1 mRNA, and LRP mRNA by reverse-transcription polymerase chain reaction. There was a correlation between the mean T/Bd Tc-99m MIBI uptake and Pgp expression of the tumors (P = 0.001, Spearman rho = - 0.702). There was a correlation between the WR of Tc-99m MIBI from the tumor and Pgp expression of the tumor (P = 0.000, Spearman rho = 0.875). Washout rate of Tc-99m MIBI was not related to the levels of MRP1 mRNA (P = 0.93, Spearman rho = 0.02) or LRP mRNA (P = 0.47, Spearman rho = 0.177). Increased WR of Tc-99m MIBI is related in Pgp over expression of the tumor. Tc-99m MIBI single photon emission computed tomography imaging may be a functional probe of overexpression of Pgp in patients with lung carcinoma. However, Tc-99m MIBI single photon emission computed tomography imaging cannot be used to identify the MDR involved in the MRP1 or LRP in these patients.

All-trans retinoic acid increases the expression of oxidative myosin heavy chain through the PPARδ pathway in bovine muscle cells derived from satellite cells.

PubMed

Kim, Jongkyoo; Wellmann, Kimberly B; Smith, Zachary K; Johnson, Bradley J

2018-04-24

All-trans retinoic acid (ATRA) has been associated with various physiological phenomenon in mammalian adipose tissue and skeletal muscle. We hypothesized that ATRA may affect skeletal muscle fiber type in bovine satellite cell culture through various transcriptional processes. Bovine primary satellite cell (BSC) culture experiments were conducted to determine dose effects of ATRA on expression of genes and protein levels related to skeletal muscle fiber type and metabolism. The semimembranosus from crossbred steers (n = 2 steers), aged approximately 24 months, were used to isolate BSC for 3 separate assays. Myogenic differentiation was induced using 3% horse serum upon cultured BSC with increasing doses (0, 1, 10, 100, 1000 nM) of ATRA. After 96 h of incubation, cells were harvested and used to measure the gene expression of protein kinase B (Akt), AMP-activated protein kinase alpha (AMPK), glucose transporter 4 (GLUT4), myogenin, lipoprotein lipase (LPL), myosin heavy chain (MHC) I, MHC IIA,MHC IIX, insulin like growth factor -1 (IGF-1), Peroxisome proliferator activated receptor gamma (PPARγ), PPARδ, and Smad transcription factor 3 (SMAD3) mRNA relative to ribosomal protein subunit 9 (RPS9). The mRNA expression of LPL was increased (P < 0.05) with 100 and 1000nM of ATRA. Expression of GLUT4 was altered (P < 0.05) by ATRA. The treatment of ATRA (1000nM) also increased (P < 0.05) mRNA gene expression of SMAD3. The gene expression of both PPARδ and PPARγ were increased (P < 0.05) with 1000nM of ATRA. Protein level of PPARδ was also affected (P < 0.05) by 1000nM of ATRA and resulted in a greater (P < 0.05) protein level of PPARδ compared to CON. All-trans retinoic acid (10nM) increased gene expression of MHC I (P < 0.05) compared to CON. Expression of MHC IIA was also influenced (P < 0.05) by ATRA. The mRNA expression of MHC IIX was decreased (P < 0.05) with 100 and 1000nM of ATRA.In muscle cells, ATRA may cause muscle fibers to transition towards the MHC isoform that prefers oxidative metabolism, as evidenced by increased expression of genes associated with the MHC I isoform. These changes in MHC isoforms appeared to be brought about by changing PPARδ gene expression and protein levels.

LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients

PubMed Central

Duzkale, Hatice; Schweighofer, Carmen D.; Coombes, Kevin R.; Barron, Lynn L.; Ferrajoli, Alessandra; O'Brien, Susan; Wierda, William G.; Pfeifer, John; Majewski, Tadeusz; Czerniak, Bogdan A.; Jorgensen, Jeffrey L.; Medeiros, L. Jeffrey; Freireich, Emil J; Keating, Michael J.

2011-01-01

We previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients. PMID:21310924

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

Expression profile of HSP genes during different seasons in goats (Capra hircus).

PubMed

Dangi, Satyaveer Singh; Gupta, Mahesh; Maurya, Divakar; Yadav, Vijay Prakash; Panda, Rudra Prasanna; Singh, Gyanendra; Mohan, Nitai Haridas; Bhure, Sanjeev Kumar; Das, Bikash Chandra; Bag, Sadhan; Mahapatra, Ramkrishna; Taru Sharma, Guttalu; Sarkar, Mihir

2012-12-01

The present study has demonstrated the expression of HSP60, HSP70, HSP90, and UBQ in peripheral blood mononuclear cells (PBMCs) during different seasons in three different age groups (Groups I, II, and III with age of 0-2, 2-5, and >5 years, respectively) of goats of tropical and temperate regions. Real-time polymerase chain reaction was applied to investigate mRNA expression of examined factors. Specificity of the desired products was documented using analysis of the melting temperature and high-resolution gel electrophoresis to verify that the transcripts are of the exact molecular size predicted. The mRNA expression of HSP60, HSP90, and UBQ was significantly higher (P < 0.05) in all age groups during peak summer season as compared with peak winter season in both tropical and temperate region goats. HSP70 mRNA expression was significantly higher (P < 0.05) during summer season as compared with winter season in tropical region goats. However, in the temperate region, in goats from all the three age groups studied, a non-significant difference of HSP70 expression between summer and winter seasons was noticed. In conclusion, results demonstrate that (1) HSP genes are expressed in caprine PBMCs and (2) higher expression of HSPs during thermal stress suggest possible involvement of them to ameliorate deleterious effect of thermal stress so as to maintain cellular integrity and homeostasis in goats.

Senescence-Induced Alterations of Laminin Chain Expression Modulate Tumorigenicity of Prostate Cancer Cells1

PubMed Central

Sprenger, Cynthia C T; Drivdahl, Rolf H; Woodke, Lillie B; Eyman, Daniel; Reed, May J; Carter, William G; Plymate, Stephen R

2008-01-01

Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs) are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM α4 and β2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM α4 or β2 chain or both chains. Increased expression of either the LM α4 or β2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer. PMID:19048114

Truncated midkine as a marker of diagnosis and detection of nodal metastases in gastrointestinal carcinomas.

PubMed Central

Aridome, K.; Takao, S.; Kaname, T.; Kadomatsu, K.; Natsugoe, S.; Kijima, F.; Aikou, T.; Muramatsu, T.

1998-01-01

Midkine (MK) is a growth factor identified as a product of a retinoic acid-responsive gene. A truncated form of MK mRNA, which lacks a sequence encoding the N-terminally located domain, was recently found in cancer cells. We investigated the expression of the truncated MK mRNA in specimens of 47 surgically removed human gastrointestinal organs using polymerase chain reaction. Truncated MK was not detected in all of the 46 corresponding non-cancerous regions. On the other hand, this short MK mRNA was expressed in the primary tumours in 12 of 16 gastric cancers, 8 of 13 colorectal carcinomas, five of nine hepatocellular carcinomas, two of two oesophageal carcinomas and one ampullary duodenal cancer. In addition, truncated MK was detectable in all of the 14 lymph node metastases but in none of three metastatic sites in the liver, suggesting that truncated MK mRNA could become a good marker of nodal metastases in gastrointestinal tract. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9716029

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2015-08-01

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

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.

Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

PubMed

Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

2001-01-01

Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

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

PubMed Central

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

2007-01-01

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

Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

PubMed

Lu, B; Yang, X J; Chen, K; Yang, D J; Yan, J Q

2009-12-15

Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

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

PubMed

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

2012-12-01

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

Expression of a chemokine by ciliary body epithelium in horses with naturally occurring recurrent uveitis and in cultured ciliary body epithelial cells.

PubMed

Gilger, Brian C; Yang, Ping; Salmon, Jacklyn H; Jaffe, Glenn J; Allen, Janice B

2002-07-01

To determine whether a chemokine (RANTES)-like protein expressed by ciliary epithelium plays a role in uveitis. 3 clinically normal horses intradermal, 5 eyes from 5 horses with recurrent uveitis, and 10 normal eyes from 5 age- and sex-matched horses. Cross-reactivity and sensitivity of recombinant human (rh)-regulated upon activation, normal T-cell expressed and secreted (RANTES) protein were evaluated in horses by use of intradermal hypersensitivity reactions and a chemotaxis assay. Aqueous humor and ciliary body of eyes from clinically normal horses and horses with uveitis were examined for RANTES expression by use of an ELISA and reverse transcription-polymerase chain reaction (RT-PCR). Expression of RANTES mRNA and protein content of primary cultures of equine ciliary pigmented epithelial cells (RT-PCR) and culture supernatant (ELISA) were measured 6 or 24 hours, respectively, after cultures were stimulated with interleukin-1beta and tumor necrosis factor-alpha. Strong reactions to intradermal hypersensitivity testing and significant chemotaxis of equine leukocytes to rh-RANTES wereas observed. Aqueous humor of eyes from horses with uveitis contained increased concentrations of rh-RANTES-like protein (mean +/- SD, 45.9+/-31.7 pg/ml), compared with aqueous humor from clinically normal horses (0 pg/ml). Ciliary body from horses with uveitis expressed RANTES mRNA, whereas ciliary body from clinically normal horses had low mRNA expression. Stimulated ciliary pigmented epithelial cells expressed increased amounts of rh-RANTES-like protein (506.1+/-298.3 pg/ml) and mRNA, compared with unstimulated samples. Ciliary epithelium may play a role in recruitment and activation of leukocytes through expression of RANTES.

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

PubMed

Bucking, Carol; Wood, Chris M

2012-04-01

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

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

PubMed

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

1995-09-01

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

Comparative effects of conjugated linoleic acid (CLA) and linoleic acid (LA) on the oxidoreduction status in THP-1 macrophages.

PubMed

Rybicka, Marta; Stachowska, Ewa; Gutowska, Izabela; Parczewski, Miłosz; Baśkiewicz, Magdalena; Machaliński, Bogusław; Boroń-Kaczmarska, Anna; Chlubek, Dariusz

2011-04-27

The aim of this study was to investigate the effect of conjugated linoleic acids (CLAs) on macrophage reactive oxygen species synthesis and the activity and expression of antioxidant enzymes, catalase (Cat), glutathione peroxidase (GPx), and superoxide dismutase (SOD). The macrophages were obtained from the THP-1 monocytic cell line. Cells were incubated with the addition of cis-9,trans-11 CLA or trans-10,cis-12 CLA or linoleic acid. Reactive oxygen species (ROS) formation was estimated by flow cytometry. Enzymes activity was measured spectrophotometrically. The antioxidant enzyme mRNA expression was estimated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was based on nonparametric statistical tests [Friedman analysis of variation (ANOVA) and Wilcoxon signed-rank test]. cis-9,trans-11 CLA significantly increased the activity of Cat, while trans-10,cis-12 CLA notably influenced GPx activity. Both isomers significantly decreased mRNA expression for Cat. Only trans-10,cis-12 significantly influenced mRNA for SOD-2 expression. The CLAs activate processes of the ROS formation in macrophages. Adverse metabolic effects of each isomer action were observed.

Mangiferin positively regulates osteoblast differentiation and suppresses osteoclast differentiation

PubMed Central

Sekiguchi, Yuusuke; Mano, Hiroshi; Nakatani, Sachie; Shimizu, Jun; Kataoka, Aya; Ogura, Kana; Kimira, Yoshifumi; Ebata, Midori; Wada, Masahiro

2017-01-01

Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3-E1 osteoblastic cells and osteoclast-like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST-1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mangiferin significantly increased the mRNA level of runt-related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate-resistant acid phosphatase-positive multinuclear cells. RT-PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast-associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3-E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes. PMID:28627701

Mangiferin positively regulates osteoblast differentiation and suppresses osteoclast differentiation.

PubMed

Sekiguchi, Yuusuke; Mano, Hiroshi; Nakatani, Sachie; Shimizu, Jun; Kataoka, Aya; Ogura, Kana; Kimira, Yoshifumi; Ebata, Midori; Wada, Masahiro

2017-08-01

Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3‑E1 osteoblastic cells and osteoclast‑like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST‑1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription‑polymerase chain reaction (RT‑PCR) demonstrated that mangiferin significantly increased the mRNA level of runt‑related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate‑resistant acid phosphatase‑positive multinuclear cells. RT‑PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast‑associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3‑E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes.

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2017-01-01

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

Unprecedented multiplicity of Ig transmembrane and secretory mRNA forms in the cartilaginous fish.

PubMed

Rumfelt, Lynn L; Diaz, Marilyn; Lohr, Rebecca L; Mochon, Evonne; Flajnik, Martin F

2004-07-15

In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (C(H)) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgW(short)) having an unusual Sec tail, which is orthologous to skate IgX(short) cDNA. The IgW and IgW(short) Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-05-01

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

Ascorbic acid transported by sodium-dependent vitamin C transporter 2 stimulates steroidogenesis in human choriocarcinoma cells.

PubMed

Wu, Ximei; Iguchi, Takuma; Itoh, Norio; Okamoto, Kousuke; Takagi, Tatsuya; Tanaka, Keiichi; Nakanishi, Tsuyoshi

2008-01-01

Reduced vitamin C [ascorbic acid (AA)], which is taken up into cells by sodium-dependent vitamin C transporter (SVCT) 1 and 2, is believed to be important for hormone synthesis, but its role in generating placental steroids needed to maintain pregnancy and fetal development is not clear. To determine the steroidogenic effect of AA and the role of SVCT2 in AA-induced steroidogenesis, we tested the effects of AA treatment and SVCT2 knockdown on steroidogenesis in human choriocarcinoma cell lines. AA treatment of JEG-3, BeWo, and JAR cells for 48-h dose dependently increased progesterone and estradiol levels. In JEG-3 cells, AA increased the mRNA expression of P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase, key enzymes for steroidogenesis. Stable knockdown of SVCT2 in JEG-3 cells by retrovirally mediated RNA interference decreased the maximal velocity of AA uptake by approximately 50%, but apparent affinity values were not affected. SVCT2 knockdown in JEG-3 cells significantly suppressed the AA-induced mRNA expression of placental P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase. This suppression of the AA-induced mRNA expression of steroidogenic enzymes subsequently decreased progesterone and estradiol production. In addition, inhibition of MAPK kinase-ERK signaling, which is a major pathway for AA-regulated gene expression, failed to affect AA-induced steroidogenesis. Our observations indicate that SVCT2-mediated AA uptake into cells is necessary for AA-induced steroidogenesis in human choriocarcinoma cell, but MAPK kinase-ERK signaling is not involved in AA-induced steroidogenesis.

Fiber transformation and replacement in low-frequency stimulated rabbit fast-twitch muscles.

PubMed

Schuler, M; Pette, D

1996-08-01

The fast-to-slow conversion of rabbit skeletal muscles by chronic low-frequency (10 Hz, 12 h daily) stimulation involves (1) sequential fast-to-slow fiber-type transitions in the order of type IID-->type IIA-->type I, and (2) the replacement of deteriorating fast-twitch glycolytic fibers by new fibers derived from satellite cells and myotubes. These two processes were analyzed in 30- and 60-day stimulated extensor digitorum longus and tibialis anterior muscles. Fast-to-slow transforming fibers were identified by myofibrillar actomyosin histochemistry as type C fibers and immunohistochemically by their reaction with monoclonal antibodies specific to slow and fast myosin heavy chain isoforms. In situ hybridization of mRNA specific to the myosin heavy chain I isoform identified all fibers expressing slow myosin, i.e., type I and C fibers. The fraction of transforming fibers ranged between 35% and 50% in 30-day stimulated muscles. The percentage of type I fibers (20%) was threefold elevated in extensor digitorum longus muscle, but unaltered (3.5%) in tibialis anterior muscle, suggesting that fast-to-slow fiber conversion was more advanced in the former than in the latter. Fiber replacement was indicated by the finding that the fiber populations of both muscles contained 15% myotubes or small fibers with central nuclei. In situ hybridization revealed that myotubes and small regenerating fibers uniformly expressed myosin heavy chain I mRNA. Similarly, high percentages of slow-myosin-expressing myotubes and small fibers were found in 60-day stimulated muscles.

Methylation pattern of IFNG in periapical granulomas and radicular cysts.

PubMed

Campos, Kelma; Gomes, Carolina Cavaliéri; de Fátima Correia-Silva, Jeane; Farias, Lucyana Conceição; Fonseca-Silva, Thiago; Bernardes, Vanessa Fátima; Pereira, Cláudia Maria; Gomez, Ricardo Santiago

2013-04-01

Interferon-γ plays an important role in the pathogenesis of periapical lesions, and the methylation of IFNG has been associated with transcriptional inactivation. The purpose of the present study was to investigate IFNG promoter methylation in association with gene transcription and protein levels in periapical granulomas and radicular cysts. Methylation-specific polymerase chain reaction was used to assess the DNA methylation pattern of the IFNG gene in 16 periapical granulomas and 13 radicular cyst samples. The transcription levels of IFNG mRNA were verified by quantitative real-time polymerase chain reaction, and protein expression was evaluated by immunohistochemistry. All the periapical lesion samples exhibited partial or total methylation of the IFNG gene. In addition, an increased methylation profile was found in radicular cysts compared with periapical granulomas. Increased IFNG mRNA expression was observed in the partially methylated periapical lesion samples relative to the samples that were completely methylated. The present study provides the first evidence of the possible impact of IFNG methylation on IFNG transcription in periapical lesions. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2016-06-01

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

[Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of macrophage colony stimulating factor in mouse osteoblasts].

PubMed

Yu, Yaqiong; Qiu, Lihong; Guo, Jiajie; Qu, Liu; Xu, Liya; Zhong, Ming

2014-09-01

To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of macrophage colony stimulating factor (M-CSF) mRNA and protein in MC3T3-E1 cells and the role of nucler factor-κB (NF-κB) in the process. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (0-50 mg/L) and 10 mg/L Pe-LPS for different hours (0-24 h). The expression of M-CSF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsordent assay (ELISA). The cells untreated by Pe-LPS served as control. The expression of M- CSF mRNA and protein was also detected in 10 mg/L Pe- LPS treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor. The groups were divided as follows, control group, BAY group (10 µmol/L BAY 11-7082 treated alone MC3T3-E1 cells), Pe-LPS group (10 mg/L Pe-LPS stimulated MC3T3-E1 cells for 6 h), BAY combine with Pe-LPS group (10 µmol/L BAY 11-7082 pretreated cells for 1 h and 10 mg/L of Pe-LPS stimulated MC3T3-E1 cells for 6 h). The level of M- CSF mRNA and protein increased significantly after treatment with different concentrations of Pe-LPS (0-50 mg/L), which indicated that Pe-LPS induced osteoblasts to express M-CSF mRNA and protein in dose dependent manners. The expression of M-CSF protein increased from (35 ± 2) ng/L (control group) to (170 ± 8) ng/L (50 mg/L group). Maximal induction of M-CSF mRNA expression was found in the MC3T3- E1 cells treated with 10 mg/L Pe-LPS for 6 h. After 6 h, the expression of M-CSF mRNA decreased gradually. The expression of M-CSF protein also increased with the treatment of 10 mg/L Pe-LPS for 10 h [(122 ± 4) ng/L]. After 10 h, the expression of M-CSF protein decreased gradually. The mRNA and proteins of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. There was no significant difference between BAY group and the control. Pe-LPS may induce the expression of M-CSF mRNA and protein in MC3T3-E1 cells through the signaling of NF-κB.

Peculiar Expression of CD3-Epsilon in Kidney of Ginbuna Crucian Carp.

PubMed

Miyazawa, Ryuichiro; Murata, Norifumi; Matsuura, Yuta; Shibasaki, Yasuhiro; Yabu, Takeshi; Nakanishi, Teruyuki

2018-01-01

TCR/CD3 complex is composed of the disulfide-linked TCR-αβ heterodimer that recognizes the antigen as a peptide presented by the MHC, and non-covalently paired CD3γε- and δε-chains together with disulfide-linked ζ-chain homodimers. The CD3 chains play key roles in T cell development and T cell activation. In the present study, we found nor or extremely lower expression of CD3ε in head- and trunk-kidney lymphocytes by flow cytometric analysis, while CD3ε was expressed at the normal level in lymphocytes from thymus, spleen, intestine, gill, and peripheral blood. Furthermore, CD4-1 + and CD8α + T cells from kidney express Zap-70, but not CD3ε, while the T cells from other tissues express both Zap-70 and CD3ε, although expression of CD3ε was low. Quantitative analysis of mRNA expression revealed that the expression level of T cell-related genes including tcrb, cd3 ε, zap-70 , and lck in CD4-1 + and CD8α + T cells was not different between kidney and spleen. Western blot analysis showed that CD3ε band was detected in the cell lysates of spleen but not kidney. To be interested, CD3ε-positive cells greatly increased after 24 h in in vitro culture of kidney leukocytes. Furthermore, expression of CD3ε in both transferred kidney and spleen leukocytes was not detected or very low in kidney, while both leukocytes expressed CD3ε at normal level in spleen when kidney and spleen leukocytes were injected into the isogeneic recipient. Lower expression of CD3ε was also found in kidney T lymphocytes of goldfish and carp. These results indicate that kidney lymphocytes express no or lower level of CD3ε protein in the kidney, although the mRNA of the gene was expressed. Here, we discuss this phenomenon from the point of function of kidney as reservoir for T lymphocytes in teleost, which lacks lymph node and bone marrow.

Overexpression of Both ERG11 and ABC2 Genes Might Be Responsible for Itraconazole Resistance in Clinical Isolates of Candida krusei

PubMed Central

He, Xiaoyuan; Zhao, Mingfeng; Chen, Jinyan; Wu, Rimao; Zhang, Jianlei; Cui, Rui; Jiang, Yanyu; Chen, Jie; Cao, Xiaoli; Xing, Yi; Zhang, Yuchen; Meng, Juanxia; Deng, Qi; Sui, Tao

2015-01-01

Objective To study the main molecular mechanisms responsible for itraconazole resistance in clinical isolates of Candida krusei. Methods The 14α-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole-susceptible (S) C.krusei at the mRNA level. Results We found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones (P = 0.015 and P = 0.002 respectively). ABC2 gene mRNA levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P = 0.007 in SDD compared with S, P = 0.016 in SDD with R, and P<0.001 in S with R respectively). While ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in a C.krusei (CK10) resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates. Conclusions There are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations may not be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates. PMID:26308936

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture

PubMed Central

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

2001-01-01

The addition of cyclosporin A (500 ng ml−1) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 × 10−7m) was added to the medium, a partial fast-to-slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation. PMID:11351029

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

PubMed

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

2017-11-06

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

Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of β(E) pre-mRNA splicing determine disease severity.

PubMed

Tubsuwan, Alisa; Munkongdee, Thongperm; Jearawiriyapaisarn, Natee; Boonchoy, Chanikarn; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

2011-09-01

Thalassaemia is characterized by the reduced or absent production of globins in the haemoglobin molecule leading to imbalanced α-globin/non α-globin chains. HbE, the result of a G to A mutation in codon 26 of the HBB (β-globin) gene, activates a cryptic 5' splice site in codon 25 leading to a reduction of correctly spliced β(E) -globin (HBB:c.79G>A) mRNA and consequently β(+) -thalassaemia. A wide range of clinical severities in bothα- and β-thalassaemia syndromes, from nearly asymptomatic to transfusion-dependent, has been observed. The correlation between clinical heterogeneity in various genotypes of thalassaemia and the levels of globin gene expression and β(E) -globin pre-mRNA splicing were examined using multiplex quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and allele-specific RT-qPCR. The α-globin/non α-globin mRNA ratio was demonstrated to be a good indicator for disease severity among different thalassaemia disorders. However, the α-globin/non α-globin mRNA ratio ranged widely in β-thalassaemia/HbE patients, with no significant difference between mild and severe phenotypes. Interestingly, the correctly to aberrantly spliced β(E) -globin mRNA ratio in 30% of mild β-thalassaemia/HbE patients was higher than that of the severe patients. The splicing process of β(E) -globin pre-mRNA differs among β-thalassaemia/HbE patients and serves as one of the modifying factors for disease severity. © 2011 Blackwell Publishing Ltd.

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

PubMed

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

2016-01-01

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

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

PubMed

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

2005-06-01

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

Decreased FOXP3 mRNA expression in children with atopic asthma and IgE-mediated food allergy.

PubMed

Krogulska, Aneta; Polakowska, Ewa; Wąsowska-Królikowska, Krystyna; Małachowska, Beata; Młynarski, Wojciech; Borowiec, Maciej

2015-11-01

The role of T regulatory lymphocytes has been investigated in various allergic diseases. However, the precise relation between the phenotype and severity of allergic diseases and the changes in FOXP3 mRNA expression are not fully understood. To compare the expression of FOXP3 mRNA in children with asthma with and without concomitant food allergy (FA) with healthy children and children with only FA. The study included 82 children: 15 with atopic asthma and IgE-dependent FA, 27 with atopic asthma without FA, 20 with IgE-dependent FA without asthma, and 20 healthy children without atopy. Reverse transcription was performed using a commercially available High Capacity cDNA Archive Kit (Applied Biosystems, Carlsbad, California). Analysis was carried out with a 7900HT real-time polymerase chain reaction system (Applied Biosystems). The average level of the FOXP3 gene expression in children with allergy was significantly lower compared with healthy children (2.2 ± 1.3 vs 4.2 ± 4.2; P = .014). The lowest mean level of FOXP3 mRNA expression (1.9 ± 1.6) was recorded in children with asthma and FA, and the highest level (4.2 ± 4.2) was recorded in healthy children without atopy (P = .036). A milder course of asthma or the degree of allergic reaction after a food challenge was associated with higher FOXP3 mRNA expression. Significantly lower levels of FOXP3 gene expression, observed more commonly in children with asthma and IgE-dependent FA than in healthy controls, were associated with a more severe clinical course. Therefore, FOXP3 expression could serve as an indicator of severe asthma with concomitant atopic conditions such as IgE-dependent FA. Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Medium-chain triglycerides promote macrophage reverse cholesterol transport and improve atherosclerosis in ApoE-deficient mice fed a high-fat diet.

PubMed

Zhang, Xinsheng; Zhang, Yong; Liu, Yinghua; Wang, Jin; Xu, Qing; Yu, Xiaoming; Yang, Xueyan; Liu, Zhao; Xue, Changyong

2016-09-01

We previously observed that medium-chain triglycerides (MCTs) could reduce body fat mass and improve the metabolism of cholesterol. We hypothesized that MCTs can improve atherosclerosis by promoting the reverse cholesterol transport (RCT) process. Therefore, the objective of this study was to investigate the roles of MCTs in macrophage RCT and the progression of atherosclerosis. To test this hypothesis, 30 4-week-old ApoE-deficient (ApoE(-/-)) mice were randomly divided into 2 groups and fed a diet of 2% MCTs or long-chain triglycerides (LCTs) for 16 weeks. Ten age- and sex-matched C57BL/6J mice were fed a diet of 2% LCTs as the control. Macrophage-to-feces RCT was assessed in vivo by intraperitoneal injection of RAW 264.7 macrophages containing (3)H-labeled cholesterol, and atherosclerotic plaques were measured. The mRNA and protein expressions were determined by reverse transcriptase polymerase chain reaction and Western blot analyses, respectively. There was a greater decrease in body fat mass, atherosclerotic plaques, and an improvement in serum lipid profiles. In addition, the MCT mice group showed an increase in (3)H-tracer in the feces and a decrease in the liver. Significantly higher levels of mRNA and protein expression of hepatic ATP-binding cassette transporter A1, ATP-binding cassette transporter G5, cholesterol 7α-hydroxylase, and intestinal ATP-binding cassette transporter G8, as well as lower levels of expression of intestinal Niemann-Pick C1-like 1, were found in the MCT group. These results suggest that MCTs could obviously promote macrophage RCT and improve atherosclerosis in ApoE(-/-) mice, indicating that MCTs have the potential to prevent cardiovascular disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Hormonal regulation of β-myosin heavy chain expression in the mouse left ventricle.

PubMed

Patrizio, Mario; Musumeci, Marco; Piccone, Ambra; Raggi, Carla; Mattei, Elisabetta; Marano, Giuseppe

2013-03-01

We investigated the influence of sex hormones on the expression of α- and β-cardiac myosin heavy chain isoforms (α-MHC and β-MHC) in C57bl/6 mice of both sexes under physiological and pathological conditions. In the left ventricles (LVs) of fertile female mice, β-MHC expression was tenfold higher compared with the age-matched males, whereas no difference was found in α-MHC expression. These differences disappeared after ovariectomy or in immature mice. We also found a sex-related difference in expression of β-adrenoceptors (β1-AR), as mRNA levels of this gene were 40% lower in fertile females compared with males of the same age but did not differ in prepubertal or ovariectomized animals. Interestingly, the deletion of both β1- and β2-ARs abolished sex difference of β-MHC expression, as mRNA levels in the LVs of knockout males were increased and reached values comparable to those of knockout females. Moreover, the β1-AR antagonist metoprolol induced about a threefold increase in β-MHC expression in adult male mice. The capability of gender to regulate β-MHC expression was also evaluated in the presence of hemodynamic overload. Thoracic aortic coarctation (TAC) produced cardiac hypertrophy along with a 12-fold increase in β-MHC and a 50% decrease in β1-AR expression in males but not in females, thus abolishing the gender difference observed in sham animals for such genes. By contrast, TAC did not change β2-AR expression. In conclusion, our results show that the expression of β-MHC and β1-AR in the LVs undergo gender-related and correlated changes under both physiological and pathological conditions and suggest a role of β1-AR-mediated signaling.

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

PubMed Central

Chen, C.; Yang, R.L.

2013-01-01

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer. PMID:23903687

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

PubMed

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

2016-11-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2018-05-07

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

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

PubMed Central

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

2015-01-01

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

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

PubMed Central

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

2012-01-01

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

Roles of Heparan Sulfate Sulfation in Dentinogenesis*

PubMed Central

Hayano, Satoru; Kurosaka, Hiroshi; Yanagita, Takeshi; Kalus, Ina; Milz, Fabian; Ishihara, Yoshihito; Islam, Md. Nurul; Kawanabe, Noriaki; Saito, Masahiro; Kamioka, Hiroshi; Adachi, Taiji; Dierks, Thomas; Yamashiro, Takashi

2012-01-01

Cell surface heparan sulfate (HS) is an essential regulator of cell signaling and development. HS traps signaling molecules, like Wnt in the glycosaminoglycan side chains of HS proteoglycans (HSPGs), and regulates their functions. Endosulfatases Sulf1 and Sulf2 are secreted at the cell surface to selectively remove 6-O-sulfate groups from HSPGs, thereby modifying the affinity of cell surface HSPGs for its ligands. This study provides molecular evidence for the functional roles of HSPG sulfation and desulfation in dentinogenesis. We show that odontogenic cells are highly sulfated on the cell surface and become desulfated during their differentiation to odontoblasts, which produce tooth dentin. Sulf1/Sulf2 double null mutant mice exhibit a thin dentin matrix and short roots combined with reduced expression of dentin sialophosphoprotein (Dspp) mRNA, encoding a dentin-specific extracellular matrix precursor protein, whereas single Sulf mutants do not show such defective phenotypes. In odontoblast cell lines, Dspp mRNA expression is potentiated by the activation of the Wnt canonical signaling pathway. In addition, pharmacological interference with HS sulfation promotes Dspp mRNA expression through activation of Wnt signaling. On the contrary, the silencing of Sulf suppresses the Wnt signaling pathway and subsequently Dspp mRNA expression. We also show that Wnt10a protein binds to cell surface HSPGs in odontoblasts, and interference with HS sulfation decreases the binding affinity of Wnt10a for HSPGs, which facilitates the binding of Wnt10a to its receptor and potentiates the Wnt signaling pathway, thereby up-regulating Dspp mRNA expression. These results demonstrate that Sulf-mediated desulfation of cellular HSPGs is an important modification that is critical for the activation of the Wnt signaling in odontoblasts and for production of the dentin matrix. PMID:22351753

Cytokine analysis in lesions refractory to endodontic treatment.

PubMed

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

2011-12-01

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

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

PubMed

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

2016-07-01

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

Interleukin-8 mRNA expression in synovial fluid of canine stifle joints with osteoarthritis.

