Geminivirus vectors for high-level expression of foreign proteins in plant cells.

Science.gov (United States)

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

SAMSN1 is highly expressed and associated with a poor survival in glioblastoma multiforme.

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Yong Yan

Full Text Available OBJECTIVES: To study the expression pattern and prognostic significance of SAMSN1 in glioma. METHODS: Affymetrix and Arrystar gene microarray data in the setting of glioma was analyzed to preliminarily study the expression pattern of SAMSN1 in glioma tissues, and Hieratical clustering of gene microarray data was performed to filter out genes that have prognostic value in malignant glioma. Survival analysis by Kaplan-Meier estimates stratified by SAMSN1 expression was then made based on the data of more than 500 GBM cases provided by The Cancer Genome Atlas (TCGA project. At last, we detected the expression of SAMSN1 in large numbers of glioma and normal brain tissue samples using Tissue Microarray (TMA. Survival analysis by Kaplan-Meier estimates in each grade of glioma was stratified by SAMSN1 expression. Multivariate survival analysis was made by Cox proportional hazards regression models in corresponding groups of glioma. RESULTS: With the expression data of SAMSN1 and 68 other genes, high-grade glioma could be classified into two groups with clearly different prognoses. Gene and large sample tissue microarrays showed high expression of SAMSN1 in glioma particularly in GBM. Survival analysis based on the TCGA GBM data matrix and TMA multi-grade glioma dataset found that SAMSN1 expression was closely related to the prognosis of GBM, either PFS or OS (P<0.05. Multivariate survival analysis with Cox proportional hazards regression models confirmed that high expression of SAMSN1 was a strong risk factor for PFS and OS of GBM patients. CONCLUSION: SAMSN1 is over-expressed in glioma as compared with that found in normal brains, especially in GBM. High expression of SAMSN1 is a significant risk factor for the progression free and overall survival of GBM.

Expression of a highly basic peroxidase gene in NaCl-adapted tomato cell suspensions.

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Medina, M I; Botella, M A; Quesada, M A; Valpuesta, V

1997-05-05

A tomato peroxidase gene, TPX2, that is only weakly expressed in the roots of young tomato seedlings is highly expressed in tomato suspension cells adapted to high external NaCl concentration. The protein encoded by this gene, with an isolectric point value of approximately 9.6, is found in the culture medium of the growing cells. Our data suggest that the expression of TPX2 in the salt-adapted cells is not the result of the elicitation imposed by the in vitro culture or the presence of high NaCl concentration in the medium.

High-throughput Microarray Detection of Vomeronasal Receptor Gene Expression in Rodents

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Xiaohong Zhang

2010-11-01

Full Text Available We performed comprehensive data mining to explore the vomeronasal receptor (V1R & V2R repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on computational prediction from gene sequence data, thereby establishing that these genes are indeed part of the vomeronasal system, especially the V2Rs. 168 V1Rs and 98 V2Rs were detected to be highly enriched in mouse vomeronasal organ (VNO, and 108 V1Rs and 87 V2Rs in rat VNO. We monitored the expression profile of mouse VR genes in other non-VNO tissues with the result that some VR genes were re-designated as VR-like genes based on their non-olfactory expression pattern. Temporal expression profiles for mouse VR genes were characterized and their patterns were classified, revealing the developmental dynamics of these so-called pheromone receptors. We found numerous patterns of temporal expression which indicate possible behavior-related functions. The uneven composition of VR genes in certain patterns suggests a functional differentiation between the two types of VR genes. We found the coherence between VR genes and transcription factors in terms of their temporal expression patterns. In situ hybridization experiments were performed to evaluate the cell number change over time for selected receptor genes.

MMSET is highly expressed and associated with aggressiveness in neuroblastoma

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Hudlebusch, Heidi Rye; Skotte, Julie; Santoni-Rugiu, Eric

2011-01-01

tumor types as well. We have performed immunohistochemical staining of tissue microarrays and found that MMSET protein is frequently and highly expressed in neuroblastoma (MMSET positive in 75% of neuroblastomas, n=164). The expression level of MMSET in neuroblastomas was significantly associated...... with poor survival, negative prognostic factors, and metastatic disease. Moreover, a subset of neuroblastomas for which pre- and post-chemotherapy biopsies were available displayed a strong decrease in MMSET protein levels after chemotherapy. In agreement with neuroblastomas becoming more differentiated...... after treatment, we show that retinoic acid-induced differentiation of human neuroblastoma cells in vitro also leads to a strong decrease in MMSET levels. Furthermore, we demonstrate that the high levels of MMSET in normal neural progenitor cells are strongly downregulated during differentiation...

Design of chimeric expression elements that confer high-level gene activity in chromoplasts.

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Caroca, Rodrigo; Howell, Katharine A; Hasse, Claudia; Ruf, Stephanie; Bock, Ralph

2013-02-01

Non-green plastids, such as chromoplasts, generally have much lower activity of gene expression than chloroplasts in photosynthetically active tissues. Suppression of plastid genes in non-green tissues occurs through a complex interplay of transcriptional and translational control, with the contribution of regulation of transcript abundance versus translational activity being highly variable between genes. Here, we have investigated whether the low expression of the plastid genome in chromoplasts results from inherent limitations in gene expression capacity, or can be overcome by designing appropriate combinations of promoters and translation initiation signals in the 5' untranslated region (5'-UTR). We constructed chimeric expression elements that combine promoters and 5'-UTRs from plastid genes, which are suppressed during chloroplast-to-chromoplast conversion in Solanum lycopersicum (tomato) fruit ripening, either just at the translational level or just at the level of mRNA accumulation. These chimeric expression elements were introduced into the tomato plastid genome by stable chloroplast transformation. We report the identification of promoter-UTR combinations that confer high-level gene expression in chromoplasts of ripe tomato fruits, resulting in the accumulation of reporter protein GFP to up to 1% of total cellular protein. Our work demonstrates that non-green plastids are capable of expressing genes to high levels. Moreover, the chimeric cis-elements for chromoplasts developed here are widely applicable in basic and applied research using transplastomic methods. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

High Expression of Osteopontin and CD44v6 in Odontogenic Keratocysts

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Yi-Ping Wang

2009-04-01

Conclusion: Binding of OPN to osteoclast cell membrane receptor integrin αv can activate the osteoclasts and increase their osteolytic activity. In addition, binding of OPN to OKC lining epithelial cell membrane receptor CD44v6 can enhance the motility, migration, invasion and spread of lining epithelial cells into the surrounding cancellous bone. Therefore, we suggest that the local aggressive behavior and high osteolytic ability of OKCs in the jawbone can be explained at least partially by high expression of OPN and CD44v6 in lining epithelial cells of OKCs and high expression of integrin αv in osteoclasts.

High bone sialoprotein (BSP expression correlates with increased tumor grade and predicts a poorer prognosis of high-grade glioma patients.

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Tao Xu

Full Text Available OBJECTIVES: To investigate the expression and prognostic value of bone sialoprotein (BSP in glioma patients. METHODS: We determined the expression of BSP using real-time RT-PCR and immunohistochemistry in tissue microarrays containing 15 normal brain and 270 glioma samples. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were performed by the stepwise forward Cox regression model. RESULTS: Both BSP mRNA and protein levels were significantly elevated in high-grade glioma tissues compared with those of normal brain and low-grade glioma tissues, and BSP expression positively correlated with tumor grade (P<0.001. Univariate and multivariate analysis showed high BSP expression was an independent prognostic factor for a shorter progression-free survival (PFS and overall survival (OS in both grade III and grade IV glioma patients [hazard ratio (HR = 2.549 and 3.154 for grade III glioma, and HR = 1.637 and 1.574 for grade IV glioma, respectively]. Patients with low BSP expression had a significantly longer median OS and PFS than those with high BSP expression. Small extent of resection and lineage of astrocyte served as independent risk factors of both shorter PFS and OS in grade III glioma patients; GBM patients without O(6-methylguanine (O(6-meG DNA methyltransferase (MGMT methylation and Karnofsky performance score (KPS less than 70 points were related to poor prognosis. Lack of radiotherapy related to shorter OS but not affect PFS in both grade III and grade IV glioma patients. CONCLUSION: High BSP expression occurs in a significant subset of high-grade glioma patients and predicts a poorer outcome. The study identifies a potentially useful molecular marker for the categorization and targeted therapy of gliomas.

Evidence against the energetic cost hypothesis for the short introns in highly expressed genes

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Niu Deng-Ke

2008-05-01

Full Text Available Abstract Background In animals, the moss Physcomitrella patens and the pollen of Arabidopsis thaliana, highly expressed genes have shorter introns than weakly expressed genes. A popular explanation for this is selection for transcription efficiency, which includes two sub-hypotheses: to minimize the energetic cost or to minimize the time cost. Results In an individual human, different organs may differ up to hundreds of times in cell number (for example, a liver versus a hypothalamus. Considered at the individual level, a gene specifically expressed in a large organ is actually transcribed tens or hundreds of times more than a gene with a similar expression level (a measure of mRNA abundance per cell specifically expressed in a small organ. According to the energetic cost hypothesis, the former should have shorter introns than the latter. However, in humans and mice we have not found significant differences in intron length between large-tissue/organ-specific genes and small-tissue/organ-specific genes with similar expression levels. Qualitative estimation shows that the deleterious effect (that is, the energetic burden of long introns in highly expressed genes is too negligible to be efficiently selected against in mammals. Conclusion The short introns in highly expressed genes should not be attributed to energy constraint. We evaluated evidence for the time cost hypothesis and other alternatives.

VEGF expression and microvascular density in relation to high-risk ...

African Journals Online (AJOL)

Bassma M. El Sabaa

2012-01-13

Jan 13, 2012 ... Eleven cases were low grade and 19 were high-grade cases. VEGF expression .... increasing microvascular permeability,26 degradation of extra- ...... soluble receptors in pre-invasive, invasive and recurrent cervical cancer.

High Expression of SQSTM1/p62 Protein Is Associated with Poor Prognosis in Epithelial Ovarian Cancer

International Nuclear Information System (INIS)

Iwadate, Reiko; Inoue, Jun; Tsuda, Hitoshi; Takano, Masashi; Furuya, Kenichi; Hirasawa, Akira; Aoki, Daisuke; Inazawa, Johji

2014-01-01

High expression of SQSTM1/p62 (p62) protein, which functions as a hub for various cellular signaling pathways, has been detected in several human cancers. However, the clinicopathological impact of high p62 expression is largely unknown in epithelial ovarian cancer (EOC). Here, the expression level of p62 in primary EOCs (n=266) was assessed by immunohistochemistry, and its clinical significance was analyzed. Univariate and multivariate analyses were used to determine the impact of p62 expression on overall survival. p62 was expressed in the cytoplasm (Cyto) and/or nucleus (Nuc) in primary EOCs, and an expression subtype (Cyto High /Nuc Low ), showing high expression in the cytoplasm but low expression in the nucleus, was significantly correlated with serous carcinoma (P<0.001), advanced stage (P=0.005), presence of residual tumor (P<0.001), and low overall survival rate (P=0.013). Furthermore, in serous carcinomas (n=107), the p62 Cyto High /Nuc Low subtype was significantly correlated with low overall survival rate (P=0.019) as an independent factor (P=0.044). Thus, our findings suggest that high expression of cytoplasmic p62 may be a novel prognostic biomarker in EOC, particularly in serous carcinoma

An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

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Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

2015-09-01

A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

Hypoxia promotes IL-32 expression in myeloma cells, and high expression is associated with poor survival and bone loss.

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Zahoor, Muhammad; Westhrin, Marita; Aass, Kristin Roseth; Moen, Siv Helen; Misund, Kristine; Psonka-Antonczyk, Katarzyna Maria; Giliberto, Mariaserena; Buene, Glenn; Sundan, Anders; Waage, Anders; Sponaas, Anne-Marit; Standal, Therese

2017-12-26

Multiple myeloma (MM) is a hematologic cancer characterized by expansion of malignant plasma cells in the bone marrow. Most patients develop an osteolytic bone disease, largely caused by increased osteoclastogenesis. The myeloma bone marrow is hypoxic, and hypoxia may contribute to MM disease progression, including bone loss. Here we identified interleukin-32 (IL-32) as a novel inflammatory cytokine expressed by a subset of primary MM cells and MM cell lines. We found that high IL-32 gene expression in plasma cells correlated with inferior survival in MM and that IL-32 gene expression was higher in patients with bone disease compared with those without. IL-32 was secreted from MM cells in extracellular vesicles (EVs), and those EVs, as well as recombinant human IL-32, promoted osteoclast differentiation both in vitro and in vivo. The osteoclast-promoting activity of the EVs was IL-32 dependent. Hypoxia increased plasma-cell IL-32 messenger RNA and protein levels in a hypoxia-inducible factor 1α-dependent manner, and high expression of IL-32 was associated with a hypoxic signature in patient samples, suggesting that hypoxia may promote expression of IL-32 in MM cells. Taken together, our results indicate that targeting IL-32 might be beneficial in the treatment of MM bone disease in a subset of patients.

Parallel Gene Expression Differences between Low and High Latitude Populations of Drosophila melanogaster and D. simulans.

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Zhao, Li; Wit, Janneke; Svetec, Nicolas; Begun, David J

2015-05-01

Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3' UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes.

High endothelin-converting enzyme-1 expression independently predicts poor survival of patients with esophageal squamous cell carcinoma.

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Wu, Ching-Fang; Lee, Ching-Tai; Kuo, Yao-Hung; Chen, Tzu-Haw; Chang, Chi-Yang; Chang, I-Wei; Wang, Wen-Lun

2017-09-01

Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis ( p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression ( p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression ( p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

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Yoichiro Ito

Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

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Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

2015-01-01

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

Expression Profiling during Arabidopsis/Downy Mildew Interaction Reveals a Highly-Expressed Effector That Attenuates Responses to Salicylic Acid

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Asai, Shuta; Caillaud, Marie-Cécile; Furzer, Oliver J.; Ishaque, Naveed; Wirthmueller, Lennart; Fabro, Georgina; Shirasu, Ken; Jones, Jonathan D. G.

2014-01-01

Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome. PMID:25329884

Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

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Shuta Asai

2014-10-01

Full Text Available Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

Low expression of CysLT1R and high expression of CysLT2R mediate good prognosis in colorectal cancer

DEFF Research Database (Denmark)

Magnusson, Cecilia; Mezhybovska, Maryna; Lörinc, Ester

2010-01-01

Colorectal cancer is the third most common cancer type in the Western world. In search of new treatment possibilities, the inflammation mediators, know as cysteinyl leukotrienes (CysLTs), have been shown to regulate intestinal epithelial cell survival and proliferation via the CysLT(1)R, and cell...... microarray of 329 colorectal patients. We found that high nuclear expression of CysLT(1)R is associated with a poor prognosis, whereas high nuclear expression of CysLT(2)R is associated with a good prognosis. We also observed that patients with colorectal tumours characterised by high CysLT(1)R but low Cys...

Increased expression of PPARγ in high fat diet-induced liver steatosis in mice

International Nuclear Information System (INIS)

Inoue, Mitsutaka; Ohtake, Takaaki; Motomura, Wataru; Takahashi, Nobuhiko; Hosoki, Yayoi; Miyoshi, Shigeki; Suzuki, Yasuaki; Saito, Hiroyuki; Kohgo, Yutaka; Okumura, Toshikatsu

2005-01-01

The present study was performed to examine a hypothesis that peroxisome proliferator-activated receptor γ (PPARγ) is implicated in high fat diet-induced liver steatosis. Mice were fed with control or high fat diet containing approximately 10% or 80% cholesterol, respectively. Macroscopic and microscopic findings demonstrated that lipid accumulation in the liver was observed as early as 2 weeks after high fat diet and that high fat diet for 12 weeks developed a fatty liver phenotype, establishing a novel model of diet-induced liver steatosis. Gene profiling with microarray and real-time PCR studies demonstrated that among genes involved in lipid metabolism, adipogenesis-related genes, PPARγ and its targeted gene, CD36 mRNA expression was specifically up-regulated in the liver by high fat diet for 2 weeks. Immunohistochemical study revealed that PPARγ protein expression is increased in the nuclei of hepatocytes by high fat diet. It was also shown that protein expression of cAMP response element-binding protein (CREB), an upstream molecule of PPARγ, in the liver was drastically suppressed by high fat diet. All these results suggest for the first time that the CREB-PPARγ signaling pathway may be involved in the high fat diet-induced liver steatosis

A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

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Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

2017-08-01

To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

Prediction of highly expressed genes in microbes based on chromatin accessibility

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Ussery David W

2007-02-01

Full Text Available Abstract Background It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed genes in microbial genomes. We compare these predictions with those based on codon adaptation index (CAI values, and also with experimental data for 6 different microbial genomes, with a particular interest in experimental data from Escherichia coli. Moreover, position preference is examined further in 328 sequenced microbial genomes. Results We find that absolute gene expression levels are correlated with the position preference in many microbial genomes. It is postulated that in these regions, the DNA may be more accessible to the transcriptional machinery. Moreover, ribosomal proteins and ribosomal RNA are encoded by DNA having significantly lower position preference values than other genes in fast-replicating microbes. Conclusion This insight into DNA structure-dependent gene expression in microbes may be exploited for predicting the expression of non-translated genes such as non-coding RNAs that may not be predicted by any of the conventional codon usage bias approaches.

Expression and Effects of High-Mobility Group Box 1 in Cervical Cancer

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Xiaoao Pang

2014-05-01

Full Text Available We investigated the significance of high- mobility group box1 (HMGB1 and T-cell-mediated immunity and prognostic value in cervical cancer. HMGB1, forkhead/winged helix transcription factor p3 (Foxp3, IL-2, and IL-10 protein expression was analyzed in 100 cervical tissue samples including cervical cancer, cervical intraepithelial neoplasia (CIN, and healthy control samples using immunohistochemistry. Serum squamous cell carcinoma antigen (SCC-Ag was immunoradiometrically measured in 32 serum samples from 37 cases of squamous cervical cancer. HMGB1 and SCC-Ag were then correlated to clinicopathological characteristics. HMGB1 expression tends to increase as cervical cancer progresses and it was found to be significantly correlated to FIGO stage and lymph node metastasis. These findings suggest that HMGB1 may be a useful prognostic indicator of cervical carcinoma. In addition, there were significant positive relationships between HMGB1 and FOXP3 or IL-10 expression (both p < 0.05. In contrast, HMGB1 and IL-2 expression was negatively correlated (p < 0.05. HMGB1 expression may activate Tregs or facilitate Th2 polarization to promote immune evasion of cervical cancer. Elevated HMGB1 protein in cervical carcinoma samples was associated with a high recurrence of HPV infection in univariate analysis (p < 0.05. HMGB1 expression and levels of SCC-Ag were directly correlated in SCC (p < 0.05. Thus, HMGB1 may be a useful biomarker for patient prognosis and cervical cancer prediction and treatment.