PubMed

de Bruin, T; de Rooster, H; van Bree, H; Cox, E

2005-12-15

The objective of this study was to examine and compare the presence of interleukin (IL)-8 mRNA in canine stifle osteoarthritis (OA) differing in etiopathogenesis. Synovial fluid (SF) samples were collected from 24 clinically normal stifle joints and 46 diseased stifle joints (32 stifle joints with cranial cruciate ligament rupture (CCLR), 2 joints with CCLR and patella luxation (PL), 7 joints with medial PL and 5 joints with primary OA). The samples were centrifuged to collect synovial fluid cells for RNA extraction. Reverse transcription polymerase chain reaction (RT-PCR) was performed to obtain cDNA from all samples. Canine IL-8 mRNA expression was determined using real time PCR. Synovial fluid glass smears were made of all samples and coloured with H&E for differential cell counts. All stifle joints were radiographed and graded for the severity of OA. Sixty-one percent (28/46) of the samples from canine stifle OA had IL-8 mRNA expression in contrast to 4% (1/24) in the control stifle joints. This difference in prevalence is highly significant. There were no statistically significant pairwise differences among the mean ranks of the various OA groups for the absolute amount of IL-8 mRNA expression. Neither was there a link between the severity of OA (determined by radiographic evaluation) and the presence of IL-8 in the SF nor any significant difference in the absolute amount of IL-8 between the different OA grades. No statistical difference was found in differential cell counts between IL-8-positive and -negative SF samples. IL-8 cannot be used as a specific joint disease marker since IL-8 expression is found in OA differing in etiopathogenesis. It might, however, relate to the ongoing inflammation within the joint.

Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken

PubMed Central

Wang, Yong; He, Jianzhong; Yang, Wenxuan; Muhantay, Gemenggul; Chen, Ying; Xing, Jinming; Liu, Jianzhu

2015-01-01

This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time. PMID:26323394

Cytokine expression in patients with bladder pain syndrome/interstitial cystitis ESSIC type 3C.

PubMed

Logadottir, Yr; Delbro, Dick; Fall, Magnus; Gjertsson, Inger; Jirholt, Pernilla; Lindholm, Catharina; Peeker, Ralph

2014-11-01

Bladder wall nitric oxide production in patients with bladder pain syndrome type 3C is increased compared to undetectable nitric oxide in patients with nonHunner bladder pain syndrome and healthy controls. However, the underlying mechanism/s of the increased nitric oxide production is largely unknown. We compared mRNA expression of a select group of cytokines in patients with bladder pain syndrome/interstitial cystitis type 3C and in pain-free controls. Cold cup biopsies from 7 patients with bladder pain syndrome type 3C and 6 healthy subjects were analyzed. mRNA expression of IL-4, 6, 10 and 17A, iNOS, TNF-α, TGF-β and IFN-γ was estimated by real-time polymerase chain reaction. IL-17 protein expression was determined by immunohistochemistry. Mast cells were labeled with tryptase to evaluate cell appearance and count. IL-6, 10 and 17A, and iNOS mRNA levels as well as the number of mast cells infiltrating the bladder mucosa were significantly increased in patients with bladder pain syndrome type 3C compared to healthy controls. TNF-α, TGF-β and IFN-γ mRNA levels were similar in patients and controls. IL-17A expression at the protein level was up-regulated and localized to inflammatory cells and urothelium in patients with bladder pain syndrome type 3C. Patients with bladder pain syndrome/interstitial cystitis had increased mRNA levels of IL-17A, 10 and 6, and iNOS. IL-17A might be important in the inflammatory process. To our knowledge the increase in IL-17A is a novel finding that may have new treatment implications. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Effects of spaceflight on murine skeletal muscle gene expression

PubMed Central

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

2009-01-01

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

Various dietary fats differentially change the gene expression of neuropeptides involved in body weight regulation in rats.

PubMed

Dziedzic, B; Szemraj, J; Bartkowiak, J; Walczewska, A

2007-05-01

Various high-fat diets are obesogenic but not to the same extent. The aim of the present study was to investigate the effects of saturated fat n-6 and n-3 polyunsaturated fatty acids (PUFAs) on the central neuropeptidergic system in adult rats. Using reverse transcriptase-polymerase chain reaction and in situ hybridisation, we evaluated the net effect of feeding in these fats, comparing the effects of a high- to low-fat diet, and the diversity of the effects of these fats in the same amount within the diet. We also determined plasma lipids, glucose, insulin and leptin concentrations. Six-week feeding with high-saturated fat evoked hyperpahagia and the largest weight gain compared to both high-PUFA diets. Rats fed high-saturated fat were found to have decreased neuropeptide Y (NPY) mRNA expression in the arcuate nucleus (ARC) and the compact zone of the dorsomedial nucleus (DMHc), unchanged pro-opiomelanocortin (POMC), galanin-like peptide (GALP) mRNA expression in the ARC, as well as melanin-concentrating hormone (MCH) and prepro-orexin (preORX) mRNA expression in the lateral hypothalamus, compared to low-saturated fed rats. By contrast, feeding with both high-PUFA diets increased POMC and GALP mRNA expression in the ARC compared to the corresponding low-fat diet and the high-saturated fat diet. Furthermore, feeding with both low-PUFA diets reduced NPY mRNA expression compared to the low-saturated fat diet exclusively in the DMHc. Uniquely, the high n-3 PUFA feeding halved MCH and preORX mRNA expression in the lateral hypothalamus compared to the other high-fat and low n-3 PUFA diets. In rats fed three high-fat diets, plasma insulin and leptin concentrations were significantly increased and the type of fat had no effect on these hormone levels. Rats fed high-saturated fat had both hyperglycaemia and hypertriacylglycerolemia and rats fed high n-3 PUFA only had hyperglycaemia. The present study demonstrates that various forms of dietary fat differentially change the expression of neuropeptide genes involved in energy homeostasis.

Placental expression of D6 decoy receptor in preeclampsia

PubMed Central

Cho, Geum Joon; Lee, Eun Sung; Jin, Hye Mi; Lee, Ji Hye; Kim, Yeun Sun; Seol, Hyun-Joo; Hong, Soon-Cheol; Kim, Hai-Joong

2015-01-01

Objective The purpose of this study was to investigate the expression of the D6 decoy receptor that can bind chemokines and target them for degradation, resulting in inhibition of inflammation in placentas from preeclamptic and normal pregnancies. Methods The current study was carried out in 35 pregnant women (23 patients with preeclampsia and 12 healthy, normotensive pregnant women) during the third trimester of pregnancy. The expressions of D6 decoy receptor in the placenta were determined with real time reverse transcriptase polymerase chain reaction and western blotting. Results The mRNA and protein of D6 decoy receptor were detected in all of placentas from preeclamptic and normal pregnancies. Placental D6 decoy receptor mRNA expression was significantly lower in patients with preeclampsia than in patients with normal pregnancies. Western blot analyses revealed decreased protein expression in cases of preeclampsia. Conclusion The expression of the D6 decoy receptor in preeclamptic placentas was significantly lower than in normal placentas. Further studies are needed to clarify the underlying mechanisms that link decreased expression of placental D6 decoy receptor and preeclampsia. PMID:26430656

Biological significance of PinX1 telomerase inhibitor in esophageal carcinoma treatment

PubMed Central

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

2016-01-01

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

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

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.

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

PubMed

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

2013-06-01

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

Heart failure-associated changes in RNA splicing of sarcomere genes.

PubMed

Kong, Sek Won; Hu, Yong Wu; Ho, Joshua W K; Ikeda, Sadakatsu; Polster, Sean; John, Ranjit; Hall, Jennifer L; Bisping, Egbert; Pieske, Burkert; dos Remedios, Cristobal G; Pu, William T

2010-04-01

Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared with control hearts. We used the Affymetrix Exon array to globally evaluate mRNA splicing in left ventricular myocardial RNA from controls (n=15) and patients with ischemic cardiomyopathy (n=15). We observed a broad and significant decrease in mRNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ischemic cardiomyopathy and control samples, suggesting distinct changes in mRNA splicing between groups. Reverse transcription-polymerase chain reaction validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of 4 key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C, gamma (FLNC), was significantly altered in ischemic cardiomyopathy and in dilated cardiomyopathy and aortic stenosis. In aortic stenosis samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with >98% accuracy. Our data indicate that mRNA splicing is broadly altered in human heart disease and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes.

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

PubMed Central

Bittker, Joshua A.

2012-01-01

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

Granulocyte colony-stimulating factor (G-CSF) production in hemorrhagic shock requires both the ischemic and resuscitation phase.

PubMed

Hierholzer, C; Kelly, E; Billiar, T R; Tweardy, D J

1997-01-01

Granulocyte colony-stimulating factor (G-CSF) is the cytokine that is critical for polymorphonuclear neutrophilic granulocyte (PMN) production as well as being a potent agonist of PMN activation. We have recently reported that in the lung and the liver of rats resuscitated after hemorrhagic shock (HS) G-CSF mRNA expression is induced. It is not known if both phases of HS, the ischemic and the reperfusion phase, are required for G-CSF mRNA induction. The present study was designed to test the hypothesis that the upregulation of G-CSF mRNA expression is the consequence of HS followed by resuscitation and that ischemia alone is insufficient to induce G-CSF mRNA expression in the affected organs. Male Sprague-Dawley rats were subjected to resuscitated and unresuscitated shock protocols of varying severity. Control animals were subjected to anesthesia and all surgical preparations except for hemorrhage. Lungs and livers were isolated and their RNA extracted. Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrated that G-CSF mRNA was induced in the lung and liver of shock animals above the level observed in control animals. Upregulation of G-CSF mRNA relative to controls occurred only in animals undergoing resuscitated HS and not in ones subjected to unresuscitated HS. These results indicate that G-CSF production specific for the hemorrhage component of shock is dependent on resuscitation. As a consequence, the production of this cytokine may be decreased through modifications in the resuscitation protocols.

Analysis of Class I Major Histocompatibility Complex Gene Transcription in Human Tumors Caused by Human Papillomavirus Infection

PubMed Central

Gameiro, Steven F.; Zhang, Ali; Ghasemi, Farhad; Barrett, John W.; Mymryk, Joe S.

2017-01-01

Oncoproteins from high-risk human papillomaviruses (HPV) downregulate the transcription of the class I major histocompatibility complex (MHC-I) antigen presentation apparatus in tissue culture model systems. This could allow infected or transformed cells to evade the adaptive immune response. Using data from over 800 human cervical and head & neck tumors from The Cancer Genome Atlas (TCGA), we determined the impact of HPV status on the mRNA expression of all six MHC-I heavy chain genes, and the β2 microglobulin light chain. Unexpectedly, these genes were all expressed at high levels in HPV positive (HPV+) cancers compared with normal control tissues. Indeed, many of these genes were expressed at significantly enhanced levels in HPV+ tumors. Similarly, the transcript levels of several other components of the MHC-I peptide-loading complex were also high in HPV+ cancers. The coordinated expression of high mRNA levels of the MHC-I antigen presentation apparatus could be a consequence of the higher intratumoral levels of interferon γ in HPV+ carcinomas, which correlate with signatures of increased infiltration by T- and NK-cells. These data, which were obtained from both cervical and oral tumors in large human cohorts, indicates that HPV oncoproteins do not efficiently suppress the transcription of the antigen presentation apparatus in human tumors. PMID:28891951

Arginine Promotes Slow Myosin Heavy Chain Expression via Akirin2 and the AMP-Activated Protein Kinase Signaling Pathway in Porcine Skeletal Muscle Satellite Cells.

PubMed

Chen, Xiaoling; Guo, Yafei; Jia, Gang; Zhao, Hua; Liu, Guangmang; Huang, Zhiqing

2018-05-09

This study aimed to investigate the effect of arginine on the expression of slow myosin heavy chain (MyHC) I and its underlying mechanism in porcine skeletal muscle satellite cells. Our results showed that arginine upregulated the mRNA (1.54 ± 0.08; p < 0.01) and protein (2.01 ± 0.01; p < 0.001) levels of MyHC I. We also showed that arginine upregulated the expression of Akirin2 (1.35 ± 0.1; p < 0.05) and increased the NO content (1.56 ± 0.04; p < 0.001). Akirin2 siRNA abolished arginine-induced upregulation of MyHC I and the increase of the NO content. In addition, arginine significantly increased the phospho-AMP-activated protein kinase (AMPK)/AMPK level (1.33 ± 0.06; p < 0.05), the AMPK content (79.55 ± 0.13; p < 0.001), and the AMPKα2 mRNA level (2.03 ± 0.20; p < 0.01). AMPKα2 silencing or AMPK inhibitor Compound C abolished arginine-induced upregulation of MyHC I. Our results provide, for the first time, evidence for the involvement of Akirin2 and the AMPK signaling pathway in arginine-induced MyHC I expression in porcine skeletal muscle satellite cells.

Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions.

PubMed

Häkkinen, T; Luoma, J S; Hiltunen, M O; Macphee, C H; Milliner, K J; Patel, L; Rice, S Q; Tew, D G; Karkola, K; Ylä-Herttuala, S

1999-12-01

We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.

Tissue-specific regulation of medium-chain acyl-CoA dehydrogenase gene by thyroid hormones in the developing rat.

PubMed

Djouadi, F; Riveau, B; Merlet-Benichou, C; Bastin, J

1997-05-15

During development, gene expression of medium-chain acyl-CoA dehydrogenase (MCAD), a nuclear-encoded mitochondrial enzyme that catalyses the first step of medium-chain fatty acid beta-oxidation, is highly regulated in tissues in accordance with fatty acid utilization, but the factors involved in this regulation are largely unknown. To investigate a possible role of thyroid hormones, rat pups were made hypothyroid by the administration of propylthiouracyl to the mother from day 12 of gestation, and their kidneys, heart and liver were removed on postnatal day 16 to determine MCAD mRNA abundance, protein level and enzyme activity. Similar experiments were run in 3,3',5-tri-iodothyronine (T3)-replaced hypothyroid (1 microg of T3/100 g body weight from postnatal day 5 to 15) and euthyroid pups. Hypothyroidism led to an increase in MCAD mRNA abundance in kidney and a decrease in abundance in heart, but had no effect in liver. The protein levels and enzyme activity were lowered in hypothyroid heart and kidney, suggesting that hypothyroidism affects post-transcriptional steps of gene expression in the kidney. All the effects of hypothyroidism were completely reversed in both heart and kidney by T3 replacement. Injection of a single T3 dose into 16-day-old euthyroid rats also led to tissue-specific changes in mRNA abundance. Nuclear run-on assays performed from hypothyroid and hypothyroid plus T3 rats showed that T3 stimulates MCAD gene transcription in heart and represses it in the kidney. These results indicate that the postnatal rise in circulating T3 is essential to the developmental regulation of the MCAD gene in vivo.

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

PubMed Central

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

2015-01-01

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

Stimulation of EphB2/ephrin-B1 signalling by tumour necrosis factor alpha in human dental pulp stem cells.

PubMed

Zhu, Lifang; Dissanayaka, Waruna Lakmal; Green, David William; Zhang, Chengfei

2015-04-01

The aim of this study was to investigate whether in vitro stimulation of dental pulp stem cells (DPSCs) by tumour necrosis factor alpha (TNF-α) would induce secretion of EphB2/ephrin-B1 signalling. Dental pulp stem cells isolated from human dental pulp were treated with TNF-α (5-100 ng/ml) over 2-48 h. EphB2/ephrin-B1 mRNA and protein levels were measured by real-time polymerase chain reaction (RT-PCR) and western blot analysis respectively. Additionally, DPSCs were pre-incubated with TNF-α receptor neutralizing antibodies or infected with nuclear factor-kappa B (NF-ĸB) inhibitor, p38 MAPK inhibitor, Jun N-terminal kinase (JNK) inhibitor and MEK inhibitor before TNF-α treatment. Results were analysed by one-way ANOVA. Tumour necrosis factor alpha increased EphB2 mRNA expression in DPSCs at concentrations up to 20 ng/ml and ephrin-B1 at concentrations up to 40 ng/ml (P < 0.05). Its mRNA expression reached maximum at 24 h when treated with TNF-α at 20 ng/ml (P < 0.05). EphB2/ephrin-B1 protein expression levels were high at 16 and 24 h as shown by western blotting. Neutralizing antibodies for TNFR1/2 receptors down-regulated EphB2/ephrin-B1 mRNA expression (P < 0.05) and ephrin-B1 protein expression, but not EphB2 protein expression. JNK-inhibitor inhibited EphB2 mRNA expression only (P < 0.05). EphB2/ephrin-B1 were invoked in DPSCs with TNF-α treatment via the JNK-dependent pathway, but not NF-ĸB, p38 MAPK or MEK signalling. © 2015 John Wiley & Sons Ltd.

[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.

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

PubMed

Amendola, R

1994-11-01

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

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.

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.

[Effect of CD-14 and toll like receptors on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis].

PubMed

Jia, Ge; Qiu, Li-Hong; Li, Ren; Lü, You; Yu, Ya-Qiong; Zhong, Ming

2011-09-01

To evaluate the effect of cluster of differentiation 14 (CD-14) and Toll like receptors (TLR) on the expression of interleukin-6 (IL-6) mRNA induced by Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS). MC3T3-E1 cells were treated with 10 mg/L Pe-LPS for different hours, and the cells uninvolved by anything as the blank group. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-liked immunosorbent assay (ELISA). The expression of CD-14, TLR-2 and TLR-4 mRNA was observed at different time point (0 - 24 h) by RT-PCR. The protein of CD-14, TLR-2 and TLR-4 was analyzed with a flow cytometer. MC3T3-E1 cells were pretreated with anti-CD-14, anti-TLR-2 and anti-TLR-4 antibody for 1 h, and then cells were stimulated with 10 mg/L Pe-LPS for 6 h. The expression of IL-6 mRNA was examined by RT-PCR. Statistical analysis was performed using one-way ANOVA Dunnett-t test with SPSS 11.0 software package. The IL-6 mRNA and proteins increased significantly after treatment with Pe-LPS. When MC3T3-E1 cells treated by Pe-LPS for 6 h, the expression of proteins soared from (11.696 ± 0.672) ng/L to (36.534 ± 0.574) ng/L (P < 0.01); In the control group, the CD-14 and TLR-4 mRNA are ambly-expression, and the ratios of CD-14 and TLR-4 positive cells were (39.038 ± 3.131)% and (11.438 ± 0.385)% respectively in MC3T3-E1. After treatment by Pe-LPS, the expression of CD-14 and TLR-4 mRNA increased significantly, and the ratios of CD-14 and TLR-4 positive cells markedly increased to (62.407 ± 1.800)% and (21.367 ± 2.271)%. TLR-2 expression did not change apparently after Pe-LPS treatment. The expression of IL-6 mRNA was partly inhibited by anti-CD-14 or anti-TLR-4 antibody, but not by TLR-2. Pe-LPS can induce the expression of IL-6 in osteoblast MC3T3-E1 through CD-14 and TLR-4, but not TLR-2.

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

PubMed

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

2009-06-01

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

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.

Effects of birth trauma and estrogen on urethral elastic fibers and elastin expression.

PubMed

Lin, Guiting; Ning, Hongxiu; Wang, Guifang; Banie, Lia; Lue, Tom F; Lin, Ching-Shwun

2010-10-01

To investigate the effects of birth trauma and estrogen on urethral elastic fibers and elastin expression. Pregnant rats were subjected to sham operation (Delivery-only), DVDO (delivery, vaginal distension and ovariectomy), or DVDO + E₂ (estrogen). At 2, 4, 8, or 12 weeks, their urethras were harvested for elastic fiber staining and reverse transcription-polymerase chain reaction analysis. Urethral cells were treated with transforming growth factor- β1 (TGFβ1) and/or estrogen and analyzed for elastin mRNA expression. Urethral cells were also examined for the activities of Smad1- and Smad3/4-responsive elements in response to TGFβ1 and estrogen. At 8 weeks post-treatment, the urethras of DVDO rats had fewer and shorter elastic fibers when compared with Delivery-only rats, and those of DVDO + E₂ rats had fewer and shorter elastic fibers when compared with DVDO rats. Elastin mRNA was expressed at low levels in Delivery-only rats and at increasingly higher levels in DVDO rats at 2, 4, and 8 weeks but at sharply lower levels in DVDO + E₂ rats when compared with DVDO rats at 8 weeks. Urethral cells expressed increasingly higher levels of elastin mRNA in response to increasing concentrations of TGFβ1 up to 1 ng/mL. At this TGFβ1 concentration, urethral cells expressed significantly lower levels of elastin mRNA when treated with estrogen before or after TGFβ1 treatment. Both Smad1- and Smad3/4-responsive elements were activated by TGFβ1 and such activation was suppressed by estrogen. Birth trauma appears to activate urethral elastin expression via TGFβ1 signaling. Estrogen interferes with this signaling, resulting in improper assembly of elastic fibers. Copyright © 2010 Elsevier Inc. All rights reserved.

Transfer of the α5(IV) Collagen Chain Gene to Smooth Muscle Restores in Vivo Expression of the α6(IV) Collagen Chain in a Canine Model of Alport Syndrome

PubMed Central

Harvey, Scott J.; Zheng, Keqin; Jefferson, Barbara; Moak, Peter; Sado, Yoshikazu; Naito, Ichiro; Ninomiya, Yoshifumi; Jacobs, Robert; Thorner, Paul S.

2003-01-01

X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the α5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant α5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine α5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the α1(IV) and α2(IV), but not the α3(IV) to α6(IV) collagen chains. By Northern blotting, an α5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the α1(IV) and α2(IV) chains but the recombinant α5(IV) chain was not incorporated. These findings are consistent with the concept that the α5(IV) chain requires one or more of the α3(IV), α4(IV), or α6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the α5(IV) transgene was injected into bladder smooth muscle that lacks both the α5(IV) and α6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the α5(IV) and α6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant α5(IV) chain is capable of restoring expression of a second α(IV) chain that requires the presence of the α5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome. PMID:12598321

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2016-08-01

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

Decreased activity and expression of intestinal oligopeptide transporter PEPT1 in rats with hyperthyroidism in vivo.

PubMed

Ashida, Kayoko; Katsura, Toshiya; Saito, Hideyuki; Inui, Ken-ichi

2004-06-01

To examine the effect of thyroid hormone status on PEPT1 in vivo, the activity and expression of PEPT1 in the small intestine were examined in euthyroid and hyperthyroid rats. Hyperthyroidism was induced by treating rats with L-thyroxine (12 mg/L) in the drinking water for 21 days. Transport activity was measured by everted small intestinal preparations and in situ intestinal loop technique. Expressions of PEPT1 mRNA and protein were evaluated by competitive polymerase chain reaction and Western blotting, respectively. The uptake of [14C]glycylsarcosine by everted small intestinal preparations was significantly decreased in hyperthyroid rats, whereas that of methyl-alpha-D-[14C(U)]-glucopyranoside was not altered. Kinetic analysis showed that the Vmax value for [14C]glycylsarcosine uptake was significantly decreased in hyperthyroid rats, whereas the Km value was not affected. The mean portal vein concentrations after intrajejunal administration of [14C]glycylsarcosine were also decreased in hyperthyroid rats. Moreover, hyperthyroidism caused a significant decrease in the expression of PEPT1 mRNA in the small intestine, whereas the expression of Na+/glucose cotransporter (SGLT1) mRNA was not changed. The level of PEPT1 protein was also decreased in the small intestine of hyperthyroid rats. These results indicate that in hyperthyroid rats, the activity and expression of PEPT1 were decreased in the small intestine.

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

PubMed

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

2014-07-01

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

ZEB1 is Estrogen Responsive In Vitro in Human Foreskin Cells and is Over Expressed in Penile Skin in Patients With Severe Hypospadias

PubMed Central

Qiao, Liang; Tasian, Gregory E.; Zhang, Haiyang; Cunha, Gerald R.; Baskin, Laurence

2012-01-01

Purpose We determined the effect of estrogen on ZEB1 in vitro and tested the hypothesis that ZEB1 is over expressed in the penile skin of subjects with hypospadias. Materials and Methods Hs68 cells, a fibroblast cell line derived from human foreskin, were exposed to 0, 1, 10 and 100 nM estrogen, and the expression level of ZEB1 was assessed using reverse transcription real-time polymerase chain reaction, Western blot and immunocytochemical analysis. Next, preputial skin was prospectively collected from case and control subjects at hypospadias repair (37 cases) and circumcision (11). Hypospadias was classified as severe (13 cases) or mild (24) based on the position of the urethral meatus. ZEB1 expression was quantified using reverse transcription real-time polymerase chain reaction, Western blot and immunohistochemical analysis. Results Estrogen increased ZEB1 expression at the mRNA and protein levels in Hs68 cells in a concentration dependent fashion (p <0.01). Subjects with severe hypospadias had significantly higher ZEB1 mRNA levels and protein expression compared to controls or subjects with mild hypospadias (both p <0.01). Subjects with severe hypospadias had increased expression of ZEB1 in the basal layers of the preputial epidermis. Conclusions Estrogen increases ZEB1 expression in a human foreskin fibroblast cell line in vitro. Furthermore, ZEB1 is significantly over expressed in the penile skin of subjects with severe hypospadias. We propose that ZEB1 overexpression may contribute to development of hypospadias and may mediate the effect of estrogen on developing external male genitalia. PMID:21421232

Posttranscriptional regulation of albumin gene expression by branched-chain amino acids in rats with acute liver injury.

PubMed

Kuwahata, Masashi; Kuramoto, Yasuko; Tomoe, Yuka; Sugata, Emi; Segawa, Hiroko; Ito, Mikiko; Oka, Tatsuzo; Miyamoto, Ken-Ichi

2004-12-24

We previously demonstrated that the integration of albumin mRNA into functional polysomes was regulated by the supply of branched-chain amino acids (BCAA) in the liver of galactosamine-treated rats. To study the mechanism of this regulation, we investigated interaction between rat liver proteins and albumin transcripts. When albumin transcript was incubated with ribosome salt wash (RSW) extracts prepared from liver, a specific RNA-protein complex (p65) formed. Competition experiments showed that a pyrimidine-rich sequence in the coding region of albumin mRNA was required for the formation of p65. The level of p65 was increased in the RSW extracts prepared from liver of galactosamine-treated rats infused with a standard amino acid formula, compared with a BCAA-enriched amino acid formula. The protein in p65 appears to be polypyrimidine tract-binding protein (PTB), because the formation of p65 was reduced in the RSW extracts pre-incubated with anti-PTB antibody. In cell-free translation analysis, immunodepletion of PTB from rabbit reticulocyte lysate caused an increase in albumin translation. These results suggest that binding of PTB to albumin mRNA suppresses its translation. A supply of BCAA may interfere with this binding and improve the translation efficiency of albumin mRNA in injured liver.

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

NASA Astrophysics Data System (ADS)

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

2017-06-01

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

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions

PubMed Central

Bradbury, Andrew R. M.; Trinklein, Nathan D.; Wilkinson, Ian C.; Tandon, Atul K.; Anderson, Stephen; Bladen, Catherine L.; Jones, Brittany; Aldred, Shelley Force; Bestagno, Marco; Burrone, Oscar; Maynard, Jennifer; Ferrara, Fortunato; Görnemann, Janina; Glanville, Jacob; Wolf, Philipp; Frenzel, Andre; Wong, Julin; Koh, Xin Yu; Eng, Hui-Yan; Lane, David; Lefranc, Marie-Paule; Clark, Mike

2018-01-01

ABSTRACT Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use. PMID:29485921

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum

PubMed Central

Montalvillo, Enrique; Bernardo, David; Martínez-Abad, Beatriz; Allegretti, Yessica; Fernández-Salazar, Luis; Calvo, Carmen; Chirdo, Fernando G.; Garrote, José A.; Arranz, Eduardo

2015-01-01

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum. PMID:26529008

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum.

PubMed

Montalvillo, Enrique; Bernardo, David; Martínez-Abad, Beatriz; Allegretti, Yessica; Fernández-Salazar, Luis; Calvo, Carmen; Chirdo, Fernando G; Garrote, José A; Arranz, Eduardo

2015-10-30

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum.

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

PubMed

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

2018-05-11

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

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

PubMed

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

2017-07-01

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

Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

PubMed Central

Szczyglowski, K; Hamburger, D; Kapranov, P; de Bruijn, F J

1997-01-01

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation. PMID:9276951

Expression of monocarboxylate transporter 1 (MCT1) in the dog intestine.

PubMed

Shimoyama, Yumiko; Kirat, Doaa; Akihara, Yuko; Kawasako, Kazufumi; Komine, Misa; Hirayama, Kazuko; Matsuda, Kazuya; Okamoto, Minoru; Iwano, Hidetomo; Kato, Seiyu; Taniyama, Hiroyuki

2007-06-01

In this study, the expression and distribution of monocarboxyolate transporter 1 (MCT1) along the intestines (duodenum, jejunum, ileum, cecum, colon and rectum) of dogs were investigated at both the mRNA and protein levels. The expression of MCT1 protein and its distribution were confirmed by Western blotting and immunohistochemical staining using the antibody for MCT1. We identified mRNA coding for MCT1 and a 43-kDa band of MCT1 protein in all regions from the duodenum to the rectum. Immunoreactive staining for MCT1 was also observed in epithelial cells throughout the intestines. MCT1 immunoreactivity was greater in the large intestine than in the small intestine. MCT1 protein was predominantly expressed on the basolateral membranes along intestinal epithelial cells, suggesting that MCT1 may play an important role in lactate efflux and transport of short-chain fatty acids (SCFAs) to the bloodstream across the basolateral membranes of the dog intestine.

FAS and FAS-L Genotype and Expression in Patients With Recurrent Pregnancy Loss

PubMed Central

Banzato, Priscilla Chamelete Andrade; Daher, Silvia; Traina, Évelyn; Torloni, Maria Regina; Gueuvoghlanian-Silva, Bárbara Yasmin; Puccini, Renata Fiorini; Pendeloski, Karen Priscilla Tezotto

2013-01-01

We assessed FAS and FAS-L gene polymorphisms and messenger RNA (mRNA) levels in patients with recurrent pregnancy loss (RPL). This case–control study compared 129 women with RPL with 235 healthy multiparous women (control group). Genomic DNA and total mRNA were extracted from whole blood, and polymorphisms genotyping was performed by polymerase chain reaction (PCR). Messenger RNA expression levels were analyzed by real-time PCR. Data were analyzed by chi-square and Fisher exact tests; P < .05 was considered significant. There were no significant differences in the FAS (670 A/G) genotype or allelic frequencies between the RPL and control groups. We found significant differences in the FAS-L (844 C/T) genotype and allelic frequencies between women with RPL and controls. Patients with RPL had significantly higher FAS-L expression. Our data suggest that FAS-L gene polymorphism is associated with increased susceptibility to RPL. Moreover, women with RPL seem to abnormally express FAS-FAS-L molecules. PMID:23420824

S-Enantiomer of 19-Hydroxyeicosatetraenoic Acid Preferentially Protects Against Angiotensin II-Induced Cardiac Hypertrophy.

PubMed

Shoieb, Sherif M; El-Kadi, Ayman O S

2018-06-07

We have recently demonstrated that the racemic mixture of 19-hydroxyeicosatetraenoic acid (19-HETE) protects against angiotensin II (Ang II) induced cardiac hypertrophy. Therefore, the purpose of this study was to investigate whether R- or S-enantiomer of 19-HETE confers cardioprotection against Ang II-induced cellular hypertrophy in RL-14 and H9c2 cells. Both cell lines were treated with vehicle or 10 μM Ang II in the absence and presence of 20 &#[mu]M 19(R)-HETE or 19(S)-HETE for 24 h. Thereafter, the level of mid-chain HETEs was determined using liquid chromatography - mass spectrometry (LC/MS). Gene and protein expression levels were measured using real-time PCR and Western blot analysis, respectively. The results showed that both 19(R)-HETE and 19(S)-HETE significantly decreased the metabolite formation rate of midchain HETEs namely 8-, 9-, 12- and 15-HETE compared to control group while the level of 5-HETE was selectively decreased by S-enantiomer. Moreover, both 19(R)-HETE and 19(S)-HETE significantly inhibited the catalytic activity of CYP1B1 and decreased the protein expression of 5- and 12-lipoxxygenase (LOX) as well as cyclooxygenase-2 (COX-2). Notably, the decrease in 15-LOX protein expression was only mediated by 19(S)-HETE. Moreover, both enantiomers protected against Ang II-induced cellular hypertrophy as evidenced by a significant decrease in mRNA expression of β/α-myosin heavy chain ratio, ANP, IL-6 and IL-8. Our data demonstrated that S-enantiomer of 19-HETE preferentially protected against Ang II-induced cellular hypertrophy via decreasing the level of mid-chain HETEs, inhibiting catalytic activity of CYP1B1, decreasing protein expression of LOX and COX-2 enzymes and decreasing mRNA expression of IL-6 and IL-8. The American Society for Pharmacology and Experimental Therapeutics.

l-Arginine Attenuates Cardiac Dysfunction, But Further Down-Regulates α-Myosin Heavy Chain Expression in Isoproterenol-Induced Cardiomyopathy.