A mammalianized synthetic nitroreductase gene for high-level expression

International Nuclear Information System (INIS)

Grohmann, Maik; Paulmann, Nils; Fleischhauer, Sebastian; Vowinckel, Jakob; Priller, Josef; Walther, Diego J

2009-01-01

The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy. We performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays. In our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect. Our results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans

High epitope expression levels increase competition between T cells.

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Almut Scherer

2006-08-01

Full Text Available Both theoretical predictions and experimental findings suggest that T cell populations can compete with each other. There is some debate on whether T cells compete for aspecific stimuli, such as access to the surface on antigen-presenting cells (APCs or for specific stimuli, such as their cognate epitope ligand. We have developed an individual-based computer simulation model to study T cell competition. Our model shows that the expression level of foreign epitopes per APC determines whether T cell competition is mainly for specific or aspecific stimuli. Under low epitope expression, competition is mainly for the specific epitope stimuli, and, hence, different epitope-specific T cell populations coexist readily. However, if epitope expression levels are high, aspecific competition becomes more important. Such between-specificity competition can lead to competitive exclusion between different epitope-specific T cell populations. Our model allows us to delineate the circumstances that facilitate coexistence of T cells of different epitope specificity. Understanding mechanisms of T cell coexistence has important practical implications for immune therapies that require a broad immune response.

High-level transient expression of recombinant protein in lettuce.

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Joh, Lawrence D; Wroblewski, Tadeusz; Ewing, Nicholas N; VanderGheynst, Jean S

2005-09-30

Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta. Copyright 2005 Wiley Periodicals, Inc

A Free-Choice High-Fat High-Sugar Diet Alters Day-Night Per2 Gene Expression in Reward-Related Brain Areas in Rats.

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Blancas-Velazquez, Aurea Susana; Unmehopa, Unga A; Eggels, Leslie; Koekkoek, Laura; Kalsbeek, Andries; Mendoza, Jorge; la Fleur, Susanne E

2018-01-01

Under normal light-dark conditions, nocturnal rodents consume most of their food during the dark period. Diets high in fat and sugar, however, may affect the day-night feeding rhythm resulting in a higher light phase intake. In vitro and in vivo studies showed that nutrients affect clock-gene expression. We therefore hypothesized that overconsuming fat and sugar alters clock-gene expression in brain structures important for feeding behavior. We determined the effects of a free-choice high-fat high-sugar (fcHFHS) diet on clock-gene expression in rat brain areas related to feeding and reward and compared them with chow-fed rats. Consuming a fcHFHS diet for 6 weeks disrupted day-night differences in Per2 mRNA expression in the nucleus accumbens (NAc) and lateral hypothalamus but not in the suprachiasmatic nucleus, habenula, and ventral tegmental area. Furthermore, short-term sugar drinking, but not fat feeding, upregulates Per2 mRNA expression in the NAc. The disruptions in day-night differences in NAc Per2 gene expression were not accompanied by altered day-night differences in the mRNA expression of peptides related to food intake. We conclude that the fcHFHS diet and acute sugar drinking affect Per2 gene expression in areas involved in food reward; however, this is not sufficient to alter the day-night pattern of food intake.

High Expression of High-Mobility Group Box 1 in Menstrual Blood: Implications for Endometriosis.

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Shimizu, Keiko; Kamada, Yasuhiko; Sakamoto, Ai; Matsuda, Miwa; Nakatsuka, Mikiya; Hiramatsu, Yuji

2017-11-01

Endometriosis is a benign gynecologic disease characterized by the presence of ectopic endometrium and associated with inflammation and immune abnormalities. However, the molecular basis for endometriosis is not well understood. To address this issue, the present study examined the expression of high-mobility group box (HMGB) 1 in menstrual blood to investigate its role in the ectopic growth of human endometriotic stromal cells (ESCs). A total of 139 patients were enrolled in this study; 84 had endometriosis and 55 were nonendometriotic gynecological patients (control). The HMGB1 levels in various fluids were measured by enzyme-linked immunosorbent assay. Expression of receptor for advanced glycation end products (RAGE) in eutopic and ectopic endometrium was assessed by immunohistochemistry, and RAGE and vascular endothelial growth factor ( VEGF) messenger RNA expression in HMGB1- and lipopolysaccharide (LPS)-treated ESCs was evaluated by real-time polymerase chain reaction. The HMGB1 concentration was higher in menstrual blood than in serum or peritoneal fluid ( P endometriosis following retrograde menstruation when complexed with other factors such as LPS by inducing inflammation and angiogenesis.

High-level expression of the native barley alpha-amylase/subtilisin inhibitor in Pichia pastoris

DEFF Research Database (Denmark)

Micheelsen, Pernille Ollendorff; Ostergaard, Peter Rahbek; Lange, Lene

2008-01-01

An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed...... and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants...

Identification of a Polyomavirus microRNA Highly Expressed in Tumors

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Chen, Chun Jung; Cox, Jennifer E.; Azarm, Kristopher; Wylie, Karen N.; Woolard, Kevin D.; Pesavento, Patricia A.; Sullivan, Christopher S.

2014-01-01

Polyomaviruses (PyVs) are associated with tumors including Merkel cell carcinoma (MCC). Several PyVs encode microRNAs (miRNAs) but to date no abundant PyV miRNAs have been reported in tumors. To better understand the function of the Merkel cell PyV (MCPyV) miRNA, we examined phylogenetically-related viruses for miRNA expression. We show that two primate PyVs and the more distantly-related raccoon PyV (RacPyV) encode miRNAs that share genomic position and partial sequence identity with MCPyV miRNAs. Unlike MCPyV miRNA in MCC, RacPyV miRNA is highly abundant in raccoon tumors. RacPyV miRNA negatively regulates reporters of early viral (T antigen) transcripts, yet robust viral miRNA expression is tolerated in tumors. We also identify raccoon miRNAs expressed in RacPyV-associated neuroglial brain tumors, including several likely oncogenic miRNAs (oncomiRs). This work describes the first PyV miRNA abundantly expressed in tumors and is consistent with a possible role for both host and viral miRNAs in RacPyV-associated tumors. PMID:25514573

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

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Bottomley Stephen P

2006-03-01

Full Text Available Abstract Background In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. Results The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. Conclusion The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high

High SRPX2 protein expression predicts unfavorable clinical outcome in patients with prostate cancer

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Zhang, Meng; Li, Xiaoli; Fan, Zhirui; Zhao, Jing; Liu, Shuzheng; Zhang, Mingzhi; Li, Huixiang; Goscinski, Mariusz Adam; Fan, Huijie; Suo, Zhenhe

2018-01-01

Background Sushi repeat-containing protein X-linked 2 (SRPX2) is overexpressed in a variety of different tumor tissues and correlated with poor prognosis in patients. Little research focuses on the role of SRPX2 expression in prostate cancer (PCa), and the clinicopathological significance of the protein expression in this tumor is relatively unknown. However, our previous transcriptome data from those cancer stem-like cells indicated the role of SRPX2 in PCa. Materials and methods In this study, RT-PCR and Western blotting were firstly used to examine the SRPX2 expression in three PCa cell lines including LNCaP, DU145, and PC3, and then SRPX2 protein expression was immunohistochemically investigated and statistically analyzed in a series of 106 paraffin-embedded PCa tissue specimens. Results Significantly lower levels of SRPX2 expression were verified in the LNCaP cells, compared with the expression in the aggressive DU145 and PC3 cells, in both mRNA and protein levels. Immunohistochemically, there were variable SRPX2 protein expressions in the clinical samples. Moreover, high levels of SRPX2 expression in the PCa tissues were significantly associated with Gleason score (P=0.008), lymph node metastasis (P=0.009), and distant metastasis (P=0.021). Furthermore, higher levels of SRPX2 expression in the PCa tissues were significantly associated with shorter overall survival (OS) (P<0.001). Conclusion Our results demonstrate that SRPX2 is highly expressed in aggressive PCa cells in vitro, and its protein expression in PCa is significantly associated with malignant clinical features and shorter OS, strongly indicating its prognostic value in prostate cancers. PMID:29881288

High Dietary Fat Selectively Increases Catalase Expression within Cardiac Mitochondria*

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Rindler, Paul M.; Plafker, Scott M.; Szweda, Luke I.; Kinter, Michael

2013-01-01

Obesity is a predictor of diabetes and cardiovascular disease. One consequence of obesity is dyslipidemia characterized by high blood triglycerides. It has been proposed that oxidative stress, driven by utilization of lipids for energy, contributes to these diseases. The effects of oxidative stress are mitigated by an endogenous antioxidant enzyme network, but little is known about its response to high fat utilization. Our experiments used a multiplexed quantitative proteomics method to measure antioxidant enzyme expression in heart tissue in a mouse model of diet-induced obesity. This experiment showed a rapid and specific up-regulation of catalase protein, with subsequent assays showing increases in activity and mRNA. Catalase, traditionally considered a peroxisomal protein, was found to be present in cardiac mitochondria and significantly increased in content and activity during high fat feeding. These data, coupled with the fact that fatty acid oxidation enhances mitochondrial H2O2 production, suggest that a localized catalase increase is needed to consume excessive mitochondrial H2O2 produced by increased fat metabolism. To determine whether the catalase-specific response is a common feature of physiological conditions that increase blood triglycerides and fatty acid oxidation, we measured changes in antioxidant expression in fasted versus fed mice. Indeed, a similar specific catalase increase was observed in mice fasted for 24 h. Our findings suggest a fundamental metabolic process in which catalase expression is regulated to prevent damage while preserving an H2O2-mediated sensing of diet composition that appropriately adjusts insulin sensitivity in the short term as needed to prioritize lipid metabolism for complete utilization. PMID:23204527

High expression of testes-specific protease 50 is associated with poor prognosis in colorectal carcinoma.

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Lei Zheng

Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50 is normally expressed in testes and abnormally expressed in breast cancer, but whether TSP50 is expressed in colorectal carcinoma (CRC and its clinical significance is unclear. We aimed to detect TSP50 expression in CRC, correlate it with clinicopathological factors, and assess its potential diagnostic and prognostic value. METHODOLOGY/PRINCIPAL FINDINGS: TSP50 mRNAs and proteins were detected in 7 CRC cell lines and 8 CRC specimens via RT-PCR and Western blot analysis. Immunohistochemical analysis of TSP50, p53 and carcinoembryonic antigen (CEA with tissue microarrays composed of 95 CRCs, 20 colorectal adenomas and 20 normal colorectal tissues were carried out and correlated with clinicopathological characteristics and disease-specific survival for CRC patients. There was no significant correlation between the expression levels of TSP50 and p53 (P = 0.751 or CEA (P = 0.663. Abundant expression of TSP50 protein was found in CRCs (68.4% while it was poorly expressed in colorectal adenomas and normal tissues (P<0.0001. Thus, CRCs can be distinguished from them with high specificity (92.5% and positive predictive value (PPV, 95.6%. The survival of CRC patients with high TSP50 expression was significantly shorter than that of the patients with low TSP50 expression (P = 0.010, specifically in patients who had early-stage tumors (stage I and II; P = 0.004. Multivariate Cox regression analysis indicated that high TSP50 expression was a statistically significant independent risk factor (hazard ratio  = 2.205, 95% CI = 1.214-4.004, P = 0.009. CONCLUSION: Our data demonstrate that TSP50 is a potential effective indicator of poor survival for CRC patients, especially for those with early-stage tumors.

Mannose receptor is highly expressed by peritoneal dendritic cells in endometriosis.

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Izumi, Gentaro; Koga, Kaori; Takamura, Masashi; Makabe, Tomoko; Nagai, Miwako; Urata, Yoko; Harada, Miyuki; Hirata, Tetsuya; Hirota, Yasushi; Fujii, Tomoyuki; Osuga, Yutaka

2017-01-01

To characterize peritoneal dendritic cells (DCs) in endometriosis and to clarify their role in its etiology. Experimental. University hospital. Sixty-three women (35 patients with endometriosis and 28 control women) who had undergone laparoscopic surgery. Peritoneal DCs from endometriosis and control samples were analyzed for the expression of cell surface markers. Monocyte-derived dendritic cells (Mo-DCs) were cultured with dead endometrial stromal cells (dESCs) to investigate changes in phagocytic activity and cytokine expression. Cell surface markers and cytokine expression and identification with the use of flow cytometry or reverse-transcription polymerase chain reaction (RT-PCR). Changes in cytokine expression and phagocytic activity of Mo-DCs cultured with dESCs and d-mannan were measured with the use of flow cytometry and RT-PCR. The proportion of mannose receptor (MR)-positive myeloid DC type 1 was higher in endometriosis samples than in control samples. The blocking of MR reduced phagocytosis of dESCs by Mo-DCs. Mo-DCs cultured with dESCs expressed higher levels of interleukin (IL) 1β and IL-6 than control samples. Peritoneal DCs in endometriosis tissue express high levels of MR, which promotes phagocytosis of dead endometrial cells and thereby contributes to the etiology of endometriosis. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Sperm protein 17 is highly expressed in endometrial and cervical cancers

International Nuclear Information System (INIS)

Li, Fang-qiu; Liu, Qun; Han, Yan-ling; Wu, Bo; Yin, Hong-lin

2010-01-01

Sperm protein 17 (Sp17) is a highly conserved mammalian protein in the testis and spermatozoa and has been characterized as a tumor-associated antigen in a variety of human malignancies. Many studies have examined the role of Sp17 in tumorigenesis and the migration of malignant cells. It has been proposed as a useful target for tumor-vaccine strategies and a novel marker to define tumor subsets and predict drug response. This study aimed to investigate the expression of Sp17 in endometrial and cervical cancer specimens, its possible correlation with the pathological characteristics, and its value in the diagnosis and immunotherapy of the related cancers. The monoclonal antibodies against human Sp17 were produced as reagents for the analysis and immunohistochemistry was used to study two major kinds of paraffin-embedded gynecological cancer specimens, including 50 cases of endometrial cancer (44 adenous and 6 adenosquamous) and 31 cases of cervical cancer (15 adenous and 16 squamous). Normal peripheral endometrial and cervical tissues were used as controls. Sp17 was found in 66% (33/50) of the patients with endometrial cancer and 61% (19/31) of those with cervical cancer. Its expression was found in a heterogeneous pattern in the cancer tissues. The expression was not correlated with the histological subtype and grade of malignancy, but the staining patterns were different in endometrial and cervical cancers. The hyperplastic glands were positive for Sp17 in the normal peripheral endometrial and cervical tissues in 10% (8/81) of the patients. Sp17 is highly expressed in human endometrial and cervical cancers in a heterogeneous pattern. Although the expression frequency of Sp17 is not correlated with the histological subtype, the staining pattern may help to define endometrial and cervical cancers. Sp17 targeted immunotherapy of tumors needs more accurate validation

High expression of HEF1 is associated with poor prognosis in urinary bladder carcinoma

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Zhang Q

2014-07-01

Full Text Available Qi Zhang,1 Hui-Ju Wang,2 Da-Hong Zhang,1 Guo-Qing Ru,3 Xu-Jun He,2 Ying-Yu Ma2 1Department of Urology, 2Key Laboratory of Gastroenterology of Zhejiang Province, 3Department of Pathology, Zhejiang Provincial People's Hospital, Hangzhou, People's Republic of China Abstract: Human enhancer of filamentation 1 (HEF1 is a multidomain scaffolding protein that has been thought to play an important role in the tumor progression of various cancers. HEF1 expression has not previously been reported in urinary bladder carcinoma, and little is known about its prognostic significance. The aim of this study was to evaluate the expression patterns of HEF1 in urinary bladder carcinoma and to investigate its prognostic significance. HEF1 expression was analyzed by immunohistochemistry using tissue microarray. A significant relationship between HEF1 expression and sex, tumor size, number of tumors, invasion depth, lymph node metastasis, and distant metastasis was found, and high expression of HEF1 was associated with worse overall survival when compared to low expression of HEF1. Multivariate analysis showed that HEF1 expression was an independent prognostic factor for overall survival in urinary bladder carcinoma. We investigated HEF1 expression in urinary bladder carcinoma and found that high HEF1 expression was associated with advanced stage, large tumor size, and shortened progression-free survival. Although the biologic function of HEF1 in urinary bladder carcinoma remains unknown, the expression of HEF1 can provide new prognostic information for disease progression. Keywords: human enhancer of filamentation 1, progression-free survival, immunohistochemistry, metastasis, bladder cancer

Histone H1x is highly expressed in human neuroendocrine cells and tumours

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Warneboldt, Julia; Haller, Florian; Horstmann, Olaf; Danner, Bernhard C; Füzesi, László; Doenecke, Detlef; Happel, Nicole

2008-01-01

Histone H1x is a ubiquitously expressed member of the H1 histone family. H1 histones, also called linker histones, stabilize compact, higher order structures of chromatin. In addition to their role as structural proteins, they actively regulate gene expression and participate in chromatin-based processes like DNA replication and repair. The epigenetic contribution of H1 histones to these mechanisms makes it conceivable that they also take part in malignant transformation. Based on results of a Blast data base search which revealed an accumulation of expressed sequence tags (ESTs) of H1x in libraries from neuroendocrine tumours (NETs), we evaluated the expression of H1x in NETs from lung and the gastrointestinal tract using immunohistochemisty. Relative protein and mRNA levels of H1x were analysed by Western blot analysis and quantitative real-time RT-PCR, respectively. Since several reports describe a change of the expression level of the replacement subtype H1.0 during tumourigenesis, the analysis of this subtype was included in this study. We found an increased expression of H1x but not of H1.0 in NET tissues in comparison to corresponding normal tissues. Even though the analysed NETs were heterogenous regarding their grade of malignancy, all except one showed a considerably higher protein amount of H1x compared with corresponding non-neoplastic tissue. Furthermore, double-labelling of H1x and chromogranin A in sections of pancreas and small intestine revealed that H1x is highly expressed in neuroendocrine cells of these tissues. We conclude that the high expression of histone H1x in NETs is probably due to the abundance of this protein in the cells from which these tumours originate

Intermittent Hypoxia Is Associated With High Hypoxia Inducible Factor-1α but Not High Vascular Endothelial Growth Factor Cell Expression in Tumors of Cutaneous Melanoma Patients

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Isaac Almendros

2018-04-01

Full Text Available Epidemiological associations linking between obstructive sleep apnea and poorer solid malignant tumor outcomes have recently emerged. Putative pathways proposed to explain that these associations have included enhanced hypoxia inducible factor (HIF-1α and vascular endothelial growth factor (VEGF cell expression in the tumor and altered immune functions via intermittent hypoxia (IH. Here, we examined relationships between HIF-1α and VEGF expression and nocturnal IH in cutaneous melanoma (CM tumor samples. Prospectively recruited patients with CM tumor samples were included and underwent overnight polygraphy. General clinical features, apnea–hypopnea index (AHI, desaturation index (DI4%, and CM characteristics were recorded. Histochemical assessments of VEGF and HIF-1α were performed, and the percentage of positive cells (0, <25, 25–50, 51–75, >75% was blindly tabulated for VEGF expression, and as 0, 0–5.9, 6.0–10.0, >10.0% for HIF-1α expression, respectively. Cases with HIF-1α expression >6% (high expression were compared with those <6%, and VEGF expression >75% of cells was compared with those with <75%. 376 patients were included. High expression of VEGF and HIF-1α were seen in 88.8 and 4.2% of samples, respectively. High expression of VEGF was only associated with increasing age. However, high expression of HIF-1α was significantly associated with age, Breslow index, AHI, and DI4%. Logistic regression showed that DI4% [OR 1.03 (95% CI: 1.01–1.06] and Breslow index [OR 1.28 (95% CI: 1.18–1.46], but not AHI, remained independently associated with the presence of high HIF-1α expression. Thus, IH emerges as an independent risk factor for higher HIF-1α expression in CM tumors and is inferentially linked to worse clinical CM prognostic indicators.

Reduced Variance of Gene Expression at Numerous Loci in a Population of Chickens Selected for High Feather Pecking

DEFF Research Database (Denmark)

Hughes, A L; Buitenhuis, A J

2010-01-01

among populations with respect to mean expression scores, but numerous transcripts showed reduced variance in expression scores in the high FP population in comparison to control and low FP populations. The reduction in variance in the high FP population generally involved transcripts whose expression...

MicroRNA-130a is highly expressed in the esophageal mucosa of achalasia patients.

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Shoji, Hiroyuki; Isomoto, Hajime; Yoshida, Akira; Ikeda, Haruo; Minami, Hitomi; Kanda, Tsutomu; Urabe, Shigetoshi; Matsushima, Kayoko; Takeshima, Fuminao; Nakao, Kazuhiko; Inoue, Haruhiro

2017-08-01

Esophageal achalasia is considered as a risk factor of esophageal cancer. The etiologies of esophageal achalasia remain unknown. Peroral endoscopic myotomy (POEM) has recently been established as a minimally invasive method with high curability. The aims of the present study were to identify the microRNAs (miRs) specific to esophageal achalasia, to determine their potential target genes and to assess their alteration following POEM. RNA was extracted from biopsy samples from middle esophageal mucosa and analyzed using a microarray. Differentially expressed miRs in achalasia patients compared with control samples were identified and analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlations between specific miR expression levels and the patients' clinical background were also investigated. In addition, alterations of selected miR expression levels before and after POEM were analyzed. The results of RT-qPCR analysis demonstrated that the miR-130a expression levels were significantly higher in patients with achalasia (Pachalasia. However, no significant change in miR-130a expression was observed between before and after POEM. In conclusion, miR-130a is highly expressed in the esophageal mucosa of patients with achalasia and may be a biomarker of esophageal achalasia.

Validation of the Amsterdam Dynamic Facial Expression Set--Bath Intensity Variations (ADFES-BIV: A Set of Videos Expressing Low, Intermediate, and High Intensity Emotions.

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Tanja S H Wingenbach

Full Text Available Most of the existing sets of facial expressions of emotion contain static photographs. While increasing demand for stimuli with enhanced ecological validity in facial emotion recognition research has led to the development of video stimuli, these typically involve full-blown (apex expressions. However, variations of intensity in emotional facial expressions occur in real life social interactions, with low intensity expressions of emotions frequently occurring. The current study therefore developed and validated a set of video stimuli portraying three levels of intensity of emotional expressions, from low to high intensity. The videos were adapted from the Amsterdam Dynamic Facial Expression Set (ADFES and termed the Bath Intensity Variations (ADFES-BIV. A healthy sample of 92 people recruited from the University of Bath community (41 male, 51 female completed a facial emotion recognition task including expressions of 6 basic emotions (anger, happiness, disgust, fear, surprise, sadness and 3 complex emotions (contempt, embarrassment, pride that were expressed at three different intensities of expression and neutral. Accuracy scores (raw and unbiased (Hu hit rates were calculated, as well as response times. Accuracy rates above chance level of responding were found for all emotion categories, producing an overall raw hit rate of 69% for the ADFES-BIV. The three intensity levels were validated as distinct categories, with higher accuracies and faster responses to high intensity expressions than intermediate intensity expressions, which had higher accuracies and faster responses than low intensity expressions. To further validate the intensities, a second study with standardised display times was conducted replicating this pattern. The ADFES-BIV has greater ecological validity than many other emotion stimulus sets and allows for versatile applications in emotion research. It can be retrieved free of charge for research purposes from the

Validation of the Amsterdam Dynamic Facial Expression Set--Bath Intensity Variations (ADFES-BIV): A Set of Videos Expressing Low, Intermediate, and High Intensity Emotions.

Science.gov (United States)

Wingenbach, Tanja S H; Ashwin, Chris; Brosnan, Mark

2016-01-01

Most of the existing sets of facial expressions of emotion contain static photographs. While increasing demand for stimuli with enhanced ecological validity in facial emotion recognition research has led to the development of video stimuli, these typically involve full-blown (apex) expressions. However, variations of intensity in emotional facial expressions occur in real life social interactions, with low intensity expressions of emotions frequently occurring. The current study therefore developed and validated a set of video stimuli portraying three levels of intensity of emotional expressions, from low to high intensity. The videos were adapted from the Amsterdam Dynamic Facial Expression Set (ADFES) and termed the Bath Intensity Variations (ADFES-BIV). A healthy sample of 92 people recruited from the University of Bath community (41 male, 51 female) completed a facial emotion recognition task including expressions of 6 basic emotions (anger, happiness, disgust, fear, surprise, sadness) and 3 complex emotions (contempt, embarrassment, pride) that were expressed at three different intensities of expression and neutral. Accuracy scores (raw and unbiased (Hu) hit rates) were calculated, as well as response times. Accuracy rates above chance level of responding were found for all emotion categories, producing an overall raw hit rate of 69% for the ADFES-BIV. The three intensity levels were validated as distinct categories, with higher accuracies and faster responses to high intensity expressions than intermediate intensity expressions, which had higher accuracies and faster responses than low intensity expressions. To further validate the intensities, a second study with standardised display times was conducted replicating this pattern. The ADFES-BIV has greater ecological validity than many other emotion stimulus sets and allows for versatile applications in emotion research. It can be retrieved free of charge for research purposes from the corresponding author.

Andrographolide suppresses high glucose-induced fibronectin expression in mesangial cells via inhibiting the AP-1 pathway.

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Lan, Tian; Wu, Teng; Gou, Hongju; Zhang, Qianqian; Li, Jiangchao; Qi, Cuiling; He, Xiaodong; Wu, Pingxiang; Wang, Lijing

2013-11-01

Mesangial cells (MCs) proliferation and accumulation of glomerular matrix proteins such as fibronectin (FN) are the early features of diabetic nephropathy, with MCs known to upregulate matrix protein synthesis in response to high glucose. Recently, it has been found that andrographolide has renoprotective effects on diabetic nephropathy. However, the molecular mechanism underlying these effects remains unclear. Cell viability and proliferation was evaluated by MTT. FN expression was examined by immunofluorescence and immunoblotting. Activator protein-1 (AP-1) activation was assessed by immunoblotting, luciferase reporter and electrophoretic mobility shift assays. Andrographolide significantly decreased high glucose-induced cell proliferation and FN expression in MCs. Exposure of MCs to high glucose markedly stimulated the expression of phosphorylated c-jun, whereas the stimulation was inhibited by andrographolide. Plasmid pAP-1-Luc luciferase reporter assay showed that andrographolide blocked high glucose-induced AP-1 transcriptional activity. EMSA assay demonstrated that increased AP-1 binding to an AP-1 binding site at -1,029 in the FN gene promoter upon high glucose stimulation, and the binding were disrupted by andrographolide treatment. These data indicate that andrographolide suppresses high glucose-induced FN expression by inhibiting AP-1-mediated pathway. © 2013 Wiley Periodicals, Inc.

Effect of high fat diet on pulmonary expression of parathyroid hormone-related protein and its downstream targets

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Learta Oruqaj

2016-10-01

Full Text Available Aims: Parathyroid hormone-related protein (PTHrP is involved in lung development and surfactant production. The latter one requires a paracrine interaction between type II alveolar cells and lipofibroblasts in which leptin triggers PTHrP-induced effects. Whether increased plasma leptin levels, as they occur in high fat diet, modify the expression of PTHrP remains unclear. Furthermore, the effect of high fat diet under conditions of forced pulmonary remodelling such as response to post myocardial infarction remains to be defined. Materials and methods: C57 bl/6 mice were randomized to either normal diet or high fat diet at an age of 6 weeks. Seven months later, the mice were euthanized and the lung was removed and frozen in fluid nitrogen until use. Samples were analyzed by real-time RT-PCR and western blot. Leptin deficient mice were used to investigate the effect of leptin on pulmonary expression of PTHrP more directly. A subgroup of mice with and without high fat diet underwent in vivo ischemia (45 min and reperfusion (4 weeks. Finally, experiments were repeated with prolonged high-fat diet. Key findings: High fat diet increased plasma leptin levels by 30.4% and the pulmonary mRNA expression of PTHrP (1,447-fold, PTH-1 receptor (4.21-fold, and PTHrP-downstream targets ADRP (7.54-fold and PPARγ (5.27-fold. Pulmonary PTHrP expression was reduced in leptin deficient mice by 88% indicating leptin dependent regulation. High fat diet further improved changes in pulmonary adaptation caused by ischemia/reperfusion (1.48-fold increased PTH-1 receptor protein expression. These effects were lost during prolonged high fat diet. Significance: This study established that physiological regulation of leptin plasma levels by high fat diet affects the pulmonary PTHrP expression and of PTHrP downstream targets. Modification of pulmonary expression of PTH-1 receptors by high fat diet after myocardial infarction suggests that the identified interaction may

Effects of high temperature on photosynthesis and related gene expression in poplar

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2014-01-01

Background High temperature, whether transitory or constant, causes physiological, biochemical and molecular changes that adversely affect tree growth and productivity by reducing photosynthesis. To elucidate the photosynthetic adaption response and examine the recovery capacity of trees under heat stress, we measured gas exchange, chlorophyll fluorescence, electron transport, water use efficiency, and reactive oxygen-producing enzyme activities in heat-stressed plants. Results We found that photosynthesis could completely recover after less than six hours of high temperature treatment, which might be a turning point in the photosynthetic response to heat stress. Genome-wide gene expression analysis at six hours of heat stress identified 29,896 differentially expressed genes (15,670 up-regulated and 14,226 down-regulated), including multiple classes of transcription factors. These interact with each other and regulate the expression of photosynthesis-related genes in response to heat stress, controlling carbon fixation and changes in stomatal conductance. Heat stress of more than twelve hours caused reduced electron transport, damaged photosystems, activated the glycolate pathway and caused H2O2 production; as a result, photosynthetic capacity did not recover completely. Conclusions This study provides a systematic physiological and global gene expression profile of the poplar photosynthetic response to heat stress and identifies the main limitations and threshold of photosynthesis under heat stress. It will expand our understanding of plant thermostability and provides a robust dataset for future studies. PMID:24774695

High-dimensional gene expression profiling studies in high and low responders to primary smallpox vaccination.

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Haralambieva, Iana H; Oberg, Ann L; Dhiman, Neelam; Ovsyannikova, Inna G; Kennedy, Richard B; Grill, Diane E; Jacobson, Robert M; Poland, Gregory A

2012-11-15

The mechanisms underlying smallpox vaccine-induced variations in immune responses are not well understood, but are of considerable interest to a deeper understanding of poxvirus immunity and correlates of protection. We assessed transcriptional messenger RNA expression changes in 197 recipients of primary smallpox vaccination representing the extremes of humoral and cellular immune responses. The 20 most significant differentially expressed genes include a tumor necrosis factor-receptor superfamily member, an interferon (IFN) gene, a chemokine gene, zinc finger protein genes, nuclear factors, and histones (P ≤ 1.06E(-20), q ≤ 2.64E(-17)). A pathway analysis identified 4 enriched pathways with cytokine production by the T-helper 17 subset of CD4+ T cells being the most significant pathway (P = 3.42E(-05)). Two pathways (antiviral actions of IFNs, P = 8.95E(-05); and IFN-α/β signaling pathway, P = 2.92E(-04)), integral to innate immunity, were enriched when comparing high with low antibody responders (false discovery rate, < 0.05). Genes related to immune function and transcription (TLR8, P = .0002; DAPP1, P = .0003; LAMP3, P = 9.96E(-05); NR4A2, P ≤ .0002; EGR3, P = 4.52E(-05)), and other genes with a possible impact on immunity (LNPEP, P = 3.72E(-05); CAPRIN1, P = .0001; XRN1, P = .0001), were found to be expressed differentially in high versus low antibody responders. We identified novel and known immunity-related genes and pathways that may account for differences in immune response to smallpox vaccination.

High SHIP2 Expression Indicates Poor Survival in Colorectal Cancer

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Ju Yang

2014-01-01

Full Text Available SH2-containing inositol 5′-phosphatase 2 (SHIP2, which generally regulates insulin signaling, cytoskeleton remodeling, and receptor endocytosis, has been suggested to play a significant role in tumor development and progression. However, the associations between SHIP2 expression and the clinical features to evaluate its clinicopathologic significance in colorectal cancer (CRC have not been determined yet. In the present study, one-step quantitative real-time polymerase chain reaction (qPCR test and immunohistochemistry (IHC analysis with CRC tissue microarrays (TMA were employed to evaluate the mRNA and protein expression of SHIP2 in CRC. The results showed that SHIP2 expression in the mRNA and protein levels was significantly higher in CRC tissues than that in corresponding noncancerous tissues (both P<0.05. The expression of SHIP2 protein in CRC was related to lymph node metastasis (P=0.036, distant metastasis (P=0.001, and overall survival (P=0.009. Kaplan-Meier method and Cox multifactor analysis suggested that high SHIP2 protein level (P=0.040 and positive distant metastasis (P=0.048 were critically associated with the unfavorable survival of CRC patients. The findings suggested that SHIP2 may be identified as a useful prognostic marker in CRC and targeting CRC may provide novel strategy for CRC treatment.

Sequence and expression analyses of ethylene response factors highly expressed in latex cells from Hevea brasiliensis.

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Piyanuch Piyatrakul

Full Text Available The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors.

Differential gene expression in patients with anal fistula reveals high levels of prolactin recepetor

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Song Yi-Huan

2017-01-01

Full Text Available Background/Aim. There are limited data examining variations in the local expression of inflammatory mediators in anal fistulas where it is anticipated that an improved understanding of the inflammatory milieu might lead to the potential therapeutic option of instillation therapy in complicated cases. The aim of the present study was to examine prolactin receptors (PRLR as inflammatory markers and to correlate their expression with both the complexity of anal fistulas and the likelihood of fistula recurrence. Methods. Microarray was used to screen the differentially expressed gene profile of anal fistula using anal mucosa samples with hemorrhoids with ageand sex-matched patients as controls and then a prospective analysis of 65 patients was conducted with anal fistulas. PRLR immunohistochemistry was performed to define expression in simple, complex and recurrent anal fistula cases. The quantitative image comparison was performed combining staining intensity with cellular distribution in order to create high and low score PRLR immunohistochemical groupings. Results. A differential expression profile of 190 genes was found. PRLR expression was 2.91 times lower in anal fistula compared with control. Sixty-five patients were assessed (35 simple, 30 complex cases. Simple fistulas showed significantly higher PRLR expression than complex cases with recurrent fistulae showing overall lower PRLR expression than de novo cases (p = 0.001. These findings were reflected in measurable integrated optical density for complex and recurrent cases (complex cases, 8.31 ± 4.91 x 104 vs simple cases, 12.30 ± 6.91 x 104; p < 0.01; recurrent cases, 7.21 ± 3.51 x 104 vs primarily healing cases, 8.31 ± 4.91 x 104; p < 0.05. In univariate regression analysis, low PRLR expression correlated with fistula complexity; a significant independent effect maintained in multivariate analysis odds ratio [(OR low to high PRLR expression = 9.52; p = 0.001]. Conclusion. PRLR

Expression of the potential therapeutic target CXXC5 in primary acute myeloid leukemia cells - high expression is associated with adverse prognosis as well as altered intracellular signaling and transcriptional regulation.

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Bruserud, Øystein; Reikvam, Håkon; Fredly, Hanne; Skavland, Jørn; Hagen, Karen-Marie; van Hoang, Tuyen Thy; Brenner, Annette K; Kadi, Amir; Astori, Audrey; Gjertsen, Bjørn Tore; Pendino, Frederic

2015-02-20

The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication.

LncRNA Expression Profile of Human Thoracic Aortic Dissection by High-Throughput Sequencing.

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Sun, Jie; Chen, Guojun; Jing, Yuanwen; He, Xiang; Dong, Jianting; Zheng, Junmeng; Zou, Meisheng; Li, Hairui; Wang, Shifei; Sun, Yili; Liao, Wangjun; Liao, Yulin; Feng, Li; Bin, Jianping

2018-01-01

In this study, the long non-coding RNA (lncRNA) expression profile in human thoracic aortic dissection (TAD), a highly lethal cardiovascular disease, was investigated. Human TAD (n=3) and normal aortic tissues (NA) (n=3) were examined by high-throughput sequencing. Bioinformatics analyses were performed to predict the roles of aberrantly expressed lncRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to validate the results. A total of 269 lncRNAs (159 up-regulated and 110 down-regulated) and 2, 255 mRNAs (1 294 up-regulated and 961 down-regulated) were aberrantly expressed in human TAD (fold-change> 1.5, PTAD than in NA. The predicted binding motifs of three up-regulated lncRNAs (ENSG00000248508, ENSG00000226530, and EG00000259719) were correlated with up-regulated RUNX1 (R=0.982, PTAD. These findings suggest that lncRNAs are novel potential therapeutic targets for human TAD. © 2018 The Author(s). Published by S. Karger AG, Basel.