PubMed

Kralova, Eva; Doka, Gabriel; Pivackova, Lenka; Srankova, Jasna; Kuracinova, Kristina; Janega, Pavol; Babal, Pavel; Klimas, Jan; Krenek, Peter

2015-10-01

In view of previously reported increased capacity for nitric oxide production, we suggested that l-arginine (ARG), the nitric oxide synthase (NOS) substrate, supplementation would improve cardiac function in isoproterenol (ISO)-induced heart failure. Male Wistar rats were treated with ISO for 8 days (5 mg/kg/day, i.p.) or vehicle. ARG was given to control (ARG) and ISO-treated (ISO+ARG) rats in water (0.4 g/kg/day). ISO administration was associated with 40% mortality, ventricular hypertrophy, decreased heart rate, left ventricular dysfunction, fibrosis and ECG signs of ischaemia. RT-PCR showed increased mRNA levels of cardiac hypertrophy marker atrial natriuretic peptide, but not BNP, decreased expression of myosin heavy chain isoform MYH6 and unaltered expression of pathological MYH7. ISO increased the protein levels of endothelial nitric oxide synthase, but at the same time it markedly up-regulated mRNA and protein levels of gp91phox, a catalytical subunit of superoxide-producing NADPH oxidase. Fibrosis was markedly increased by ISO. ARG treatment moderately ameliorated left ventricular dysfunction, but was without effect on cardiac hypertrophy and fibrosis. Combination of ISO and ARG led to a decrease in cav-1 expression, a further increase in MYH7 expression and a down-regulation of MYH6 that inversely correlated with gp91phox mRNA levels. Although ARG, at least partially, improved ISO-impaired basal left ventricular systolic function, it failed to reduce cardiac hypertrophy, fibrosis, oxidative stress and mortality. The protection of contractile performance might be related to increased capacity for nitric oxide production and the up-regulation of MYH7 which may compensate for the marked down-regulation of the major MYH6 isoform. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

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

PubMed

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

2016-10-01

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

Immunomodulatory Effects of Deokgu Thermomineral Water Balneotherapy on Oxazolone-Induced Atopic Dermatitis Murine Model

PubMed Central

Lee, Young Bok; Kim, Su Jin; Park, Sae Mi; Lee, Kyung Ho; Han, Hyung Jin; Yu, Dong Soo; Woo, So Youn; Yun, Seong Taek; Hamm, Se-Yeong; Kim, Hong Jig

2016-01-01

Background Although the therapeutic mechanism of balneotherapy for atopic dermatitis has not been clarified, many atopic patients who visit thermomineral springs have shown clinical improvements. Objective This study was aimed to evaluate the immunomodulatory effect of thermomineral water balneotherapy on the atopic dermatitis murine model. Methods The oxazolone-induced atopic dermatitis murine model was used to evaluate the therapeutic effect of balneotherapy with Deokgu thermomineral water compared with distilled water. Histologic evaluation and confocal microscopic imaging were performed to analyze the lesional expression of cluster-of-differentiation (CD)4 and forkhead box p3 (Foxp3). Lesional mRNA expression of interleukin (IL) 33, thymic stromal lymphopoietin (TSLP), and Foxp3 was evaluated by real-time reverse transcription polymerase chain reaction. Results Compared with the distilled water bath group, confocal microscopic evaluation of CD4 and Foxp3 merged images showed increased expression of regulatory T cells in the thermomineral balneotherapy group. The lesional mRNA level of IL-33 showed a reduced trend in the thermomineral balneotherapy group, whereas the level of mRNA of Foxp3 was increased. TSLP showed a decreased trend in both distilled water and thermomineral water bath groups. There was a trend of reduced expression in lesional IL-33 mRNA but increased cell count of CD4+ Foxp3+ regulatory T cells in thermomineral balneotherapy compared with distilled water bath. Conclusion Therefore, thermomineral balneotherapy can be an effective and safe adjuvant therapeutic option for atopic dermatitis. PMID:27081266

Immunomodulatory Effects of Deokgu Thermomineral Water Balneotherapy on Oxazolone-Induced Atopic Dermatitis Murine Model.

PubMed

Lee, Young Bok; Kim, Su Jin; Park, Sae Mi; Lee, Kyung Ho; Han, Hyung Jin; Yu, Dong Soo; Woo, So Youn; Yun, Seong Taek; Hamm, Se-Yeong; Kim, Hong Jig; Kim, Jin-Wou

2016-04-01

Although the therapeutic mechanism of balneotherapy for atopic dermatitis has not been clarified, many atopic patients who visit thermomineral springs have shown clinical improvements. This study was aimed to evaluate the immunomodulatory effect of thermomineral water balneotherapy on the atopic dermatitis murine model. The oxazolone-induced atopic dermatitis murine model was used to evaluate the therapeutic effect of balneotherapy with Deokgu thermomineral water compared with distilled water. Histologic evaluation and confocal microscopic imaging were performed to analyze the lesional expression of cluster-of-differentiation (CD)4 and forkhead box p3 (Foxp3). Lesional mRNA expression of interleukin (IL) 33, thymic stromal lymphopoietin (TSLP), and Foxp3 was evaluated by real-time reverse transcription polymerase chain reaction. Compared with the distilled water bath group, confocal microscopic evaluation of CD4 and Foxp3 merged images showed increased expression of regulatory T cells in the thermomineral balneotherapy group. The lesional mRNA level of IL-33 showed a reduced trend in the thermomineral balneotherapy group, whereas the level of mRNA of Foxp3 was increased. TSLP showed a decreased trend in both distilled water and thermomineral water bath groups. There was a trend of reduced expression in lesional IL-33 mRNA but increased cell count of CD4(+) Foxp3(+) regulatory T cells in thermomineral balneotherapy compared with distilled water bath. Therefore, thermomineral balneotherapy can be an effective and safe adjuvant therapeutic option for atopic dermatitis.

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

PubMed

Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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

2004-10-11

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

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

PubMed

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

2018-03-21

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

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

PubMed

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

2016-08-15

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

Effects of dietary biotin supplementation on glucagon production, secretion, and action.

PubMed

Lazo-de-la-Vega-Monroy, Maria-Luisa; Larrieta, Elena; Tixi-Verdugo, Wilma; Ramírez-Mondragón, Rafael; Hernández-Araiza, Ileana; German, Michael S; Fernandez-Mejia, Cristina

Despite increasing evidence that pharmacologic concentrations of biotin modify glucose metabolism, to our knowledge there have not been any studies addressing the effects of biotin supplementation on glucagon production and secretion, considering glucagon is one of the major hormones in maintaining glucose homeostasis. The aim of this study was to investigate the effects of dietary biotin supplementation on glucagon expression, secretion, and action. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 wk postweaning. Glucagon gene mRNA expression was measured by the real-time polymerase chain reaction. Glucagon secretion was assessed in isolated islets and by glucagon concentration in plasma. Glucagon action was evaluated by glucagon tolerance tests, phosphoenolpyruvate carboxykinase (Pck1) mRNA expression, and glycogen degradation. Compared with the control group, glucagon mRNA and secretion were increased from the islets of the biotin-supplemented group. Fasting plasma glucagon levels were higher, but no differences between the groups were observed in nonfasting glucagon levels. Despite the elevated fasting glucagon levels, no differences were found in fasting blood glucose concentrations, fasting/fasting-refeeding glucagon tolerance tests, glycogen content and degradation, or mRNA expression of the hepatic gluconeogenic rate-limiting enzyme, Pck1. These results demonstrated that dietary biotin supplementation increased glucagon expression and secretion without affecting fasting blood glucose concentrations or glucagon tolerance and provided new insights into the effect of biotin supplementation on glucagon production and action. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of zinc deficiency and supplementation on leptin and leptin receptor expression in pregnant mice.

PubMed

Ueda, Hidenori; Nakai, Taketo; Konishi, Tatsuya; Tanaka, Keiichi; Sakazaki, Fumitoshi; Min, Kyong-Son

2014-01-01

Leptin is an adipose-derived hormone that primarily regulates energy balance in response to nutrition. Human placental cells produce leptin, whereas murine placental cells produce soluble leptin receptors (Ob-R). However, the roles of these proteins during pregnancy have not been elucidated completely. As an essential metal, zinc (Zn) is central to insulin biosynthesis and energy metabolism. In the present study, the effects of Zn deficiency and supplementation on maternal plasma leptin and soluble Ob-R regulation in pregnant mice placentas were examined using enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and Western blotting. Nutritional Zn deficiency significantly reduced plasma insulin concentrations and fetal and placental weights in pregnant mice. Plasma leptin concentrations in pregnant mice also increased 20- to 40-fold compared with those in non-pregnant mice. Although dietary Zn deficiency and supplementation did not affect plasma leptin concentrations in non-pregnant mice, Zn-deficient pregnant mice had significantly reduced plasma leptin concentrations and adipose leptin mRNA expression. In contrast, Zn-supplemented pregnant mice had increased plasma leptin concentrations without increased adipose leptin mRNA expression. Placental soluble Ob-R mRNA expression also decreased in Zn-deficient mice and tended to increase in Zn-supplemented mice. These results indicate that Zn influences plasma leptin concentrations by modulating mRNA expression of soluble Ob-R in the placenta, and leptin in visceral fat during pregnancy. These data suggest that both adipose and placenta-derived leptin system are involved in the regulation of energy metabolism during fetal growth.

Association of mRNA expression of iron metabolism-associated genes and progression of non-alcoholic steatohepatitis in rats.

PubMed

Higuchi, Teruhisa; Moriyama, Mitsuhiko; Fukushima, Akiko; Matsumura, Hiroshi; Matsuoka, Shunichi; Kanda, Tatsuo; Sugitani, Masahiko; Tsunemi, Akiko; Ueno, Takahiro; Fukuda, Noboru

2018-05-25

Excess iron is associated with non-alcoholic steatohepatitis (NASH). mRNA expression of duodenal cytochrome b, divalent metal transporter 1, ferroportin 1, hepcidin, hephaestin and transferrin receptor 1 in liver were higher in high fat, high cholesterol-containing diet (HFCD) group than in normal diet (ND) group. mRNA levels of divalent metal transporter 1 and transferrin receptor 1, which stimulate iron absorption and excretion, were enhanced in small intestine. Epithelial mucosa of small intestine in HFCD group was characterized by plasma cell and eosinophil infiltration and increased vacuoles. Iron absorption was enhanced in this NASH model in the context of chronic inflammation of small intestinal epithelial cells, consequences of intestinal epithelial cell impairment caused by HFCD. Iron is transported to hepatocytes via portal blood, and abnormalities in iron absorption and excretion occur in small intestine from changes in iron transporter expression, which also occurs in NASH liver. Knockdown of hepcidin antimicrobial peptide led to enhanced heavy chain of ferritin expression in human hepatocytes, indicating association between hepcidin production and iron storage in hepatocytes. Iron-related transporters in liver and lower/upper portions of small intestine play critical roles in NASH development. Expression of iron metabolism-related genes in liver and small intestine was analyzed in stroke-prone spontaneously hypertensive rats (SHR-SP), which develop NASH. Five-week-old SHR-SP fed ND or HFCD were examined. mRNA and protein levels of iron metabolism-related genes in liver and small intestine from 12- and 19-week-old rats were evaluated by real-time RT-PCR and immunohistochemistry or Western blot.

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

PubMed

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

2018-06-01

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

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.

LEFPS1, a Tomato Farnesyl Pyrophosphate Gene Highly Expressed during Early Fruit Development1

PubMed Central

Gaffe, Joel; Bru, Jean-Philippe; Causse, Mathilde; Vidal, Alain; Stamitti-Bert, Linda; Carde, Jean-Pierre; Gallusci, Philippe

2000-01-01

Farnesyl pyrophosphate synthase (FPS) catalyzes the synthesis of farnesyl pyrophosphate, a key intermediate in sterol and sesquiterpene biosynthesis. Using a polymerase chain reaction-based approach, we have characterized LeFPS1, a tomato (Lycoperscion esculentum cv Wva 106) fruit cDNA, which encodes a functional FPS. We demonstrate that tomato FPSs are encoded by a small multigenic family with genes located on chromosomes 10 and 12. Consistent with farnesyl pyrophosphate requirement in sterol biosynthesis, FPS genes are ubiquitously expressed in tomato plants. Using an LeFPS1 specific probe, we show that the corresponding gene can account for most of FPS mRNA in most plant organs, but not during young seedling development, indicating a differential regulation of FPS genes in tomato. FPS gene expression is also under strict developmental control: FPS mRNA was mainly abundant in young organs and decreased as organs matured with the exception of fruits that presented a biphasic accumulation pattern. In this latter case in situ hybridization studies have shown that FPS mRNA is similarly abundant in all tissues of young fruit. Taken together our results suggest that several FPS isoforms are involved in tomato farnesyl pyrophosphate metabolism and that FPS genes are mostly expressed in relation to cell division and enlargement. PMID:10938353

Comparison of the age-related porcine endogenous retrovirus (PERV) expression using duplex RT-PCR

PubMed Central

Moon, Hyoung Joon; Kim, Hye Kwon; Park, Seong Jun; Lee, Chul Seung; Song, Dae Sub; Kang, Bo Kyu

2009-01-01

Porcine endogenous retroviruses (PERVs) are members of family Retroviridae, genus Gamma retrovirus, and transmitted by both horizontally and vertically like other endogenous retroviruses (ERVs). PERV was initially described in the 1970s having inserted its gene in the host genome of different pig breeds, and three classes, PERV-A, PERV-B, and PERV-C are known. The therapeutic use of living cells, tissues, and organs from animals called xenotransplantation might relieve the limited supply of allografts in the treatment of organ dysfunction. Because of ethical considerations, compatible organ sizes, and physiology, the pig has been regarded as an alternative source for xenotransplantation. Sensitive duplex reverse transcription-polymerase chain reaction protocols for simultaneously detecting PERV gag mRNA and porcine glyceraldehydes 3-phosphate dehydrogenase mRNA in one tube was established. To compare the age-related PERV expression patterns of the lung, liver, spleen, kidney, heart, and pancreas in commercial pigs, 20 pigs from four age groups (5 heads each in 10 days-, 40 days-, 70 days-, and 110 days-old, respectively) were used in this study. The expression patterns of PERV were statistically different among age groups in lung, liver, and kidney (ANOVA, p < 0.05). These data may support in the selection of appropriate donor pigs expressing low levels of PERV mRNA. PMID:19934597

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

PubMed

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

2015-06-01

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

Estrogen receptors and biologic response in rat parathyroid tissue and C cells.

PubMed Central

Naveh-Many, T; Almogi, G; Livni, N; Silver, J

1992-01-01

The expression of the PTH and calcitonin genes is dramatically decreased by 1,25(OH)2D3 in vivo, and the PTH gene expression is increased by hypocalcemia. We have now studied the effect of estrogens on the expression of these genes in vivo. 17 beta-Estradiol, given to ovariectomized rats, led to a fourfold increase in PTH mRNA and calcitonin mRNA levels. These effects occurred 24 h after single injections of 37-145 nmol estradiol, or after constant infusions of 12 pmol/d for 1 or 2 wk, where there was no effect on serum calcium levels. The estrogen receptor mRNA was demonstrated in the thyroparathyroid tissue by polymerase chain reaction. The estrogen binding was localized to the parathyroid and C cells by immunohistochemistry. Uterus weight was increased by repeated larger doses (73 nmol/d x 7) of estradiol, but not by the small doses (12 pmol/d for 1 or 2 wk) which were effective on the PTH and calcitonin genes, suggesting a sensitive endocrine effect. These results confirm that the parathyroid and C cells are target organs for estrogen, leading to an increased expression of PTH and calcitonin, which by their combined anabolic effect on bone would help prevent osteoporosis. Images PMID:1469095

Infrasound-induced changes on sexual behavior in male rats and some underlying mechanisms.

PubMed

Zhuang, Zhiqiang; Pei, Zhaohui; Chen, Jingzao

2007-01-01

To investigate some bioeffects of infrasound on copulation as well as underlying mechanisms, we inspected the changes of sexual behavior, serum testosterone concentration and mRNA expression levels of steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) in testes of rats exposed to infrasound of 8Hz at 90 or 130dB for 1, 7, 14 and 21 days (2h/day), respectively. Rats exposed to 90dB exhibited significant decrement in sexual behavior, serum testosterone levels and mRNA expression levels of StAR and P450scc at the time point of 1 day but not at the rest time points, and no significantly change of SF-1 mRNA expression was observed over the period of 21 days in spite of mild fluctuation. Rats exposed to 130dB exhibited significant decrement in all aspects above, which became more profound with prolonged exposure. Our conclusion is that adverse bioeffects of infrasound on reproduction depend on some exposure parameters, the mechanism of which could involve in the decreased expression of some key enzymes or regulator for testosterone biosynthesis. Copyright © 2006. Published by Elsevier B.V.

[The Role of 5-Aza-CdR on Methylation of Promoter in RASSF1A Gene in Endometrial Carcinoma].

PubMed

Huang, Li-ping; Chen, Chen; Wang, Xue-ping; Liu, Hui

2015-05-01

To explore the effect of demethylating drug 5-Aza-2'-deoxycytidine (5-Aza-CdR) on methtylation status of the Ras-association domain familylA gene (RASSF1A) in human endometrial carcinoma. Randomly'assign the human endometrial carcinoma cell line HEC-1-B into groups and use demethylating drug 5-Aza-CdR of different concentration to treat them. Then Methylation-specific polymerase chain reaction (MSP), real-time PCR, Western blot, TUNEL technology were used to analyze methylation status of RASSF1A promoter CpG islands, RASSF1A mRNA expression, RASSF1A protein expression and apoptosis of HEC-1-B cell. High DNA methylation in RASSF1A gene promoter region, low RASSF1A mRNA level and protein expression and out of control of human endometrial carcinoma cell HEC-1-B apoptosis were observed. 5-Aza-CdR of different concentration could reverse RASSF1A gene's methylation status, recover the expression of mRNA and protein, and control the growth of HEC-1-B by inducing apoptosis. Aberrant methylation of RASSF1A in endometrial cancer as a therapeutic target, demethylating agent 5-Aza-CdR could be an effective way of gene therapy.

Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.

PubMed

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

2016-11-17

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

Amlodipine at high dose increases preproendothelin-1 expression in the ventricles and aorta of normotensive rats.

PubMed

Krenek, Peter; Morel, Nicole; Kyselovic, Jan; Wibo, Maurice

2004-04-01

High doses of dihydropyridine calcium channel blockers can activate the sympathetic nervous system and the renin-angiotensin system. Both noradrenaline and angiotensin II stimulate preproendothelin-1 gene expression, yet the effects of high doses of dihydropyridines on preproendothelin-1 expression in vivo remain unknown. To investigate the effects of high doses of dihydropyridines on preproendothelin-1 expression in the ventricles and aorta of normotensive rats. Sprague-Dawley rats were treated with amlodipine 5 or 20 mg/kg per day (Amlo 5 or Amlo 20) in drinking water for 5 days or 5 weeks. Systolic blood pressure and heart rate were measured by tail-cuff plethysmography. Gene expression was examined by reverse transcriptase polymerase chain reaction. Amlo 5 increased heart rate during the first week only and had no effect on blood pressure and ventricular weight and gene expression. Amlo 20 reduced blood pressure transiently and increased heart rate consistently. It did not change relative left ventricular weight (corrected for body weight) after 5 days, but increased it after 5 weeks; it increased relative right ventricular weight at both time points. Aorta weight (mg/mm) was decreased after 5 weeks of treatment with both dosages of amlodipine. Preproendothelin-1 mRNA levels were increased by Amlo 20 in the ventricles and aorta and, concomitantly, renin mRNA was increased in the kidney. Less consistently, interleukin-6 mRNA also increased in ventricles, whereas cardiotrophin-1 mRNA remained unchanged. The sensitivity of isolated aorta to the contractile effect of noradrenaline was decreased by Amlo 5, but not by Amlo 20. In Sprague-Dawley rats, high-dose amlodipine, while promoting neurohormonal activation, induced overexpression of preproendothelin-1 mRNA in the ventricles and aorta. Endothelin-1 overexpression could contribute to the lack of inhibitory effect of high-dose amlodipine on ventricular mass in normotensive rats.

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

PubMed Central

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

2011-01-01

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

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.

In vivo hair growth-promoting effect of rice bran extract prepared by supercritical carbon dioxide fluid.

PubMed

Choi, Jae-Suk; Jeon, Min-Hee; Moon, Woi-Sook; Moon, Jin-Nam; Cheon, Eun Jin; Kim, Joo-Wan; Jung, Sung Kyu; Ji, Yi-Hwa; Son, Sang Wook; Kim, Mi-Ryung

2014-01-01

The potential hair growth-promoting activity of rice bran supercritical CO2 extract (RB-SCE) and major components of RB-SCE, linoleic acid, policosanol, γ-oryzanol, and γ-tocotrienol, were evaluated with the histological morphology and mRNA expression levels of cell growth factors using real-time reverse transcriptase-polymerase chain reaction (PCR) in C57BL/6 mice. RB-SCE showed hair growth-promoting potential to a similar extent as 3% minoxidil, showing that the hair follicles were induced to be in the anagen stage. The numbers of the hair follicles were significantly increased. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and keratinocyte growth factor (KGF) were also significantly increased and that of transforming growth factor-β (TGF-β) decreased in RB-SCE-treated groups. Among the major components of RB-SCE, linoleic acid and γ-oryzanol induced the formation of hair follicles according to examination of histological morphology and mRNA expression levels of cell growth factors. In conclusion, our results demonstrate that RB-SCE, particularly linoleic acid and γ-oryzanol, promotes hair growth and suggests RB-SCE can be applied as hair loss treatment.

Impact of haloperidol and quetiapine on the expression of genes encoding antioxidant enzymes in human neuroblastoma SH-SY5Y cells.

PubMed

Schmidt, Andreas Johannes; Hemmeter, Ulrich Michael; Krieg, Jürgen-Christian; Vedder, Helmut; Heiser, Philip

2009-05-01

Antipsychotics are known to alter antioxidant activities in vivo. Therefore, the aim of the present study was to examine in the human neuroblastoma SH-SY5Y cell line the impact of a typical (haloperidol) and an atypical (quetiapine) antipsychotic on the expression of genes encoding the key enzymes of the antioxidant metabolism (Cu, Zn superoxide dismutase; Mn superoxide dismutase; glutathione peroxidase; catalase) and enzymes of the glutathione metabolism (gamma-glutamyl cysteine synthetase, glutathione-S-transferase, gamma-glutamyltranspeptidase, glutathione reductase). The cells were incubated for 24h with 0.3, 3, 30 and 300microM haloperidol and quetiapine, respectively; mRNA levels were measured by polymerase chain reaction. In the present study, we observed mostly significant decreases of mRNA contents. With respect to the key pathways, we detected mainly effects on the mRNA levels of the hydrogen peroxide detoxifying enzymes. Among the enzymes of the glutathione metabolism, glutathione-S-transferase- and gamma-glutamyltranspeptidase-mRNA levels showed the most prominent effects. Taken together, our results demonstrate a significantly reduced expression of genes encoding for antioxidant enzymes after treatment with the antipsychotics, haloperidol and quetiapine.

Diesel Exhaust Particles Enhance MUC4 Expression in NCI-H292 Cells and Nasal Epithelial Cells via the p38/CREB Pathway.

PubMed

Park, Il-Ho; Kang, Ju-Hyung; Kim, Jin Ah; Shin, Jae-Min; Lee, Heung-Man

2016-01-01

Diesel exhaust particles (DEPs), the major contributors to air pollution, induce inflammatory responses in the nasal epithelium. Overproduction of airway mucins is an important pathogenic finding in inflammatory airway diseases. The aims of the present study were to determine the effect of DEPs on the expression of the mucin gene MUC4 and to investigate the underlying mechanism of DEP-induced MUC4 expression in NCI-H292 cells and primary nasal epithelial cells (PNECs). NCI-H292 cells were stimulated for 24 h with DEPs. Messenger RNA (mRNA) and protein expression of MUC4 was determined by real-time reverse transcription (RT) polymerase chain reaction (PCR) and Western blotting. NCI-H292 cells were exposed to 3 mitogen-activated protein kinase inhibitors (U0126, SB203580, and SP600125) and a CREB (cAMP response element-binding protein) inhibitor prior to stimulation with DEPs, and MUC4 expression was examined by RT-PCR and Western blotting. PNECs were pretreated with a p38 inhibitor and CREB inhibitor prior to stimulation with DEPs, and MUC4 expression was then determined by RT-PCR and/or Western blotting. DEPs significantly increased the expression of MUC4 mRNA and protein. MUC4 mRNA and protein expression was inhibited by pretreatment with p38 and CREB inhibitors in NCI-H292 stimulated with DEPs. p38 and CREB inhibitors also blocked the expression of MUC4 mRNA and protein in DEP-stimulated PNECs. We demonstrated that DEPs stimulated the expression of MUC4 via the p38/CREB pathway in NCI-H292 cells and PNECs. The results of the present study pave the way for further studies on the role of MUC4 in DEP-induced hypersecretion in airway epithelium. © 2017 S. Karger AG, Basel.

Triptolide Upregulates Myocardial Forkhead Helix Transcription Factor p3 Expression and Attenuates Cardiac Hypertrophy

PubMed Central

Ding, Yuan-Yuan; Li, Jing-Mei; Guo, Feng-Jie; Liu, Ya; Tong, Yang-Fei; Pan, Xi-Chun; Lu, Xiao-Lan; Ye, Wen; Chen, Xiao-Hong; Zhang, Hai-Gang

2016-01-01

The forkhead/winged helix transcription factor (Fox) p3 can regulate the expression of various genes, and it has been reported that the transfer of Foxp3-positive T cells could ameliorate cardiac hypertrophy and fibrosis. Triptolide (TP) can elevate the expression of Foxp3, but its effects on cardiac hypertrophy remain unclear. In the present study, neonatal rat ventricular myocytes (NRVM) were isolated and stimulated with angiotensin II (1 μmol/L) to induce hypertrophic response. The expression of Foxp3 in NRVM was observed by using immunofluorescence assay. Fifty mice were randomly divided into five groups and received vehicle (control), isoproterenol (Iso, 5 mg/kg, s.c.), one of three doses of TP (10, 30, or 90 μg/kg, i.p.) for 14 days, respectively. The pathological morphology changes were observed after Hematoxylin and eosin, lectin and Masson’s trichrome staining. The levels of serum brain natriuretic peptide (BNP) and troponin I were determined by enzyme-linked immunosorbent assay and chemiluminescence, respectively. The mRNA and protein expressions of α- myosin heavy chain (MHC), β-MHC and Foxp3 were determined using real-time PCR and immunohistochemistry, respectively. It was shown that TP (1, 3, 10 μg/L) treatment significantly decreased cell size, mRNA and protein expression of β-MHC, and upregulated Foxp3 expression in NRVM. TP also decreased heart weight index, left ventricular weight index and, improved myocardial injury and fibrosis; and decreased the cross-scetional area of the myocardium, serum cardiac troponin and BNP. Additionally, TP markedly reduced the mRNA and protein expression of myocardial β-MHC and elevated the mRNA and protein expression of α-MHC and Foxp3 in a dose-dependent manner. In conclusion, TP can effectively ameliorate myocardial damage and inhibit cardiac hypertrophy, which is at least partly related to the elevation of Foxp3 expression in cardiomyocytes. PMID:27965581

Metformin Reduces Hepatic Expression of SIRT3, the Mitochondrial Deacetylase Controlling Energy Metabolism

PubMed Central

Buler, Marcin; Aatsinki, Sanna-Mari; Izzi, Valerio; Hakkola, Jukka

2012-01-01

Metformin inhibits ATP production in mitochondria and this may be involved in the anti-hyperglycemic effects of the drug. Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates the function of the electron transport chain and maintains basal ATP yield. We hypothesized that metformin treatment could diminish mitochondrial ATP production through downregulation of SIRT3 expression. Glucagon and cAMP induced SIRT3 mRNA in mouse primary hepatocytes. Metformin prevented SIRT3 induction by glucagon. Moreover, metformin downregulated constitutive expression of SIRT3 in primary hepatocytes and in the liver in vivo. Estrogen related receptor alpha (ERRα) mediates regulation of Sirt3 gene by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). ERRα mRNA expression was regulated in a similar manner as SIRT3 mRNA by glucagon, cAMP and metformin. However, a higher metformin concentration was required for downregulation of ERRα than SIRT3. ERRα siRNA attenuated PGC-1α mediated induction of SIRT3, but did not affect constitutive expression. Overexpression of the constitutively active form of AMP-activated protein kinase (AMPK) induced SIRT3 mRNA, indicating that the SIRT3 downregulation by metformin is not mediated by AMPK. Metformin reduced the hepatocyte ATP level. This effect was partially counteracted by SIRT3 overexpression. Furthermore, metformin decreased mitochondrial SIRT3 protein levels and this was associated with enhanced acetylation of several mitochondrial proteins. However, metformin increased mitochondrial mass in hepatocytes. Altogether, our results indicate that metformin attenuates mitochondrial expression of SIRT3 and suggest that this mechanism is involved in regulation of energy metabolism by metformin in the liver and may contribute to the therapeutic action of metformin. PMID:23166782

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

PubMed

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

2015-08-01

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

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

PubMed

Xu, Qianghua; Qin, Ye

2012-09-01

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

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.

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

PubMed

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

2014-05-01

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

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

PubMed

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

2012-01-01

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

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

PubMed Central

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

2018-01-01

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

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

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

Yano, Hiroyuki; Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593; Hamanaka, Ryoji

Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Realmore » time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.« less

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

PubMed Central

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

2007-01-01

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

Macrophage colony-stimulating factor induces prolactin expression in rat pituitary gland.

PubMed

Hoshino, Satoya; Kurotani, Reiko; Miyano, Yuki; Sakahara, Satoshi; Koike, Kanako; Maruyama, Minoru; Ishikawa, Fumio; Sakatai, Ichiro; Abe, Hiroyuki; Sakai, Takafumi

2014-06-01

We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/ M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-α mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.

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

PubMed Central

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

2013-01-01

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

The anti-oestrogen fulvestrant (ICI 182,780) reduces the androgen receptor expression, ERK1/2 phosphorylation and cell proliferation in the rat ventral prostate.

PubMed

Fernandes, S A F; Gomes, G R O; Siu, E R; Damas-Souza, D M; Bruni-Cardoso, A; Augusto, T M; Lazari, M F M; Carvalho, H F; Porto, C S

2011-10-01

This study proposed to investigate further the role of oestrogens during pubertal growth of rat ventral prostate, by analysing the effect of anti-oestrogen fulvestrant (ICI 182,780) on the expression of androgen (AR) and oestrogen receptors (ESR1 and ESR2), mitogen-activated protein kinase (ERK1/2) phosphorylation, and expression of Ki-67, a biomarker for cell proliferation. Ventral prostates were obtained from 90-day-old rats treated once a week for 2 months with vehicle (control) or ICI 182,780 (10 mg/rat, s.c.). Transcripts for AR, ESR1 and ESR2 were evaluated by quantitative real-time polymerase chain reaction. Expression of AR, ESR1, ESR2, total and phospho-ERK1/2 was analysed by Western blot or immunofluorescence. Ki-67-positive cells and myosin heavy chain were detected by immunohistochemistry. Cylindrical epithelial cells slightly taller, epithelial dysplasia and an increase in smooth muscle layer were observed in the ventral prostate from ICI 182,780-treated rats. ICI 182,780 did not change the mRNA, but decreased the protein levels for AR in the ventral prostate. The expression of ESR1 (mRNA and protein) was upregulated by ICI 182,780, but no changes were observed on ESR2 expression (mRNA and protein). ICI 182,780 decreased the phosphorylation state of ERK1/2, with no changes in total ERK1/2 levels. Ki-67-positive cells in the ventral prostate were also decreased by ICI 182,780. In conclusion, ICI 182,780 induces downregulation of AR expression and may block the translocation of ESR1 and ESR2 from the nucleus to the plasma membrane, decreasing ERK1/2 phosphorylation and prostatic epithelial cell proliferation. These findings provide a basis for physiological roles of oestrogen in the ventral prostate. Further studies with fulvestrant are necessary in benign prostate hyperplasia and prostatic cancer models. © 2010 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.

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

PubMed Central

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

2014-01-01

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

Effect of cyclophilin A on gene expression in human pancreatic cancer cells.

PubMed

Li, Min; Wang, Hao; Li, Fei; Fisher, William E; Chen, Changyi; Yao, Qizhi

2005-11-01

We previously found that cyclophilin A (CypA) is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. In this study, we further investigated the effect of CypA on gene expression of several key molecules that are involved in pancreatic cancer cell proliferation. Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3) and human pancreatic ductal epithelial (HPDE) cells were used. The messenger RNA (mRNA) levels of CypA, CypB, CD147, neuropilins (NRPs), vascular endothelial growth factor (VEGF), and VEGF receptors upon the treatment of exogenous recombinant human CypA were determined by real-time reverse-transcription polymerase chain reaction. Exogenous human recombinant CypA reduced the mRNA levels of NRP-1 and VEGF, but not endogenous CypA, CypB, and CD147, in Panc-1, MIA PaCa-2, and BxPC-3 cells. In contrast, HPDE cells showed a decrease of endogenous CypA and CD147 mRNA, but not detectable changes of CypB, NRPs, and VEGF mRNA levels upon exogenous CypA treatment. These data show that exogenous CypA downregulates NRP-1 and VEGF expression in pancreatic cancer cells. This effect is different in normal HPDE cells. Thus, soluble CypA may affect cell growth of pancreatic cancer.