Expressed microRNA associated with high rate of egg production in chicken ovarian follicles.

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Wu, N; Gaur, U; Zhu, Q; Chen, B; Xu, Z; Zhao, X; Yang, M; Li, D

2017-04-01

MicroRNA (miRNA) is a highly conserved class of small noncoding RNA about 19-24 nucleotides in length that function in a specific manner to post-transcriptionally regulate gene expression in organisms. Tissue miRNA expression studies have discovered a myriad of functions for miRNAs in various aspects, but a role for miRNAs in chicken ovarian tissue at 300 days of age has not hitherto been reported. In this study, we performed the first miRNA analysis of ovarian tissues in chickens with low and high rates of egg production using high-throughput sequencing. By comparing low rate of egg production chickens with high rate of egg production chickens, 17 significantly differentially expressed miRNAs were found (P chickens with high rates of egg production, suggesting that these miRNAs have an important role in ovary development and reproductive management of chicken. Furthermore, we uncovered that a significantly up-regulated miRNA-gga-miR-200a-3p-is ubiquitous in reproduction-regulation-related pathways. This miRNA may play a special central role in the reproductive management of chicken, and needs to be further studied for confirmation. © 2016 Stichting International Foundation for Animal Genetics.

Effects of dietary high fructose corn syrup on regulation of energy intake and leptin gene expression in rats

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Guadalupe López-Rodríguez

2015-12-01

Full Text Available OBJECTIVE: To evaluate in Wistar rats the effect of chronic use of high fructose corn syrup on serum lipids, body weight, energy intake regulation, and expression of associated genes. METHODS: For 11 weeks, male rats were fed a standard diet with either water (control or 15% high fructose corn syrup solution, or fed a high-fat diet. The rats' food intake and body weight were measured weekly. Expression of leptin and fatty acid synthase genes was quantified in their brain and adipose tissue upon sacrifice at age 119 days using real-time polymerase chain reaction. RESULTS: The intake of 15% high fructose corn syrup did not affect the rats' weight, only the rats on the high-fat diet gained significant weight. The rats in both diets had lower levels of leptin expression and high levels of fatty acid synthase in the brain, which were associated with high serum triglycerides. CONCLUSION: Fifteen percent high fructose corn syrup intake and the high-fat diet reduced leptin gene expression in the brain of Wistar rats, with differential effects on weight gain.

Validation of the Amsterdam Dynamic Facial Expression Set – Bath Intensity Variations (ADFES-BIV): A Set of Videos Expressing Low, Intermediate, and High Intensity Emotions

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Wingenbach, Tanja S. H.

2016-01-01

Most of the existing sets of facial expressions of emotion contain static photographs. While increasing demand for stimuli with enhanced ecological validity in facial emotion recognition research has led to the development of video stimuli, these typically involve full-blown (apex) expressions. However, variations of intensity in emotional facial expressions occur in real life social interactions, with low intensity expressions of emotions frequently occurring. The current study therefore developed and validated a set of video stimuli portraying three levels of intensity of emotional expressions, from low to high intensity. The videos were adapted from the Amsterdam Dynamic Facial Expression Set (ADFES) and termed the Bath Intensity Variations (ADFES-BIV). A healthy sample of 92 people recruited from the University of Bath community (41 male, 51 female) completed a facial emotion recognition task including expressions of 6 basic emotions (anger, happiness, disgust, fear, surprise, sadness) and 3 complex emotions (contempt, embarrassment, pride) that were expressed at three different intensities of expression and neutral. Accuracy scores (raw and unbiased (Hu) hit rates) were calculated, as well as response times. Accuracy rates above chance level of responding were found for all emotion categories, producing an overall raw hit rate of 69% for the ADFES-BIV. The three intensity levels were validated as distinct categories, with higher accuracies and faster responses to high intensity expressions than intermediate intensity expressions, which had higher accuracies and faster responses than low intensity expressions. To further validate the intensities, a second study with standardised display times was conducted replicating this pattern. The ADFES-BIV has greater ecological validity than many other emotion stimulus sets and allows for versatile applications in emotion research. It can be retrieved free of charge for research purposes from the corresponding author

High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

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Bonvini, P; Zin, A; Alaggio, R; Pawel, B; Bisogno, G; Rosolen, A

2013-01-01

Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. Methods: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT–PCR, western blotting, IHC, and copy number analysis. Results: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (Ptumour size (PALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included. Conclusion: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour. PMID:24149177

Maternal low protein diet and postnatal high fat diet increases adipose imprinted gene expression

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Maternal and postnatal diet can alter Igf2 gene expression and DNA methylation. To test whether maternal low protein and postnatal high fat (HF) diet result in alteration in Igf2 expression and obesity, we fed obese-prone Sprague-Dawley rats 8% (LP) or 20% (NP) protein for 3 wk prior to breeding and...

Freedom of Expression Laws and the College Press: Lessons Learned from the High Schools.

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Paxton, Mark

This paper examines two recent attempts to enact state freedom of expression laws for public college and university students and discusses the prospects for such laws in the context of state scholastic freedom of expression laws covering high school journalists in six states. It examines the case of Kincaid v. Gibson, which decided that…

The influence of bovine milk high or low in isoflavones on hepatic gene expression in mice

DEFF Research Database (Denmark)

Skaanild, Mette Tingleff; Nielsen, Tina Skau

2012-01-01

Isoflavones have generated much attention due to their potential positive effects in various diseases. Phytoestrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phytoestrogens. The aim of this study was to analyze the changes...... in hepatic gene expression after dietary intake of milk high and low in isoflavones. In addition to pelleted feed female NMRI mice were offered water, water added either 17β-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array q......PCR kit. It was revealed that Tween 80 and 17β-estradiol upregulated both phase I and phase II genes to the same extent whereas equol alone, high and low isoflavone milk did not alter the expression of phase I genes but decreased the expression of phase II genes. This study shows that dietary isoflavones...

Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer

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Hansen, Torben Frøstrup; Andersen, Claus Lindbjerg; Nielsen, Boye Schnack

2011-01-01

was to analyse the possible relationship between miRNA-126, VEGFR-2 and angiogenesis in tumour tissue from patients with colorectal cancer (CRC). Tumour tissue was obtained from 81 patients. The miRNA-126 and VEGFR-2 gene expression levels were analysed by PCR and the protein concentrations of VEGFR-2 were...... the median as the cut-off. The median gene expression levels of VEGFR-2 were significantly lower in the tumours expressing low levels of miRNA-126, 0.30 (95% CI, 0.24‑0.36), compared to those expressing high levels of miRNA-126, 0.48 (95% CI, 0.28-0.60), p=0.02. A positive association was observed with VEGFR...... analysed by ELISA. Angiogenesis, visualised by the endothelial cell marker CD105 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridisation was performed for miRNA-126. Tumours were classified as low or high miRNA‑126-expressing using...

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

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Yacoby, I.; Tegler, L. T.; Pochekailov, S.; Zhang, S.; King, P. W.

2012-04-01

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

Effects of BST and high energy diet on gene expression in mammary parenchyma of dairy heifers

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Betina Joyce Lew

2013-07-01

Full Text Available The objective of this study was to determine the effects of dietary energy and recombinant bovine somatotropin (bST injection to identify genes that might control mammogenesis. Total RNA was extracted from the parenchymal tissue of 32 heifers randomly assigned to one of four treatments: two diets (a standard diet and a high energy, high protein diet, each with or without bST. To perform microarray experiments, RNA samples were pooled (2 animals/pool before reverse transcription and labeling with Cy3 or Cy5. A 4-node loop design was used to examine the differential gene expression among treatments using a bovine-specific cDNA microarray (National Bovine Functional Genomics Consortium Library, NBFGC containing 18,263 unique expressed sequence tags (EST. Significance levels of differential gene expression among treatments were assessed using a mixed model approach. Injection of bST altered the expression of 12 % of the genes on NBFGC slide related to tissue development, whereas 6% were altered by diet. Administration of bST increases the expression of genes positively related to cell proliferation and mammary parenchyma to a greater extent than a high energy diet.

LCR/MEL: A versatile system for high-level expression of heterologous proteins in erythroid cells.

NARCIS (Netherlands)

M. Needham; C. Gooding; K. Hudson; M. Antoniou (Michael); F.G. Grosveld (Frank); M. Hollis

1992-01-01

textabstractWe have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin

Masked expression of life-history traits in a highly variable environment

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DeBoer, Jason A.; Fontaine, Joseph J.; Chizinski, Christopher J.; Pope, Kevin L.

2015-01-01

Differing life-history strategies may act as a constraint on reproductive expression that ultimately limits the ability of individual species to respond to changes in the magnitude or frequency of environmental variation, and potentially underlies the variation often inherent in phenotypic and evolved responses to anthropogenic change. Alternatively, if there are environmental cues that predict reproductive potential, differential expression of life-history strategies may represent differences in the adaptive capacity to optimize current reproductive value given variation in environmental conditions. We compared several aspects of walleye Sander vitreus spawning ecology at two reservoirs that differ in environmental variability (i.e., annual water-level fluctuation) to identify the capacity of phenotypic expression and the corresponding association with age. Despite significant differences in female body and liver masses between reservoirs that differ in environmental variability, we found no difference in reproductive investment measured by egg size and fecundity. Walleye in a highly variable environment appear to exhibit reproductive traits more typical of a short-lived life-history strategy, which may be resultant from the interaction of environmental and anthropogenic pressures. This finding emphasizes the need to identify the degree to which life-history expression represents physiological constraints versus ecological optimization, particularly as anthropogenic change continues to alter environmental conditions. 

Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase.

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Romero, Lucía Virginia; Targovnik, Alexandra Marisa; Wolman, Federico Javier; Cascone, Osvaldo; Miranda, María Victoria

2011-05-01

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots. © Springer Science+Business Media B.V. 2011

Cyclophosphamide Alters the Gene Expression Profile in Patients Treated with High Doses Prior to Stem Cell Transplantation

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El-Serafi, Ibrahim; Abedi-Valugerdi, Manuchehr; Potácová, Zuzana; Afsharian, Parvaneh; Mattsson, Jonas; Moshfegh, Ali; Hassan, Moustapha

2014-01-01

Background Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. Methods We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. Results Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. Conclusion This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression. PMID:24466173

High expression of L-type amino acid transporter 1 as a prognostic marker in bile duct adenocarcinomas

International Nuclear Information System (INIS)

Yanagisawa, Nobuyuki; Hana, Kiyomi; Nakada, Norihiro; Ichinoe, Masaaki; Koizumi, Wasaburo; Endou, Hitoshi; Okayasu, Isao; Murakumo, Yoshiki

2014-01-01

Oncocytic L-type amino acid transporter (LAT) 1 may be a prognostic indicator and target of new molecular therapeutic agents against malignancies. To investigate whether LAT1 expression influence the outcomes of patients with bile duct cancer, the expression of LAT1, LAT2, CD98, and Ki-67 was investigated immunohistochemically in 134 surgically resected bile duct adenocarcinomas, including 84 distal extrahepatic bile duct adenocarcinomas, 21 hilar cholangiocarcinomas, 15 intrahepatic cholangiocarcinomas, and 14 ampullary adenocarcinomas. LAT1 expression was weakly correlated with CD98 expression and Ki-67 labeling index (LI). Kaplan–Meier analysis showed a significant difference in prognosis between patients with bile duct adenocarcinomas having LAT1-high and -low scores, whereas LAT2 and CD98 expression and Ki-67 LI were not predictive of poor prognosis. Prognosis tended to be worse in patients having tumors with LAT1-high/LAT2-low than LAT1-low/LAT2-high scores (P = 0.0686). Multivariable analyses revealed that LAT1 expression, surgical margin, pT stage were independent prognostic factors. In conclusion, aberrant overexpression of LAT1 in bile duct adenocarcinoma predicts poor prognosis, suggesting that LAT1 may be a potential target of anticancer therapy

Emotional Experience, Expression, and Regulation of High-Quality Japanese Elementary School Teachers

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Hosotani, Rika; Imai-Matsumura, Kyoko

2011-01-01

The present study investigates the emotional experience, expression, and regulation processes of high-quality Japanese elementary school teachers while they interact with children, in terms of teachers' emotional competence. Qualitative analysis of interview data demonstrated that teachers had various emotional experiences including self-elicited…

FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

International Nuclear Information System (INIS)

Pašaliç, Z; Greif, P A; Jurinoviç, V; Mulaw, M; Kakadia, P M; Tizazu, B; Fröhlich-Archangelo, L; Krause, A; Bohlander, S K

2011-01-01

The t(10;11)(p13;q14) translocation results in the fusion of the CALM (clathrin assembly lymphoid myeloid leukemia protein) and AF10 genes. This translocation is observed in acute myeloblastic leukemia (AML M6), acute lymphoblastic leukemia (ALL) and malignant lymphoma. Using a yeast two-hybrid screen, the four and a half LIM domain protein 2 (FHL2) was identified as a CALM interacting protein. Recently, high expression of FHL2 in breast, gastric, colon, lung as well as in prostate cancer was shown to be associated with an adverse prognosis. The interaction between CALM and FHL2 was confirmed by glutathione S-transferase-pulldown assay and co-immunoprecipitation experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294–335 of CALM. The transcriptional activation capacity of FHL2 was reduced by CALM, but not by CALM/AF10, which suggests that regulation of FHL2 by CALM might be disturbed in CALM/AF10-positive leukemia. Extremely high expression of FHL2 was seen in acute erythroid leukemia (AML M6). FHL2 was also highly expressed in chronic myeloid leukemia and in AML with complex aberrant karyotype. These results suggest that FHL2 may play an important role in leukemogenesis, especially in the case of AML M6

Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

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Mandy Y M Lo

Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

Machine learning in computational biology to accelerate high-throughput protein expression

DEFF Research Database (Denmark)

Sastry, Anand; Monk, Jonathan M.; Tegel, Hanna

2017-01-01

and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide...... the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. Availability and implementation: We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template...

Requirements for growth and IL-10 expression of highly purified human T regulatory cells

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Bonacci, Benedetta; Edwards, Brandon; Jia, Shuang; Williams, Calvin; Hessner, Martin J.; Gauld, Stephen; Verbsky, James

2013-01-01

Human regulatory T cells (TR) cells have potential for the treatment of a variety of immune mediated diseases but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human TR cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9D4) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting TR cell proliferation, and under these conditions the proliferative capacity of TR cells was comparable to conventional CD4 lymphocytes. Blocking TGF-β activity abrogated IL-10 expression from these cells, while addition of TGF-β resulted in IL-10 production. These data demonstrate that highly purified populations of TR cells are anergic even in the presence of high doses of IL-2. Furthermore, antigen presenting cells provide proper co-stimulation to overcome the anergic phenotype of TR cells, and under these conditions they are highly sensitive to IL-2. In addition, these data demonstrate for the first time that TGF-β is critical to enable human TR cells to express IL-10. PMID:22562448

Myostatin expression, lymphocyte population, and potential cytokine production correlate with predisposition to high-fat diet induced obesity in mice.

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Jeri-Anne Lyons

2010-09-01

Full Text Available A strong relationship exists between increased inflammatory cytokines and muscle insulin resistance in obesity. This study focused on identifying a relationship between metabolic propensity and myostatin expression in muscle and spleen cells in response to high-fat diet intake. Using a comparative approach, we analyzed the effects of high-fat diet intake on myostatin and follistatin expression, spleen cell composition, and potential cytokine expression in high-fat diet induced obesity (HFDIO resistant (SWR/J and susceptible (C57BL/6 mice models. Results demonstrated overall increased myostatin expression in muscle following high-fat diet intake in HFDIO-susceptible mice, while myostatin expression levels decreased initially in muscle from high-fat diet fed resistant mice. In HFDIO-resistant mice, myostatin expression decreased in spleen, while myostatin increased in spleen tissue from HFDIO-susceptible mice. Proinflammatory cytokine (IL-17, IL-1β, and IFNγ potential increased in splenocytes from HFDIO-susceptible mice. In comparison, C57BL/6 mice fed a high-fat diet exhibited higher frequencies of CD4(+/CD44(hi and CD8(+/CD44(hi cells in the spleen compared to control fed mice. Together, these results suggest that susceptibility to high-fat diet induced obesity could be influenced by local myostatin activity in a tissue-specific manner and that splenocytes exhibit differential cytokine production in a strain-dependent manner. This study sets the stage for future investigations into the interactions between growth, inflammation, and metabolism.

High expression of ZNF703 independent of amplification indicates worse prognosis in patients with luminal B breast cancer

International Nuclear Information System (INIS)

Reynisdottir, Inga; Arason, Adalgeir; Einarsdottir, Berglind O; Gunnarsson, Haukur; Staaf, Johan; Vallon-Christersson, Johan; Jonsson, Goran; Ringnér, Markus; Agnarsson, Bjarni A; Olafsdottir, Kristrun; Fagerholm, Rainer; Einarsdottir, Thorbjorg; Johannesdottir, Gudrun; Johannsson, Oskar Thor; Nevanlinna, Heli; Borg, Ake; Barkardottir, Rosa Bjork

2013-01-01

Amplification of 8p12-p11 is relatively common in breast cancer and several genes within the region have been suggested to affect breast tumor progression. The aim of the study was to map the amplified 8p12-p11 region in a large set of breast tumors in an effort to identify the genetic driver and to explore its impact on tumor progression and prognosis. Copy number alterations (CNAs) were mapped in 359 tumors, and gene expression data from 577 tumors (359 tumors included) were correlated with CNA, clinical–pathological factors, and protein expression (39 tumors). 8p12-p11 was amplified in 11.4% of tumors. The smallest region of amplification harbored one full-length gene, ZNF703. ZNF703 mRNA expression was significantly higher in estrogen receptor (ER)-positive than ER-negative tumors (P = 2 × 10 −16 ), a reflection of high expression in luminal tumors. Forty-eight percent of tumors with ZNF703 amplification were luminal B tumors in which the best correlation between DNA copy number and mRNA was seen (P = 1.2 × 10 −7 ) as well as correlation between mRNA and protein expression (P = 0.02). High ZNF703 mRNA correlated with poor survival in patients with ER-positive luminal B tumors (log rank P = 0.04). Furthermore, high ZNF703 mRNA expression correlated with poor outcome in patients with ZNF703 copy number neutral, ER-positive, luminal B tumors (log rank P = 0.004). The results support ZNF703 as the driver gene of the 8p12 amplification and suggest that independent of amplification, high expression of the gene affects prognosis in luminal B tumors. Our mapping of 8p12-p11 and analyses of ZNF703 mRNA and protein expression in breast tumors support ZNF703 as an oncogene in luminal B tumors. High ZNF703 expression, independent of the amplification, correlated with worse prognosis for the breast cancer patients with ER-positive luminal tumors, particularly of the luminal B subtype

A high resolution atlas of gene expression in the domestic sheep (Ovis aries).

Science.gov (United States)

Clark, Emily L; Bush, Stephen J; McCulloch, Mary E B; Farquhar, Iseabail L; Young, Rachel; Lefevre, Lucas; Pridans, Clare; Tsang, Hiu G; Wu, Chunlei; Afrasiabi, Cyrus; Watson, Mick; Whitelaw, C Bruce; Freeman, Tom C; Summers, Kim M; Archibald, Alan L; Hume, David A

2017-09-01

Sheep are a key source of meat, milk and fibre for the global livestock sector, and an important biomedical model. Global analysis of gene expression across multiple tissues has aided genome annotation and supported functional annotation of mammalian genes. We present a large-scale RNA-Seq dataset representing all the major organ systems from adult sheep and from several juvenile, neonatal and prenatal developmental time points. The Ovis aries reference genome (Oar v3.1) includes 27,504 genes (20,921 protein coding), of which 25,350 (19,921 protein coding) had detectable expression in at least one tissue in the sheep gene expression atlas dataset. Network-based cluster analysis of this dataset grouped genes according to their expression pattern. The principle of 'guilt by association' was used to infer the function of uncharacterised genes from their co-expression with genes of known function. We describe the overall transcriptional signatures present in the sheep gene expression atlas and assign those signatures, where possible, to specific cell populations or pathways. The findings are related to innate immunity by focusing on clusters with an immune signature, and to the advantages of cross-breeding by examining the patterns of genes exhibiting the greatest expression differences between purebred and crossbred animals. This high-resolution gene expression atlas for sheep is, to our knowledge, the largest transcriptomic dataset from any livestock species to date. It provides a resource to improve the annotation of the current reference genome for sheep, presenting a model transcriptome for ruminants and insight into gene, cell and tissue function at multiple developmental stages.

A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

DEFF Research Database (Denmark)

Peng, Nan; Deng, Ling; Mei, Yuxia

2012-01-01

Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression...... to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high...... levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

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Yu, Jingwen; Wu, Yanqing; Yang, Peixin

2016-05-01

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses

High TUG1 expression is associated with chemotherapy resistance and poor prognosis in esophageal squamous cell carcinoma.

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Jiang, Lin; Wang, Wenchao; Li, Guoli; Sun, Canlin; Ren, Zhenqin; Sheng, Haihui; Gao, Hengjun; Wang, Chaofu; Yu, Hong

2016-08-01

Long noncoding RNAs (lncRNAs) play critical roles in diverse biological processes such as tumorigenesis and metastasis. Taurine upregulated gene 1 (TUG1) is a cancer-related lncRNA that is associated with chromatin-modifying complexes and plays an important role in gene regulation. In this study, we determined the expression patterns of TUG1 in esophageal squamous cell carcinoma (ESCC) and evaluated its clinical significance. The expression level of TUG1 was examined in 218 pairs of ESCC and adjacent non-cancerous tissues by using quantitative real-time polymerase chain reaction. The relationship between TUG1 expression and clinical features and prognosis was statistically analyzed. The expression level of TUG1 was significantly upregulated in ESCC tissues compared with paired adjacent normal tissues. High TUG1 expression was significantly correlated with chemotherapy resistance. Survival analysis showed that patients with high TUG1 expression had poor prognosis, especially for cases with well and moderate differentiation, ulcerative type, smaller size, and chemotherapy-sensitive tumors. Our findings suggest that elevated TUG1 expression is related to chemotherapy resistance and may help predict a poor prognostic outcome of ESCC. TUG1 may provide a potential therapeutic target for ESCC.

High interleukin-6 mRNA expression is a predictor of relapse in colon cancer

DEFF Research Database (Denmark)

Olsen, Jesper; Kirkeby, Lene T; Olsen, Jørgen

2015-01-01

AIM: To investigate the expression of interleukin-6 (IL6) in colon cancer tissue, and to examine if the risk of relapse is influenced by IL6 expression. MATERIALS AND METHODS: Fresh-frozen biopsies from tumor and normal adjacent tissues were taken from patients with colon cancer during surgery...... for clinicopathological characteristics (Hazard Ratio=2.16, 95% CI=1.07-4.40; pcolon cancer tissue at the transcriptional level and is significantly associated with increased risk of relapse....... to normal adjacent tissue (pcancer stage. We found a significant association between high IL6 expression and risk of relapse (Hazard Ratio=2.23, 95% CI=1.10-4.53; p

Gene expression profiles following high-dose exposure to gamma radiation in salmonella enterica serovar typhimurium

International Nuclear Information System (INIS)

Lim, Sang Yong; Jung, Sun Wook; Joe, Min Ho; Kim, Dong Ho

2008-01-01

Microarrays can measure the expression of thousands of genes to identify the changes in expression between different biological states. To survey the change of whole Salmonella genes after a relatively high dose of gamma radiation (1 kGy), transcriptome dynamics were examined in the cells by using DNA microarrays. At least 75 genes were induced and 89 genes were reduced two-fold or more after irradiation. Several genes located in pSLT plasmid, cyo operon, and Gifsy prophage were induced along with many genes encoding uncharacterized proteins.While, the expression of genes involved in the virulence of Salmonella as well as metabolic functions were decreased. Although the radiation response as a whole could not be illustrated by using DNA microarrays, the data suggest that the response to high dose of irradiation might be more complex than the SOS response

Gene expression profiles following high-dose exposure to gamma radiation in salmonella enterica serovar typhimurium

Energy Technology Data Exchange (ETDEWEB)

Lim, Sang Yong; Jung, Sun Wook; Joe, Min Ho; Kim, Dong Ho [Radiation Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

2008-08-15

Microarrays can measure the expression of thousands of genes to identify the changes in expression between different biological states. To survey the change of whole Salmonella genes after a relatively high dose of gamma radiation (1 kGy), transcriptome dynamics were examined in the cells by using DNA microarrays. At least 75 genes were induced and 89 genes were reduced two-fold or more after irradiation. Several genes located in pSLT plasmid, cyo operon, and Gifsy prophage were induced along with many genes encoding uncharacterized proteins.While, the expression of genes involved in the virulence of Salmonella as well as metabolic functions were decreased. Although the radiation response as a whole could not be illustrated by using DNA microarrays, the data suggest that the response to high dose of irradiation might be more complex than the SOS response.

High MRPS23 expression contributes to hepatocellular carcinoma proliferation and indicates poor survival outcomes.

Science.gov (United States)

Pu, Meng; Wang, Jianlin; Huang, Qike; Zhao, Ge; Xia, Congcong; Shang, Runze; Zhang, Zhuochao; Bian, Zhenyuan; Yang, Xishegn; Tao, Kaishan

2017-07-01

Hepatocellular carcinoma is one of the most prevalent neoplasms and the leading cause of cancer-related mortality worldwide. Mitochondrial ribosomal protein S23 is encoded by a nuclear gene and participates in mitochondrial protein translation. Mitochondrial ribosomal protein S23 overexpression has been found in many types of cancer. In this study, we explored mitochondrial ribosomal protein S23 expression in primary hepatocellular carcinoma tissues compared with matched adjacent non-tumoral liver tissues using mitochondrial ribosomal protein S23 messenger RNA and protein levels collected from public databases and clinical samples. Immunohistochemistry was performed to analyze the relationship between mitochondrial ribosomal protein S23 and various clinicopathological features. The results indicated that mitochondrial ribosomal protein S23 was significantly overexpressed in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 expression was correlated with the tumor size and tumor-metastasis-node stage. Moreover, patients with high mitochondrial ribosomal protein S23 expression levels presented poorer survival rates. Mitochondrial ribosomal protein S23 was an independent prognostic factor for survival, especially at the early stage of hepatocellular carcinoma. In addition, the downregulation of mitochondrial ribosomal protein S23 decreased the proliferation of hepatocellular carcinoma in vitro and in vivo. In conclusion, we verified for the first time that mitochondrial ribosomal protein S23 expression was upregulated in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 levels can predict poor clinical outcomes in hepatocellular carcinoma, and this protein plays a key role in tumor proliferation. Therefore, mitochondrial ribosomal protein S23 may be a potential therapeutic target for hepatocellular carcinoma.

Cloning, high-level expression, purification and crystallization of peptide deformylase from Leptospira interrogans.

Science.gov (United States)

Li, Yikun; Ren, Shuangxi; Gong, Weimin

2002-05-01

A new peptide deformylase (PDF; EC 3.5.1.27) gene from Leptospira interrogans was identified and cloned into expression plasmid pET22b(+) and was highly expressed in Escherichia coli BL21(DE3). With DEAE-Sepharose anion-exchange chromatography followed by Superdex G-75 size-exclusion chromatography, 60 mg of PDF from L. interrogans was purified from 1 l of cell culture. Crystallization screening of the purified enzyme resulted in two crystal forms, from one of which a 3 A resolution X-ray diffraction data set has been collected.

High expression of nuclear survivin and Aurora B predicts poor overall survival in patients with head and neck squamous cell cancer

Energy Technology Data Exchange (ETDEWEB)

Erpolat, O.P.; Akmansu, M. [Medical School of Gazi Univ., Besevler-Ankara (Turkey). Dept. of Radiation Oncology; Gocun, P.U.; Karakus, E.; Akyol, G. [Medical School of Gazi Univ., Besevler-Ankara (Turkey). Dept. of Pathology

2012-03-15

Survivin is one of the apoptosis inhibitor proteins. Together with Aurora B, it also plays a role in regulating several aspects of mitosis. High expression of these markers is correlated with malignant behavior of various cancers and resistance to therapy. Our aim was to evaluate the prognostic role of these markers in head and neck cancers. We evaluated the expression of Aurora B and survivin in tissue specimens of 58 patients with head and neck squamous cell carcinoma using immunohistochemistry. Patients who showed high expression of cytoplasmic and nuclear survivin and Aurora B had significantly shorter overall survival (p = 0.036, p < 0.000, p = 0.032, respectively). In multivariate analysis, high expression of nuclear survivin was the only independent negative prognostic factor (p = 0.024). Moreover, it was found that high co-expression of nuclear survivin and Aurora B had a negative effect on survival in univariate (p < 0.000) and multivariate (p < 0.000) analyses. The negative prognostic values of high expression of Aurora B and high co-expression of nuclear survivin and Aurora B on survival were shown. These findings suggest that co-expression of nuclear survivin and Aurora B can be useful diagnostic markers and therapeutic targets for head and neck squamous cell carcinoma. However, further studies with a larger number of patients in a more homogeneous disease group are needed to confirm the conclusion.

Secretory leukocyte protease inhibitor expression and high-risk HPV infection in anal lesions of HIV positive patients

Science.gov (United States)

NUOVO, Gerard J.; GRINSZTEJN, Beatriz; FRIEDMAN, Ruth K.; VELOSO, Valdiléa G.; CUNHA, Cynthia B.; COUTINHO, José R.; VIANNA-ANDRADE, Cecilia; OLIVEIRA, Nathalia S.; WOODHAM, Andrew W.; DA SILVA, Diane M.; KAST, W. Martin

2016-01-01

Objective The aim of the current study was to evaluate secretory leukocyte protease inhibitor (SLPI) expression in anal biopsies from HIV-positive (HIV+) individuals, and compare that to anal intraepithelial neoplasia (AIN) diagnoses and human papillomavirus (HPV) status. Design This is a cross-sectional study of a cohort of 54 HIV+ (31 males and 23 females) from an AIDS clinic in Rio de Janeiro, Brazil. Methods The study material consisted of anorectal tissue biopsies obtained from HIV+ subjects, which were used to construct tissue microarray paraffin blocks for immunohistochemical analysis of SLPI expression. Biopsies were evaluated by an expert pathologist and classified as low-grade anal intraepithelial neoplasia (AIN1), high-grade anal intraepithelial neoplasia (AIN2/3), or normal squamous epithelium. Additionally, DNA from the biopsies was extracted and analyzed for the presence of low- or high-risk HPV DNA. Results Histologically normal squamous epithelium from the anorectal region showed strong positive SLPI staining in 17/20 (85%) samples. In comparison, 9/17 (53%) dysplastic squamous epithelial samples from AIN1 patients showed strong SLPI staining, and only 5/17 (29%) samples from AIN2-3 patients exhibited strong SPLI staining, which both were significantly fewer than those from normal tissue (p=0.005). Furthermore, there was a significantly higher proportion of samples in which oncogenic high-risk HPV genotypes were detected in low SLPI expressing tissues than that in tissues with high SLPI expression (p=0.040). Conclusion Taken together these results suggest that low SLPI expression is associated with high-risk HPV infections in the development of AIN. PMID:27149102

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

Directory of Open Access Journals (Sweden)

Bekim Sadikovic

Full Text Available Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

An Ultra-High Fluorescence Enhancement and High Throughput Assay for Revealing Expression and Internalization of Chemokine Receptor CXCR4.

Science.gov (United States)

He, Hua; Wang, Xiaojuan; Cheng, Tiantian; Xia, Yongqing; Lao, Jun; Ge, Baosheng; Ren, Hao; Khan, Naseer Ullah; Huang, Fang

2016-04-18

Revealing chemokine receptor CXCR4 expression, distribution, and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy. We here describe a sensitive and high-throughput immunoassay for determining CXCR4 expression and distribution in cancer cells. The assay is accessible to a wide range of users in an ordinary lab only by dip-coating poly(styrene-co-N-isopropylacrylamide) spheres on the glass substrate. The self- assembled spheres form three-dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 by a factor of up to 1000. CXCR4 in cells is detected by using the sandwich immunoassay, where the primary antibody recognizes CXCR4 and the secondary antibody is labeled with CF647. With the newly established assay, we quantified the total expression of CXCR4, its distribution on the cell membrane and cytoplasm, and revealed their internalization level upon SDF-1α activation in various cancer cells, even for those with extremely low expression level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Emotional Facial and Vocal Expressions during Story Retelling by Children and Adolescents with High-Functioning Autism

Science.gov (United States)

Grossman, Ruth B.; Edelson, Lisa R.; Tager-Flusberg, Helen

2013-01-01

Purpose: People with high-functioning autism (HFA) have qualitative differences in facial expression and prosody production, which are rarely systematically quantified. The authors' goals were to qualitatively and quantitatively analyze prosody and facial expression productions in children and adolescents with HFA. Method: Participants were 22…

Rad51 expression levels predict synthetic lethality and metastatic potential in high grade breast cancers

International Nuclear Information System (INIS)

Wiegmans, A.P.; Al-Ejeh, F.; Khanna, K.K.

2012-01-01

Among women with breast cancer, 30-40% will develop metastatic disease and only achieve an overall survival of less than 5 years. Despite new-targeted therapy, breast tumors that harbour similar histology or molecular phenotype differ in their response to treatment. To uncover potential new therapeutic targets and improve outcome, we performed data mining of cancer micro array databases. We found that high expression of the homologous recombination protein, RAD51, was significantly associated with high-grade breast cancer, aggressive subtypes and increased risk of metastasis. We confirmed using immunohistochemistry that RAD5 1 was highly expressed in metastatic tumours and high-grade triple negative, HER2+ and luminal-B tumours. This provided a rationale for targeting RAD5 1 in high-grade, therapy-resistant breast cancers. Here, we report for the first time preclinical evaluation of RAD5 1 as a therapeutic target. We found that, in-vitro high RAD5 expressing cell lines were resistant to PARP inhibitor while knockdown reversed this resistance. In-vivo, knockdown of RAD5 1 inhibited metastatic progression using a syngeneic breast cancer model and the seeding of human xenografts to distant sites, including brain and lung. Concurrent PARP inhibition reduced primary tumor growth and delayed metastasis supporting synthetic lethality in-vivo. Together these insights provide pre-clinical data demonstrating RAD5 1 as a new biomarker and potential therapeutic target against aggressive metastatic breast cancer. (author)

Fanconi anemia genes are highly expressed in primitive CD34+ hematopoietic cells

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Brodeur Isabelle

2003-06-01

Full Text Available Abstract Background Fanconi anemia (FA is a complex recessive genetic disease characterized by progressive bone marrow failure (BM and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. Methods Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. Results We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34- stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. Conclusions These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34+ cells correlates with a role at the CD34+ differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism.

High expression of BAG3 predicts a poor prognosis in human medulloblastoma.

Science.gov (United States)

Yang, Dong; Zhou, Ji; Wang, Hao; Wang, Yutao; Yang, Ge; Zhang, Yundong

2016-10-01

Bcl2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock protein (Hsp) 70, regulates various physiological and pathological processes. However, its role in human medulloblastoma has not been clarified. First of all, the expression of BAG3 was examined in formalin-fixed, paraffin-embedded specimens by immunohistochemical staining. And then, the prognostic role of BAG3 was analyzed in 51 medulloblastoma samples. Finally, the roles of BAG3 in the proliferation, migration, and invasion of Daoy medulloblastoma cell were investigated using a specific short hairpin RNA (shRNA). The expression of BAG3 in medulloblastoma tissues was higher than nontumorous samples. Furthermore, BAG3 overexpression significantly correlated with poor prognosis of patients with medulloblastoma. The overall survival and tumor-free survival in patients with BAG3 low expression were higher than high expression. Univariate and multivariate analysis showed that BAG3 overexpression was an independent prognostic marker for medulloblastoma. After the BAG3 knockdown, the Daoy cells exhibited decreased the ability to proliferate and form neurosphere. The preliminary mechanism study showed that overexpression of BAG3 might facilitate the cell cycle transition from G1 to S phase by modulating the cyclin-dependent kinase 2 (CDK2) and cyclin E expression. Additionally, we found that BAG3 might enhance the medulloblastoma cell migratory and invasive ability. In summary, BAG3 overexpression may regulate the survival and invasive properties of medulloblastoma and may serve as a potential therapy target for medulloblastoma.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

Science.gov (United States)

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

A High-Fructose-High-Coconut Oil Diet Induces Dysregulating Expressions of Hippocampal Leptin and Stearoyl-CoA Desaturase, and Spatial Memory Deficits in Rats.