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.

Apoptosis and Inflammation Associated Gene Expressions in Monocrotaline-Induced Pulmonary Hypertensive Rats after Bosentan Treatment

PubMed Central

Hong, Young Mi; Kwon, Jung Hyun; Choi, Shinkyu

2014-01-01

Background and Objectives Vascular wall remodeling in pulmonary hypertension can be caused by an aberration in the normal balance between proliferation and apoptosis of endothelial cell in the pulmonary artery. The objective of this study was to evaluate the effect of bosentan on apoptosis in monocrotaline (MCT)-induced pulmonary hypertension. Materials and Methods Sprague-Dawley rats were divided into three groups: control (C) group, M group (MCT 60 mg/kg) and B group (MCT 60 mg/kg plus bosentan 20 mg/day orally). Gene expressions of Bcl (B cell leukemia/lymphoma)-2, caspase-3, complement component (C)-6, vascular endothelial growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were analyzed by real time polymerase chain reaction and western blot analysis. Results The messenger ribonucleic acid (mRNA) expressions of caspase-3 and VEGF were significantly increased in the M group compared with the C group, and significantly decreased in the B group compared with the M group in week 4. mRNA expression of IL-6 was significantly decreased in weeks 1, 2, and 4 in the B group compared with the M group. mRNA expression of TNF-α was significantly decreased on day 5 and in weeks 1 and 2 in the B group compared with the M group. Conclusion Bosentan may have potential for preventing apoptosis and inflammation. PMID:24653739

Presence and Expression of Microbial Genes Regulating Soil Nitrogen Dynamics Along the Tanana River Successional Sequence

NASA Astrophysics Data System (ADS)

Boone, R. D.; Rogers, S. L.

2004-12-01

We report on work to assess the functional gene sequences for soil microbiota that control nitrogen cycle pathways along the successional sequence (willow, alder, poplar, white spruce, black spruce) on the Tanana River floodplain, Interior Alaska. Microbial DNA and mRNA were extracted from soils (0-10 cm depth) for amoA (ammonium monooxygenase), nifH (nitrogenase reductase), napA (nitrate reductase), and nirS and nirK (nitrite reductase) genes. Gene presence was determined by amplification of a conserved sequence of each gene employing sequence specific oligonucleotide primers and Polymerase Chain Reaction (PCR). Expression of the genes was measured via nested reverse transcriptase PCR amplification of the extracted mRNA. Amplified PCR products were visualized on agarose electrophoresis gels. All five successional stages show evidence for the presence and expression of microbial genes that regulate N fixation (free-living), nitrification, and nitrate reduction. We detected (1) nifH, napA, and nirK presence and amoA expression (mRNA production) for all five successional stages and (2) nirS and amoA presence and nifH, nirK, and napA expression for early successional stages (willow, alder, poplar). The results highlight that the existing body of previous process-level work has not sufficiently considered the microbial potential for a nitrate economy and free-living N fixation along the complete floodplain successional sequence.

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

PubMed

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

2018-06-01

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

Expression and Functional Activities of Selected Sulfotransferase Isoforms in BeWo Cells and Primary Cytotrophoblast Cells

PubMed Central

Mitra, Pallabi; Audus, Kenneth L.

2009-01-01

Several cytosolic sulfotransferase enzyme isoforms are functional in placenta but there is limited information available on the utility of cultured trophoblast cells for studying sulfation. The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. The objective of this work was to examine the mRNA expression and enzyme activities of four sulfotransferase isoforms reported to be functional in human placenta (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine mRNA expression. Enzyme activities were assessed using the following substrates: 4-nitrophenol for SULT1A1, dopamine for SULT1A3, 17β-estradiol for SULT1E1, and dehydroepiandrosterone for SULT2A1. For 4-nitrophenol and dopamine sulfation, apparent Km values, response to inhibitors (2,6-dichloro-4-nitrophenol and sodium chloride), and thermal stability profiles indicated that 4-nitrophenol and dopamine sulfation in BeWo cells were being mediated by SULT1A1 and SULT1A3, respectively. SULT1A1 and SULT1A3 were also functional in the cytotrophoblast cells. Both at the protein and at the mRNA levels, SULT1A1 was more abundant in BeWo cells in comparison to the primary cytotrophoblast cells. SULT1E1 and SULT2A1 mRNA were not detected in the cytotrophoblasts. SULT1E1 mRNA was weakly expressed in BeWo but there was negligible functional activity. Although SULT2A1 mRNA was abundantly expressed in BeWo, Western blot and enzyme activities revealed that the protein is not expressed in BeWo cells. The results suggest that the BeWo cells and the cytotrophoblast cells can be used to examine the roles of SULT1A1 and SULT1A3 in placental metabolism. PMID:19646966

ERG Oncoprotein Expression in Prostate Cancer: Clonal Progression of ERG-Positive Tumor Cells and Potential for ERG-Based Stratification

DTIC Science & Technology

2010-01-01

staining results as ERG positive or negative. Analysis of ERG mRNA by branched-chain DNA ( bDNA ) signal amplification One 4-mm thick section was selected...patients treated with radical prostatectomy by using bDNA assay as described in Materials and Methods. Consecutive tissue slides from whole-mounted FFPE

Coexpression of an unusual form of the EWS-WT1 fusion transcript and interleukin 2/15 receptor betamRNA in a desmoplastic small round cell tumour.

PubMed

Nakanishi, Y; Oinuma, T; Sano, M; Fuchinoue, F; Komatsu, K; Seki, T; Obana, Y; Tabata, M; Kikuchi, K; Shimamura, M; Ohmori, K; Nemoto, N

2006-10-01

The beta chain of the interleukin 2/15 receptor (IL-2/15Rbeta) is induced by the expression of the EWS-WT1. A case of desmoplastic small round cell tumour (DSRCT) expressing only an unusual EWS-WT1 treated by us is reported here. To characterise an unusual form of EWS-WT1. Frozen tissue sections of the axillary tumour were examined using a laser-assisted microdissection technique and reverse transcriptase polymerase chain reaction. The novel fusion of exon 8 of EWS and the defective exon 10 of WT1 (-KTS) was detected. Although it was an unusual form, the coexpression of the present EWS-WT1, IL-2/15Rbeta and Janus kinase (JAK1) mRNA was detected in the tumour cells. IL-2 and signal transducers and activators of transcription (STAT5) mRNA were detected in both tumour and stromal cells. The induction of the IL-2/15 receptor signalling pathway may contribute to tumorigenesis in DSRCT through a paracrine or an autocrine system, even though the EWS-WT1 was an unusual form.

Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca(2+)-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban: an insight into the mechanism of atrial electrical remodeling.

PubMed

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

1999-04-01

We investigated the gene expression of calcium-handling genes including L-type calcium channel, sarcoplasmic reticular calcium adenosine triphosphatase (Ca(2+)-ATPase), ryanodine receptor, calsequestrin and phospholamban in human atrial fibrillation. Recent studies have demonstrated that atrial electrical remodeling in atrial fibrillation is associated with intracellular calcium overload. However, the changes of calcium-handling proteins remain unclear. A total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The messenger ribonucleic acid (mRNA) amount of the genes was measured by reverse transcription-polymerase chain reaction and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase. The mRNA of L-type calcium channel and of Ca(2+)-ATPase was significantly decreased in patients with persistent atrial fibrillation for more than 3 months (0.36+/-0.26 vs. 0.90+/-0.88 for L-type calcium channel; 0.69+/-0.42 vs. 1.21+/-0.68 for Ca(2+)-ATPase; both p < 0.05, all data in arbitrary unit). We further demonstrated that there was no spatial dispersion of the gene expression among the four atrial tissue sampling sites. Age, gender and underlying cardiac disease had no significant effects on the gene expression. In contrast, the mRNA levels of ryanodine receptor, calsequestrin and phospholamban showed no significant change in atrial fibrillation. L-type calcium channel and the sarcoplasmic reticular Ca(2+)-ATPase gene were down-regulated in atrial fibrillation. These changes may be a consequence of, as well as a contributory factor for, atrial fibrillation.

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

PubMed

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

2011-10-01

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

Biphasic Modulation of NOS Expression, Protein and Nitrite Products by Hydroxocobalamin Underlies Its Protective Effect in Endotoxemic Shock: Downstream Regulation of COX-2, IL-1β, TNF-α, IL-6, and HMGB1 Expression

PubMed Central

Sampaio, André L. F.; Dalli, Jesmond; Brancaleone, Vincenzo; D'Acquisto, Fulvio; Perretti, Mauro; Wheatley, Carmen

2013-01-01

Background. NOS/•NO inhibitors are potential therapeutics for sepsis, yet they increase clinical mortality. However, there has been no in vivo investigation of the (in vitro) •NO scavenger, cobalamin's (Cbl) endogenous effects on NOS/•NO/inflammatory mediators during the immune response to sepsis. Methods. We used quantitative polymerase chain reaction (qPCR), ELISA, Western blot, and NOS Griess assays, in a C57BL/6 mouse, acute endotoxaemia model. Results. During the immune response, pro-inflammatory phase, parenteral hydroxocobalamin (HOCbl) treatment partially inhibits hepatic, but not lung, iNOS mRNA and promotes lung eNOS mRNA, but attenuates the LPS hepatic rise in eNOS mRNA, whilst paradoxically promoting high iNOS/eNOS protein translation, but relatively moderate •NO production. HOCbl/NOS/•NO regulation is reciprocally associated with lower 4 h expression of TNF-α, IL-1β, COX-2, and lower circulating TNF-α, but not IL-6. In resolution, 24 h after LPS, HOCbl completely abrogates a major late mediator of sepsis mortality, high mobility group box 1 (HMGB1) mRNA, inhibits iNOS mRNA, and attenuates LPS-induced hepatic inhibition of eNOS mRNA, whilst showing increased, but still moderate, NOS activity, relative to LPS only. experiments (LPS+D-Galactosamine) HOCbl afforded significant, dose-dependent protection in mice Conclusions. HOCbl produces a complex, time- and organ-dependent, selective regulation of NOS/•NO during endotoxaemia, corollary regulation of downstream inflammatory mediators, and increased survival. This merits clinical evaluation. PMID:23781123

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

PubMed Central

Niessner, M; Volk, B A

1995-01-01

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

Intermittent fasting favored the resolution of Salmonella typhimurium infection in middle-aged BALB/c mice.

PubMed

Campos-Rodríguez, Rafael; Godínez-Victoria, Marycarmen; Reyna-Garfias, Humberto; Arciniega-Martínez, Ivonne Maciel; Reséndiz-Albor, Aldo Arturo; Abarca-Rojano, Edgar; Cruz-Hernández, Teresita Rocío; Drago-Serrano, Maria Elisa

2016-02-01

Intermittent fasting (IF) reportedly increases resistance and intestinal IgA response to Salmonella typhimurium infection in mature mice. The aim of this study was to explore the effect of aging on the aforementioned improved immune response found with IF. Middle-aged male BALB/c mice were submitted to IF or ad libitum (AL) feeding for 40 weeks and then orally infected with S. typhimurium. Thereafter, infected animals were all fed AL (to maximize their viability) until sacrifice on day 7 or 14 post-infection. We evaluated body weight, bacterial load (in feces, Peyer's patches, spleen and liver), total and specific intestinal IgA, lamina propria IgA+ plasma cells, plasma corticosterone, and messenger RNA (mRNA) expression of α-chain, J-chain, and the polymeric immunoglobulin receptor (pIgR) in liver and intestinal mucosa. In comparison with the infected AL counterpart, the infected IF group (long-term IF followed by post-infection AL feeding) generally had lower intestinal and systemic bacterial loads as well as higher total IgA on both post-infection days. Both infected groups showed no differences in corticosterone levels, body weight, or food and caloric intake. The increase in intestinal IgA was associated with enhanced pIgR mRNA expression in the intestine (day 7) and liver. Thus, to maintain body weight and caloric intake, IF elicited metabolic signals that possibly induced the increased hepatic and intestinal pIgR mRNA expression found. The increase in IgA probably resulted from intestinal IgA transcytosis via pIgR. This IgA response along with phagocyte-induced killing of bacteria in systemic organs (not measured) may explain the resolution of the S. typhimurium infection.

Reversal of subcellular remodelling by losartan in heart failure due to myocardial infarction

PubMed Central

Babick, Andrea; Chapman, Donald; Zieroth, Shelley; Elimban, Vijayan; Dhalla, Naranjan S

2012-01-01

This study tested the reversal of subcellular remodelling in heart failure due to myocardial infarction (MI) upon treatment with losartan, an angiotensin II receptor antagonist. Twelve weeks after inducing MI, rats were treated with or without losartan (20 mg/kg; daily) for 8 weeks and assessed for cardiac function, cardiac remodelling, subcellular alterations and plasma catecholamines. Cardiac hypertrophy and lung congestion in 20 weeks MI-induced heart failure were associated with increases in plasma catecholamine levels. Haemodynamic examination revealed depressed cardiac function, whereas echocardiographic analysis showed impaired cardiac performance and marked increases in left ventricle wall thickness and chamber dilatation at 20 weeks of inducing MI. These changes in cardiac function, cardiac remodelling and plasma dopamine levels in heart failure were partially or fully reversed by losartan. Sarcoplasmic reticular (SR) Ca2+-pump activity and protein expression, protein and gene expression for phospholamban, as well as myofibrillar (MF) Ca2+-stimulated ATPase activity and α-myosin heavy chain mRNA levels were depressed, whereas β-myosin heavy chain expression was increased in failing hearts; these alterations were partially reversed by losartan. Although SR Ca2+-release activity and mRNA levels for SR Ca2+-pump were decreased in failing heart, these changes were not reversed upon losartan treatment; no changes in mRNA levels for SR Ca2+-release channels were observed in untreated or treated heart failure. These results suggest that the partial improvement of cardiac performance in heart failure due to MI by losartan treatment is associated with partial reversal of cardiac remodelling as well as partial recovery of SR and MF functions. PMID:22947202

mRNA Regulation of Cardiac Iron Transporters and Ferritin Subunits in a Mouse Model of Iron Overload

PubMed Central

Brewer, Casey J.; Wood, Ruth I.; Wood, John C.

2014-01-01

Iron cardiomyopathy is the leading cause of death in iron overload. Men have twice the mortality rate of women, though the cause is unknown. In hemojuvelin-knockout mice, a model of the disease, males load more cardiac iron than females. We postulated that sex differences in cardiac iron import cause differences in cardiac iron concentration. RT-PCR was used to measure mRNA of cardiac iron transporters in hemojuvelin-knockout mice. No sex differences were discovered among putative importers of non-transferrin bound iron (L-type and T-type calcium channels, ZRT/IRT-like protein 14 zinc channels). Transferrin-bound iron transporters were also analyzed; these are controlled by the iron regulatory element/iron regulatory protein (IRE/IRP) system. There was a positive relationship between cardiac iron and ferroportin mRNA in both sexes, but it was significantly steeper in females (p<0.05). Transferrin receptor 1 and divalent metal transporter 1 were more highly expressed in females than males (p<0.01 and p<0.0001, respectively), consistent with their lower cardiac iron levels, as predicted by IRE/IRP regulatory pathways. Light-chain (L) ferritin showed a positive correlation with cardiac iron that was nearly identical in males and females (R2=0.41, p<0.01 and R2=0.56, p<0.05, respectively), while heavy-chain (H) ferritin was constitutively expressed in both sexes. This represents the first report of IRE/IRP regulatory pathways in the heart. Transcriptional regulation of ferroportin was suggested in both sexes, creating a potential mechanism for differential set points for iron export. Constitutive H-ferritin expression suggests a logical limit to cardiac iron buffering capacity at levels known to produce heart failure in humans. PMID:25220979

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

PubMed

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

2017-01-01

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

Osteogensis imperfecta type I is commonly due to a COLIAI null allel of type I collagen

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

Willing, M.C.; Pruchno, C.J.; Atkinson, M.

Dermal fibroblasts from most individuals with osteogenesis imperfecta (OI) type I produce about half the normal amount of type I procollagen, as a result of decreased synthesis of one of its constituent chains, pro[alpha](I). To test the hypothesis that decreased synthesis of pro[alpha](I) chains results from mutations in the COL1A1 gene, the authors used primer extension with nucleotide-specific chain termination to measure the contribution of individual COL1A1 alleles to the mRNA pool in fibroblasts from affected individuals. A polymorphic Mn/I restriction endonuclease site in the 3'-untranslated region of COL1A1 was used to distinguish the transcripts of the two alleles inmore » heterozygous individuals. Twenty-three individuals from 21 unrelated families were studied. In each case there was marked diminution in steady-state mRNA levels from one COL1A2 allele. Loss of an allele through deletion or rearrangement was not the cause of the diminished COL1A1 mRNA levels. Primer extension with nucleotide-specific chain termination allows identification of the mutant COL1A1 allele in cell strains that are heterozygous for an expressed polymorphism. It is applicable to sporadic cases, to small families, and to large families in whom key individuals are uninformative at the polymorphic sites used in linkage analysis, making it a useful adjunct to the biochemical screening of collagenous proteins for OI. 40 refs., 3 figs., 1 tab.« less

Traumatic brain injury decreases serotonin transporter expression in the rat cerebrum.

PubMed

Abe, Keiichi; Shimada, Ryo; Okada, Yoshikazu; Kibayashi, Kazuhiko

2016-04-01

An association has been postulated between traumatic brain injury (TBI) and depression. The serotonin transporter (SERT) regulates the concentration of serotonin in the synaptic cleft and represents a molecular target for antidepressants. We hypothesized that SERT expression in the brain changes following TBI. We performed immunohistochemistry, real-time polymerase chain reaction analysis for mRNA and western blot analysis for protein to examine the time-dependent changes in SERT expression in the cerebrum during the first 14 days after TBI, using a controlled cortical impact model in rats. SERT immunoreactivity in neuronal fibres within the area adjacent to the cortical contusion decreased 1 to 14 days after injury. Significantly decreased SERT mRNA and protein expression were noted in the area adjacent to the cortical contusion 7 days after injury. There were no significant changes in SERT expression in the cingulum of the injured brain. The findings of this study indicate that TBI decreases SERT expression in the cerebral cortex. The decreased levels of SERT expression after TBI may result in decreased serotonin neurotransmission in the brain and indicate a possible relationship with depression following TBI.

Development of recombinant canine adenovirus type-2 expressing the Gn glycoprotein of Seoul virus.

PubMed

Yuan, Ziguo; Zhang, Xiuxiang; Zhang, Shoufeng; Liu, Ye; Gao, Shengyan; Zhang, Fei; Xu, Huijuan; Wang, Xiaohu; Hu, Rongliang

2008-05-01

Seoul virus glycoprotein Gn is a major structural protein and candidate antigen of hantavirus that induces a highly immunogenic response for hantavirus vaccine. In this study, a replication-competent recombinant canine adenovirus type-2 expressing Gn was constructed by the in vitro ligation method. The Gn expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the SV40 early mRNA polyadenylation signal, was cloned into the SspI site of the E3 region which is not essential for proliferation of CAV-2. Expression of Gn was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Kasid, A.; Morecki, S.; Aebersold, P.

Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration andmore » expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.« less

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

PubMed

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

2010-12-01

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

Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type II collagen and aggrecan mRNA expression.

PubMed

Smith, R L; Lin, J; Trindade, M C; Shida, J; Kajiyama, G; Vu, T; Hoffman, A R; van der Meulen, M C; Goodman, S B; Schurman, D J; Carter, D R

2000-01-01

The normal loading of joints during daily activities causes the articular cartilage to be exposed to high levels of intermittent hydrostatic pressure. This study quantified effects of intermittent hydrostatic pressure on expression of mRNA for important extracellular matrix constituents. Normal adult bovine articular chondrocytes were isolated and tested in primary culture, either as high-density monolayers or formed aggregates. Loaded cells were exposed to 10 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for periods of 2, 4, 8, 12, and 24 hrs. Other cells were intermittently loaded for a period of 4 hrs per day for 4 days. Semiquantitative reverse transcription polymerase chain reaction assays were used to assess mRNA signal levels for collagen types II and I and aggrecan. The results showed that type II collagen mRNA signal levels exhibited a biphasic pattern, with an initial increase of approximately five-fold at 4 and 8 hrs that subsequently decreased by 24 hrs. In contrast, aggrecan mRNA signal increased progressively up to three-fold throughout the loading period. Changing the loading profile to 4 hrs per day for 4 days increased the mRNA signal levels for type II collagen nine-fold and for aggrecan twenty-fold when compared to unloaded cultures. These data suggest that specific mechanical loading protocols may be required to optimally promote repair and regeneration of diseased joints.

Effect of ration size on fillet fatty acid composition, phospholipid allostasis and mRNA expression patterns of lipid regulatory genes in gilthead sea bream (Sparus aurata).

PubMed

Benedito-Palos, Laura; Calduch-Giner, Josep A; Ballester-Lozano, Gabriel F; Pérez-Sánchez, Jaume

2013-04-14

The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA.

[Effects of lipopolysaccharides from Porphyromonas endodontalis on the expression of MIP-1α in mouse osteoblasts].

PubMed

Yu, Ya-Qiong; Li, Xiao-Lin; Qiu, Li-Hong; Guo, Jia-Jie; Yang, Di; Guo, Yan

2017-06-01

To investigate the effects of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of macrophageinflammatoryprotein-1α (MIP-1α) mRNA and protein levels in MC3T3-E1 cells and the influence of curcumin in the process. MC3T3-E1 cells were treated with 20 mg/L P.e-LPS for different times (0-48 h). The expression of MIP-1α mRNA and protein was detected by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and enzyme linked immunosorbent assay(ELISA). MC3T3-E1 cells were pretreated with inhibitor of (curcumin) for 1 h, and then treated with 20 mg/L P.e-LPS. The expression of MIP-1α was also detected by real-time RT-PCR and ELISA.Statistical analysis was performed using one-way ANOVA and Dunnett's t test with SPSS 13.0 software package. In the observation time (0-48 h), the impact of 20 P.e-LPS mg/L on induction of MIP-1α in MC3T3-El cells exhibited a time-dependent manner. The expression of MIP-1α mRNA and protein decreased significantly after pretreatment with 10 μmol/L curcumin for 1 h. The results suggest that P.e-LPS may mediate MIP-1α expression in MC3T3-E1 cells, and curcumin has a significant inhibitory effect on this process.

Expression profile of BRCA1 and BRCA2 genes in premenopausal Mexican women with breast cancer: clinical and immunohistochemical correlates.

PubMed

Loredo-Pozos, Gloria; Chiquete, Erwin; Oceguera-Villanueva, Antonio; Panduro, Arturo; Siller-López, Fernando; Ramos-Márquez, Martha E

2009-01-01

Low BRCA1 gene expression is associated with increased invasiveness and influences the response of breast carcinoma (BC) to chemotherapeutics. However, expression of BRCA1 and BRCA2 genes has not been completely characterized in premenopausal BC. We analyzed the clinical and immunohistochemical correlates of BRCA1 and BRCA2 expression in young BC women. We studied 62 women (mean age 38.8 years) who developed BC before the age of 45 years. BRCA1 and BRCA2 mRNA expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HER-2 and p53 proteins by immunohistochemistry. Body mass index (BMI) > or = 27 (52%) and a declared family history of BC (26%) were the main risk factors. Ductal infiltrative adenocarcinoma was found in 86% of the cases (tumor size >5 cm in 48%). Disease stages I-IV occurred in 2, 40, 55, and 3%, respectively (73% implicating lymph nodes). Women aged < or = 35 years (24%) had more family history of cervical cancer, stage III/IV disease, HER-2 positivity, and lower BRCA1 expression than older women (P < 0.05). BRCA1 and BRCA2 expression correlated in healthy, but not in tumor tissues (TT). Neither BRCA1 nor BRCA2 expression was associated with tumor histology, differentiation, nodal metastasis or p53 and HER-2 expression. After multivariate analysis, only disease stage explained BRCA1 mRNA levels in the lowest quartile. Premenopausal BC has aggressive clinical and molecular characteristics. Low BRCA1 mRNA expression is associated mainly with younger ages and advanced clinical stage of premenopausal BC. BRCA2 expression is not associated with disease severity in young BC women.

[Effect of Leonurus Heterophyllus Sweet on tissue factor transcription and expression in human umbilical vein endothelial cells in vitro].

PubMed

Zheng, Lian; Fang, Chi-hua

2007-06-01

To investigate the effect of Leonurus Heterophyllus Sweet, (LHS) on tissue factor (TF) transcription and expression induced by thrombin in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with different concentrations of LHS and the TF mRNA expression was detected by reverse transcript-polymerase chain reaction (RT-PCR). LHS treatment of HUVECs at different concentrations and for different times resulted in significant differences in TF expression (Plt;0.01). The transcription of TF in LHS-treated cells was significantly different from that of the blank control group (Plt;0.01). LHS can decrease the expression of TF and intervene with TF transcription in HUVECs in vitro.

Impact of gastro-esophageal reflux on mucin mRNA expression in the esophageal mucosa.

PubMed

van Roon, Aafke H C; Mayne, George C; Wijnhoven, Bas P L; Watson, David I; Leong, Mary P; Neijman, Gabriëlle E; Michael, Michael Z; McKay, Andrew R; Astill, David; Hussey, Damian J

2008-08-01

Changes in the expression of mucin genes in the esophageal mucosa associated with uncomplicated gastro-esophageal reflux disease have not been evaluated even though such changes could be associated with reflux-induced mucosal damage. We therefore sought to identify reflux-induced changes in mucin gene expression using a cell line and biopsies from the esophageal mucosa in patients with and without reflux. MUC-1, MUC-3, MUC-4, and MUC-5AC gene expressions were investigated in the HET-1A cell line following exposure to acid (pH 4) and/or bile (120 muM of a bile salt milieu), and in esophageal mucosal biopsies from controls, subjects with non-erosive gastro-esophageal reflux, and subjects with reflux associated with ulcerative esophagitis (erosive). The mucosal biopsies were also evaluated for IL-6 mRNA expression (inflammatory marker) and CK-14 mRNA expression (mucosal basal cell layer marker). Gene expression was determined using real-time reverse transcriptase-polymerase chain reaction analysis. In the cell line studies, there were differences in mRNA levels for all of the evaluated mucins following treatment with either acid or the acid and bile combination. In the studies which evaluated tissue specimens, IL-6 and CK-14 mRNA levels increased according to degree of reflux pathology. The expression of MUC-1 and MUC-4 in mucosa from patients with erosive reflux was lower than in subjects without reflux and in patients with non-erosive reflux, whereas the expression of MUC-3 and MUC-5AC was increased (although these differences did not reach significance at p < 0.05). When mRNA expression data for tissue samples from all groups were combined, significant correlations were identified between IL-6 vs. CK-14 and IL-6 vs. MUC-3, MUC-3 vs. CK-14 and MUC-3 vs. MUC-5AC, and for MUC-1 vs. MUC-5AC. The correlation between IL-6 and CK-14 was also significant within the control and non-erosive reflux groups. The correlation between IL-6 and MUC-3 was significant within the control and erosive reflux groups, and the correlation between MUC-1 and MUC-5AC was significant within the erosive reflux group. The results of this study suggest that the profile of mucin expression in the esophageal mucosa is influenced by the pH and composition of the gastro-esophageal reflux. Further work should explore the response of these genes to acid and bile reflux, and their role in the etiology of mucosal damage in gastro-esophageal reflux.

Cardiomyogenic Differentiation in Cardiac Myxoma Expressing Lineage-Specific Transcription Factors

PubMed Central

Kodama, Hiroaki; Hirotani, Takashi; Suzuki, Yusuke; Ogawa, Satoshi; Yamazaki, Kazuto

2002-01-01

We investigated five cases of cardiac myxoma and one case of cardiac undifferentiated sarcoma by light and electron microscopy, in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction for cardiomyocyte-specific transcription factors, Nkx2.5/Csx, GATA-4, MEF2, and eHAND. Conventional light microscopy revealed that cardiac myxoma and sarcoma cells presented variable cellular arrangements and different histological characteristics. Ultrastructurally, some of the myxoma cells exhibited endothelium-like or immature mesenchymal cell differentiation. Immunohistochemistry for Nkx2.5/Csx, GATA-4, and eHAND was slightly to intensely positive in all myxoma cases. MEF2 immunoreactivity was observed in all cases including the case of sarcoma, thus suggesting myogenic differentiation of myxoma or sarcoma cells. In situ hybridization for Nkx2.5/Csx also revealed that all myxoma cells, but not sarcoma cells, expressed mRNA of the cardiac homeobox gene, Nkx2.5/Csx. Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively. In conclusion, cardiac myxoma cells were found to express various amounts of cardiomyocyte-specific transcription factor gene products at the mRNA and protein levels, thus suggesting cardiomyogenic differentiation. These results support the concept that cardiac myxoma might arise from mesenchymal cardiomyocyte progenitor cells. PMID:12163362

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2017-09-01

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

In vitro cytokine expression by peripheral mononuclear cells in herbal drug-induced skin eruption.

PubMed

Norisugi, Osamu; Yoshihisa, Yoko; Shimizu, Kyoko; Shimizu, Tadamichi

2014-01-01

Herbal medicine is widely used worldwide and is associated with side-effects such as skin eruptions. Herbal drugs are often produced by combining multiple crude drugs, mostly of plant origin. Determining which medi-cinal plants are associated with the herbal drugs that induce skin eruptions can therefore be difficult. This study investigated mRNA expression of several cytokines in peripheral mononuclear cells (PBMCs) from two patients with herbal drug-induced skin eruptions; one reacted to keishi-bukuryo-gan (KBG), composed of 5 medicinal plants, and the other patient reacted to senna. PBMCs (1×106) from the 2 patients were cultured for 24 h with the supernatant from the medicinal plants from KBG or senna in various concentrations, and a reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed. A high mRNA level of interleukin (IL)-4 and IL-5 was detected in PBMCs stimulated by KBG and two of its components. Senna stimulated a high level of IL-4 and IL-5 mRNA levels in PBMCs from patient with senna-induced drug reaction.

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion.

PubMed

Yang, Ying; Zhou, Li-bin; Liu, Shang-quan; Tang, Jing-feng; Li, Feng-yin; Li, Rong-ying; Song, Huai-dong; Chen, Ming-dao

2005-08-01

To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion. Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY)1 and NPY5, neurotensin (NT)1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA). Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

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.

Letrozole increases ovarian growth and Cyp17a1 gene expression in the rat ovary

PubMed Central

Ortega, Israel; Sokalska, Anna; Villanueva, Jesus A.; Cress, Amanda B.; Wong, Donna H.; Stener-Victorin, Elisabet; Stanley, Scott D.; Duleba, Antoni J.

2012-01-01

Objective To evaluate the effects of letrozole on ovarian size and steroidogenesis in vivo, as well as on proliferation and steroidogenesis of theca-interstitial cells alone and in coculture with granulosa cells using an in vitro model. Design In vivo and in vitro studies. Setting Research laboratory. Animal(s) Immature Sprague-Dawley female rats. Intervention(s) In vivo effects of letrozole were studied in intact rats receiving either letrozole (90-day continuous-release SC pellets, 400 µg/d) or placebo pellets (control group). In in vitro experiments, theca cells were cultured alone or in coculture with granulosa cells in the absence or presence of letrozole. Main Outcome Measure(s) Deoxyribonucleic acid synthesis was determined by thymidine incorporation assay; steroidogenesis by mass spectrometry; and steroidogenic enzyme messenger RNA (mRNA) expression by polymerase chain reaction. Result(s) In vivo, letrozole induced an increase in ovarian size compared with the control group and also induced a profound increase of androgen, LH levels, and Cyp17a1 mRNA expression. Conversely, a decrease in Star, Cyp11a1, and Hsd3b1 transcripts was observed in letrozole-exposed rats. In vitro, letrozole did not alter either theca cell proliferation or Cyp17a1 mRNA expression. Similarly, letrozole did not affect Cyp17a1 transcripts in granulosa-theca cocultures. Conclusion(s) These findings suggest that letrozole exerts potent, but indirect, effect on growth of rat ovary and dramatically increases androgen levels and Cyp17a1 mRNA expression, the key enzyme regulating the androgen biosynthesis pathway. The present findings reveal novel mechanisms of action of letrozole in the rat ovary. PMID:23200686

Influence of trichloroacetic acid peeling on the skin stress response system.