Science.gov (United States)

Lin, Ching-I; Shen, Chu-Fu; Hsu, Tsui-Han; Lin, Shyh-Hsiang

2017-06-16

We investigated the effects of high-fructose-high-fat diets with different fat compositions on metabolic parameters, hippocampal-dependent cognitive function, and brain leptin (as well as stearoyl-CoA desaturase (SCD1) mRNA expressions). Thirty-two male Wistar rats were divided into 3 groups, a control group ( n = 8), a high-fructose soybean oil group (37.5% of fat calories, n = 12), and a high-fructose coconut oil group (37.5% of fat calories, n = 12) for 20 weeks. By the end of the study, the coconut oil group exhibited significantly higher serum fasting glucose, fructosamine, insulin, leptin, and triglyceride levels compared to those of the control and soybean oil groups. However, hippocampal leptin expression and leptin receptor mRNA levels were significantly lower, while SCD1 mRNA was significantly higher in rats fed the high-fructose-high-coconut oil diet than in rats fed the other experimental diets. In addition, the coconut oil group spent significantly less time in the target quadrant on the probe test in the Morris water maze (MWM) task. Rats fed the high-fructose-high-coconut oil diet for 20 weeks were prone to develop hyperglycemia, hyperinsulinemia, hyperleptinemia, and hypertriglyceridemia. These metabolic consequences may contribute to hippocampal-dependent memory impairment, accompanied by a lower central leptin level, and a higher SCD1 gene expression in the brain.

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila.

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Tharadra, Sana Khalid; Medina, Adriana; Ray, Anandasankar

2013-01-01

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.

Early diffusion of gene expression profiling in breast cancer patients associated with areas of high income inequality.

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Ponce, Ninez A; Ko, Michelle; Liang, Su-Ying; Armstrong, Joanne; Toscano, Michele; Chanfreau-Coffinier, Catherine; Haas, Jennifer S

2015-04-01

With the Affordable Care Act reducing coverage disparities, social factors could prominently determine where and for whom innovations first diffuse in health care markets. Gene expression profiling is a potentially cost-effective innovation that guides chemotherapy decisions in early-stage breast cancer, but adoption has been uneven across the United States. Using a sample of commercially insured women, we evaluated whether income inequality in metropolitan areas was associated with receipt of gene expression profiling during its initial diffusion in 2006-07. In areas with high income inequality, gene expression profiling receipt was higher than elsewhere, but it was associated with a 10.6-percentage-point gap between high- and low-income women. In areas with low rates of income inequality, gene expression profiling receipt was lower, with no significant differences by income. Even among insured women, income inequality may indirectly shape diffusion of gene expression profiling, with benefits accruing to the highest-income patients in the most unequal places. Policies reducing gene expression profiling disparities should address low-inequality areas and, in unequal places, practice settings serving low-income patients. Project HOPE—The People-to-People Health Foundation, Inc.

A preliminary study on action mechanisms of surviving expression in cell apoptosis induced by high-LET radiation

International Nuclear Information System (INIS)

Jin Xiaodong; Li Qiang; Gong Li; Wu Qingfeng; Li Ping; Dai Zhongying; Liu Xinguo; Tao Jiajun

2010-01-01

It has been proven that over-expression of surviving in cancerous cell lines is related to the radioresistance of cells to high-LET radiation in previous work. In this study, action mechanisms of surviving gene in apoptosis induced by high-LET radiation were investigated. We found that inhibiting surviving by siRNA had no notable influence on Bcl-2 and Bax expressions induced by carbon ions. Surviving depressed cell apoptosis through the inhibition of the activities of caspase-3 and -9 possibly in cell apoptosis induced by high-LET radiation. (authors)

High pressure-sensitive gene expression in Lactobacillus sanfranciscensis

Directory of Open Access Journals (Sweden)

R.F. Vogel

2005-08-01

Full Text Available Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK, while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

Optimal specific radioactivity of anti-HER2 Affibody molecules enables discrimination between xenografts with high and low HER2 expression levels

Energy Technology Data Exchange (ETDEWEB)

Tolmachev, Vladimir [Uppsala University, Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala (Sweden); Uppsala University, Department of Medical Sciences, Nuclear Medicine, Uppsala (Sweden); Waallberg, Helena [Royal Institute of Technology, School of Biotechnology, Stockholm (Sweden); Sandstroem, Mattias [Uppsala University Hospital, Section of Hospital Physics, Department of Oncology, Uppsala (Sweden); Hansson, Monika; Wennborg, Anders [Affibody AB, Stockholm (Sweden); Orlova, Anna [Uppsala University, Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala (Sweden)

2011-03-15

Overexpression of the HER2 receptor is a biomarker for predicting those patients who may benefit from trastuzumab therapy. Radiolabelled Affibody molecules can be used to visualize HER2 expression in tumour xenografts with high sensitivity. However, previous studies demonstrated that the difference in uptake in xenografts with high and low HER2 expression levels is not proportional to the difference in expression levels. We hypothesized that discrimination between tumours with high and low HER2 expression may be improved by increasing the injected dose (reducing the specific activity) of the tracer. The influence of injected dose of anti-HER2 {sup 111}In-DOTA-Z{sub HER2} {sub 342} Affibody molecule on uptake in SKOV-3 (high HER2 expression) and LS174T (low expression) xenografts was investigated. The optimal range of injected doses enabling discrimination between xenografts with high and low expression was determined. To verify this, tumour uptake was measured in mice carrying both SKOV-3 and LS174T xenografts after injection of either 1 or 15 {mu}g {sup 111}In-DOTA-Z{sub HER2:342}. An increase in the injected dose caused a linear decrease in the radioactivity accumulation in the LS174T xenografts (low HER2 expression). For SKOV-3 xenografts, the dependence of the tumour uptake on the injected dose was less dramatic. The injection of 10-30 {mu}g {sup 111}In-DOTA-Z{sub HER2:342} per mouse led to the largest difference in uptake between the two types of tumour. Experiments in mice bearing two xenografts confirmed that the optimized injected dose enabled better discrimination of expression levels. Careful optimization of the injected dose of Affibody molecules is required for maximum discrimination between xenografts with high and low levels of HER2 expression. This information has potential relevance for clinical imaging applications. (orig.)

Using Improved Entropy-Cloud Model to Select High-Speed Railway Express Freight Train Service Sites

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Wencheng Huang

2017-01-01

Full Text Available High-speed railway (HSR express freight train service sites selection is critical to the development of China’s Third Party Logistics market. In this paper, we formulate an improved entropy-cloud model based approach to solve the HSR express sites selection problem for the first time. The basic data of the indicators, for example, existing railway network conditions, traffic environment, express freight market demand, and policy, will be used as the inputs. We apply improved entropy method to obtain each subindicator’s weight. The cloud model will be used to select and classify each station under evaluation.

Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

DEFF Research Database (Denmark)

Vester-Christensen, Malene Bech; Abou Hachem, Maher; Næsted, Henrik

2010-01-01

biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha...

Grape powder consumption affects the expression of neurodegeneration-related brain proteins in rats chronically fed a high-fructose-high-fat diet.

Science.gov (United States)

Liao, Hsiang; Chou, Liang-Mao; Chien, Yi-Wen; Wu, Chi-Hao; Chang, Jung-Su; Lin, Ching-I; Lin, Shyh-Hsiang

2017-05-01

Abnormal glucose metabolism in the brain is recognized to be associated with cognitive decline. Because grapes are rich in polyphenols that produce antioxidative and blood sugar-lowering effects, we investigated how grape consumption affects the expression and/or phosphorylation of neurodegeneration-related brain proteins in aged rats fed a high-fructose-high-fat (HFHF) diet. Wistar rats were maintained on the HFHF diet from the age of 8 weeks to 66 weeks, and then on an HFHF diet containing either 3% or 6% grape powder as an intervention for 12 weeks. Western blotting was performed to measure the expression/phosphorylation levels of several cortical and hippocampal proteins, including amyloid precursor protein (APP), tau, phosphatidylinositol-3-kinase (PI3K), extracellular signal-regulated kinase (ERK), receptor for advanced glycation end products (RAGEs), erythroid 2-related factor 2 (Nrf2) and brain-derived neurotrophic factor (BDNF). Inclusion of up to 6% grape powder in the diet markedly reduced RAGE expression and tau hyperphosphorylation, but upregulated the expression of Nrf2 and BDNF, as well as the phosphorylation of PI3K and ERK, in the brain tissues of aged rats fed the HFHF diet. Thus, grape powder consumption produced beneficial effects in HFHF-diet-fed rats, exhibiting the potential to ameliorate changes in neurodegeneration-related proteins in the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis.

Science.gov (United States)

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.

Transient depletion of T cells with high LFA-1 expression from peripheral circulation during acute Plasmodium falciparum malaria

DEFF Research Database (Denmark)

Hviid, L; Theander, T G; Abdulhadi, N H

1991-01-01

with high expression of the surface marker LFA-1 (CD11a/CD18) during the clinical episode, in addition to the functional changes described above. Two donors that did not show phenotypic changes were furthermore characterized by having an unabated proliferative response and normal plasma IL 2R levels. All...... peripheral CD3+ T lymphocytes expressed LFA-1, which had a clearly bimodal distribution on these cells. The T cell subpopulation having high LFA-1 expression (LFA-1++) was composed of both memory and unprimed T cells, according to their expression of CD45RA and CD45R0. Analysis of expression of membrane...

Gene expression patterns in the ventral tegmental area relate to oestrus behaviour in high-producing dairy cows

NARCIS (Netherlands)

Wyszynska-Koko, J.; Wit, de A.A.C.; Beerda, B.; Veerkamp, R.F.; Pas, te M.F.W.

2011-01-01

Reduced oestrus behaviour expression or its absence (silent oestrus) results in subfertility in high-producing dairy cows. Insight into the genomic regulation of oestrus behaviour is likely to help alleviate reproduction problems. Here, gene expression was recorded in the ventral tegmental area

High RBM3 expression is associated with an improved survival and oxaliplatin response in patients with metastatic colorectal cancer.

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Christina Siesing

Full Text Available High expression of the RNA-binding motif protein 3 (RBM3 has been shown to correlate, with prolonged survival in several malignant diseases and with the benefit of platinum-based chemotherapy in ovarian cancer. The aim of this study was to evaluate RBM3 in metastatic colorectal cancer (mCRC as a prognostic factor for overall survival and in relation to benefit of first-line chemotherapy.Immunohistochemical staining was conducted and evaluated in tumours from 455 mCRC patients. Kaplan-Meier analysis and Cox regression proportional hazards models were used to access the impact of RBM3 expression on overall survival (OS and progression-free survival (PFS.High RBM3 expression, both nuclear and cytoplasmic, was an independent prognostic factor for prolonged OS (hazard ratio [HR] 0.67, 95% confidence interval [CI] 0.50-0.90 and HR 0.66, 95% CI 0.48-0.91, respectively. PFS was significantly longer in patients with high RBM3 expression who had received first-line oxaliplatin based treatment, compared to those who had received irinotecan based treatment, both regarding nuclear and cytoplasmic expression (p-value 0.020 and 0.022 respectively.High RBM3 expression is an independent predictor of prolonged survival in mCRC patients, in particular in patients treated with first-line oxaliplatin based chemotherapy.

[High-level expression of heterologous protein based on increased copy number in Saccharomyces cerevisiae].

Science.gov (United States)

Zhang, Xinjie; He, Peng; Tao, Yong; Yang, Yi

2013-11-04

High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering

High-level transfer and long-term expression of the human beta-globin gene in a mouse transplant model.

Science.gov (United States)

Raftopoulos, H; Ward, M; Bank, A

1998-06-30

Insertion of a normally functioning human beta-globin gene into the hematopoietic stem cells (HSC) of patients with beta-thalassemia may be an effective approach to the therapy of this disorder. Safe, efficient gene transfer and long-term, high-level expression of the transferred human beta-globin gene in animal models are prerequisites for HSC somatic gene therapy. We have recently shown for the first time that, using a modified beta-globin retroviral vector in a mouse transplant model, long-term, high-level expression of a transferred human beta-globin gene is possible. The human beta-globin gene continues to be detected up to eight months post-transplantation of beta-globin-transduced hematopoietic cells into lethally irradiated mice. The transferred human beta-globin gene is detected in three of five mice surviving long-term (> 4 months) transplanted with bone marrow cells transduced with high-titer virus. The unrearranged 5.1 kb human beta-globin gene-containing provirus is seen by Southern blotting in two of these mice. More importantly, long-term expression of the transferred gene is seen in two mice at levels of 5% and 20% that of endogenous murine beta-globin. We document stem cell transduction by showing continued high-level expression of the human beta-globin gene in secondarily transplanted recipient mice. These results provide evidence of HSC transduction with a human beta-globin gene in animals and demonstrate that retroviral-mediated unrearranged human beta-globin gene transfer leads to a high level of human beta-globin gene expression in the long term for the first time. A gene therapy strategy may be a feasible therapeutic approach to the beta-thalassemias if consistent human beta-globin gene transfer and expression into HSC can be achieved.

High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

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D’Urzo Nunzia

2013-02-01

Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

Analytical expression for the phantom generated bremsstrahlung background in high energy electron beams

International Nuclear Information System (INIS)

Sorcini, B.B.; Hyoedynmaa, S; Brahme, A.

1995-01-01

Qualification of the bremsstrahlung photon background generated by an electron beam in a phantom is important for accurate high energy electron beam dosimetry in radiation therapy. An analytical expression has been derived for the background of phantom generated bremsstrahlung photons in plane parallel electron beams normally incident on phantoms of any atomic number between 4 and 92 (Be, C, H 2 O, Al, Cu, Ag, Pb and U). The expression can be used with fairly good accuracy in the energy range between 1 and 50 MeV. The expression is globally based on known scattering power and radiation and collision stopping power data for the phantom material at the mean energy of the incident electrons. The depth dose distribution due to the bremsstrahlung generated in the phantom is derived by folding the bremsstrahlung energy fluence with a simple analytical one-dimensional photon energy deposition kernel. The energy loss of the primary electrons and the generation, attenuation and absorption of bremsstrahlung photons are taken into account in the analytical formula. The photon energy deposition kernel is used to account for the bremsstrahlung produced at one depth that will contribute to the down stream dose. A simple analytical expression for photon energy deposition kernel is consistent with the classical analytical relation describing the photon depth dose distribution. From the surface to the practical range the photon dose increases almost linearly due to accumulation and buildup of the photon produced at different phantom layers. At depths beyond the practical range a simple exponential function can be use to describe the bremsstrahlung attenuation in the phantom. For comparison Monte Carlo calculated distributions using ITS3 Monte Carlo Code were used. Good agreement is found between the analytical expression and Monte Carlo calculation. Deviations of 5% from Monte Carlo calculated bremmstrahlung background are observed for high atomic number materials. The method can

Comparative analysis of codon usage patterns and identification of predicted highly expressed genes in five Salmonella genomes

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Mondal U

2008-01-01

Full Text Available Purpose: To anlyse codon usage patterns of five complete genomes of Salmonella , predict highly expressed genes, examine horizontally transferred pathogenicity-related genes to detect their presence in the strains, and scrutinize the nature of highly expressed genes to infer upon their lifestyle. Methods: Protein coding genes, ribosomal protein genes, and pathogenicity-related genes were analysed with Codon W and CAI (codon adaptation index Calculator. Results: Translational efficiency plays a role in codon usage variation in Salmonella genes. Low bias was noticed in most of the genes. GC3 (guanine cytosine at third position composition does not influence codon usage variation in the genes of these Salmonella strains. Among the cluster of orthologous groups (COGs, translation, ribosomal structure biogenesis [J], and energy production and conversion [C] contained the highest number of potentially highly expressed (PHX genes. Correspondence analysis reveals the conserved nature of the genes. Highly expressed genes were detected. Conclusions: Selection for translational efficiency is the major source of variation of codon usage in the genes of Salmonella . Evolution of pathogenicity-related genes as a unit suggests their ability to infect and exist as a pathogen. Presence of a lot of PHX genes in the information and storage-processing category of COGs indicated their lifestyle and revealed that they were not subjected to genome reduction.

High Expression of PHGDH Predicts Poor Prognosis in Non–Small Cell Lung Cancer

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Jinhong Zhu

2016-12-01

Full Text Available Tumors have exceptionally high demands for energy and anabolism because of their rapid growth. The de novo serine synthesis pathway initiated by phosphoglycerate dehydrogenase (PHGDH has been recognized as a hallmark of metabolic adaption in carcinogenesis. The oncogenic role and prognostic value of PHGDH have been investigated in multiple cancer types, including breast cancer, melanoma, cervical cancer, and colon cancer. Due to the importance of PHGDH in cancer, we attempted to determine the clinical significance of PHGDH in 319 patients with non–small cell lung cancer (NSCLC. We evaluated the mRNA and protein expression levels of PHGDH gene, using quantitative reverse transcriptase polymerase chain reaction and tissue array–based immunohistochemistry, respectively. Significantly increased PHGDH expression in mRNA and protein levels was identified in tumor tissues versus matched adjacent nontumor tissues. More interestingly, immunohistochemical expression of PHGDH was significantly associated with lymph node metastasis (P = .021 and TNM stage (P = .016. Kaplan-Meier survival analysis indicated that NSCLC patients with low levels of PHGDH outperformed patients with high levels of PHGDH regarding 5-year overall survival. Significantly longer survival in the former suggested the prognostic implication of PHGDH in NSCLC. Multivariate survival analysis using Cox regression model demonstrated that high PHGDH levels and advanced TNM stage (III + IV were independent predictors of prognosis in NSCLC. Moreover, bioinformatics analysis confirmed the increase in PHGDH transcripts (data from The Cancer Genome Atlas and its prognostic value (Kaplan-Meier plotter in NSCLC. In conclusion, this study suggested the clinical implication of PHGDH in NSCLC. PHGDH may be a promising therapeutic target in NSCLC.

Perception of emotional facial expressions in individuals with high Autism-spectrum Quotient (AQ

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Ervin Poljac

2012-10-01

Full Text Available Autism is characterized by difficulties in social interaction, communication, restrictive and repetitive behaviours and specific impairments in emotional processing. The present study employed The Autism Spectrum Quotient (Baron-Cohen et al. 2006 to quantify autistic traits in a group of 260 healthy individuals and to investigate whether this measure is related to the perception of facial emotional expressions. The emotional processing of twelve participants that scored significantly higher than the average on the AQ was compared to twelve participants with significantly lower AQ scores. Perception of emotional expressions was estimated by The Facial Recognition Task (Montagne et al. 2007. There were significant differences between the two groups with regard to accuracy and sensitivity of the perception of emotional facial expressions. Specifically, the group with high AQ score was less accurate and needed higher emotional content to recognize emotions of anger, disgust, happiness and sadness. This result implies a selective impairment that might be helpful in understanding the psychopathology of autism spectrum disorders.

Recombinant myostatin reduces highly expressed microRNAs in differentiating C2C12 cells

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Zachary A. Graham

2017-03-01

Full Text Available Myostatin is small glycopeptide that is produced and secreted by skeletal muscle. It is a potent negative regulator of muscle growth that has been associated with conditions of frailty. In C2C12 cells, myostatin limits cell differentiation. Myostatin acts through activin receptor IIB, activin receptor-like kinase (ALK and Smad transcription factors. microRNAs (miRNA are short, 22 base pair nucleotides that bind to the 3′ UTR of target mRNA to repress translation or reduce mRNA stability. In the present study, expression in differentiating C2C12 cells of the myomiRs miR-1 and 133a were down-regulated following treatment with 1 µg of recombinant myostatin at 1 d post-induction of differentiation while all myomiRs (miR-1, 133a/b and 206 were upregulated by SB431542, a potent ALK4/5/7 inhibitor which reduces Smad2 signaling, at 1 d and all, with the exception of miR-206, were upregulated by SB431542 at 3 d. The expression of the muscle-enriched miR-486 was greater following treatment with SB431542 but not altered by myostatin. Other highly expressed miRNAs in skeletal muscle, miR-23a/b and 145, were altered only at 1 d post-induction of differentiation. miR-27b responded differently to treatments at 1 d, where it was upregulated, as compared to 3 d, where it was downregulated. Neither myostatin nor SB431542 altered cell size or cell morphology. The data indicate that myostatin represses myomiR expression in differentiating C2C12 cells and that inhibition of Smad signaling with SB431542 can result in large changes in highly expressed miRNAs in differentiating myoblasts.

Effect of high energy intake on carcass composition and hypothalamic gene expression in Bos indicus heifers

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Juliane Diniz-Magalhães

Full Text Available ABSTRACT The objective of this study was to evaluate the effect of high or low energy intake on carcass composition and expression of hypothalamic genes related to the onset of puberty. Twenty-four prepubertal Nellore heifers, 18-20- months-old, with 275.3±18.0 kg body weight (BW, and 4.9±0.2 (1-9 scale body condition score (BCS were randomly assigned to two treatments: high-energy diet (HE and low-energy diet (LE. Heifers were housed in two collective pens and fed diets formulated to promote average daily gain of 0.4 (LE or 1.2 kg (HE BW/day. Eight heifers from each treatment were slaughtered after the first corpus luteum detection - considered as age of puberty. The 9-10-11th rib section was taken and prepared for carcass composition analyses. Samples from hypothalamus were collected, frozen in liquid nitrogen, and stored at −80 °C. Specific primers for targets (NPY, NPY1R, NPY4R, SOCS3, OXT, ARRB1, and IGFPB2 and control (RPL19 and RN18S1 genes were designed for real-time PCR and then the relative quantification of target gene expression was performed. High-energy diets increased body condition score, cold carcass weight, and Longissimus lumborum muscle area and decreased age at slaughter. High-energy diets decreased the expression of NPY1R and ARRB1 at 4.4-fold and 1.5-fold, respectively. In conclusion, the hastening of puberty with high energy intake is related with greater body fatness and lesser hypothalamic expression of NPY1 receptor and of β-arrestin1, suggesting a less sensitive hypothalamus to the negative effects of NPY signaling.

Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c(4) by high-cell-density fed-batch cultivation of Pseudomonas putida

DEFF Research Database (Denmark)

Thuesen, Marianne Hallberg; Nørgaard, Allan; Hansen, Anne Merete

2003-01-01

The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein...

High-throughput sequencing of microRNA transcriptome and expression assay in the sturgeon, Acipenser schrenckii.

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Lihong Yuan

Full Text Available Sturgeons are considered as living fossils and have very high evolutionary, economical and conservation values. The multiploidy of sturgeon that has been caused by chromosome duplication may lead to the emergence of new microRNAs (miRNAs involved in the ploidy and physiological processes. In the present study, we performed the first sturgeon miRNAs analysis by RNA-seq high-throughput sequencing combined with expression assay of microarray and real-time PCR, and aimed to discover the sturgeon-specific miRNAs, confirm the expressed pattern of miRNAs and illustrate the potential role of miRNAs-targets on sturgeon biological processes. A total of 103 miRNAs were identified, including 58 miRNAs with strongly detected signals (signal >500 and P≤0.01, which were detected by microarray. Real-time PCR assay supported the expression pattern obtained by microarray. Moreover, co-expression of 21 miRNAs in all five tissues and tissue-specific expression of 16 miRNAs implied the crucial and particular function of them in sturgeon physiological processes. Target gene prediction, especially the enriched functional gene groups (369 GO terms and pathways (37 KEGG regulated by 58 miRNAs (P<0.05, illustrated the interaction of miRNAs and putative mRNAs, and also the potential mechanism involved in these biological processes. Our new findings of sturgeon miRNAs expand the public database of transcriptome information for this species, contribute to our understanding of sturgeon biology, and also provide invaluable data that may be applied in sturgeon breeding.

High-throughput sequencing of microRNA transcriptome and expression assay in the sturgeon, Acipenser schrenckii.

Science.gov (United States)

Yuan, Lihong; Zhang, Xiujuan; Li, Linmiao; Jiang, Haiying; Chen, Jinping

2014-01-01

Sturgeons are considered as living fossils and have very high evolutionary, economical and conservation values. The multiploidy of sturgeon that has been caused by chromosome duplication may lead to the emergence of new microRNAs (miRNAs) involved in the ploidy and physiological processes. In the present study, we performed the first sturgeon miRNAs analysis by RNA-seq high-throughput sequencing combined with expression assay of microarray and real-time PCR, and aimed to discover the sturgeon-specific miRNAs, confirm the expressed pattern of miRNAs and illustrate the potential role of miRNAs-targets on sturgeon biological processes. A total of 103 miRNAs were identified, including 58 miRNAs with strongly detected signals (signal >500 and P≤0.01), which were detected by microarray. Real-time PCR assay supported the expression pattern obtained by microarray. Moreover, co-expression of 21 miRNAs in all five tissues and tissue-specific expression of 16 miRNAs implied the crucial and particular function of them in sturgeon physiological processes. Target gene prediction, especially the enriched functional gene groups (369 GO terms) and pathways (37 KEGG) regulated by 58 miRNAs (P<0.05), illustrated the interaction of miRNAs and putative mRNAs, and also the potential mechanism involved in these biological processes. Our new findings of sturgeon miRNAs expand the public database of transcriptome information for this species, contribute to our understanding of sturgeon biology, and also provide invaluable data that may be applied in sturgeon breeding.

High contrast pet imaging of GRPR expression in prostate cancer using cobalt-labeled bombesin antagonist RM26

DEFF Research Database (Denmark)

Mitran, Bogdan; Thisgaard, Helge; Rosenström, Ulrika

2017-01-01

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer.The aim of the current study was to develop a55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with57Co and55Co...

Expression profiling on high-density DNA grids to detect novel targets in dendritic cells

International Nuclear Information System (INIS)

Weissmann, M.

2000-10-01

Gene expression analyzes on a large scale using DNA microarrays is a novel approach to study transcription of thousands of genes in parallel. By comparing gene expression profiles of different cell-types and of cells in different activation, novel regulatory networks will be identified that are unique to a cell-type and hence, important in its biological function. Among the differentially expressed genes many novel drug targets will be found. The Genetic department of the Novartis Research Institute was following this approach to identify novel genes, which are critical in the antigen presenting function of DCs and could become promising drug targets. Drugs that modulate effector functions of DCs towards induction of energy or tolerance in T-cells could be useful in the treatment of chronic inflammatory or autoimmune diseases. By using specific robotics equipment high-density cDNA grids on nylon membranes have been produced for hybridizations with various radioactive labeled DNA probes. By our format, based on 384 well plates and limited by the resolution power of our current image analysis software, 27.648 cDNA clones, bacterial colonies or pure DNA, were spotted on one filter. For RNA profiling, we generated filters containing a collection of genes expressed in peripheral blood DCs or monocytes and characterized by oligonucleotide fingerprinting (ONF) as being differentially expressed. The gene collection contained many unknown genes. Sequence analysis of to date 18.000 cDNA clones led to an estimate of 5.000 non-redundant genes being represented in the collection. 10 % of them are either completely unknown or homologous to rare ESTs (expressed sequence tags) in the public EST database. These clones occurred predominantly in small fingerprint clusters and were therefore assumed to be rarely expressed in DCs or monocytes. Some of those genes may become novel drug targets if their expression is DC specific or induced by external stimuli driving DCs into

Preferential cytotoxicity of bortezomib toward highly malignant human liposarcoma cells via suppression of MDR1 expression and function

International Nuclear Information System (INIS)

Hu, Yamei; Wang, Lingxian; Wang, Lu; Wu, Xuefeng; Wu, Xudong; Gu, Yanhong; Shu, Yongqian; Sun, Yang; Shen, Yan; Xu, Qiang

2015-01-01

Liposarcoma is the most common soft tissue sarcoma with a high risk of relapse. Few therapeutic options are available for the aggressive local or metastatic disease. Here, we report that the clinically used proteasome inhibitor bortezomib exhibits significantly stronger cytotoxicity toward highly malignant human liposarcoma SW872-S cells compared with its parental SW872 cells, which is accompanied by enhanced activation of apoptotic signaling both in vitro and in vivo. Treatment of cells with Jun-N-terminal kinase (JNK) inhibitor SP60015 or the translation inhibitor cycloheximide ameliorated this enhanced apoptosis. Bortezomib inhibited MDR1 expression and function more effectively in SW872-S cells than in SW872 cells, indicating that the increased cytotoxicity relies on the degree of proteasome inhibition. Furthermore, the pharmacological or genetic inhibition of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2, which is highly expressed in SW872-S cells, resulted in partial reversal of cell growth inhibition and increase of MDR1 expression in bortezomib-treated SW872-S cells. These results show that bortezomib exhibits preferential cytotoxicity toward SW872-S cells possibly via highly expressed SERCA2-associated MDR1 suppression and suggest that bortezomib may serve as a potent agent for treating advanced liposarcoma. - Highlights: • We compare the cytotoxicity of different drugs between SW872-S and SW872 cells. • Highly malignant liposarcoma cells SW872-S show hypersensitivity to bortezomib. • Apoptotic signaling is robustly enhanced in bortezomib-treated SW872-S cells. • Bortezomib has strong suppression on MDR1 expression and function in SW872-S cells. • Inhibition of SERCA2 protects SW872-S cells from bortezomib

Preferential cytotoxicity of bortezomib toward highly malignant human liposarcoma cells via suppression of MDR1 expression and function

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Hu, Yamei; Wang, Lingxian; Wang, Lu; Wu, Xuefeng; Wu, Xudong [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 (China); Gu, Yanhong; Shu, Yongqian [Department of Clinical Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029 (China); Sun, Yang [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 (China); Shen, Yan, E-mail: shenyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 (China); Xu, Qiang, E-mail: molpharm@163.com [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 (China)

2015-02-15

Liposarcoma is the most common soft tissue sarcoma with a high risk of relapse. Few therapeutic options are available for the aggressive local or metastatic disease. Here, we report that the clinically used proteasome inhibitor bortezomib exhibits significantly stronger cytotoxicity toward highly malignant human liposarcoma SW872-S cells compared with its parental SW872 cells, which is accompanied by enhanced activation of apoptotic signaling both in vitro and in vivo. Treatment of cells with Jun-N-terminal kinase (JNK) inhibitor SP60015 or the translation inhibitor cycloheximide ameliorated this enhanced apoptosis. Bortezomib inhibited MDR1 expression and function more effectively in SW872-S cells than in SW872 cells, indicating that the increased cytotoxicity relies on the degree of proteasome inhibition. Furthermore, the pharmacological or genetic inhibition of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2, which is highly expressed in SW872-S cells, resulted in partial reversal of cell growth inhibition and increase of MDR1 expression in bortezomib-treated SW872-S cells. These results show that bortezomib exhibits preferential cytotoxicity toward SW872-S cells possibly via highly expressed SERCA2-associated MDR1 suppression and suggest that bortezomib may serve as a potent agent for treating advanced liposarcoma. - Highlights: • We compare the cytotoxicity of different drugs between SW872-S and SW872 cells. • Highly malignant liposarcoma cells SW872-S show hypersensitivity to bortezomib. • Apoptotic signaling is robustly enhanced in bortezomib-treated SW872-S cells. • Bortezomib has strong suppression on MDR1 expression and function in SW872-S cells. • Inhibition of SERCA2 protects SW872-S cells from bortezomib.

[A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

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Mo, Qianzhen; Mai, Rongjia; Yang, Zhixiao; Chen, Minfang; Yang, Tiezhao; Lai, Huafang; Yang, Peiliang; Chen, Qiang; Zhou, Xiaohong

2012-06-01

To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

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Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

Decreased FOXD3 Expression Is Associated with Poor Prognosis in Patients with High-Grade Gliomas.

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Wei Du

Full Text Available The transcription factor forkhead box D3 (FOXD3 plays important roles in the development of neural crest and has been shown to suppress the development of various cancers. However, the expression and its potential biological roles of FOXD3 in high-grade gliomas (HGGs remain unknown.The mRNA and protein expression levels of FOXD3 were examined using real-time quantitative PCR and western blotting in 23 HGG and 13 normal brain samples, respectively. Immunohistochemistry was used to validate the expression FOXD3 protein in 184 HGG cases. The association between FOXD3 expression and the prognosis of HGG patients were analyzed using Kaplan-Meier survival curves and Cox proportional hazards regression models. In addition, we further examined the effects of FOXD3 on the proliferation and serum starvation-induced apoptosis of glioma cells.In comparison to normal brain tissues, FOXD3 expression was significantly decreased in HGG tissues at both mRNA and protein levels. Immunohistochemistry further validated the expression of FOXD3 in HGG tissues. Moreover, low FOXD3 expression was significantly associated with poor prognosis in HGG patients. Depletion of FOXD3 expression promoted glioma cell proliferation and inhibited serum starvation-induced apoptosis, whereas overexpression of FOXD3 inhibited glioma cell proliferation and promoted serum starvation-induced apoptosis.Our results indicated that FOXD3 might serve as an independent prognostic biomarker and a potential therapeutic target for HGGs, which warrant further investigation.

Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery.

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Panavas, Tadas; Lu, Jin; Liu, Xuesong; Winkis, Ann-Marie; Powers, Gordon; Naso, Michael F; Amegadzie, Bernard

2011-09-01

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity. Copyright © 2011 Elsevier Inc. All rights reserved.

High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli

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Huang Yadong

2010-02-01

Full Text Available Abstract Background Fibroblast growth factor 21 (FGF21 is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21 in Escherichia coli (E. coli is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier by polymerase chain reaction (PCR, and expressed the fused gene in E. coli BL21(DE3. Results By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC was shown to be higher than 96% with low endotoxin level (in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ injection. Conclusions This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.

Long-term high-level expression of human beta-globin occurs following transplantation of transgenic marrow into irradiated mice.

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Himelstein, A; Ward, M; Podda, S; de la Flor Weiss, E; Costantini, F; Bank, A

1993-03-01

When the human beta-globin gene is transferred into the bone marrow cells of live mice, its expression is very low. To investigate the reason for this, we transferred the bone marrow of transgenic mice containing and expressing the human beta-globin into irradiated recipients. We demonstrate that long-term high level expression of the human beta-globin gene can be maintained in the marrow and blood of irradiated recipients following transplantation. Although expression decreased over time in most animals because of host marrow reconstitution, the ratio of human beta-globin transgene expression to endogenous mouse beta-globin gene expression in donor-derived erythroid cells remained constant over time. We conclude that there is no inherent limitation to efficient expression of an exogenous human beta-globin gene in mouse bone marrow cells following marrow transplantation.

HPV and high-risk gene expression profiles predict response to chemoradiotherapy in head and neck cancer, independent of clinical factors

International Nuclear Information System (INIS)

Jong, Monique C. de; Pramana, Jimmy; Knegjens, Joost L.; Balm, Alfons J.M.; Brekel, Michiel W.M. van den; Hauptmann, Michael; Begg, Adrian C.; Rasch, Coen R.N.

2010-01-01

Purpose: The purpose of this study was to combine gene expression profiles and clinical factors to provide a better prediction model of local control after chemoradiotherapy for advanced head and neck cancer. Material and methods: Gene expression data were available for a series of 92 advanced stage head and neck cancer patients treated with primary chemoradiotherapy. The effect of the Chung high-risk and Slebos HPV expression profiles on local control was analyzed in a model with age at diagnosis, gender, tumor site, tumor volume, T-stage and N-stage and HPV profile status. Results: Among 75 patients included in the study, the only factors significantly predicting local control were tumor site (oral cavity vs. Pharynx, hazard ratio 4.2 [95% CI 1.4-12.5]), Chung gene expression status (high vs. Low risk profile, hazard ratio 4.4 [95% CI 1.5-13.3]) and HPV profile (negative vs. Positive profile, hazard ratio 6.2 [95% CI 1.7-22.5]). Conclusions: Chung high-risk expression profile and a negative HPV expression profile were significantly associated with increased risk of local recurrence after chemoradiotherapy in advanced pharynx and oral cavity tumors, independent of clinical factors.

Cellular Stress Response Gene Expression During Upper and Lower Body High Intensity Exercises.