PubMed

Kimura, Ayako; Kanazawa, Nobuo; Li, Hong-Jin; Yonei, Nozomi; Yamamoto, Yuki; Furukawa, Fukumi

2011-08-01

Although trichloroacetic acid (TCA) peeling is widely applied for cosmetic treatment of photodamaged skin, the entire biological mechanisms have yet to be determined. The skin stress response system (SSRS) involves corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) products that are locally-generated in response to locally-provided stressors or pro-inflammatory cytokines. This system would restrict tissue damage and restore local homeostasis. To determine the influence of TCA peeling on the SSRS in vitro and in vivo, expressions of POMC, melanocortin receptor 1 (MC1R), CRH and CRH receptor 1 (CRHR1) mRNA were examined by reverse transcription polymerase chain reaction in Pam212 murine keratinocytes, murine plantar and healthy human abdominal skin specimens after TCA treatment. In addition, their protein expressions as well as those of POMC-derived peptides were examined immunohistochemically. After TCA treatment, transient upregulation of POMC and MC1R mRNA expressions was observed in both murine and human skin, as well as in Pam212. Enhanced POMC protein, recovery of once-impaired MC1R protein, and no enhancement of POMC-derived peptide productions were revealed immunohistochemically in both murine and human epidermis. In contrast, neither expression levels of CRH and CRHR1 mRNA nor epidermal protein were enhanced after TCA application in murine and human skin, except for induction of human CRH mRNA expression. These results suggest that TCA activates the SSRS by inducing POMC and MC1R productions of keratinocytes in the CRH-independent manner, and that the biological effects of POMC itself are responsible for the TCA-induced epidermal SSRS activation. © 2010 Japanese Dermatological Association.

Changes in oestrogen receptor alpha expression in the nucleus of the solitary tract of the rat over the oestrous cycle and following ovariectomy.

PubMed

Spary, E J; Maqbool, A; Batten, T F C

2010-06-01

Oestrogen is capable of modulating autonomic outflow and baroreflex function via actions on groups of neurones in the brainstem. We investigated the presence of oestrogen receptor (ER) alpha in a part of the nucleus of the solitary tract (NTS) associated with central cardiovascular control, aiming to determine whether ERalpha mRNA and protein expression is correlated with levels of circulating oestrogen during the oestrous cycle. Polymerase chain reaction (PCR) detected ERalpha mRNA in the NTS at each stage of the oestrous cycle, from ovariectomised, sham-operated and male rats. Real-time PCR showed variations in ERalpha mRNA expression during the oestrous cycle, with the highest levels seen in oestrus, and lowest levels in metoestrus (P < 0.05 versus oestrus) and proestrus (P < 0.05 versus oestrus). Expression in males was lower than in dioestrus and oestrus females (P < 0.05). After ovariectomy, ERalpha mRNA levels were decreased compared to sham-operated animals (P < 0.01). Confocal fluorescence immunohistochemistry with stereological analysis showed that numbers of ERalpha immunoreactive cell nuclei per mm(3) of tissue in the caudal NTS were significantly greater in proestrus than in other groups of rats (P < 0.05). There were also differences among the groups in the extent of colocalisation of ERalpha in neurones immunoreactive for tyrosine hydroxylase and nitric oxide synthase. These results imply a complex pattern of region-specific oestrogen signalling in the NTS and suggest that ERalpha expression in this important autonomic nucleus may be related to circulating oestrogen levels. This may have consequences for the regulation of autonomic tone and baroreflex sensitivity when oestrogen levels decline, for example following menopause.

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

PubMed

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

2010-04-01

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

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

Proteoglycans in Leiomyoma and Normal Myometrium

PubMed Central

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

2015-01-01

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

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions.

PubMed

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions

PubMed Central

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity. PMID:25191551

Effects and mechanism of integrin-β1 gene expression inhibited by shRNA in invasion of pancreatic carcinoma PANC-1 cells.

PubMed

Yu, Feng; Li, Hua; Bu, Xuefeng; Zhang, Yongjun

2012-01-01

To investigate the effects of integrin-β1 gene expression inhibited by shRNA on invasion of pancreatic carcinoma PANC-1 cells in vitro. The eukaryotic expression plasmid of short hairpin RNA (shRNA) targeting integrin-β1 gene (integrin-β1-shRNA) was constructed and transfected into PANC-1 cells. The expressions of integrin-β1 mRNA and protein were detected by real-time quantitative polymerase chain reaction (PCR) and western blot assay, respectively. The invasive ability of PANC-1 cells was observed with a transwell cell culture chamber and the expressions of MMP-2 and MMP-9 were assayed. Compared to the untransfected group, recombinant expression plasmid integrin-β1-shRNA resulted in reduction of integrin-β1 mRNA and protein by 78.58%±7.24% and 92.88%±3.18%, respectively and the average number of invading PANC-1 cells were decreased from 52±5 to 21±4 (p<0.01) and the expressions of MMP-2 and MMP-9 were inhibited. Recombinant expression plasmid integrin-β1- shRNA can inhibit the expression of integrin-β1 gene and suppress the invasion of PANC-1 cells in vitro significantly.

[The role of Leptin on neuron apoptosis in mice with cerebral ischemia/reperfusion injury].

PubMed

Yan, Guang-tao; Si, Yi-ling; Zhang, Jin-ying; Deng, Zi-hui; Xue, Hui

2011-06-01

To study the effect of Leptin on neuron apoptosis in mice with cerebral ischemia injury and its mechanism. Seventy-five mice were randomly divided into three groups. Focal cerebral ischemia/reperfusion injury model in mice was reproduced by middle cerebral artery occlusion for 2 hours followed by reperfusion. In Leptin intervention group mice were given Leptin 1 μg/g during cerebral ischemia by intraperitoneal injection. Mice in the model group were given equal amount of phosphate buffer saline. After reperfusion for 24 hours, the neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The mRNA and protein expression of apoptosis relative gene caspase-3 and bcl-2 were determined by reverse transcription-polymerase chain reaction (RT-PCR) and immuno histochemistry. Most of neuron necrosis was observed in cerebral ischemia center in model group. Compared with sham-operation group, neuron apoptosis rate, mRNA and protein expression of caspase-3 and bcl-2 in model group increased significantly [apoptosis rate: (68.65 ± 0.79)% vs. (4.40 ± 0.00)%, caspase-3 mRNA: 2.563 ± 0.250 vs. 0.153 ± 0.020, bcl-2 mRNA: 0.337 ± 0.100 vs. 0.125 ± 0.030, caspase-3 protein (absorbance value, A value): 0.57 ± 0.05 vs. 0.37 ± 0.03, bcl-2 protein (A value): 0.51 ± 0.04 vs. 0.35 ± 0.01, all P<0.01]. The apoptosis rate of penumbra neurons was reduced in Leptin intervention group significantly compared with model group [(42.30 ± 8.45)% vs. (68.65 ± 0.79)%, P<0.01]. Compared with model group, the mRNA and protein expression of caspase-3 in Leptin intervention group were reduced significantly [caspase-3 mRNA: 2.267 ± 0.040 vs. 2.563 ± 0.250, caspase-3 protein (A value): 0.45 ± 0.04 vs. 0.57 ± 0.05, P>0.05 and P<0.01], and the mRNA and protein expression of bcl-2 in Leptin intervention group upregulated significantly [bcl-2 mRNA: 0.662 ± 0.040 vs. 0.337 ± 0.100, bcl-2 protein (A value): 0.76 ± 0.09 vs. 0.51 ± 0.04, both P<0.01]. Leptin could reduce apoptosis of neurons through down-regulation of the expression of caspase-3 and up-regulation of the expression of bcl-2. The results suggest that Leptin plays a neuroprotective role in cerebral ischemia injury.

Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma.

PubMed

Elloul, Sivan; Elstrand, Mari Bukholt; Nesland, Jahn M; Tropé, Claes G; Kvalheim, Gunnar; Goldberg, Iris; Reich, Reuven; Davidson, Ben

2005-04-15

It was demonstrated previously that the Snail family of transcription factors and Smad-interacting protein 1 (Sip1) regulate E-cadherin and matrix metalloproteinase 2 (MMP-2) expression, cellular morphology, and invasion in carcinoma. For the current study, the authors analyzed the relation between the expression of Snail, Slug, and Sip1; the expression of MMP-2 and E-cadherin; and clinical parameters in patients with metastatic ovarian and breast carcinoma. One hundred one fresh-frozen, malignant effusions from patients who were diagnosed with gynecologic carcinomas (78 ovarian carcinomas and 23 breast carcinomas) were studied for mRNA expression of Snail, Slug, Sip1, MMP-2, and E-cadherin using reverse transcriptase-polymerase chain reaction analysis. Snail mRNA and E-cadherin protein expression levels also were studied in ovarian carcinoma effusions using in situ hybridization and immunocytochemistry. The results were analyzed for possible correlation with clinicopathologic parameters in both tumor types. E-cadherin mRNA expression was lower in breast carcinoma (P = 0.001), whereas Snail expression was higher (P = 0.003). The Snail/E-cadherin ratio (P < 0.001) and the Sip1/E-cadherin ratio (P = 0.002) were higher in breast carcinomas. Sip1 mRNA expression (P < 0.001) and Slug mRNA expression (P < 0.001) were correlated with the expression of MMP-2 in ovarian carcinomas. The Sip1/E-cadherin ratio was higher in primary ovarian carcinomas at the time of diagnosis compared with postchemotherapy ovarian carcinoma effusions (P = 0.003), higher in Stage IV tumors compared with Stage III tumors (P = 0.049), and higher in pleural effusions compared with peritoneal effusions (P = 0.044). In a univariate survival analysis of patients with ovarian carcinoma, a high Sip1/E-cadherin ratio predicted poor overall survival (P = 0.018). High E-cadherin mRNA expression predicted better disease-free survival (P = 0.023), with a similar trend for a low Slug/E-cadherin ratio (P = 0.07). High Snail mRNA expression predicted shorter effusion-free survival (P = 0.008), disease-free survival (P = 0.03), and overall survival (P = 0.008) in patients with breast carcinoma. Transcription factors that regulate E-cadherin were expressed differentially in metastatic ovarian and breast carcinoma. Snail may predict a poor outcome in patients who have breast carcinoma metastatic to effusions. E-cadherin expression generally was conserved in effusions from patients with ovarian carcinoma, but the subset of patients with postulated Sip1-induced repression of this adhesion molecule had a significantly worse outcome. This finding was in agreement with the stronger suppression of E-cadherin by Snail and Sip1 in breast carcinoma effusions, a clinical condition associated with extremely poor survival. (c) 2005 American Cancer Society.

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

PubMed

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

2016-08-01

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

Gene expression of fatty acid transport and binding proteins in the blood-brain barrier and the cerebral cortex of the rat: differences across development and with different DHA brain status.

PubMed

Pélerin, Hélène; Jouin, Mélanie; Lallemand, Marie-Sylvie; Alessandri, Jean-Marc; Cunnane, Stephen C; Langelier, Bénédicte; Guesnet, Philippe

2014-11-01

Specific mechanisms for maintaining docosahexaenoic acid (DHA) concentration in brain cells but also transporting DHA from the blood across the blood-brain barrier (BBB) are not agreed upon. Our main objective was therefore to evaluate the level of gene expression of fatty acid transport and fatty acid binding proteins in the cerebral cortex and at the BBB level during the perinatal period of active brain DHA accretion, at weaning, and until the adult age. We measured by real time RT-PCR the mRNA expression of different isoforms of fatty acid transport proteins (FATPs), long-chain acyl-CoA synthetases (ACSLs), fatty acid binding proteins (FABPs) and the fatty acid transporter (FAT)/CD36 in cerebral cortex and isolated microvessels at embryonic day 18 (E18) and postnatal days 14, 21 and 60 (P14, P21 and P60, respectively) in rats receiving different n-3 PUFA dietary supplies (control, totally deficient or DHA-supplemented). In control rats, all the genes were expressed at the BBB level (P14 to P60), the mRNA levels of FABP5 and ACSL3 having the highest values. Age-dependent differences included a systematic decrease in the mRNA expressions between P14-P21 and P60 (2 to 3-fold), with FABP7 mRNA abundance being the most affected (10-fold). In the cerebral cortex, mRNA levels varied differently since FATP4, ACSL3 and ACSL6 and the three FABPs genes were highly expressed. There were no significant differences in the expression of the 10 genes studied in n-3 deficient or DHA-supplemented rats despite significant differences in their brain DHA content, suggesting that brain DHA uptake from the blood does not necessarily require specific transporters within cerebral endothelial cells and could, under these experimental conditions, be a simple passive diffusion process. Copyright © 2014 Elsevier Ltd. All rights reserved.

Short Communication: Effect of heat stress on heat-shock protein (Hsp60) mRNA expression in rainbow trout Oncorhynchus mykiss.

PubMed

Shi, H N; Liu, Z; Zhang, J P; Kang, Y J; Wang, J F; Huang, J Q; Wang, W M

2015-05-18

The enhanced expression of heat shock proteins (hsps) in organisms can be detected in response to many kinds of stressor. For fish, high temperature is an important stressor, and hsp expression is associated with differences in environmental temperature. In this study, rainbow trout (Oncorhynchus mykiss) that were accustomed to an aquatic temperature of 18°C were exposed to an elevated temperature (25°C), and hsp60 expression in the gill, liver, spleen, heart, and head kidney was quantified using real-time polymerase chain reaction in unstressed and heat-stressed animals. The fish responded to heat stress in a time- and tissue-specific manner. Cardiac hsp60 mRNA levels were largely unchanged, and the greatest induction of hsp60 in heat-stressed animals was recorded in the liver, suggesting that protein damage and the consequent requirement for the Hsp60 protein are probably greater in hepatic tissue. Therefore, fish must be provided with optimal temperature conditions in order to realize their potential growth and maximize fish farm profits.

Coordinated Expression of Cyclin-dependent Kinase-4 and its Regulators in Human Oral Tumors

PubMed Central

POI, MING J.; KNOBLOCH, THOMAS J.; SEARS, MARTA T.; UHRIG, LANA K.; WARNER, BLAKE M.; WEGHORST, CHRISTOPHER M.; LI, JUNAN

2014-01-01

Background/Aim While aberrant expression of cyclin-dependent kinase-4 (CDK4) has been found in squamous cell carcinoma of the head and neck (SCCHN), the associations between CDK4 and its regulators, namely, cyclin D1, cyclin E, gankyrin, SEI1, and BMI1 in gene expression remain to be explored. Herein we investigated the mRNA profiles of these oncogenes and their interrelations in different oral lesion tissues. Materials and Methods Thirty SCCHN specimens and patient-matched high at-risk mucosa (HARM) and 16 healthy control specimens were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. Results The mRNA levels of CDK4, cyclin D1, gankyrin, SEI1, BMI1 were significantly elevated in both HARM and SCCHN (in comparison with control specimens), and statistically significant correlations were found among these markers in gene expression. Conclusion Up-regulation of CDK4 and its regulators takes place in oral cancer progression in a coordinate manner, and HARM and SCCHN share a similar molecular signature within the CDK4-pRB pathway. PMID:24982332

Expression analysis of extracellular matrix components in brush biopsies of oral lesions.

PubMed

Driemel, Oliver; Kosmehl, Hartwig; Rosenhahn, Julia; Berndt, Alexander; Reichert, Torsten E; Zardi, Luciano; Dahse, Regine

2007-01-01

Oral brush biopsies have proved to be a promising new non-invasive methodology in the diagnosis of oral lesions. The extracellular matrix (ECM) molecules gamma2 chain of laminin-5 (L5gamma2), tenascin-c (Tn-C) and the fibronectin isoform containing EDB (EDB-fn) are involved in matrix remodeling during malignant transformation in oral carcinoma. Expression of L5gamma2, Tn-C and EDB-fn was analysed in brush biopsy-obtained cells of benign inflammatory or hyperproliferative lesions and primary oral squamous cell carcinoma (OSCC) using the Roche LightCycler 2.0 System. Oral carcinoma are detectable with mRNA resynthesis of the ECM molecules L5gamma2 and Tn-C in oral brush biopsies. EDB-fn mRNA was not detected--the stroma myofibroblasts are apparently a preferential source of EDB-fn and sampling by oral brush biopsy harvests epithelial cells and does not reach the cells which do express EDB-fn. The performance of gene expression analysis in brush biopsies is limited by a high RNase activity in the oral cavity.

Alterations of peroxisome proliferator-activated receptor γ and monocyte chemoattractant protein 1 gene expression in the nitrofen-induced hypoplastic lung.

PubMed

Gosemann, Jan-Hendrik; Doi, Takashi; Kutasy, Balazs; Friedmacher, Florian; Dingemann, Jens; Puri, Prem

2012-05-01

Peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in normal lung development. Peroxisome proliferator-activated receptor γ messenger RNA (mRNA) is detectable at 18 days of gestation in fetal rat lungs, and levels peak just before birth. Peroxisome proliferator-activated receptor γ agonists are reported to stimulate lung development, whereas inhibition of PPARγ disrupts postnatal lung maturation. Monocyte chemoattractant protein 1 (MCP-1), which is inhibited by PPARγ, is reported to disrupt late lung morphogenesis. This study was designed to investigate the hypothesis that PPARγ expression is downregulated and that MCP-1 expression is upregulated during the late stages of lung development in nitrofen-induced hypoplastic lungs. Pregnant rats were treated with nitrofen or vehicle on D9. RNA was extracted from fetal lungs (D18 and D21), and relative mRNA expression levels of PPARγ and MCP-1 were determined by reverse transcriptase-polymerase chain reaction. Immunohistochemistry was performed to evaluate protein expression/distribution of PPARγ and MCP-1. Relative mRNA expression levels of PPARγ were significantly downregulated in the nitrofen group compared with controls on D21, whereas MCP-1 levels were upregulated. Immunohistochemical study showed markedly decreased PPARγ and increased MCP-1 immunoreactivity in the nitrofen-induced hypoplastic lungs compared with controls on gestational day 21. Altered pulmonary gene expression of PPARγ and MCP-1 during late gestation may impair lung development and maturation, contributing to pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia model. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of cigarette smoke extract and nicotine on the expression of thrombomodulin and endothelial protein C receptor in cultured human umbilical vein endothelial cells

PubMed Central

Wei, Yujie; Lai, Bin; Liu, Huiliang; Li, Yi; Zhen, Wang; Fu, Ling

2018-01-01

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5–5%) and nicotine (10-3-10-9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription-quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6-h exposure, TM protein and mRNA expression levels decreased in a dose-dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking-associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences. PMID:29257196

Motor Skills Training Improves Sensorimotor Dysfunction and Increases Microtubule-Associated Protein 2 mRNA Expression in Rats with Intracerebral Hemorrhage.

PubMed

Tamakoshi, Keigo; Kawanaka, Kentaro; Onishi, Hideaki; Takamatsu, Yasuyuki; Ishida, Kazuto

2016-08-01

In this study, we examined the effects of motor skills training on the sensorimotor function and the expression of genes associated with synaptic plasticity after intracerebral hemorrhage (ICH) in rats. Male Wistar rats were subjected to ICH or sham operation. ICH was caused by the injection of collagenase into the left striatum. Rats were randomly assigned to no training, acrobatic training, and sham groups. The acrobatic group performed 5 types of acrobatic tasks from 4 to 28 days after surgery. The forelimb sensorimotor function was evaluated over time using forepaw grasping, forelimb placing, and postural instability tests. At 14 and 29 days after the lesion, we analyzed the mRNA expression levels of microtubule-associated protein 2 (MAP2), brain-derived neurotrophic factor, and growth-associated protein 43 in the bilateral sensorimotor cortex (forelimb area) by real-time reverse transcription-polymerase chain reaction. Motor skills training in ICH rats improved the sensorimotor dysfunction significantly from the early phase. The mRNA expression level of MAP2 was upregulated in the ipsilesional sensorimotor cortex by motor skills training at 29 days after the lesion. Our results suggest that sensorimotor functional recovery following motor skills training after ICH is promoted by dendritic growth in the ipsilesional sensorimotor cortex. Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease.

PubMed

Hirose, Jun; Ryan, Lawrence M; Masuda, Ikuko

2002-12-01

Excess accumulation of extracellular inorganic pyrophosphate (ePPi) in aged human cartilage is crucial in calcium pyrophosphate dihydrate (CPPD) crystal formation in cartilage matrix. Two sources of ePPi are ePPi-generating ectoenzymes (NTPPPH) and extracellular transport of intracellular PPi by ANK. This study was undertaken to evaluate the role of NTPPPH and ANK in ePPi elaboration, by investigating expression of NTPPPH enzymes (cartilage intermediate-layer protein [CILP] and plasma cell membrane glycoprotein 1 [PC-1]) and ANK in human chondrocytes from osteoarthritic (OA) articular cartilage containing CPPD crystals and without crystals. Chondrocytes were harvested from knee cartilage at the time of arthroplasty (OA with CPPD crystals [CPPD], n = 8; OA without crystals [OA], n = 10). Normal adult human chondrocytes (n = 1) were used as a control. Chondrocytes were cultured with transforming growth factor beta1 (TGFbeta1), which stimulates ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which inhibits ePPi elaboration. NTPPPH and ePPi were measured in the media at 48 hours. Media CILP, PC-1, and ANK were determined by dot-immunoblot analysis. Chondrocyte messenger RNA (mRNA) was extracted for reverse transcriptase-polymerase chain reaction to study expression of mRNA for CILP, PC-1, and ANK. NTPPPH and ANK mRNA and protein were also studied in fresh frozen cartilage. Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with normal chondrocytes, and tended to be higher compared with OA chondrocytes. Basal expression of mRNA for CILP (chondrocytes) and ANK (cartilage) was higher in both CPPD chondrocytes and CPPD cartilage extract than in OA or normal samples. PC-1 mRNA was less abundant in CPPD chondrocytes and cartilage extract than in OA chondrocytes and extract, although the difference was not significant. CILP, PC-1, and ANK protein levels were similar in CPPD, OA, and normal chondrocytes or cartilage extracts. Both CILP and ANK mRNA expression and ePPi elaboration were stimulated by TGFbeta1 and inhibited by IGF-1 in chondrocytes from all sources. CILP and ANK mRNA expression correlates with chondrocyte ePPi accumulation around CPPD and OA chondrocytes, and all respond similarly to growth factor stimulation. These findings suggest that up-regulated CILP and ANK expression contributes to higher ePPi accumulation from CPPD crystal-forming cartilage.

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

PubMed

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

2015-10-01

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

IL-4 mRNA Is Downregulated in the Liver of Pancreatic Cancer Patients Suffering from Cachexia.

PubMed

Prokopchuk, Olga; Steinacker, Jürgen M; Nitsche, Ulrich; Otto, Stephanie; Bachmann, Jeannine; Schubert, Elaine C; Friess, Helmut; Martignoni, Marc E

2017-01-01

Interleukin-4 (IL-4) together with interleukin-13 (IL-13) play an important role in inflammation and wound repair, and are known to be upregulated in human skeletal muscle after strenuous physical exercise. Additionally, these cytokines may act as autocrine growth factors in pancreatic cancer cells. We hypothesize that IL-4, IL-13, and their corresponding receptors are involved in mechanism of cancer cachexia. Tissue samples from human skeletal muscle, white fat, liver, healthy pancreas, and pancreatic ductal adenocarcinoma were analyzed by quantitative real-time polymerase chain reaction for mRNA expression levels of IL-4, IL-13, IL-4 receptor α, and IL-13 receptor α1. We demonstrate for the first time that liver IL-4 mRNA is downregulated in vivo in patients with pancreatic cancer and cachexia. Additionally, IL-4 mRNA in the liver inversely correlated with musculus psoas thickness. We speculate that suppression of IL-4 is involved in cancer cachexia, although the exact mechanisms have to be further elucidated.

Anatomical and histological profiling of orphan G-protein-coupled receptor expression in gastrointestinal tract of C57BL/6J mice.

PubMed

Ito, Junko; Ito, Masahiko; Nambu, Hirohide; Fujikawa, Toru; Tanaka, Kenichi; Iwaasa, Hisashi; Tokita, Shigeru

2009-11-01

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors and regulate a variety of physiological and disease processes. Although the roles of many non-odorant GPCRs have been identified in vivo, several GPCRs remain orphans (oGPCRs). The gastrointestinal (GI) tract is the largest endocrine organ and is a promising target for drug discovery. Given their close link to physiological function, the anatomical and histological expression profiles of benchmark GI-related GPCRs, such as the cholecystokinin-1 receptor and GPR120, and 106 oGPCRs were investigated in the mucosal and muscle-myenteric nerve layers in the GI tract of C57BL/6J mice by quantitative real-time polymerase chain reaction. The mRNA expression patterns of these benchmark molecules were consistent with previous in situ hybridization and immunohistochemical studies, validating the experimental protocols in this study. Of 96 oGPCRs with significant mRNA expression in the GI tract, several oGPCRs showed unique expression patterns. GPR85, GPR37, GPR37L1, brain-specific angiogenesis inhibitor (BAI) 1, BAI2, BAI3, and GPRC5B mRNAs were preferentially expressed in the muscle-myenteric nerve layer, similar to GPCRs that are expressed in both the central and enteric nerve systems and that play multiple regulatory roles throughout the gut-brain axis. In contrast, GPR112, trace amine-associated receptor (TAAR) 1, TAAR2, and GPRC5A mRNAs were preferentially expressed in the mucosal layer, suggesting their potential roles in the regulation of secretion, immunity, and epithelial homeostasis. These anatomical and histological mRNA expression profiles of oGPCRs provide useful clues about the physiological roles of oGPCRs in the GI tract.

The HGF Receptor c-Met Is Overexpressed in Esophageal Adenocarcinoma1

PubMed Central

Herrera, Luis J; El-Hefnawy, Talal; Queiroz de Oliveira, Pierre E; Raja, Siva; Finkelstein, Sydney; Gooding, William; Luketich, James D; Godfrey, Tony E; Hughes, Steven J

2005-01-01

Abstract The hepatocyte growth factor (HGF) receptor, Met, has established oncogenic properties; however, its expression and function in esophageal adenocarcinoma (EA) remain poorly understood. We aimed to determine the expression and potential alterations in Met expression in EA. Met expression was investigated in surgical specimens of EA, Barrett's esophagus (BE), and normal esophagus (NE) using immunohistochemistry (IHC) and quantitative reverse transcriptase polymerase chain reaction. Met expression, phosphorylation, and the effect of COX-2 inhibition on expression were examined in EA cell lines. IHC demonstrated intense Met immunoreactivity in all (100%) EA and dysplastic BE specimens. In contrast, minimal immunostaining was observed in BE without dysplasia or NE specimens. Met mRNA and protein levels were increased in three EA cell lines, and Met protein was phosphorylated in the absence of serum. Sequence analysis found the kinase domain of c-met to be wild type in all three EA cell lines. HGF mRNA expression was identified in two EA cell lines. In COX-2-overexpressing cells, COX-2 inhibition decreased Met expression. Met is consistently overexpressed in EA surgical specimens and in three EA cell lines. Met dysregulation occurs early in Barrett's dysplasia to adenocarcinoma sequence. Future study of Met inhibition as a potential biologic therapy for EA is warranted. PMID:15720819

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

Effects of tiletamine on the adenosine monophosphate-activated protein kinase signaling pathway in the rat central nervous system.

PubMed

Su, Li-Xue; Shi, Xing-Xing; Yang, Peng; Chen, Hao; Li, Xin; Fan, Hong-Gang; Wang, Hong-Bin

2017-10-01

The dissociative anesthetic tiletamine, which acts on the central nervous system (CNS), is widely used in veterinary medicine and animal experiments. Recent studies indicate that adenosine 5'-monophosphate activated protein kinase (AMPK) plays a key role in the analgesic action of tiletamine. In the present study, the effects of tiletamine on the AMPK signaling pathway in rats were investigated. Sprague-Dawley rats were injected intraperitoneally with tiletamine and executed at 10, 20, 40 and 60min post injection. The cerebral cortex, hippocampus, thalamus, cerebellum and brainstem were immediately taken out to evaluate the mRNA and protein phosphorylation levels of liver kinase B1 (LKB1), AMPKα and eIF4E-binding protein 1 (4EBP1) using quantitative real-time polymerase chain reaction and western blot analysis. Tiletamine increased AMPK mRNA expression in the rat brain (P<0.01). Increased mRNA expression of AMPK was accompanied by an increase in phosphorylation of LKB1, resulting in significant decreases in the phosphorylation levels of 4EBP1 in the corresponding brain regions (P<0.01). In summary, the findings indicate that tiletamine regulates the mRNA expression and protein phosphorylation levels of LKB1, AMPK and 4EBP1 in the CNS, suggesting that the analgesic effect of the anesthetic is mediated, at least in part, by the AMPK signaling pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combined therapeutic effect and molecular mechanisms of metformin and cisplatin in human lung cancer xenografts in nude mice.

PubMed

Chen, Yu-Qin; Chen, Gang

2015-01-01

This work was aimed at studying the inhibitory activity of metformin combined with the commonly used chemotherapy drug cisplatin in human lung cancer xenografts in nude mice. We also examined the combined effects of these drugs on the molecular expression of survivin, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor-C (VEGF-C), and vascular endothelial growth factor receptor-3 (VEGFR-3) to determine the mechanism of action and to explore the potential applications of the new effective drug therapy in lung cancer. The nude mice model of lung cancer xenografts was established, and mice were randomly divided into the metformin group, the cisplatin group, the metformin + cisplatin group, and the control group. The animals were killed 42 days after drug administration, and the tumor tissues were then sampled to detect the messenger ribonucleic acid (mRNA) and protein expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the cisplatin group and the combined treatment group were lower than that in the control group (P < 0.05). In the metformin group, the expression of MMP-2 protein and mRNA was lower than that in the control group (P < 0.05). The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the combined treatment group were lower than that in the cisplatin group and the metformin group (P < 0.05). Metformin inhibited the expression of MMP-2, cisplatin and the combined treatment inhibited the expression of survivin, MMP-2, VEGF-C, and VEGFR-3, and the combined treatment of metformin with cisplatin resulted in enhanced anti-tumor efficacy.

Embryotrophic factor-3 from human oviductal cells affects the messenger RNA expression of mouse blastocyst.

PubMed

Lee, Y L; Lee, K F; Xu, J S; Kwok, K L; Luk, J M; Lee, W M; Yeung, W S B

2003-02-01

Our previous results showed that embryotrophic factor-3 (ETF-3) from human oviductal cells increased the size and hatching rate of mouse blastocysts in vitro. The present study investigated the production of ETF-3 by an immortalized human oviductal cell line (OE-E6/E7) and the effects of ETF-3 on the mRNA expression of mouse embryos. The ETF-3 was purified from primary oviductal cell conditioned media using sequential liquid chromatographic systems, and antiserum against ETF-3 was raised. The ETF-3-supplemented Chatot-Ziomek-Bavister medium was used to culture Day 1 MF1 x BALB/c mouse embryos for 4 days. The ETF-3 treatment significantly enhanced the mouse embryo blastulation and hatching rate. The antiserum, at concentrations of 0.03-3%, abolished the embryotrophic effect of ETF-3. Positive ETF-3 immunoreactivity was detected in the primary oviductal cells, OE-E6/E7, and blastocysts derived from ETF-3 treatment. Vero cells (African Green Monkey kidney cell line), fibroblasts, and embryos cultured in control medium did not possess ETF-3 immunoreactivity. The mRNA expression patterns of the treated embryos were studied at the blastocyst stage by mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The DDRT-PCR showed that some of the mRNAs were differentially expressed after ETF-3 treatment. Twelve of the differentially expressed mRNAs that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expression of seven of these mRNAs (ezrin, heat shock 70-kDa protein, cytochrome c oxidase subunit VIIa-L precursor, proteinase-activated receptor 2, eukaryotic translation initiation factor 2beta, cullin 1, and proliferating cell nuclear antigen) was confirmed by semiquantitative RT-PCR. In conclusion, immortalized oviductal cells produce ETF-3, which influences mRNA expression of mouse blastocyst.

7-Ketocholesterol inhibits isocitrate dehydrogenase 2 expression and impairs endothelial function via microRNA-144.

PubMed

Fu, Xiaodong; Huang, Xiuwei; Li, Ping; Chen, Weiyu; Xia, Min

2014-06-01

Oxysterol is associated with the induction of endothelial oxidative stress and impaired endothelial function. Mitochondria play a central role in oxidative energy metabolism and the maintenance of proper redox status. The purpose of this study was to determine the effects and mechanisms of 7-ketocholesterol (7-KC) on isocitrate dehydrogenase 2 (IDH2) and its impact on endothelial function in both human aortic endothelial cells (HAECs) and C57BL/6J mice. HAECs treated with 7-KC showed significant reductions of IDH2 mRNA and protein levels and enzyme activity, leading to decreased NADPH concentration and an increased ratio of reduced-to-oxidized glutathione in the mitochondria. 7-KC induced the expression of a specific microRNA, miR-144, which in turn targets and downregulates IDH2. In silico analysis predicted that miR-144 could bind to the 3'-untranslated region of IDH2 mRNA. Overexpression of miR-144 decreased the expression of IDH2 and the levels of NADPH. A complementary finding is that a miR-144 inhibitor increased the mRNA and protein expression levels of IDH2. Furthermore, miR-144 level was elevated in HAECs in response to 7-KC. Anti-Ago1/2 immunoprecipitation coupled with a real-time polymerase chain reaction assay revealed that 7-KC increased the functional targeting of miR-144/IDH2 mRNA in HAECs. Infusion of 7-KC in vivo decreased vascular IDH2 expression and impaired vascular reactivity via miR-144. 7-KC controls miR-144 expression, which in turn decreases IDH2 expression and attenuates NO bioavailability to impair endothelial homeostasis. The newly identified 7-KC-miR-144-IDH2 pathway may contribute to atherosclerosis progression and provides new insight into 7-KC function and microRNA biology in cardiovascular disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Anti-proliferative and apoptotic effects of the novel taspine derivative tas41 in the Caco-2 cell line.

PubMed

Zhang, Yanmin; Zhang, Jie; Dai, Bingling; Wang, Nan; He, Langchong

2011-05-01

Taspine was screened and isolated for the first time from Radix et Rhizoma Leonticis. Tas41 is a novel taspine derivative. We investigated the effects of tas41 on proliferation of the Caco-2 cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a fluorescence-activated cell sorter (FACS), enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and western blotting (WB). Changes in the cell cycle, apoptosis, activation of caspase-3, caspase-8 and caspase-9, and expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) were investigated after Caco-2 cells were treated with tas41. At the same time, expressions of apoptosis protein bcl-2 and bax were determined. Tas41 was found to induce apoptosis in a concentration-dependent manner as confirmed by DNA fragmentation analysis, TUNEL assay and flow cytometry. Protein and mRNA expressions of EGF, VEGF, CDK2, bcl-2 and bax were evaluated by ELISA, WB and RT-PCR. Tas41 had a better anti-proliferative effect than taspine on Caco-2 cells. A DNA ladder and apoptosis was observed, and the increased apoptotic activity by tas41 was accompanied by a decrease in the expression of VEGF protein and mRNA. The activities of caspase-3, caspase-8 and caspase-9 were significantly increased in cells treated with tas41 compared with those in the control group. In addition, protein and mRNA expressions of bcl-2 were decreased, and protein and mRNA expressions of bax were increased. These findings demonstrate that tas41 can inhibit the proliferation of, and induce apoptosis in, Caco-2 cells by activating caspase-3, caspase-8 and caspase-9, downregulating the expressions of VEGF, upregulating the ratio of bax/bcl-2. Copyright © 2011 Elsevier B.V. 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.

Abnormal regulation for progesterone production in placenta with prenatal cocaine exposure in rats.

PubMed

Wu, L; Yan, J; Qu, S C; Feng, Y Q; Jiang, X L

2012-12-01

Cocaine abuse in pregnant women is currently a significant public hygiene problem and is tightly associated with elevated risk for preterm delivery. Placental steroidogenesis especially progesterone production was essential for success and maintenance of pregnancy in humans and rodents. In the present study, we determined the impact of prenatal cocaine exposure on pathways of placental progesterone synthesis in rats. Pregnant rats were treated cocaine twice daily (15 mg/kg/day) during the third trimester, and the maternal and fetal plasma progesterone and pregnenolone concentrations were detected. We also examined both the protein and mRNA expression of some key enzymes and regulators for progesterone production in placenta. Results showed that, after maternal cocaine use during pregnancy, progesterone and pregnenolone concentrations in both maternal and fetal rats were significantly decreased. Although prenatal cocaine exposure had no effects on placental 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1) expression, protein and mRNA expression of the cholesterol side-chain cleavage enzyme (P450scc/CYP11a) in placenta was significantly inhibited. Moreover, protein and mRNA expressions of MLN64 that regulating cholesterol transport and activating protein 2γ (AP2γ/Tfap2c) that controlling P450scc/CYP11a gene expression in placenta were both decreased following maternal cocaine use in pregnancy. Collectively, this study suggested that prenatal cocaine exposure could insult the placental progesterone production in rats possibly associated with the high risk for preterm delivery. Copyright © 2012 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization, and expression analysis of a heat shock protein (HSP) 70 gene from Paphia undulata.

PubMed

Wu, Xiangwei; Tan, Jing; Cai, Mingyi; Liu, Xiande

2014-06-15

In this study, a full-length HSP70 cDNA from Paphia undulata was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length cDNA is 2,351 bp, consisting of a 5'-untranslated region (UTR) of 83 bp, a 3'-UTR of 315 bp, and an open reading frame (ORF) of 1,953 bp. This cDNA encodes 650 amino acids with an estimated molecular weight of 71.3 kDa and an isoelectric point of 5.51. Based on the amino acid sequence analysis and phylogenetic analysis, this HSP70 gene was identified as a member of the cytoplasmic HSP70 family, being the constitutive expression, and it was designated as PuHSC70. The distribution of PuHSC70 mRNA in the mantle, digestive gland, adductor muscle, gonad, gill, heart, and hemocytes suggested that PuHSC70 is ubiquitously expressed. The mRNA levels of PuHSC70 under high temperature and high salinity stresses were analyzed by real-time PCR. Under high temperature stress of 32°C, PuHSC70 mRNA in the mantle, digestive gland, gill, and heart was significantly up-regulated at 1h and 2h, and it was then progressively down-regulated. In the adductor muscle, the level of PuHSC70 mRNA gradually increased throughout the study period; the mRNA levels in the gonad and hemocytes increased significantly at 4h and 8h (P<0.05) and then decreased at 8h and 14 h, respectively, however they increased again afterwards, reaching the highest levels at 50h. Under high salinity (32 ‰) stress, the mRNA levels of PuHSC70 in the mantle and gonad were increased significantly only at 24h and 48 h (P<0.05), and at the rest of the study period they were slightly elevated. Compared with the pretreatment level, the levels of expression in the digestive gland and gill were unchanged or reduced throughout the study period. The levels of PuHSC70 mRNA in the adductor muscle, hemocytes, and heart were significantly increased, reaching a maximum at 24h, and then they gradually decreased; moreover, in the heart, the mRNA expression recovered to the pretreatment level at 50h; while in the adductor muscle and hemocytes, the expression level remained higher than that of the control. The cloning and expression analyses of PuHSC70 provide theoretical basis to further study the mechanism of physiological response to thermal and high salinity stresses. Copyright © 2014 Elsevier B.V. All rights reserved.

Evolutionarily conserved TCR binding sites, identification of T cells in primary lymphoid tissues, and surprising trans-rearrangements in nurse shark.

PubMed

Criscitiello, Michael F; Ohta, Yuko; Saltis, Mark; McKinney, E Churchill; Flajnik, Martin F

2010-06-15

Cartilaginous fish are the oldest animals that generate RAG-based Ag receptor diversity. We have analyzed the genes and expressed transcripts of the four TCR chains for the first time in a cartilaginous fish, the nurse shark (Ginglymostoma cirratum). Northern blotting found TCR mRNA expression predominantly in lymphoid and mucosal tissues. Southern blotting suggested translocon-type loci encoding all four chains. Based on diversity of V and J segments, the expressed combinatorial diversity for gamma is similar to that of human, alpha and beta may be slightly lower, and delta diversity is the highest of any organism studied to date. Nurse shark TCRdelta have long CDR3 loops compared with the other three chains, creating binding site topologies comparable to those of mammalian TCR in basic paratope structure; additionally, nurse shark TCRdelta CDR3 are more similar to IgH CDR3 in length and heterogeneity than to other TCR chains. Most interestingly, several cDNAs were isolated that contained IgM or IgW V segments rearranged to other gene segments of TCRdelta and alpha. Finally, in situ hybridization experiments demonstrate a conservation of both alpha/beta and gamma/delta T cell localization in the thymus across 450 million years of vertebrate evolution, with gamma/delta TCR expression especially high in the subcapsular region. Collectively, these data make the first cellular identification of TCR-expressing lymphocytes in a cartilaginous fish.

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

PubMed

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

2009-10-18

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

Association between p75 neurotrophin receptor gene expression and cell apoptosis in tissues surrounding hematomas in rat models of intracerebral hemorrhage.

PubMed

He, Baixiang; Bao, Gang; Guo, Shiwen; Xu, Gaofeng; Li, Qi; Wang, Ning

2012-03-15

Animal models of intracerebral hemorrhage were established by injection of autologous blood into the caudate nucleus in rats. Cell apoptosis was measured by flow cytometry and immunohistochemical staining of the p75 neurotrophin receptor. p75 neurotrophin receptor protein was detected by immunohistochemistry. p75 neurotrophin receptor mRNA was examined by quantitative real-time polymerase chain reactions. At 24 hours after modeling, cellular apoptosis occured around hematoma with upregulation of p75 neurotrophin receptor protein and mRNA was observed, which directly correlated to apoptosis. This observation indicated that p75 neurotrophin receptor upregulation was associated with cell apoptosis around hematomas after intracerebral hemorrhage.

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.

Effects of the HIF1 inhibitor, echinomycin, on growth and NOTCH signalling in leukaemia cells.

PubMed

Yonekura, Satoru; Itoh, Mai; Okuhashi, Yuki; Takahashi, Yusuke; Ono, Aya; Nara, Nobuo; Tohda, Shuji

2013-08-01

To examine the effects of echinomycin, a compound that inhibits DNA-binding activity of hypoxia-inducible factor-1 (HIF1), on leukaemia cell growth. Three acute myeloid leukaemia cell lines and three T-lymphoblastic leukaemia cell lines were cultured with echinomycin. Cell growth, mRNA and protein expression levels were examined by WST-1 assay, reverse-transcription polymerase chain reaction and immunoblotting, respectively. HIF1α protein was expressed in all cell lines under normoxia. Treatment with echinomycin suppressed cell growth and induced apoptosis in association with decreased mRNA expression of HIF1 targets, glucose transporter-1 (GLUT1) and B-cell CLL/lymphoma-2 (BCL2). Echinomycin also suppressed the protein expression of NOTCH1, cleaved NOTCH1, v-myc myelocytomatosis viral oncogene homolog (MYC), v-akt murine thymoma viral oncogene homolog-1 (AKT), phosphorylated AKT, mechanistic target of rapamycin (mTOR), and phosphorylated mTOR and increased that of cleaved caspase-3 in some cell lines. Echinomycin suppresses leukaemia cell growth in association with reduced NOTCH1 expression. This is the first report to show that HIF inhibitor treatment suppresses NOTCH1 signalling. HIF inhibitors could be novel candidates for a molecular-targeted therapy against leukaemia.

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.

OXTR and ZEB1 expression before and after progesterone dosing in pregnant women with threatened premature labor.

PubMed

Xu, Y-J; Ren, L-D; Zhai, S-S; Ran, L-M; Hu, L-L; Luo, X-H; Hong, T; Liu, R; Yu, Y-R; Ban, Y-J

2017-07-01

To investigate changes in the peripheral blood mRNA levels of oxytocin receptor (OXTR) and Zinc finger E-box-binding homeobox 1 (ZEB1) before and after progesterone dosing in pregnant women with threatened premature labor. Blood samples were collected from 30 healthy pregnant women with 28- to 33+6-week gestational age and singleton pregnancy (group A) and from 30 pregnant women with singleton pregnancy and threatened premature labor before and 48 hours after progesterone dosing (groups B and C, respectively) for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay to assess the OXTR and ZEB1 mRNA levels. The OXTR mRNA level was higher in the group B than in the groups A and C, and the ZEB1 mRNA level was lower in the group B than in the groups A and C. Notably, no significant difference was found in the mRNA level of OXTR or ZEB1 between group A and group C. The peripheral blood mRNA level of OXTR was increased, and that of ZEB1 was decreased in pregnant women with threatened premature labor. Progesterone helped to maintain pregnancy by readjusting the mRNA levels of OXTR and ZEB1.

Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model.

PubMed

Baptista, Maria João; Recamán, Mónica; Melo-Rocha, Gustavo; Nogueira-Silva, Cristina; Roriz, José-Mário; Soares-Fernandes, João; Gonzaga, Silvia; Santos, Marta; Leite-Moreira, Adelino; Areias, José Carlos; Correia-Pinto, Jorge

2006-09-01

Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from beta to alpha isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model. Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, alpha and beta MHC isoforms, as well as beta-actin (housekeeping gene); and (ii) separation of MHC isoforms (alpha and beta isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained. We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 +/- 0.1; nitrofen, 1.1 +/- 0.2; CDH, 1.3 +/- 0.2) and SERCA2a (control, 1.0 +/- 0.1; nitrofen, 0.9 +/- 0.1; CDH, 1.0 +/- 0.2). Myocardial gene expressions of alpha and beta mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the alpha-to-beta ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 +/- 2.5; CDH, 17.0 +/- 2.0). Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.

miR-205-5p negatively regulates hepatic acetyl-CoA carboxylase β mRNA in lipid metabolism of Oreochromis niloticus.

PubMed

Tao, Yi-Fan; Qiang, Jun; Bao, Jing-Wen; Li, Hong-Xia; Yin, Guo-Jun; Xu, Pao; Chen, De-Ju

2018-06-20

MicroRNAs (miRNAs) are non-coding RNAs that function as post-transcriptional gene regulators and that play vital roles controlling lipid metabolism. miR-205 is an important miRNA related to adipogenesis and lipid metabolism. However, little is known about the potential role of miR-205-5p in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). In this study, we used miRanda software to search for potential miR-205-5p target genes and found a lipid-metabolism-related gene called acetyl-CoA carboxylase β (ACACβ). Quantitative real-time polymerase chain reaction data indicated that there may be a negative regulation relationship between miR-205-5p and ACACβ gene expression under HFD rearing. Using luciferase reporter assays, we verified the binding site of miR-205-5p in the 3'-untranslated region of the ACACβ mRNA. Furthermore, an in vivo functional analysis of miR-205-5p was performed by injecting GIFT juveniles with a miR-205-5p antagomir. Reduced levels of miR-205-5p in GIFT liver increased ACACβ mRNA expression 12 h post-injection. miR-205-5p suppression also increased fatty acid synthase and peroxisome proliferator-activated receptor-α mRNA levels 48 h and 120 h post-injection, respectively. Taken together, our results indicate that miR-205-5p negative regulates hepatic ACACβ mRNA expression, and may serve as an important regulator in controlling hepatic lipid metabolism in GIFT. Copyright © 2018. Published by Elsevier B.V.

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.

Mechanoregulation of human articular chondrocyte aggrecan and type II collagen expression by intermittent hydrostatic pressure in vitro.

PubMed

Ikenoue, Takashi; Trindade, Michael C D; Lee, Mel S; Lin, Eric Y; Schurman, David J; Goodman, Stuart B; Smith, R Lane

2003-01-01

This study addressed the hypothesis that duration and magnitude of applied intermittent hydrostatic pressure (IHP) are critical parameters in regulation of normal human articular chondrocyte aggrecan and type II collagen expression. Articular chondrocytes were isolated from knee cartilage and maintained as primary, high-density monolayer cultures. IHP was applied at magnitudes of 1, 5 and 10 MPa at 1 Hz for durations of either 4 h per day for one day (4 x 1) or 4 h per day for four days (4 x 4). Total cellular RNA was isolated and analyzed for aggrecan and type II collagen mRNA signal levels using specific primers and reverse transcription polymerase chain reaction (RT-PCR) nested with beta-actin primers as internal controls. With a 4x1 loading regimen, aggrecan mRNA signal levels increased 1.3- and 1.5-fold at 5 and 10 MPa, respectively, relative to beta-actin mRNA when compared to unloaded cultures. Changing the duration of loading to a 4x4 regimen increased aggrecan mRNA signal levels by 1.4-, 1.8- and 1.9-fold at loads of 1, 5 and 10 MPa, respectively. In contrast to the effects of IHP on aggrecan, type II collagen mRNA signal levels were only upregulated at loads of 5 and 10 MPa with the 4x4 loading regimen. Analysis of cell-associated protein by western blotting confirmed that IHP increased aggrecan and type II collagen in chondrocyte extracts. These data demonstrate that duration and magnitude of applied IHP differentially alter chondrocyte matrix protein expression. The results show that IHP provides an important stimulus for increasing cartilage matrix anabolism and may contribute to repair and regeneration of damaged or diseased cartilage.

Molecular pathology of pulmonary surfactants and cytokines in drowning compared with other asphyxiation and fatal hypothermia.

PubMed

Miyazato, Takako; Ishikawa, Takaki; Michiue, Tomomi; Maeda, Hitoshi

2012-07-01

Drowning involves complex fatal factors, including asphyxiation and electrolyte/osmotic disturbances, as well as hypothermia in cold water. The present study investigated the molecular pathology of pulmonary injury due to drowning, using lung specimens from forensic autopsy cases of drowning (n = 21), acute mechanical asphyxia due to neck compression and smothering (n = 24), and hypothermia (cold exposure, n = 11), as well as those of injury (n = 23), intoxication (n = 13), fire fatality (n = 18), and acute cardiac death (n = 9) for comparison. TaqMan real-time reverse transcription polymerase chain reaction was used to quantify messenger RNA (mRNA) expressions of pulmonary surfactant-associated proteins A and D (SP-A and SP-D), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10. SP-A and SP-D mRNA levels were lower for drowning, mechanical asphyxiation, fire fatality, and acute cardiac deaths than for hypothermia and injury. TNF-α, IL-1β, and IL-10 mRNA levels were higher for drowning or for drowning and injury than for other groups; there was no significant difference between fire fatality, involving airway injury due to inhalation of hot/irritant gases, and other control groups. These observations suggest characteristic molecular biological patterns of pulmonary injury involving suppression of pulmonary surfactants and activation of early-phase mediators of inflammation in drowning, with high mRNA expression levels of pulmonary surfactants in fatal hypothermia; however, there was no significant difference among these markers in immunohistochemical detection, except for SP-A. These mRNA expressions can be used as markers of pulmonary injury to assist in investigations of the pathophysiology of drowning and fatal hypothermia in combination with other biochemical and biological markers.

Effects of triclosan (TCS) on hormonal balance and genes of hypothalamus-pituitary- gonad axis of juvenile male Yellow River carp (Cyprinus carpio).

PubMed

Wang, Fan; Liu, Fei; Chen, Wanguang; Xu, Ruijie; Wang, Wei

2018-02-01

Triclosan (TCS) is a broad spectrum antimicrobial agent which has been widely dispersed and determinated in the aquatic environment. However, the effects of TCS on reproductive endocrine in male fish are poorly understood. In this study, male Yellow River carp (Cyprinus carpio) were exposed to 0, 1/5, 1/10 and 1/20 LC 50 (96 h LC 50 of TCS to carp) TCS under semi-static conditions for 42 d. Vitellogenin (Vtg), 17β-estradiol (E 2 ), testosterone(T), gonadotropin (GtH), and gonadotropin-releasing hormone (GnRH) levels were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also examined the mRNA expressions of aromatase, GtHs-β, GnRH, estrogen receptor (Er), and androgen receptor (Ar) by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). TCS induced Vtg levels of hepatopancreas, E 2 levels of serum, and inhibited Ar and Er mRNA levels, suggesting that the induction of Vtg production by TCS was indirectly caused by non-Er pathways. TCS-induced Vtg levels by interfering with the reproductive axis at plenty of latent loci of male carps: (a) TCS exposure increased the aromatase mRNA expression of hypothalamus and gonad aromatase, consequently increasing serum concentrations of E 2 to induce Vtg in hepatopancreas; (b) TCS treatment changed GtH-β and GnRH mRNA expression and secretion, causing the disturbance of reproductive endocrine; (c) TCS exposure decreased Ar mRNA levels, indicating potential Ar-mediated antiandrogen action. These mechanisms showed that TCS may induce Vtg production in male carp by non-Er-mediated pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation of natriuretic peptide receptor A and B expression by transforming growth factor-beta 1 in cultured aortic smooth muscle cells.

PubMed

Fujio, N; Gossard, F; Bayard, F; Tremblay, J

1994-06-01

Two types of natriuretic peptide receptors (NPR-A and NPR-B) are membrane guanylate cyclases whose relative expression varies in different tissues. Because natriuretic peptides have been shown to inhibit aortic smooth muscle proliferation, we investigated the regulation of NPR-A and NPR-B in these cells under different proliferative conditions. NPR subtype mRNA levels were measured by our newly developed quantitative reverse transcription-polymerase chain reaction assay using mutated NPR-A and NPR-B cRNA as internal standards. The functional impact of their expression was determined by atrial natriuretic peptide (ANP)- and C-type natriuretic peptide (CNP)-induced stimulation of cyclic GMP production. In the intact aorta, NPR-B mRNA levels were found to be 10-fold higher than those of NPR-A. This dominance was further amplified (1000-fold) in long-term cultures (10 to 15 passages) of aortic smooth muscle cells (ASMC). Higher cyclic GMP production with CNP than with ANP was observed in cultured ASMC from Wistar-Kyoto (WKY) rats. Similar stimulation by the two agonists was noted in spontaneously hypertensive rat (SHR) cells, paralleled by a 10-fold increase in NPR-A mRNA levels and ANP stimulation of cyclic GMP in hypertensive cells. The present study also evaluated NPR-A and NPR-B mRNA control by transforming growth factor-beta 1 (TGF-beta 1), an important regulator of cell proliferation that is overexpressed in SHR ASMC. TGF-beta 1 decreased both NPR-A and NPR-B mRNA levels with a predominant effect in SHR cells at high cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

PubMed

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast cell-mediated allergic reactions.

Fatty Acid Oxidation Changes and the Correlation with Oxidative Stress in Different Preeclampsia-Like Mouse Models

PubMed Central

Ding, Xiaoyan; Yang, Zi; Han, Yiwei; Yu, Huan

2014-01-01

Background Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) expression is decreased in placenta of some cases of preeclampsia (PE) which may result in free fatty acid (FFA) increased. High FFA level will induce oxidative stress, so abnormal long-chain fatty acid-oxidation may participate in the pathogenesis of PE through oxidative stress pathway. Methods PE-like groups were ApoC3 transgenic mice with abnormal fatty acid metabolism, classical PE-like models with injection of Nw-nitro-L-arginine-methyl ester (L-NA) or lipopolysaccharide (LPS) and the antiphospholipid syndrome (APS) mouse model with β2GPI injection (ApoC3+NS, ApoC3+L-NA, L-NA, LPS and β2GPI groups). The control group was wild-type mice with normal saline injection. Except for β2GPI mice, the other mice were subdivided into pre-implantation (Pre) and mid-pregnancy (Mid) subgroups by injection time. Results All PE-like groups showed hypertension and proteinuria except ApoC3+NS mice only showed hypertension. Serum FFA levels increased significantly except in LPS group compared to controls (P<0.05). LCHAD mRNA and protein expression in the liver and placenta was significantly higher for ApoC3+NS, ApoC3+L-NA and β2GPI mice and lower for L-NA mice than controls (P<0.05) but did not differ between LPS mice and controls. P47phox mRNA and protein expression in the liver significantly increased in all PE-like groups except LPS group, while P47phox expression in the placenta only significantly increased in L-NA and β2GPI groups. Conclusions Abnormal long-chain fatty acid-oxidation may play a different role in different PE-like models and in some cases participate in the pathogenesis of PE through oxidative stress pathway. PMID:25302499

Expression of platelet-derived growth factor BB, erythropoietin and erythropoietin receptor in canine and feline osteosarcoma.

PubMed

Meyer, F R L; Steinborn, R; Grausgruber, H; Wolfesberger, B; Walter, I

2015-10-01

The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Expression of platelet-derived growth factor BB, erythropoietin and erythropoietin receptor in canine and feline osteosarcoma

PubMed Central

Meyer, F.R.L.; Steinborn, R.; Grausgruber, H.; Wolfesberger, B.; Walter, I.

2015-01-01

The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation. PMID:26189892

Characterize and Gene Expression of Heat Shock Protein 90 in Marine Crab Charybdis japonica following Bisphenol A and 4-Nonylphenol Exposures

PubMed Central

Park, Kiyun

2014-01-01

Objectives Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of protein. In this study, an HSP90 gene was isolated from Asian paddle crab, Charybdis japonica, as a bio-indicator to monitor the marine ecosystem. Methods This work reports the responses of C. japonica HSP90 mRNA expression to cellular stress by endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 4-nonylphenol (NP) using real-time. reverse transcription polymerase chain reaction. Results The deduced amino acid sequence of HSP90 from C. japonica shared a high degree of homology with their homologues in other species. In a phylogenetic analysis, C. japonica HSP90 is evolutionally related with an ortholog of the other crustacean species. The expression of HSP90 gene was almost distributed in all the examined tissues of the C. japonica crab but expression levels varied among the different body parts of the crabs. We examined HSP90 mRNA expression pattern in C. japonica crabs exposed to EDCs for various exposure times. The expression of HSP90 transcripts was significantly increased in C. japonica crabs exposed to BPA and NP at different concentrations for 12, 24, 48 and 96 hours. The mRNA expression of HSP90 gene was significantly induced in a concentration- and time-dependent manner after BPA or NP exposures for 96 hours. Conclusions Taken together, expression analysis of Asian paddle crab HSP90 gene provided useful molecular information about crab responses in stress conditions and potential ways to monitor the EDCs stressors in marine environments. PMID:24955332

Characterize and Gene Expression of Heat Shock Protein 90 in Marine Crab Charybdis japonica following Bisphenol A and 4-Nonylphenol Exposures.

PubMed

Park, Kiyun; Kwak, Ihn-Sil

2014-01-01

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of protein. In this study, an HSP90 gene was isolated from Asian paddle crab, Charybdis japonica, as a bio-indicator to monitor the marine ecosystem. This work reports the responses of C. japonica HSP90 mRNA expression to cellular stress by endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 4-nonylphenol (NP) using real-time. reverse transcription polymerase chain reaction. The deduced amino acid sequence of HSP90 from C. japonica shared a high degree of homology with their homologues in other species. In a phylogenetic analysis, C. japonica HSP90 is evolutionally related with an ortholog of the other crustacean species. The expression of HSP90 gene was almost distributed in all the examined tissues of the C. japonica crab but expression levels varied among the different body parts of the crabs. We examined HSP90 mRNA expression pattern in C. japonica crabs exposed to EDCs for various exposure times. The expression of HSP90 transcripts was significantly increased in C. japonica crabs exposed to BPA and NP at different concentrations for 12, 24, 48 and 96 hours. The mRNA expression of HSP90 gene was significantly induced in a concentration- and time-dependent manner after BPA or NP exposures for 96 hours. Taken together, expression analysis of Asian paddle crab HSP90 gene provided useful molecular information about crab responses in stress conditions and potential ways to monitor the EDCs stressors in marine environments.

The Geonomic Organization of the CD28 Gene. Implications for the Regulation of CD28 mRNA Expression and Heterogeneity

DTIC Science & Technology

1990-07-01

doanrmsialecgtonptcelsvstsrtinrnsoa Partial primary structure of the alpha and beta chains of human tdomn ctmvity nat Nrento 320 ticl levsis.ti tasoa...L. Moretta. and C. MW. Croce. tlon and RNA splicing defects in five cloned j6- thalassaemia genes. 1987. Tp44 molecules Involved In antigen-independent T cell acti- Na t ure 302:59 1.

Transcription Analysis of the Beta-Glucosidase Precursor in Wild-Type and l-4i Mutant Bombyx mori (Lepidoptera: Bombycidae).

PubMed

Kang, Lequn; Huang, Fei; Wu, Fan; Zhao, Qiaoling

2015-01-01

Lethal fourth-instar larvae (l-4i) mutant of Bombyx mori, a recently discovered novel mutant, die from energy depletion due to genetic mutation. Beta-glucosidase is a common digestive enzyme that hydrolyzes cellulose in the diet to provide energy. In this study, the mRNA expression profiles of B. mori beta-glucosidase precursor (BmpreBG) were characterized by reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The transcription level of BmpreBG varied in different tissues and developmental stages, except in the pupa and moth, which are the no-diet period. Remarkably, the mRNA expression level of BmpreBG was sharply reduced in l-4i but not in the wild type, which suggested that the digestive function of the mutant was severely damaged. This was consistent with the l-4i phenotypic traits of not eating mulberries, lack of energy, and ultimate death. 5'-rapid amplification of cDNA ends showed, for the first time, that BmpreBG has a 160-bp 5'-untranslated region. These findings suggested that B. mori β-glucosidase precursor was involved in the death process of l-4i mutant larvae. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Long-chain acyl-CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle.

PubMed

Teodoro, Bruno G; Sampaio, Igor H; Bomfim, Lucas H M; Queiroz, André L; Silveira, Leonardo R; Souza, Anderson O; Fernandes, Anna M A P; Eberlin, Marcos N; Huang, Tai-Yu; Zheng, Donghai; Neufer, P Darrell; Cortright, Ronald N; Alberici, Luciane C

2017-02-01

Long-chain acyl-CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1-α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism. Long-chain acyl-CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl-CoA species toward different metabolic fates such as lipid synthesis or β-oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up-regulated by acute high-fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p-AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC-1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC-1α mRNA. In conclusion, ACSL6 drives acyl-CoA toward lipid synthesis and its downregulation improves mitochondrial biogenesis, respiratory capacity and lipid oxidation. These outcomes are associated with the activation of the AMPK/PGC1-α pathway. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

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

Underexpression of mineralocorticoid receptor in colorectal carcinomas and association with VEGFR-2 overexpression.

PubMed

Di Fabio, Francesco; Alvarado, Carlos; Majdan, Agnieszka; Gologan, Adrian; Voda, Linda; Mitmaker, Elliot; Beitel, Lenore K; Gordon, Philip H; Trifiro, Mark

2007-11-01

The human mineralocorticoid receptor (MR) is a steroid receptor widely expressed in colorectal mucosa. A significant role for the MR in the reduction of vascular endothelial growth factor receptor-2 (VEGFR-2) mRNA levels has been demonstrated in vitro. To evaluate a potential contribution of MR to colorectal carcinoma progression, we analyzed the expression of MR in relation to VEGFR-2. Fresh human colorectal cancer tissue and adjacent normal mucosa were harvested from 48 consecutive patients. MR and VEGFR-2 mRNA expression levels were determined by real-time reverse transcriptase-polymerase chain reaction and correlated with clinicopathological parameters. A decline of MR expression was observed in all carcinomas compared to normal mucosa. Expression of MR was a median of 11-fold lower in carcinoma compared to the normal mucosa, irrespective of the location, size, stage, and differentiation. MR was a median of 20-fold underexpressed in carcinomas with VEGFR-2 overexpression vs only 9-fold in carcinomas with VEGFR-2 underexpression (p = 0.035, Mann-Whitney test). These findings support the hypothesis that reduction of MR expression may be one of the early events involved in colorectal carcinoma progression. The inverse association between MR and VEGFR-2 expression in carcinoma suggests a potential tumor-suppressive function for MR.

Changes in myometrial expression of progesterone receptor membrane components 1 and 2 are associated with human parturition at term.

PubMed

Wang, Ray; Sheehan, Penelope M; Brennecke, Shaun P

2016-04-01

While the exact mechanism of human parturition remains unknown, functional progesterone withdrawal is believed to play a key regulatory role. Progesterone receptor membrane components 1 and 2 (PGRMC1, PGRMC2) are putative progesterone receptors and the aim of this project was to investigate their expression in human myometrium. Human term myometrium was obtained from the lower uterine segment incision in women undergoing elective (not-in-labour, NIL; n=11) and emergency Caesarean sections (in-labour, IL; n=10), following written consent. PGRMC1 and 2 expression was quantified using real-time reverse transcription polymerase chain reaction and western blot. Subcellular localisation was performed by immunohistochemistry and immunofluorescence. There was a significant decrease in PGRMC1 mRNA (P=0.0317) and protein expression (P=0.0151) in IL myometrium, compared with NIL myometrium. PGRMC2 mRNA expression (P=0.0151) was also decreased in IL myometrium, compared with NIL myometrium. Immunostaining studies confirmed the presence of PGRMC1 and 2 in smooth-muscle cells. Expression was perinuclear in NIL myometrium and more generalised and cytoplasmic in IL myometrium. The decrease in PGRMC1 expression and the translocation away from a perinuclear location for both PGRMC1 and 2 could contribute to a functional progesterone withdrawal that may ultimately initiate parturition.

Mechanical tensile stress effects on the expression of bone sialoprotein in bovine cementoblasts.

PubMed

Yu, Hongyou; Ren, Yijin; Sandham, Andrew; Ren, Aishu; Huang, Lan; Bai, Ding

2009-03-01

To develop a new cementoblast culture method and to detect bone sialoprotein (BSP) expression in response to high and low mechanical tensile stress in cementoblast in vitro. Cementoblasts were collected from the roots of newborn bovine teeth and were identified with cementum-derived attachment protein (CAP) antibody 3G9. Cell proliferation was evaluated by MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, and mineralization was confirmed by von Kossa staining. Mechanical tensile stress was applied in vitro to the cementoblast with the use of a uniaxial four-point bending system with 2000 or 4000 microstrains, at a frequency of 0.5 Hz for 3, 6, 12, 24, or 36 hours. BSP mRNA level was quantified by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). A large amount of cementoblast was observed to be expressing CAP. Cementoblasts had a proliferation tendency similar to that of osteoblasts but different from that of periodontal ligament (PDL) cells. Cementoblasts had the ability to become mineralized between osteoblasts and PDL cells. The mechanical tensile stress significantly up-regulated BSP mRNA expression, which reached a peak at 24 hours in both 2000 and 4000 microstrain groups (P < .01) and was tenfold and sixfold higher than that of controls, respectively. BSP expression dropped toward baseline levels at 36 hours in both groups. Mechanical tensile stress up-regulated the expression of BSP. Low mechanical tensile stress induced earlier and more intensive up-regulation of BSP mRNA; this might represent the optimal stimuli for cementoblast activity.

Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis.

PubMed

Carrarelli, Patrizia; Yen, Chih-Fen; Arcuri, Felice; Funghi, Lucia; Tosti, Claudia; Wang, Tzu-Hao; Huang, Joseph S; Petraglia, Felice

2015-09-01

To evaluate the expression pattern of activins and related growth factor messenger RNA (mRNA) levels in adenomyotic nodules and in their endometrium. Prospective study. University hospital. Symptomatic premenopausal women scheduled to undergo hysterectomy for adenomyosis. Samples from adenomyotic nodules and homologous endometria were collected. Endometrial tissue was also obtained from a control group. Quantitative real-time polymerase chain reaction (PCR) analysis and immunohistochemical localization of activin-related growth factors (activin A, activin B, and myostatin), binding protein (follistatin), antagonists (inhibin-α, cripto), and receptors (ActRIIa, ActRIIb) were performed. Myostatin mRNA levels in adenomyotic nodule were higher than in eutopic endometrium and myostatin, activin A, and follistatin concentrations were higher than in control endometrium. No difference was observed for inhibin-α, activin B, and cripto mRNA levels. Increased mRNA levels of ActRIIa and ActRIIb were observed in adenomyotic nodules compared with eutopic endometrium and control endometrium. Immunofluorescent staining for myostatin and follistatin confirmed higher protein expression in both glands and stroma of patients with adenomyosis than in controls. The present study showed for the first time that adenomyotic tissues express high levels of myostatin, follistatin, and activin A (growth factors involved in proliferation, apoptosis, and angiogenesis). Increased expression of their receptors supports the hypothesis of a possible local effect of these growth factors in adenomyosis. The augmented expression of ActRIIa, ActRIIb, and follistatin in the endometrium of these patients may play a role in adenomyosis-related infertility. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

The distribution of the orphan bombesin receptor subtype-3 in the rat CNS.

PubMed

Jennings, C A; Harrison, D C; Maycox, P R; Crook, B; Smart, D; Hervieu, G J

2003-01-01

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor that shares between 47 and 51% homology with other known bombesin receptors. The natural ligand for BRS-3 is currently unknown and little is known about the mechanisms regulating BRS-3 gene expression. Unlike other mammalian bombesin receptors that have been shown to be predominantly expressed in the CNS and gastrointestinal tract, expression of the BRS-3 receptor in the rat brain has previously not been observed. To gain further understanding of the biology of BRS-3, we have studied the distribution of BRS-3 mRNA and protein in the rat CNS. The mRNA expression pattern was studied using reverse transcription followed by quantitative polymerase chain reaction. Using immunohistological techniques, the distribution of BRS-3 protein in the rat brain was investigated using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The BRS-3 receptor was found to be widely expressed in the rat brain at both mRNA and protein levels. Particularly strong immunosignals were observed in the cerebral cortex, hippocampal formation, hypothalamus and thalamus. Other regions of the brain such as the basal ganglia, midbrain and reticular formation were also immunopositive for BRS-3. In conclusion, our neuroanatomical data provide evidence that BRS-3 is as widely expressed in the rat brain as other bombesin-like peptide receptors and suggest that this receptor may also have important roles in the CNS, mediating the functions of a so far unidentified ligand.

Branched-chain amino acids supplementation protects streptozotocin-induced insulin secretion and the correlated mechanism.

PubMed

Lu, Ming; Zhang, Xiujuan; Zheng, Dongmei; Jiang, Xiuyun; Chen, Qing

2015-01-01

Significant evidence demonstrates that oxidative stress can impair insulin secretion and contribute to the development of type 2 diabetes. Branched-chain amino acids (BCAAs) are reported to be positively related to insulin secretion. This study aimed to determine how oxidative stress affects the function of islets and whether BCAAs can ameliorate the oxidative stress, and accompanying c-jun N-terminal kinase (JNK), protein kinase D1 (PKD1), and pancreatic/duodenal homeobox-1 (PDX-1) changes induced by streptozotocin (STZ). Plasma glucose, plasma insulin, and JNK, PKD1 and PDX-1 mRNA and protein expression were measured in rats treated with STZ and BCAAs. The glucose level in STZ-induced diabetic rats was much higher than that in control animals, and the elevated plasma glucose level in diabetic rats could be significantly inhibited by BCAAs treatment. Consistent with the change in glucose levels, the levels of insulin were also affected by BCAAs treatment. The mRNA and protein expression of JNK, PDX-1, and PKD1 were significantly altered in diabetic rats compared with the control group (P<0.01) and treatment with a low dose of BCAA reversed these changes in those above markers significantly (P<0.01). The present study demonstrated that STZ-induced oxidative stress could reduce serum insulin levels and alter the JNK, PDX-1, and PKD1 expression. BCAAs restored the levels of serum insulin reversed changes in JNK, PDX-1, and PKD1 expression. © 2014 International Union of Biochemistry and Molecular Biology.

DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

PubMed

Campos, Kelma; Gomes, Carolina Cavalieri; Farias, Lucyana Conceição; Silva, Renato Menezes; Letra, Ariadne; Gomez, Ricardo Santiago

2016-01-01

Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2016-11-01

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

The role of fructose‑1,6‑bisphosphatase 1 in abnormal development of ovarian follicles caused by high testosterone concentration.

PubMed

Liu, Tao; Zhao, Han; Wang, Jianfeng; Shu, Xin; Gao, Yuan; Mu, Xiaoli; Gao, Fei; Liu, Hongbin

2017-11-01

The present study aimed to identify the molecular mechanisms underlying the effects of the fructose‑1,6‑bisphosphatase 1 (FBP1) signaling pathway within normal follicle development and in hyperandrogenism‑induced abnormal follicle growth. To achieve this, murine primary follicles, granulosa cells (GCs) and theca‑interstitial cells (TICs) were isolated, cultured in vitro and treated with a high concentration of androgens. A concentration of 1x10‑5 mol/l testosterone was considerable to induce hyperandrogenism by MTT assay. All cells were divided into four groups, as follows: Control group, testosterone group, androgen receptor antagonist‑flutamide group and flutamide + testosterone group. Flutamide was used in the present study as it blocks the effects of the androgen receptor. The mRNA expression levels of FBP1 were detected using reverse transcription‑quantitative polymerase chain reaction. The expression levels and localization of FBP1 were analyzed by western blot analysis and immunofluorescence staining. The experimental results demonstrated that androgen presence stimulated follicle development, whereas excessive testosterone inhibited development. FBP1 was identified as being mainly expressed in follicles; FBP1 protein was significantly expressed in GCs of the 14‑day‑cultured follicle, as well as in the cytoplasm and nuclei of GCs and TICs in vitro. Testosterone increased FBP1 expression during a specific range of testosterone concentrations. Testosterone increased the expression of FBP1 within GCs. Furthermore, FBP1 and phosphoenolpyruvate carboxykinase 1 (PCK1) mRNA expression was increased in GCs treated with testosterone, whereas forkhead box protein O1 (FOXO1) and peroxisome proliferator‑activated receptor γ coactivator‑1α mRNA expression was significantly decreased in the testosterone group. In TICs, testosterone and flutamide inhibited the mRNA expression levels of FOXO1 and glucose‑6‑phosphatase enzyme, and promoted the expression of PCK1. These results suggested that the FBP1 signaling pathway may serve an important role in normal follicle growth and hyperandrogenism‑induced abnormal development, which may be associated with abnormal glucose metabolism induced by high concentrations of testosterone.

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

[Effect of hydroquinone on the histone deacetylase in human bone marrow mononuclear cells].

PubMed

Hong, L L; Yu, K; Yan, Q X; Xu, X; Shi, Y F; Ge, H P

2016-03-20

To observe the activity of histone deacetylase and the mRNA expression level of HDAC1 and HDAC2 in human bone marrow mononuclear cells, which induced by hydroquinone and exposed to hydroquinone plus Trichostatin as a histone deacetylase inhibitor for 10 hours respectively. Collect the bone marrow mononuclear cells suspension,divided into control group,HQ group (3 h, 6 h, 12 h, 24 h) , HQ+TSA 10 h group and HQ 10 h group. Extract the nuclear proteins and RNA, test the activity of histone deacetylase with the colorimetric HDAC assay kit and detect the mRNA expression level of HDAC1 and HDAC2 by real-time Polymerase Chain Reaction (PCR). The HDAC activity of HQ3 h group, HQ6 h group and HQ12 h group were 1.31 times, 1.53 times and 1.148 times than that of control group respectively. And the difference was statistically significant (P<0.05). Except the HQ24 h group (P>0.05) , the HDAC1 mRNA expression of HQ3 h group, HQ6 h group and HQ12 h group were 1.173 times, 1.901 times and 2.348 times than that of control group respectively. And the difference was statistically significant (P<0.05). The HDAC2 mRNA expression of HQ6 h group and HQ12 h group were 1.426 times and 1.766 times than that of the control group respectively. And the difference was statistically significant (P<0.05). No significant difference was observed between HQ3 h group, HQ24 h group and control group (P>0.05). The cells were treated by hydroquinone plus TSA for 10 hours. The HDAC activity of HQ+TSA 10h group was reduced by 25.6% than that of HQ 10 h group (P<0.05) and rised 13.0% compared to the control group (P<0.05). And the difference was statistically significant between groups (P<0.05) .The cells were treated by hydroquinone plus TSA for 10 hours. The HDAC1 mRNA expression of the HQ+TSA 10h group is reduced by 26.9% than that of HQ10h group. The HDAC2 mRNA expression is reduced by 19.3% compared to the HQ 10h group.And the difference was statistically significant between groups (P<0.05). The HDAC1 and HDAC2 mRNA expression is obviously higher than the control group, the difference was statistically significant (P<0.05). Treatment of hydroquinone, the histone deacetylase activity and the mRNA expression of HDAC1 and HDAC2 were increased in a certain time range. The histone deacetylase inhibitor (TSA) can reduce the histone deacetylase activity and the mRNA expression level of HDAC1 and HDAC2 in the bone marrow mononuclear cell induce by hydroquinone.It can be confirmed that hematopoietic damage induced by the benzene metabolites is related to the histone acetylation modification level.

Muscarinic acetylcholine receptor expression in aganglionic bowel.

PubMed

Oue, T; Yoneda, A; Shima, H; Puri, P

2000-01-01

In Hirschsprung's disease (HD) there exists an overabundance of acetylcholine (ACh), which in turn stimulates excessive production of the enzyme acetylcholinesterase. Muscarinic ACh receptors (mAChRs) play an important role in smooth-muscle contraction. Recent studies have indicated five different subtypes of mAChRs encoded by five different genes, ml to m5. The purpose of this study was to investigate the expression of each mAChR subtype in aganglionic (AG) colon to further understand the pathophysiology of HD. Entire colon resected at the time of pull-through operation for HD was obtained from 14 patients. Specimens obtained at autopsy from 8 age-matched patients without gastrointestinal disease acted as controls. Frozen sections were used for indirect immunohistochemistry as well as in-situ hybridization. Immunohistochemistry was performed using specific antiserum against each mAChR subtype and in-situ hybridization was performed using specific oligonucleotide probes against ml to m5 subtypes. Messenger RNA (mRNA) was extracted from normoganglionic (NG) and AG bowel of HD patients and normal control bowel. Reverse transcription-polymerase chain reaction was performed to evaluate mRNA levels of each mAChR subtype. To adjust the levels of mRNA expression, a housekeeping gene G3PDH, known to be expressed normally, was used as an internal control. Strong m2 and m3 immunoreactivity was observed in the mucosal layer, smooth-muscle layers, and myenteric plexus of NG bowel, whereas ml immunoreactivity was only detected in the mucosal layer. The most striking finding was the abundance of m3-immunoreactive fibers in muscle layers of NG bowel while there was a total lack of m3 fibers in smooth-muscle of AG bowel. Intense mRNA signals encoding m2 and m3 and to a lesser degree ml were detected in NG bowel, and these signals were weak in AG bowel. Immunoreactivity and mRNA expression of m4 and m5 was not detected in NG or AG bowel. The lack of m3-immunoreactive fibers in the smooth-muscle layers of AG bowel and decreased m2 and m3 mRNA expression in AG bowel may be responsible for the motility dysfunction in the aganglionic segment.

Exercise-stimulated FGF23 promotes exercise performance via controlling the excess reactive oxygen species production and enhancing mitochondrial function in skeletal muscle.

PubMed

Li, Dong-Jie; Fu, Hui; Zhao, Ting; Ni, Min; Shen, Fu-Ming

2016-05-01

Physical exercise induces many adaptive changes in skeletal muscle and the whole body and improves metabolic characteristics. Fibroblast growth-factor 23 (FGF23) is a unique member of the FGF family that acts as a hormone regulating phosphate metabolism, calcitriol concentration, and kidney functions. The role of FGF23 in exercise and skeletal muscle is largely unknown yet. C57BL/6J mice were exercised on a motor treadmill. Mice serum FGF23 levels; FGF23 mRNA expression in various organs including the liver, heart, skeletal muscle tissue, and thyroid; and FGF23 receptor Klotho mRNA expression were examined using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and immunoblotting, respectively, after a single bout of acute exercise (60min), exhaustive exercise, and chronic prolonged exercise (60min every day for one week). C57BL/6J mice were injected with recombinant FGF23 (100mg/kg, twice per day, i.p.) or vehicle control (saline) for 3days, and then the exercise performance, reactive oxygen species (ROS), H2O2 production, and mitochondrial functional biomarkers in muscle (gene expression of sirtuin 1, PPAR-δ, PGC-1α and mitochondrial transcription factor A [TFAM], and citrate synthase activity) were assayed. Three forms of exercise, acute exercise, exhaustive exercise, and chronic exercise, increased serum FGF23 levels. However, only chronic exercise upregulated FGF23 mRNA and protein expression in skeletal muscle. FGF23 mRNA expression in the heart, liver, and thyroid was not affected. FGF23 protein was mainly located in the cytoplasm in skeletal muscle tissue and the localization of FGF23 was not altered by exercise. Exogenous FGF23 treatment significantly extended the time to exhaustion and reduced the exercise-induced ROS and H2O2 production. FGF23 treatment increased the mRNA level of PPAR-δ and citrate synthase activity, but did not influence the mRNA expression of sirtuin 1, PGC-1α, and TFAM in skeletal muscle. These results demonstrate that exercise-stimulated FGF23 promotes exercise performance via controlling the excess ROS production and enhancing mitochondrial function in skeletal muscle, which reveals an entirely novel role of FGF23 in skeletal muscle. Copyright © 2016 Elsevier Inc. All rights reserved.

Cloning and functional expression of novel cholesterol transporters ABCG1 and ABCG4 in gonadotropin-releasing hormone neurons of the tilapia.

PubMed

Phang, Y L; Soga, T; Kitahashi, T; Parhar, I S

2012-02-17

In addition to reproduction, gonadotropin-releasing hormone (GnRH) has been postulated to control cholesterol metabolism via cholesterol transport, which is carried out partly by the members of ATP-binding cassette (ABC) transporters G1 (ABCG1) and G4 (ABCG4). However, there is yet to be evidence demonstrating the relationship between these transporters with reference to GnRH neurons. In the present study, we cloned two ABCG1 messenger RNA (mRNA) variants and one ABCG4 mRNA and examined their expression in the brain including GnRH neurons (GnRH1, GnRH2, and GnRH3) in the cichlid tilapia (Oreochromis niloticus). Comparison of nucleotide sequences of the tilapia ABCG1 and ABCG4 with that of other fish species showed that both of these genes are evolutionarily conserved among fishes. ABCG1 and ABCG4 were shown to have high mRNA expressions in the CNS, pituitary, and gonads. In the brain, real-time polymerase chain reaction (PCR) showed that ABCG4 mRNA was higher than ABCG1a in all brain regions including the olfactory bulb (ABCG1=13.34, ABCG4=6796.35; P<0.001), dorsal telencephalon (ABCG1=8.64, ABCG4=10149.13; P=0.001), optic tectum (ABCG1=22.12, ABCG4=13931.04; P<0.01), cerebellum (ABCG1=8.68, ABCG4=12382.90; P<0.01), and preoptic area-midbrain-hypothalamus (ABCG1=21.36, ABCG4=13255.41; P=0.001). Similarly, although ABCG1 mRNA level is much higher in the pituitary compared with the brain, it was still significantly lower compared with ABCG4 (ABCG1=337.73, ABCG4=1157.87; P=0.01). The differential pattern of expression of ABCG1 and ABCG4 in the brain versus pituitary suggests that the two transporters are regulated by different mechanisms. Furthermore, ABCG1 and ABCG4 mRNA expressions were found in all three types of laser-captured GnRH neurons with highly similar percentage of expressions, suggesting that cholesterol efflux from GnRH neurons may require heterodimerization of both ABCG1 and ABCG4. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Uromodulin mRNA from Urinary Extracellular Vesicles Correlate to Kidney Function Decline in Type 2 Diabetes Mellitus.

PubMed

Yamamoto, Cindy M; Murakami, Taku; Oakes, Melanie L; Mitsuhashi, Masato; Kelly, Colleen; Henry, Robert R; Sharma, Kumar

2018-05-18

Extracellular vesicles (EVs) enclose mRNA derived from their cell of origin and are considered a source of potential biomarkers. We examined urinary EV mRNA from individuals with diabetic kidney disease (DKD), chronic kidney disease, type 2 diabetes (T2DM), and obese and healthy controls to determine if such biomarkers had the potential to classify kidney disease and predict patients at higher risk of renal function decline. A total of 242 participants enrolled in this study. Urinary EV mRNA from all subjects were isolated by a filter-based platform, and the expression of 8 target genes were determined by quantitative polymerase chain reaction (qPCR). Changes in estimated glomerular filtration rate (eGFR) in 161 T2DM patients were evaluated for 2 consecutive years and compared with EV RNA profiles at baseline. We observe that mild and severe DKD groups show a significant 3.2- and -4.4-fold increase in UMOD compared to healthy controls and expression increases linearly from healthy, diabetic, and DKD subjects. UMOD expression is significantly correlated to albumin creatinine ratio (ACR), eGFR, and HbA1c. Using linear discriminant analyses with mRNA from severe DKD and T2DM as training data, a multi-gene signature classified DKD and -non-DKD with a sensitivity of 93% and specificity of 73% with area under the receiver operating characteristic (ROC) curve (AUC) = 0.90. Although 6% of T2DM were determined to have a > 80% posterior probability of developing DKD based on this mRNA profile, eGFR changes observed within the 2-year follow-up did not reveal a decline in kidney function. Urinary EV UMOD mRNA levels are progressively elevated from T2DM to DKD groups and correlate with widely used eGFR and ACR diagnostic criteria. An EV mRNA signature could identify DKD with greater than 90% sensitivity and 70% specificity. © 2018 S. Karger AG, Basel.

S100A8/A9 mRNA induction in an ex vivo model of endotoxin tolerance: roles of IL-10 and IFNγ.

PubMed

Fontaine, Mathieu; Planel, Séverine; Peronnet, Estelle; Turrel-Davin, Fanny; Piriou, Vincent; Pachot, Alexandre; Monneret, Guillaume; Lepape, Alain; Venet, Fabienne

2014-01-01

Septic syndromes are the leading cause of death in intensive care units. They are characterized by the development of immune dysfunctions such as endotoxin tolerance (ET), whose intensity and duration are associated with increased risk of nosocomial infections and mortality. Alarmins S100A8 and S100A9 have been shown to be increased after septic shock. Importantly, a delayed S100A9 mRNA increase predicts hospital-acquired infection in patients. The aim of this study was to investigate the regulation of S100A8 and S100A9 mRNA expression in an ex vivo model of ET. ET was reproduced ex vivo by priming healthy peripheral blood mononuclear cells (number of donors  = 9 to 10) with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. ET was established by observing decreased TNFα and increased IL-10 transcriptomic responses to two subsequent endotoxin challenges. Interestingly, ET was associated with increased S100A8 and S100A9 mRNA expression ex vivo. We showed that IL-10 played a role in this process, since S100A8 and S100A9 mRNA increases were significantly abrogated by IL-10 blockade in the model. Conversely, treatment with rIFN-γ, a pro-inflammatory and immunostimulating molecule known to block ET induction, was able to restore normal S100A8 and S100A9 mRNA in this model. In this ex vivo model, we observed that S100A8 and S100A9 mRNA expression was significantly increased during ET. This reproduced ex vivo the observations we had previously made in septic shock patients. Interestingly, IL-10 blockade and rIFN-γ treatment partially abrogated S100A8/A9 mRNA increases in this model. Pending confirmation in larger, independent clinical studies, these preliminary results suggest that S100A8 and S100A9 mRNA levels might be used as surrogate markers of ET and as stratification tools for personalized immunotherapy in septic shock patients.

Imbalance in leptin-adiponectin levels and leptin receptor expression as chief contributors to triple negative breast cancer progression in Northeast India.

PubMed

Sultana, Rizwana; Kataki, Amal Ch; Borthakur, Bibhuti Bhusan; Basumatary, Tarun K; Bose, Sujoy

2017-07-20

Triple-Negative breast cancer (TNBC), accounts for a large percentage of breast cancer cases in India including Northeast India. TNBC has an unclear molecular aetiology and hence limited targeted therapies. Human breast is comprised of glandular, ductal, connective, and adipose tissues. Adipose tissue is composed of adipocytes. The adipocytes apart from being energy storage depots, are also active sources of adipocytokines and/or adipokines. The role of adipokines in breast cancer including TNBC has been sporadically documented. Two adipokines in particular, leptin and adiponectin, have come to be recognized for their influence on breast cancer risk and tumour biology. Therefore, the aim of this study was to understand the association of differential expression of critical adipokines and associated cellular mechanism in the susceptibility and severity of TNBC in northeast Indian population. We collected 68 TNBC and 63 controls cases and examined for serum leptin and adiponectin levels using enzyme linked immunosorbent assay (ELISA). Leptin Receptor (Ob-R) mRNA expression was determined by real-time polymerase chain reaction (RT-PCR) assay. Differential Ob-R mRNA expression and correlation with cancer stem cell (CSC) markers was evaluated, and correlated with severity. The serum leptin levels were significantly associated with TNBC severity, while the adiponectin levels were comparative. The serum leptin levels correlated inversely with the adiponetin levels. Serum leptin levels were unaffected with difference in parity. The difference in leptin levels in pre and post menopausal cases were found to be statistically non-significant. Higher leptin levels were also found to be associated obesity, mortality and recurrence. Obesity was found to be a factor for TNBC pathogenesis and severity. Increased Ob-R mRNA expression was associated with TNBC, significantly with TNBC severity, and was significantly higher in obese patients with higher grade TNBC cases. The Ob-R gene mRNA expression was significantly higher in the obese TNBC cases showing recurrence or mortality. The higher Ob-R gene mRNA expression correlated significantly with higher serum leptin levels and lower serum adiponectin levels in TNBC cases. The Ob-R mRNA expression with associated with modulation of CSC oct4 and nanog. In conclusion, the present study is first of its kind on TNBC from northeast India, indicates that adipocytokines does play a role in TNBC pathogenesis. Thus, the understanding of molecular mechanisms of both leptin and adiponectin and their interplay in TNBC offer the prospects for new therapeutic approaches targeting similar signalling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

SiRNA-Mediated Down-Regulation of CLIC4 Gene Inhibits Cell Proliferation and Accelerates Cell Apoptosis of Mouse Liver Cancer Hca-F and Hca-P Cells.

PubMed

Yu, Qiu-Yun; Zhou, Xin-Feng; Xia, Qing; Shen, Jia; Yan, Jia; Zhu, Jiu-Ting; Li, Xiang; Shu, Ming

2018-01-01

This study explored the effects involved in silencing CLIC4 on apoptosis and proliferation of mouse liver cancer Hca-F and Hca-P cells. A CLIC4-target small interfering RNA (siRNA) was designed to compound into two individual complementary oligonucleotide chains. A process of annealing and connection to a pSilencer vector was followed by transfection with Hca-F and Hca-P cells. Quantitative real-time polymerase chain reaction and Western blotting techniques were used to determine CLIC4 mRNA and protein expressions. CCK8 assay and flow cytometry were employed for analysis of the survival and apoptosis rate as well as the cell cycle in an octreotide-induced apoptosis model. Expressions of caspase 3, caspase 9, and cleaved PARP were measured using Western blotting. The CLIC4 mRNA and protein expressions in Hca-F and Hca-P cells transfected by pSilencer-CLIC4 siRNA plasmid in the blank group displayed remarkably decreased levels of expression, when compared with both the control and negative control (NC) groups. Decreased survival rates and cleaved PARP expression, increased cell apoptosis rate,expressions of caspase 3 and caspase 9 in Hca-F and Hca-P cells were detected in groups that had been cultured in a medium containing octreotide. The pSilencer-CLIC4 siRNA-2 group when compared with the control and NC groups exhibited decreased survival rates, cleaved PARP expression, increased cell apoptosis rates, and increased expressions of caspase 3 and caspase 9 of Hca-F and Hca-P cells. The results demonstrated that siRNA-induced down-regulation of CLIC4 could proliferation, while in turn promoting apoptosis of mouse liver cancer Hca-F and Hca-P cells. J. Cell. Biochem. 119: 659-668, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans

PubMed Central

2004-01-01

Numerous invertebrate species belonging to several phyla cannot synthesize sterols de novo and rely on a dietary source of the compound. SCPx (sterol carrier protein 2/3-oxoacyl-CoA thiolase) is a protein involved in the trafficking of sterols and oxidation of branched-chain fatty acids. We have isolated SCPx protein from Spodoptera littoralis (cotton leafworm) and have subjected it to limited amino acid sequencing. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.9 kb), which encodes a 57 kDa protein. Northern blotting detected two mRNA transcripts, one of 1.9 kb, encoding SCPx, and one of 0.95 kb, presumably encoding SCP2 (sterol carrier protein 2). The former mRNA was highly expressed in midgut and Malpighian tubules during the last larval instar. Furthermore, constitutive expression of the gene was detected in the prothoracic glands, which are the main tissue producing the insect moulting hormone. There was no significant change in the 1.9 kb mRNA in midgut throughout development, but slightly higher expression in the early stages. Conceptual translation of the cDNA and a database search revealed that the gene includes the SCP2 sequence and a putative peroxisomal targeting signal in the C-terminal region. Also a cysteine residue at the putative active site for the 3-oxoacyl-CoA thiolase is conserved. Southern blotting showed that SCPx is likely to be encoded by a single-copy gene. The mRNA expression pattern and the gene structure suggest that SCPx from S. littoralis (a lepidopteran) is evolutionarily closer to that of mammals than to that of dipterans. PMID:15149283

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

PubMed Central

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

2011-01-01

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

Proteoglycans in Leiomyoma and Normal Myometrium: Abundance, Steroid Hormone Control, and Implications for Pathophysiology.

PubMed

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

2016-03-01

Uterine leiomyoma are a common benign pelvic tumors composed of modified smooth muscle cells and a large amount of extracellular matrix (ECM). The proteoglycan composition of the leiomyoma ECM is thought to affect pathophysiology of the disease. To test this hypothesis, we examined the abundance (by immunoblotting) and expression (by quantitative real-time polymerase chain reaction) of the proteoglycans biglycan, decorin, and versican in leiomyoma and normal myometrium and determined whether expression is affected by steroid hormones and menstrual phase. Leiomyoma and normal myometrium were collected from women (n = 17) undergoing hysterectomy or myomectomy. In vitro studies were performed on immortalized leiomyoma (UtLM) and normal myometrial (hTERT-HM) cells with and without exposure to estradiol and progesterone. In leiomyoma tissue, abundance of decorin messenger RNA (mRNA) and protein were 2.6-fold and 1.4-fold lower, respectively, compared with normal myometrium. Abundance of versican mRNA was not different between matched samples, whereas versican protein was increased 1.8-fold in leiomyoma compared with myometrium. Decorin mRNA was 2.4-fold lower in secretory phase leiomyoma compared with proliferative phase tissue. In UtLM cells, progesterone decreased the abundance of decorin mRNA by 1.3-fold. Lower decorin expression in leiomyoma compared with myometrium may contribute to disease growth and progression. As decorin inhibits the activity of specific growth factors, its reduced level in the leiomyoma cell microenvironment may promote cell proliferation and ECM deposition. Our data suggest that decorin expression in leiomyoma is inhibited by progesterone, which may be a mechanism by which the ovarian steroids affect leiomyoma growth and disease progression. © The Author(s) 2015.

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.

Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas.

PubMed

Kim, Tae-im; Choi, Seung-il; Lee, Hyung Keun; Cho, Young Jae; Kim, Eung Kweon

2008-06-30

The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts. Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. Bcl-xL, Bax, and TGFBI mRNA expression was measured using reverse transcription polymerase chain reaction (RT-PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting. MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT-PCR analysis showed that MMC decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types. RT-PCR and immunoblotting analysis showed that MMC reduced TGFBI mRNA levels and cellular and media TGFBIp protein levels in all cell types. MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients.

Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs

PubMed Central

Fain, John N.; Company, Joseph M.; Booth, Frank W.; Laughlin, M. Harold; Padilla, Jaume; Jenkins, Nathan T.; Bahouth, Suleiman W.; Sacks, Harold S.

2013-01-01

Background Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. Methods Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16–20 weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. Results There was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. Conclusion Exercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice. PMID:23831442

Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor gamma chain.

PubMed Central

Stingl, G; Koning, F; Yamada, H; Yokoyama, W M; Tschachler, E; Bluestone, J A; Steiner, G; Samelson, L E; Lew, A M; Coligan, J E

1987-01-01

The murine epidermis is a heterogeneous epithelium composed of keratinocytes, melanocytes, Langerhans cells, and a recently described subpopulation (2-3%) of bone-marrow-derived leukocytes with a dendritic morphology and the cell surface phenotype Thy-1+, L3T4-, Lyt-2-. Previous studies have demonstrated that cell lines derived from freshly explanted Thy-1+ dendritic epidermal cells (DEC) have abundant mRNA for rearranged T-cell receptor (TCR) gamma-chain genes. Analysis of Thy-1+ DEC in situ, freshly isolated cell suspensions of Thy-1+ DEC, and long-term Thy-1+ DEC lines demonstrated that 100% of the Thy-1+ DEC reacted with a monoclonal antibody to the epsilon chain of the murine T3 complex and that 40-60% of resident Thy-1+ DEC were also reactive with an antiserum to the TCR gamma chain. Two Thy-1+ DEC lines expressed a disulfide-linked 70-kDa molecule that could be precipitated with an anti-gamma-chain antiserum and could be coprecipitated with an antiserum to the T3 delta chain; the molecule appeared as a single 34-kDa band under reducing conditions. The phenotype of Thy-1+ DEC (T3+, L3T4-, Lyt-2-, TCR gamma chain+) thus resembles that of the recently described subpopulation of murine and human lymphocytes that have been identified in the thymus, peripheral blood, and fetal blood. Images PMID:2885839

Intercellular communication in the immune system: differential expression of connexin40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations

PubMed Central

Oviedo‐orta, E; Hoy, T; Evans, W H

2000-01-01

The distribution and function of connexins (integral membrane proteins assembled into gap junction intercellular communication channels) were studied in human lymphocyte subpopulations. The expression of mRNA encoding connexins in peripheral blood and tonsil‐derived T, B and natural killer (NK) lymphocytes was examined. Connexin43 (Cx43) mRNA was expressed in peripheral blood and tonsil lymphocytes, but Cx40 mRNA expression was confined to tonsil‐derived T and B lymphocytes; Cx26, Cx32, Cx37 and Cx45 were not detected by reverse transcription–polymerase chain reaction (RT–PCR). Western blot analysis also demonstrated the presence of Cx40 and Cx43 proteins in T and B lymphocytes in a manner coincidental to the mRNA detection. Stimulation in vitro of T and B lymphocytes with phytohaemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, increased Cx40 and Cx43 protein expression. Flow cytometric analysis, using antibodies to extracellular loop amino acid sequences of connexins, confirmed the surface expression of connexins in all lymphocyte subpopulations. Assembly of connexins into gap junctions providing direct intercellular channels linking attached lymphocytes was demonstrated by using a dye transfer technique. The exchange of dye between lymphocytes was inhibited by a connexin extracellular loop mimetic peptide and α‐glycyrrhetinic acid, two reagents that restrict intercellular communication across gap junctions. Dye coupling occurred between homologous and heterologous co‐cultures of T and B lymphocytes, and was not influenced by their stimulation with PHA and LPS. The connexin mimetic peptide caused a significant decrease in the in vitro synthesis of immunoglobulin M (IgM) by T‐ and B‐lymphocyte co‐cultured populations in the presence or absence of stimulation by PHA. The results identify connexins as important cell surface components that modulate immune processes. PMID:10792506

Effect of high ovarian response on the expression of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) in peri-implantation endometrium in IVF women

PubMed Central

Xu, Li-Zhen; Gao, Min-Zhi; Yao, Li-Hua; Liang, A-Juan; Zhao, Xiao-Ming; Sun, Zhao-Gui

2015-01-01

Objective: To investigate the effect of ovarian stimulation on the expression of EG-VEGF mRNA and protein in peri-implantation endometrium in women undergoing IVF and its relation with endometrial receptivity (ER). Design: Prospective laboratory study. Setting: University hospital. Patients: Eighteen women in stimulated cycles (SC) as study subjects and 18 women in natural cycles (NC) as controls. Women in SC group were classified with two subgroups, high ovarian response (SC1, n=9) with peak serum E2>5,000 pg/mL and moderate ovarian response (SC2, n=9) with peak serum E2 1,000-5,000 pg/mL. Intervention(s): Endometrial biopsies were collected 6 days after ovulation in NC or after oocyte retrieval in SC. Main outcome measure(s): Endometrium histological dating was observed with HE staining. EG-VEGF mRNA expression levels determined by real-time polymerase chain reaction analysis, and protein levels by immunohistochemistry. Results: All endometrial samples were in the secretory phase. The endometrial development in SC1 was 1 to 2 days advanced to NC, and with dyssynchrony between glandular and stromal tissue. Immunohistochemistry analysis showed that EG-VEGF protein was predominantly expressed in the glandular epithelial cells and endothelial cells of vessels, and also presented in the stroma. The image analysis confirmed that both the gland and stroma of endometrium in SC1 had a significantly lower EG-VEGF protein expression than that in SC2 and NC endometrium. Moreover, EG-VEGF mRNA levels were significantly lower in SC1 than in NC. Both EG-VEGF protein and mRNA levels had no significant difference between SC2 and NC. Conclusion: Decreased expression of EG-VEGF in the peri-implantation is associated with high ovarian response, which may account for the impaired ER and lower implantation rate in IVF cycles. PMID:26464631

Effect of high ovarian response on the expression of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) in peri-implantation endometrium in IVF women.

PubMed

Xu, Li-Zhen; Gao, Min-Zhi; Yao, Li-Hua; Liang, A-Juan; Zhao, Xiao-Ming; Sun, Zhao-Gui

2015-01-01

To investigate the effect of ovarian stimulation on the expression of EG-VEGF mRNA and protein in peri-implantation endometrium in women undergoing IVF and its relation with endometrial receptivity (ER). Prospective laboratory study. University hospital. Eighteen women in stimulated cycles (SC) as study subjects and 18 women in natural cycles (NC) as controls. Women in SC group were classified with two subgroups, high ovarian response (SC1, n=9) with peak serum E2>5,000 pg/mL and moderate ovarian response (SC2, n=9) with peak serum E2 1,000-5,000 pg/mL. Endometrial biopsies were collected 6 days after ovulation in NC or after oocyte retrieval in SC. Endometrium histological dating was observed with HE staining. EG-VEGF mRNA expression levels determined by real-time polymerase chain reaction analysis, and protein levels by immunohistochemistry. All endometrial samples were in the secretory phase. The endometrial development in SC1 was 1 to 2 days advanced to NC, and with dyssynchrony between glandular and stromal tissue. Immunohistochemistry analysis showed that EG-VEGF protein was predominantly expressed in the glandular epithelial cells and endothelial cells of vessels, and also presented in the stroma. The image analysis confirmed that both the gland and stroma of endometrium in SC1 had a significantly lower EG-VEGF protein expression than that in SC2 and NC endometrium. Moreover, EG-VEGF mRNA levels were significantly lower in SC1 than in NC. Both EG-VEGF protein and mRNA levels had no significant difference between SC2 and NC. Decreased expression of EG-VEGF in the peri-implantation is associated with high ovarian response, which may account for the impaired ER and lower implantation rate in IVF cycles.

Mitochondrial superoxide dismutase activation with 17 β-estradiol-treated human lens epithelial cells

PubMed Central

Gottipati, Srinivas

2008-01-01

Purpose 17 β-estradiol (17β-E2) protects human lens epithelial cells against oxidative stress by preserving mitochondrial function in part via the non-genomic rapid activation of prosurvival signal transduction pathways. The study described herein examined whether 17β-E2 also elicits genomic protection by influencing the expression (and activity) of mitochondrial-associated manganese superoxide dismutase (MnSOD) as a possible parallel mechanism by which 17β-E2 protects against oxidative stress. Methods Virally-transformed human lens epithelial cells (HLE-B3) were pre-incubated with 17β-E2, and mRNA or protein lysates were collected over a time course ranging from 90 min to 24 h. Positive expression of lens epithelial cell MnSOD mRNA was determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT–PCR), and its levels were monitored by real-time PCR up to 24 h after 17β-E2 administration. Western blot analysis was used to examine the pattern of protein expression as influenced by 17β-E2 treatment. MnSOD activity as influenced by 17β-E2 was determined by measuring enzymatic activity. Results A significant rapid increase in the activity of MnSOD was observed with HLE-B3 cells by 90 min post-bolus addition of 17β-E2, which returned to control level by 240 min. Neither an increase in MnSOD mRNA nor in protein expression was detected up through 24 h. Conclusions These data demonstrate that 17β-E2 rapidly and transiently increases the activity of MnSOD but influences neither its mRNA expression nor its protein expression. The results suggest that (estrogen-activated) MnSOD plays an important role against mitochondrial oxidative stress by diminishing reactive oxygen species, thus promoting cell survival. PMID:18490963

mRNA Expression of Platelet-Derived Growth Factor Receptor-{beta} and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

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

Erben, Philipp; Horisberger, Karoline; Muessle, Benjamin

2008-12-01

Purpose: Deviant expression of platelet-derived growth factor receptor-{beta} (PDGFR{beta}) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFR{beta} and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Methods and Materials: Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFR{beta} and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG,more » 0-1). Results: The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFR{beta} ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFR{beta} ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFR{beta} and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFR{beta} ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFR{beta} and c-kit mRNA expression and the pathologic regression rate. Conclusion: Cetuximab-based chemoradiotherapy increased PDGFR{beta} levels even further compared with the pretreatment samples and deserves further investigation.« less

Cykotine mRNA expression in mouse retina after laser injury by reverse transcriptase-polymerase chain reaction (RT-PCR)

NASA Astrophysics Data System (ADS)

Schuschereba, Steven T.; Bowman, Phillip D.; Ujimore, Veronica; Hoxie, Stephen W.; Pizarro, Jose M.; Cross, Michael E.; Lund, David J.

1996-04-01

The purpose of this study was to identify cytokines produced by the retina after laser injury. With the aid of a scanning laser ophthalmoscope (SLO), right eyes of mice received lesions from a continuous wave argon laser. Left eyes served as unirradiated controls. At 2, 4, 6, 12, 24, and 48 hr after laser irradiation groups of 3 mice were euthanized and retinas fixed for histology or isolated for RNA. Messenger RNA (mRNA) was reverse-transcribed into complementary DNA (cDNA) and subjected to polymerase chain reaction for the following cytokines: tumor necrosis factor-(alpha) (TNF-(alpha) ), interleukin-1(alpha) /(Beta) (IL- 1(alpha) /(Beta) ), interleukin-6 (IL-6), transforming growth factor-(Beta) 1 (TGF- (Beta) 1), macrophage colony stimulating factor (M-CSF), inducible nitric oxide synthase (iNOS), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Histologically, lesions were confined to the photoreceptors, retinal pigment epithelium, and choroid. In laser-injured retinas, mRNA levels were elevated for IL-1(alpha) , TGF-(Beta) 1, iNOS, and G3PDH, but not TNF-(alpha) , IL-1(Beta) , or IL-6. It appears that the retina, in response to laser injury, upregulates a select number of cytokines in a time-course dependent fashion.

[Development of a diagnostic test system for early non-invasive detection of prostate cancer based on PCA3 mRNA levels in urine sediment using quantitative reverse tanscription polymerase chain reaction (qRT-PCR)].

PubMed

Pavlov, K A; Shkoporov, A N; Khokhlova, E V; Korchagina, A A; Sidorenkov, A V; Grigor'ev, M É; Pushkar', D Iu; Chekhonin, V P

2013-01-01

The wide introduction of prostatic specific antigen (PSA) determination into clinical practice has resulted in a larger number of prostate biopsies, while the lower age threshold for PSA has led to a larger number of unnecessary prostate biopsies. Hence, there is a need for new biomarkers that can detect prostate cancer. PCA3 is a noncoding messenger ribonucleic acid (mRNA) that is expressed exclusively in prostate cells. The aim of the study has been to develop a diagnostic test system for early non-invasive detection of prostate cancer based on PCA3 mRNA levels in urine sediment using quantitative reverse transcription polymerase chain reaction (qRT-PCR). As part of the study, a laboratory diagnostic test system prototype has been designed, an application methodology has been developed and specificity and sensitivity data of the method has been assessed. The diagnostic system has demonstrated its ability to detect significantly elevated levels of PCA 3/KLK 3 in samples from prostate cancer (PCa) patients compared with those from healthy men. The findings have shown relatively high diagnostic sensitivity, specificity and negative-predictive values for an early non-invasive screening of prostate cancer

Aberrant Epigenetic Alterations of Glutathione-S-Transferase P1 in Age-Related Nuclear Cataract.

PubMed

Chen, Jia; Zhou, Jing; Wu, Jian; Zhang, Guowei; Kang, Lihua; Ben, Jindong; Wang, Yong; Qin, Bai; Guan, Huaijin

2017-03-01

Oxidative damage of lens tissue contributes to the formation of age-related cataract. Pi-class glutathione-S-transferase (GSTP1) plays a role in the removal of oxidative adducts by transferring them to glutathione. To assess epigenetic regulation of GSTP1 and its potential role in age-related nuclear cataract (ARNC) pathogenesis, we evaluated GSTP1 mRNA expression, methylation, and chromatin modifications in lenses from ARNC patients. The mRNA and protein of lens GSTP1 were assayed by relative quantitative real-time polymerase chain reaction (qRT-PCR) and Western blots. Methylation of the GSTP1 promoter was determined by bisulfite genomic sequencing. Chromatin modification was detected by chromatin immunoprecipitation. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities were also assayed by enzyme-linked immunosorbent assay (ELISA)-like reaction. To assess the effect of DNA methylation on the mRNA expression of GSTP1, human lens epithelium HLE-B3 cells were treated with the demethylation compound 5-aza-dC, followed by qRT-PCR assay. GSTP1 mRNA and protein levels were significantly reduced in lens epithelium and cortex of ARNC cases versus age-matched controls. The changes corresponded to hypermethylation of the GSTP1 promoter CpG islands. The loss of GSTP1 mRNA and protein and the increased DNA promoter methylation might be correlated with the severity of the ARNC. ARNC lenses also had lower acetylation of histone proteins H3, H4, and lower methylation of H3K4, and higher methylation of H3K9. Histone modifications were not correlated with the severity of the ARNCs. DNMT and HDAC were elevated in lenses from ARNCs compared with controls. Demethylation treatment of HLE-B3 cells with 5-aza-dC enhanced the expression of GSTP1. Epigenetic alteration of GSTP1 regulates its expression in lens epithelial and cortical tissues. These changes likely contribute to the pathogenesis of ARNC.

Studies on Pentoxifylline and Tocopherol Combination for Radiation-Induced Heart Disease in Rats

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

Liu Hui; Department of Radiotherapy, Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong; Xiong Mai

Purpose: To investigate whether the application of pentoxifylline (PTX) and tocopherol l (Vit. E) could modify the development of radiation-induced heart disease and downregulate the expression of transforming growth factor (TGF)-{beta}1mRNA in rats. Methods and Materials: A total of 120 Sprague-Dawley rats were separated into four groups: control group, irradiated group, experimental group 1, and experiment group 2. Supplementation was started 3 days before irradiation; in experimental group 1, injection of PTX (15 mg/kg/d) and Vit. E (5.5 mg/kg/d) continued till the 12th week postirradiation, whereas in experimental group 2 it was continued until the 24th week postirradiation. All ratsmore » were administrated a single dose of 20 Gy irradiation to the heart except the control group. Histopathologic evaluation was performed at various time points (Days 1, 2, 4, 8, and 12 and 24th week) up to 24 weeks after irradiation. Changes of levels of TGF-{beta}1 mRNA expression were also investigated at the same time points using competitive polymerase chain reaction. Results: Compared with the irradiated group, levels of TGF-{beta}1 mRNA of the rat hearts were relatively low in the two experimental groups on the 12th week postirradiation. In experimental group 1, there was a rebound expression of TGF-{beta}1 mRNA on the 24th week postirradiation, whereas that of the experimental group 2 remained low (p < 0.05). The proportions of collagen fibers of the two experimental groups were lower than that of irradiated group (p < 0.05). A rebound could be observed in the experimental group 1. Conclusion: PTX and Vit. E downregulated the expression of TGF-{beta}1 mRNA. The irradiated rat hearts showed a marked pathologic response to the drugs. The withdrawal of drugs in the 12th week postirradiation could cause rebound effects of the development of fibrosis.« less

The IGF-1/Akt/S6 pathway and expressions of glycolytic myosin heavy chain isoforms are upregulated in chicken skeletal muscle during the first week after hatching.

PubMed

Saneyasu, Takaoki; Tsuchihashi, Tatsuya; Kitashiro, Ayana; Tsuchii, Nami; Kimura, Sayaka; Honda, Kazuhisa; Kamisoyama, Hiroshi

2017-11-01

Skeletal muscle mass is an important trait in the animal industry. We previously reported an age-dependent downregulation of the insulin-like growth factor 1 (IGF-1)/Akt/S6 pathway, major protein synthesis pathway, in chicken breast muscle after 1 week of age, despite a continuous increase of breast muscle weight. Myosin heavy chain (HC), a major protein in muscle fiber, has several isoforms depending on chicken skeletal muscle types. HC I (fast-twitch glycolytic type) is known to be expressed in adult chicken breast muscle. However, little is known about the changes in the expression levels of protein synthesis-related factors and HC isoforms in perihatching chicken muscle. In the present study, protein synthesis-related factors, such as IGF-1 messenger RNA (mRNA) levels, phosphorylation of Akt, and phosphorylated S6 content, increased in an age-dependent manner after post-hatch day (D) 0. The mRNA levels of HC I, III and V (fast-twitch glycolytic type) dramatically increased after D0. The increase ratio of breast muscle weight was approximately 1100% from D0 to D7. To our knowledge, these findings provide the first evidence that upregulation of protein synthesis pathway and transcription of fast twitch glycolytic HC isoforms play critical roles in the increase of chicken breast muscle weight during the first week after hatching. © 2017 Japanese Society of Animal Science.

Effect of transient receptor potential vanilloid 6 gene silencing on the expression of calcium transport genes in chicken osteoblasts.

PubMed

Zhang, Jie; Deng, Yifeng; Ma, Huijie; Hou, Jiafa; Zhou, ZhenLei

2015-03-01

Ca2+ plays a major role in the regulation of signal transduction. Transient receptor potential vanilloid 6 is a Ca2+-selective channel that serves as an important rate-limiting step in the facilitation of Ca2+ entry into cells, but little is known about the regulation of transient receptor potential vanilloid 6 in chickens. In this study, we evaluated the effects of transient receptor potential vanilloid 6 gene interference on the expression of calbindin-D28K, Na+/Ca2+ exchangers, and plasma membrane Ca2+ ATPase 1b to investigate the mechanism underlying the regulation of transient receptor potential vanilloid 6. Three hairpin siRNA expression vectors targeting transient receptor potential vanilloid 6 (pSIREN- transient receptor potential vanilloid 6) and a negative control (pSIREN-control) were constructed and transfected into chicken osteoblasts. The mRNA and protein expression levels were evaluated by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. The mRNA expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 45.7% (P<0.01) and 27.9% (P<0.01), respectively, 48 h after transfection with one of the three constructs (pSIREN- transient receptor potential vanilloid 6-3) compared with the level obtained in the untreated group. There was no significant difference in the mRNA expression levels of Na+/Ca2+ exchangers and plasma membrane Ca2+ ATPase 1b. The protein expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 40.2% (P<0.01) and 29.8% (P<0.01), respectively, 48 h after transfection with pSIREN-transient receptor potential vanilloid 6-3 compared with the level obtained in the untreated group. In conclusion, the vector-based transient receptor potential vanilloid 6-shRNA can efficiently suppress the mRNA and protein expression of transient receptor potential vanilloid 6 in chicken osteoblasts, and transient receptor potential vanilloid 6 regulates the expression of calbindin-D28K during Ca2+ transport. © 2015 Poultry Science Association Inc.

Measurement of messenger RNA encoding the alpha-chain, polymeric immunoglobulin receptor, and J-chain in duodenal mucosa from dogs with and without chronic diarrhea by use of quantitative real-time reverse transcription-polymerase chain reaction assays.

PubMed

Peters, Iain R; Helps, Chris R; Calvert, Emma L; Hall, Edward J; Day, Michael J

2005-01-01

To examine the difference in expression of messenger RNA (mRNA) transcripts for polymeric immunoglobulin receptor (plgR), alpha-chain, and J-chain determined by use of quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) assays in duodenal biopsy specimens obtained from dogs with and without chronic diarrhea. Biopsy specimens of the proximal portion of the duodenum were obtained endoscopically from 39 dogs evaluated because of chronic diarrhea (12 German Shepherd Dogs and 27 non-German Shepherd Dog breeds); specimens were also obtained from a control group of 7 dogs evaluated because of other gastrointestinal tract diseases and 2 dogs that were euthanatized as a result of nongastrointestinal tract disease. Dogs were anesthetized, and multiple mucosal biopsy specimens were obtained endoscopically at the level of the caudal duodenal flexure by use of biopsy forceps; in 2 control dogs, samples were obtained from the descending duodenum within 5 minutes of euthanasia. One-step QRT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase, plgR, alpha-chain, and J-chain in duodenal mucosal tissue. There was no significant difference in the level of expression of any transcript among non-German Shepherd Dog breeds without diarrhea (control group), non-German Shepherd Dog breeds with chronic diarrhea, and German Shepherd Dogs with chronic diarrhea. Conclusions and Clinical Relevance-Results indicated that the susceptibility of German Shepherd Dogs to chronic diarrhea is not a result of simple failure of transcription of the key genes that encode molecules involved in mucosal IgA secretion.

Escalating Methamphetamine Regimen Induces Compensatory Mechanisms, Mitochondrial Biogenesis, and GDNF Expression, in Substantia Nigra.

PubMed

Valian, Neda; Ahmadiani, Abolhassan; Dargahi, Leila

2017-06-01

Methamphetamine (MA) produces long-lasting deficits in dopaminergic neurons in the long-term use via several neurotoxic mechanisms. The effects of MA on mitochondrial biogenesis is less studied currently. So, we evaluated the effects of repeated escalating MA regimen on transcriptional factors involved in mitochondrial biogenesis and glial-derived neurotrophic factor (GDNF) expression in substantia nigra (SN) and striatum of rat. In male Wistar rats, increasing doses of MA (1-14 mg/kg) were administrated twice a day for 14 days. At the 1st, 14th, 28th, and 60th days after MA discontinuation, we measured the PGC1α, TFAM and NRF1 mRNA levels, indicator of mitochondrial biogenesis, and GDNF expression in SN and striatum. Furthermore, we evaluated the glial fibrillary acidic protein (GFAP) and Iba1 mRNA levels, and the levels of tyrosine hydroxylase (TH) and α-synuclein (α-syn) using immunohistochemistry and real-time polymerase chain reaction (PCR). We detected increments in PGC1α and TFAM mRNA levels in SN, but not striatum, and elevations in GDNF levels in SN immediately after MA discontinuation. We also observed increases in GFAP and Iba1 mRNA levels in SN on day 1 and increases in Iba1 mRNA on days 1 and 14 in striatum. Data analysis revealed that the number of TH + cells in the SN did not reduce in any time points, though TH mRNA levels was increased on day 1 after MA discontinuation in SN. These data show that repeated escalating MA induces several compensatory mechanisms, such as mitochondrial biogenesis and elevation in GDNF in SN. These mechanisms can reverse MA-induced neuroinflammation and prevent TH-immunoreactivity reduction in nigrostriatal pathway. J. Cell. Biochem. 118: 1369-1378, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Epigenetic regulation of matrix metalloproteinase expression in ameloblastoma

PubMed Central

2012-01-01

Background An ameloblastoma is a benign odontogenic neoplasm with aggressive behaviour and high recurrence rates. The increased expression of matrix metalloproteinases (MMPs) has been reported in ameloblastomas. In the present study, we hypothesised that epigenetic alterations may regulate MMP expression in ameloblastomas. Methods We investigated the methylation status of the genes MMP-2 and MMP-9 in addition to mRNA transcription and protein expression in ameloblastomas. Methylation analysis was performed by both methylation-specific polymerase chain reaction (MSP-PCR) and restriction enzyme digestion to evaluate the methylation profile of MMP-2 and MMP-9 in 12 ameloblastoma samples and 12 healthy gingiva fragments, which were included as controls. Furthermore, we investigated the transcription levels of the genes by quantitative reverse-transcription PCR (qRT-PCR). Zymography was performed to verify protein expression in ameloblastomas. Results The ameloblastomas showed a high frequency of unmethylated MMP-2 and MMP-9, whereas the healthy gingival samples presented a sharp prevalence of methylated MMPs. Higher expression levels of MMP-9 were found in ameloblastomas compared to healthy gingiva. However, no significant differences in the MMP-2 mRNA expression between groups was found. All ameloblastomas showed positive expression of MMP-2 and MMP-9 proteins. Conclusions Our findings suggest that expression of MMP-9 is increased in ameloblastomas and is possibly modulated by unmethylation of the gene. PMID:22866959

Over-expression of mammaglobin-B in canine mammary tumors.

PubMed

Pandey, Mamta; Sunil Kumar, B V; Gupta, Kuldip; Sethi, Ram Saran; Kumar, Ashwani; Verma, Ramneek

2018-06-15

Mammaglobin, a member of secretoglobin family has been recognized as a breast cancer associated protein. Though the exact function of the protein is not fully known, its expression has been reported to be upregulated in human breast cancer.We focused on studying the expression of mammaglobin-B gene and protein in canine mammary tumor (CMT) tissue. Expression of mammaglobin-B mRNA and protein were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. High levels of mammaglobin-B mRNA expression (6.663 ± 0.841times) was observed in CMT as compared to age and breed matched healthy controls. Further, expression of mammaglobin-B protein was detected in paraffin-embedded mammary tumor tissues from the same subjects by IHC. Mammaglobin-B protein was overexpressed only in 6.67% of healthy mammary glands while, a high level of its expression was scored in 76.7% of the CMT subjects. Moreover, no significant differences in terms of IHC score and qRT-PCR score with respect to CMT histotypes or tumor grades were observed, indicating that mammaglobin-B over-expression occurred irrespective of CMT types or grades. Overall, significantly increased expression of mammaglobin-B protein was found in CMTs with respect to healthy mammary glands, which positively correlates to its transcript. These findings suggest that overexpression of mammaglobin-B is associated with tumors of canine mammary glands.

[Expression of aquaporin-4 during brain edema in rats with thioacetamide-induced acute encephalopathy].

PubMed

Wang, Li-Qing; Zhu, Sheng-Mei; Zhou, Heng-Jun; Pan, Cai-Fei

2011-09-27

To investigate the expression of aquaporin-4 (AQP4) during brain edema in rats with thioacetamide-induced acute liver failure and encephalopathy. The rat model of acute hepatic failure and encephalopathy was induced by intraperitoneal injection of thioacetamide (TAA) at a 24-hour interval for 2 consecutive days. Thirty-two SD rats were randomly divided into the model group (n = 24) and the control group (normal saline, n = 8). And then the model group was further divided into 3 subgroups by the timepoint of decapitation: 24 h (n = 8), 48 h (n = 8) and 60 h (n = 8). Then we observed their clinical symptoms and stages of HE, indices of liver function and ammonia, liver histology and brain water content. The expression of AQP4 protein in brain tissues was measured with Western blot and the expression of AQP4mRNA with RT-PCR (reverse transcription-polymerase chain reaction). Typical clinical manifestations of hepatic encephalopathy occurred in all TAA-administrated rats. The model rats showed the higher indices of ALT (alanine aminotransferase), AST (aspartate aminotransferase), TBIL (total bilirubin) and ammonia than the control rats (P < 0.05). The brain water content was significantly elevated in TAA-administrated rats compared with the control (P < 0.05). The expressions of AQP4 protein and mRNA in brain tissues significantly increased in TAA-administrated rats (P < 0.05). In addition, the expressions of AQP4 protein and mRNA were positively correlated with brain water content (r = 0.536, P < 0.01; r = 0.566, P = 0.01). The high expression of AQP4 in rats with TAA-induced acute liver failure and encephalopathy plays a significant role during brain edema. AQP4 is one of the molecular mechanisms for the occurrence of brain edema in hepatic encephalopathy.

[Primary investigation of the relationship between glucocorticoid induced leucine zipper and inflammatory reaction].

PubMed

Bai, Xiang-jun; Li, Bo; Wang, Hai-ping; Yang, Zhao-hui; Li, Si-qi

2007-01-01

To investigate the mechanism of the action of glucocorticoid induced leucine zipper (GILZ) in inflammatory reaction. Human monocyte cell line THP-1 cells were divided into two groups and cultured in non-serum RPMI1640 medium.In one group the cells were treated with dexamethasone (DEX). Twelve hours later total RNA and total protein were abstracted in both two groups. The mRNA encoding for expression of GILZ was semiquantitatively detected by reserve transcriptase-polymerase chain reaction (RT-PCR). Protein expression of nuclear factor-KappaB (NF-KappaB) p65 and activator protein-1 (AP-1) were assessed by Western blotting. Peripheral blood of 10 trauma patients [injury severity score (ISS) >or=16 scores] were collected and the leukocytes were isolated within 24 hours after trauma. The leukocytes were divided into two groups and cultured in non-serum medium. In one group the cells were treated with DEX. Twelve hours later total RNA and total protein were abstracted in both two groups. The mRNA encoding for expression of GILZ was semiquantitatively detected by RT-PCR. Protein expression of NF-KappaB p65 and AP-1 were assessed by Western blotting. Stimulated by DEX, the expression of GILZ mRNA was increased both in THP-1 cells and the leukocytes of trauma patients compared with those of control groups (both P<0.01). Whereas, protein expressions of NF-KappaB p65 and AP-1 of THP-1 cells and leukocytes in peripheral blood of trauma patients were decreased in the stimulation groups compared with those of control groups (all P<0.01). The expression of GILZ gene is up-regulated by glucocorticoid. Overexpression of GILZ inhibits NF-KappaB and AP-1 activities, suggesting that GILZ possesses anti-inflammatory function.

Inverse correlation between HPSE gene single nucleotide polymorphisms and heparanase expression: possibility of multiple levels of heparanase regulation

PubMed Central

Ostrovsky, Olga; Korostishevsky, Michael; Shafat, Itay; Mayorov, Margarita; Ilan, Neta; Vlodavsky, Israel; Nagler, Arnon

2009-01-01

Heparanase is an endo-β-glucuronidase that specifically cleaves the saccharide chains of heparan sulfate proteoglycans. Heparanase plays important roles in processes such as angiogenesis, tumor metastasis, tissue repair and remodeling, inflammation and autoimmunity. Genetic variations of the heparanase gene (HPSE) have been associated with heparanase transcription level. The present study was undertaken to identify haplotype or single nucleotide polymorphisms (SNPs) genotype combinations that correlate with heparanase expression both at the mRNA and protein levels. For this purpose, 11 HPSE gene SNPs were genotyped among 108 healthy individuals. Five out of the eleven polymorphisms revealed an association between the SNPs and heparanase expression. SNP rs4693608 exhibited a strong evidence of association. Analysis of haplotypes distribution revealed that the combination of two SNPs (rs4693608 and rs4364254) disclosed the most significant result. This approach allowed segregation of possible genotype combinations to three groups that correlate with low (LR: GG-CC, GG-CT, GG-TT, GA-CC), intermediate (MR: GA-CT, GA-TT) and high (HR: AA-TT, AA-CT) heparanase expression. Unexpectedly, LR genotype combinations were associated with low mRNA expressions level and high heparanase concentration in plasma, while HR genotype combinations were associated with high expression of mRNA and low plasma protein level. Because the main site of activity of secreted active heparanase is the extracellular matrix and cell surface, the origin and functional significance of plasma heparanase remain to be investigated. The current study indicates that rs4693608 and rs4364254 SNPs are involved in the regulation of heparanase expression and provides the basis for further studies on the association between HPSE gene SNPs and disease outcome. PMID:19406828

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.

The role of miR-370 in fibrosis after myocardial infarction

PubMed Central

Yuan, Hui; Gao, Jie

2017-01-01

In the present study, we investigated the expression of miR-370 in the border area of infarction after myocardial infarction and its role in the process of post-infarction fibrosis. A myocardial infarction model in Sprague-Dawley rats was established. After two weeks, the mRNA levels of transforming growth factor-β1 (TGFβ1), TGFβRII, ColIa1, ColIIIa1 and miR-370 and the expression of TGFβ1, TGFβRII and α-smooth muscle actin (α-SMA) proteins in the border area of infarction were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. Cardiac fibroblasts in neonatal rat were isolated and cultured, and the changes in the above indicators were detected after AngII and miR-370 intervention. Luciferase reporter gene assay was conducted to verify whether TGFβRII was a target gene of miR-370. In the border area after myocardial infarction, the expression of miR-370 decreased, while mRNA levels of TGFβ1, TGFβRII, ColIa1 and ColIIIa1 and levels of TGFβ1, TGFβRII and α-SMA proteins were all increased. Luciferase reporter gene assay confirmed that TGFβRII was the target gene of miR-370. miR-370 reduced the expression of TGFβRII and inhibited the increased expression of TGFβRII and collagen protein caused by AngII. As well, its inhibited the differentiation effect of muscle fibroblasts while it did not inhibit the expression of TGFβ1. miR-370 inhibited the expression of TGFβRII protein by combining with TGFβRII mRNA. miR-370 also partially blocked TGFβ1-TGFβRII and induced the downstream signal transduction pathways, thus exerting anti-fibrotic effects. PMID:28350072

Effect of Maternal Obesity on Fetal Growth and Expression of Placental Fatty Acid Transporters.

PubMed

Ye, Kui; Li, Li; Zhang, Dan; Li, Yi; Wang, Hai Qing; Lai, Han Lin; Hu, Chuan Lai

2017-12-15

To explore the effects of maternal high-fat (HF) diet-induced obesity on fetal growth and the expression of placental nutrient transporters. Maternal obesity was established in rats by 8 weeks of pre-pregnancy fed HF diet, while rats in the control group were fed normal (CON) diet. Diet-induced obesity (DIO) rats and diet-induced obesity-resistant (DIR) rats were selected according to body weight gain over this period. After copulation, the CON rats were divided into two groups: switched to HF diet (CON-HF group) or maintained on the CON diet (CON-CON group). The DIO rats and DIR rats were maintained on the HF diet throughout pregnancy. Pregnant rats were euthanized at day 21 gestation, fetal and placental weights were recorded, and placental tissue was collected. Reverse transcription-polymerase chain reaction was used to determine mRNA expression of placental nutrient transporters. Protein expression was determined by Western blot. Average fetal weight of DIO dams was reduced by 6.9%, and the placentas of CON-HF and DIO dams were significantly heavier than the placentas of CON-CON and DIR dams at day 21 of gestation (p<0.05). The fetal/placental weight ratio of DIO dams was significantly reduced compared with the fetal/placental weight ratio of CON-CON dams (p<0.05). The mRNA expression of GLUT-1 and SNAT-2 were not significantly different between groups. The mRNA and protein expression levels of CD36, FATP-1, and FATP-4 in DIO dams were decreased significantly (p<0.05). Maternal obesity induced by a HF diet led to intrauterine growth retardation and down-regulated the expression of placental fatty acid transporters.

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

PubMed

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

2016-10-01

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

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