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Kochanowicz, Andrzej; Sawczyn, Stanisław; Niespodziński, Bartłomiej; Mieszkowski, Jan; Kochanowicz, Kazimierz; Żychowska, Małgorzata

2017-01-01

The aim was to compare the effect of upper and lower body high-intensity exercise on chosen genes expression in athletes and non-athletes. Fourteen elite male artistic gymnasts (EAG) aged 20.6 ± 3.3 years and 14 physically active men (PAM) aged 19.9 ± 1.0 years performed lower and upper body 30 s Wingate Tests. Blood samples were collected before, 5 and 30 minutes after each effort to assess gene expression via PCR. Significantly higher mechanical parameters after lower body exercise was observed in both groups, for relative power (8.7 ± 1.2 W/kg in gymnasts, 7.2 ± 1.2 W/kg in controls, p = 0.01) and mean power (6.7 ± 0.7 W/kg in gymnasts, 5.4 ± 0.8 W/kg in controls, p = 0.01). No differences in lower versus upper body gene expression were detected for all tested genes as well as between gymnasts and physical active man. For IL-6 m-RNA time-dependent effect was observed. Because of no significant differences in expression of genes associated with cellular stress response the similar adaptive effect to exercise may be obtained so by lower and upper body exercise.

Maternal high-fat diet associated with altered gene expression, DNA methylation, and obesity risk in mouse offspring

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Zaidi, Rabab; Shah, Shyam; Oakley, M. Elsa; Pavlatos, Cassondra; El Idrissi, Samir; Xing, Xiaoyun; Li, Daofeng; Wang, Ting; Cheverud, James M.

2018-01-01

We investigated maternal obesity in inbred SM/J mice by assigning females to a high-fat diet or a low-fat diet at weaning, mating them to low-fat-fed males, cross-fostering the offspring to low-fat-fed SM/J nurses at birth, and weaning the offspring onto a high-fat or low-fat diet. A maternal high-fat diet exacerbated obesity in the high-fat-fed daughters, causing them to weigh more, have more fat, and have higher serum levels of leptin as adults, accompanied by dozens of gene expression changes and thousands of DNA methylation changes in their livers and hearts. Maternal diet particularly affected genes involved in RNA processing, immune response, and mitochondria. Between one-quarter and one-third of differentially expressed genes contained a differentially methylated region associated with maternal diet. An offspring high-fat diet reduced overall variation in DNA methylation, increased body weight and organ weights, increased long bone lengths and weights, decreased insulin sensitivity, and changed the expression of 3,908 genes in the liver. Although the offspring were more affected by their own diet, their maternal diet had epigenetic effects lasting through adulthood, and in the daughters these effects were accompanied by phenotypic changes relevant to obesity and diabetes. PMID:29447215

Maternal high-fat diet associated with altered gene expression, DNA methylation, and obesity risk in mouse offspring.

Science.gov (United States)

Keleher, Madeline Rose; Zaidi, Rabab; Shah, Shyam; Oakley, M Elsa; Pavlatos, Cassondra; El Idrissi, Samir; Xing, Xiaoyun; Li, Daofeng; Wang, Ting; Cheverud, James M

2018-01-01

We investigated maternal obesity in inbred SM/J mice by assigning females to a high-fat diet or a low-fat diet at weaning, mating them to low-fat-fed males, cross-fostering the offspring to low-fat-fed SM/J nurses at birth, and weaning the offspring onto a high-fat or low-fat diet. A maternal high-fat diet exacerbated obesity in the high-fat-fed daughters, causing them to weigh more, have more fat, and have higher serum levels of leptin as adults, accompanied by dozens of gene expression changes and thousands of DNA methylation changes in their livers and hearts. Maternal diet particularly affected genes involved in RNA processing, immune response, and mitochondria. Between one-quarter and one-third of differentially expressed genes contained a differentially methylated region associated with maternal diet. An offspring high-fat diet reduced overall variation in DNA methylation, increased body weight and organ weights, increased long bone lengths and weights, decreased insulin sensitivity, and changed the expression of 3,908 genes in the liver. Although the offspring were more affected by their own diet, their maternal diet had epigenetic effects lasting through adulthood, and in the daughters these effects were accompanied by phenotypic changes relevant to obesity and diabetes.

High expression of BCL-2 predicts favorable outcome in non-small cell lung cancer patients with non squamous histology

International Nuclear Information System (INIS)

Anagnostou, Valsamo K; Boffa, Daniel; Gettinger, Scott; Detterbeck, Frank; Homer, Robert J; Dougenis, Dimitrios; Rimm, David L; Syrigos, Konstantinos N; Lowery, Frank J; Zolota, Vassiliki; Tzelepi, Vassiliki; Gopinath, Arun; Liceaga, Camil; Panagopoulos, Nikolaos; Frangia, Konstantina; Tanoue, Lynn

2010-01-01

Bcl-2 promotes cell survival by inhibiting adapters needed for the activation and cleavage of caspases thus blocking the proteolytic cascade that ultimately dismantles the cell. Bcl-2 has been investigated as a prognostic factor in non small cell lung cancer (NSCLC) patients with conflicting results. Here, we quantitatively assessed Bcl-2 expression in two large and independent cohorts to investigate the impact of Bcl-2 on survival. AQUA ® , a fluorescent-based method for analysis of in situ protein expression, was used to measure Bcl-2 protein levels and classify tumors by Bcl-2 expression in a cohort of 180 NSCLC patients. An independent cohort of 354 NSCLC patients was used to validate Bcl-2 classification and evaluate outcome. Fifty % and 52% of the cases were classified as high expressers in training and validation cohorts respectively. Squamous cell carcinomas were more likely to be high expressers compared to adenocarcinomas (63% vs. 45%, p = 0.002); Bcl-2 was not associated with other clinical or pathological characteristics. Survival analysis showed that patients with high BCL-2 expression had a longer median survival compared to low expressers (22 vs. 17.5 months, log rank p = 0.014) especially in the subset of non-squamous tumors (25 vs. 13.8 months, log rank p = 0.04). Multivariate analysis revealed an independent lower risk for all patients with Bcl-2 expressing tumors (HR = 0.53, 95% CI 0.37-0.75, p = 0.0003) and for patients with non-squamous tumors (HR = 0.5, 95% CI 0.31-0.81, p = 0.005). Bcl-2 expression defines a subgroup of patients with a favorable outcome and may be useful for prognostic stratification of NSCLC patients

Proanthocyanidins Prevent High Glucose-Induced Eye Malformation by Restoring Pax6 Expression in Chick Embryo

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Rui-Rong Tan

2015-08-01

Full Text Available Gestational diabetes mellitus (GDM is one of the leading causes of offspring malformations, in which eye malformation is an important disease. It has raised demand for therapy to improve fetal outcomes. In this study, we used chick embryo to establish a GDM model to study the protective effects of proanthocyanidins on eye development. Chick embryos were exposed to high glucose (0.2 mmol/egg on embryo development day (EDD 1. Proanthocyanidins (1 and 10 nmol/egg were injected into the air sac on EDD 0. Results showed that both dosages of proanthocyanidins could prevent the eye malformation and rescue the high glucose-induced oxidative stress significantly, which the similar effects were showed in edaravone. However, proanthocyanidins could not decrease the glucose concentration of embryo eye. Moreover, the key genes regulating eye development, Pax6, was down-regulated by high glucose. Proanthocyanidins could restore the suppressed expression of Pax6. These results indicated proanthocyanidins might be a promising natural agent to prevent high glucose-induced eye malformation by restoring Pax6 expression.

Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae

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Takaya, Tomohiro; Koda, Risa; Adachi, Daisuke; Ogino, Chiaki; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering; Nakashima, Kazunori [Kobe Univ. (Japan). Organization of Advanced Science and Technology; Wada, Junpei; Bogaki, Takayuki [Ozeki Co., Nishinomiya-shi, Hyogo (Japan)

2011-05-15

In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5{sup '} untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL1) and double copies (pSENSU-FHL2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL1 and 2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time. (orig.)

Early expressive and receptive language trajectories in high-risk infant siblings of children with autism spectrum disorder

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Julie Longard

2017-11-01

Full Text Available Background & aims In response to limited research on early language development in infants at high risk for Autism Spectrum Disorder (ASD, the current prospective study examined early expressive and receptive language trajectories in familial high-risk (HR infants who were and were not later diagnosed with ASD (HR-ASD and HR-N, respectively, and low-risk (LR controls with no family history of ASD. Methods Participants were 523 children (371 HR siblings, 56% boys; 152 LR controls, 52% boys followed from age 6 or 12 months to 36 months. Based on independent, best-estimate clinical diagnoses at 36 months, HR participants were classified as HR-ASD (n = 94; 69% boys, or HR-N (n = 277; 52% boys; the sample also included 152 LR controls (52% boys. Expressive and receptive language trajectories were examined based on corresponding domain standard scores on the Mullen Scales of Early Learning ( MSEL at 6, 12, 24, and 36 months. In the combined sample of HR and LR infants, semi-parametric group-based modeling was used to identify distinct trajectories in MSEL standard scores. Results A 3-group solution provided optimal fit to variation in both expressive and receptive language, with the following patterns of scores: (1 inclining from average to above average, (2 stable-average, and (3 declining from average to well below average. For both expressive and receptive language, membership in these trajectories was related to 3-year diagnostic outcomes. Conclusions Although HR-ASD, HR-N, and LR control infants were in each trajectory group, membership in the declining trajectory (expressive and/or receptive was associated with an ASD diagnosis. Implications Evidence of declining trajectories in either expressive or receptive language may be a risk marker for ASD in a high-risk sample.

High expression of ubiquitin-conjugating enzyme 2C (UBE2C) correlates with nasopharyngeal carcinoma progression

International Nuclear Information System (INIS)

Shen, Zhihua; Guo, Junli; Jie, Wei; Jiang, Xiaofan; Zeng, Chao; Zheng, Shaojiang; Luo, Botao; Zeng, Yumei; Ding, Ranran; Jiang, Hanguo; He, Qiyi

2013-01-01

Overexpression of ubiquitin-conjugating enzyme 2C (UBE2C) has been detected in many types of human cancers, and is correlated with tumor malignancy. However, the role of UBE2C in human nasopharyngeal carcinoma (NPC) is unclear. In this study, we investigated the role of aberrant UBE2C expression in the progression of human NPC. Immunohistochemical analysis was performed to detect UBE2C protein in clinical samples of NPC and benign nasopharyngeal tissues, and the association of UBE2C expression with patient clinicopathological characteristics was analyzed. UBEC2 expression profiles were evaluated in cell lines representing varying differentiated stages of NPC and immortalized nasopharyngeal epithelia NP-69 cells using quantitative RT-PCR, western blotting and fluorescent staining. Furthermore, UBE2C was knocked down using RNA interference in these cell lines and proliferation and cell cycle distribution was investigated. Immunohistochemical analysis revealed that UBE2C protein expression levels were higher in NPC tissues than in benign nasopharyngeal tissues (P<0.001). Moreover, high UBE2C protein expression was positively correlated with tumor size (P=0.017), lymph node metastasis (P=0.016) and distant metastasis (P=0.015) in NPC patients. In vitro experiments demonstrated that UBE2C expression levels were inversely correlated with the degree of differentiation of NPC cell lines, whereas UBE2C displayed low level of expression in NP-69 cells. Knockdown of UBE2C led to significant arrest at the S and G2/M phases of the cell cycle, and decreased cell proliferation was observed in poorly-differentiated CNE2Z NPC cells and undifferentiated C666-1 cells, but not in well-differentiated CNE1 and immortalized NP-69 cells. Our findings suggest that high expression of UBE2C in human NPC is closely related to tumor malignancy, and may be a potential marker for NPC progression

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

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Sustarsic, Elahu G. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Junnila, Riia K. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Kopchick, John J., E-mail: kopchick@ohio.edu [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH (United States)

2013-11-08

Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on

Maternal high-fat diet and offspring expression levels of vitamin K-dependent proteins.

Science.gov (United States)

Lanham, S A; Cagampang, F R; Oreffo, R O C

2014-12-01

Studies suggest that bone growth and development and susceptibility to vascular disease in later life are influenced by maternal nutrition during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including osteocalcin, matrix Gla protein, periostin, and growth-arrest specific- protein 6, in both bone and vascular development. We have examined whether there are alterations in these VKDPs in bone and vascular tissue from offspring of mothers subjected to a nutritional challenge: a high-fat diet during pregnancy and postnatally, using 6-week-old mouse offspring. Bone site-specific and sex-specific differences across femoral and vertebral bone in male and female offspring were observed. Overall a high-fat maternal diet and offspring diet exacerbated the bone changes observed. Sex-specific differences and tissue-specific differences were observed in VKDP levels in aorta tissue from high-fat diet-fed female offspring from high-fat diet-fed mothers displaying increased levels of Gas6 and Ggcx compared with those of female controls. In contrast, differences were seen in VKDP levels in femoral bone of female offspring with lower expression levels of Mgp in offspring of mothers fed a high-fat diet compared with those of controls. We observed a significant correlation in Mgp expression levels within the femur to measures of bone structure of the femur and vertebra, particularly in the male offspring cohort. In summary, the current study has highlighted the importance of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure.

High expression of miR-21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer

DEFF Research Database (Denmark)

Rask, Lene; Balslev, Eva; Jørgensen, Stine

2011-01-01

features, we performed in situ hybridization and semi-quantitative assessment of the miR-21 signal on 12 LN negative grade I (assumed low risk), and 12 LN positive grade II (high risk) breast cancers. miR-21 was predominantly seen in cancer associated fibroblast-like cells, with no difference in expression......Low-risk and high-risk breast cancer patients are stratified primarily according to their lymph node (LN) status and grading. However, some low-risk patients relapse, and some high-risk patients have a favorable clinical outcome, implying a need for better prognostic and predictive tests. Micro...... RNAs are often aberrantly expressed in cancer and microRNA-21 is upregulated in a variety of cancers, including breast cancer. High miR-21 levels have been associated with poor prognosis. To determine the cellular localization of miR-21 and to compare its expression levels with histopathological...

Long-term intake of a high prebiotic fiber diet but not high protein reduces metabolic risk after a high fat challenge and uniquely alters gut microbiota and hepatic gene expression.

Science.gov (United States)

Saha, Dolan C; Reimer, Raylene A

2014-09-01

A mismatch between early developmental diet and adulthood may increase obesity risk. Our objective was to determine the effects of re-matching rats to their weaning diets high in protein or fiber after transient high-fat/high-sucrose challenge in adulthood. We hypothesize that a long-term high fiber diet will be associated with a gut microbiota and hepatic gene expression reflective of reduced adiposity. Wistar rat pups were fed a control (C), high prebiotic fiber (HF), or high protein (HP) diet from 3-15 weeks of age; a high-fat/high-sucrose diet from 15-21 weeks; their respective C, HF, or HP diets from 21-25 weeks. Gut microbiota of cecal contents and hepatic gene expression were measured when rats were terminated at 25 weeks of age. HF rats had higher total bacteria, bifidobacteria and Bacteroides/Prevotella spp than C and HP at 25 weeks (P diet attenuated the typical increase in Firmicutes:Bacteroidetes ratio associated with consumption of a high fat diet. Lower hepatic cholesterol with long-term HF diet intake may be related to alterations in gut microbiota and hepatic lipid metabolism. Copyright © 2014 Elsevier Inc. All rights reserved.

High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

DEFF Research Database (Denmark)

Bach, Søren Spanner; Bassard, Jean-Étienne André; Andersen-Ranberg, Johan

2014-01-01

To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized...

Transplastomic expression of bacterial L-aspartate-alpha-decarboxylase enhances photosynthesis and biomass production in response to high temperature stress.

Science.gov (United States)

Fouad, W M; Altpeter, F

2009-10-01

Metabolic engineering for beta-alanine over-production in plants is expected to enhance environmental stress tolerance. The Escherichia coli L-aspartate-alpha-decarboxylase (AspDC) encoded by the panD gene, catalyzes the decarboxylation of L-aspartate to generate beta-alanine and carbon dioxide. The constitutive E. coli panD expression cassette was co-introduced with the constitutive, selectable aadA expression cassette into the chloroplast genome of tobacco via biolistic gene transfer and homologous recombination. Site specific integration of the E. coli panD expression cassette into the chloroplast genome and generation of homotransplastomic plants were confirmed by PCR and Southern blot analysis, respectively, following plant regeneration and germination of seedlings on selective media. PanD expression was verified by assays based on transcript detection and in vitro enzyme activity. The AspDC activities in transplastomic plants expressing panD were drastically increased by high-temperature stress. beta-Alanine accumulated in transplastomic plants at levels four times higher than in wildtype plants. Analysis of chlorophyll fluorescence on plants subjected to severe heat stress at 45 degrees C under light verified that photosystem II (PSII) in transgenic plants had higher thermotolerance than in wildtype plants. The CO(2) assimilation of transplastomic plants expressing panD was more tolerant to high temperature stress than that of wildtype plants, resulting in the production of 30-40% more above ground biomass than wildtype control. The results presented indicate that chloroplast engineering of the beta-alanine pathway by over-expression of the E. coli panD enhances thermotolerance of photosynthesis and biomass production following high temperature stress.

Effect of lycium barbarum polysaccharides on high glucose-induced retinal ganglion cell apoptosis, gene expression and delayed rectifier potassium current

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Xiao-Fei Ma

2017-05-01

Full Text Available Objective: To study the effect of lycium barbarum polysaccharides (LBP on high glucoseinduced retinal ganglion cell apoptosis, gene expression and delayed rectifier potassium current. Methods: RGC-5 retinal ganglion cell lines were cultured and divided into control group, high glucose group and LBP group that were treated with normal DMEM, highglucose DMEM as well as high-glucose DMEM containing 500 ng/mL LBP respectively. After treatment, the Annexin V-FITC/PI kits were used to measure the number of apoptotic cells, fluorescence quantitative PCR kits were used to determine the expression of apoptosis genes and antioxidant genes, and patch clamp was used to test delayed rectifier potassium current. Results: 12, 24, 36 and 48 h after intervention, the number of apoptotic cells of high glucose group was significantly higher than that of control group, and the number of apoptotic cells of LBP group was significantly lower than that of high glucose group (P<0.05; 24 and 48 h after intervention, c-fos, c-jun, caspase-3, caspase-9, Nrf-2, NQO1 and HO-1 mRNA expression as well as potassium current amplitude (IK and maximum conductance (Gmax of high glucose group were significantly higher than those of control group while half maximum activation voltage (V1/2 was significantly lower than that of control group (P<0.05; c-fos, c-jun, caspase-3 and caspase-9 mRNA expression as well as IK and Gmax of LBP group were significantly lower than those of high glucose group, while Nrf-2, NQO1 and HO-1 mRNA expression as well as V1/2 of LBP group were significantly higher than those of high glucose group (P<0.05. Conclusions: LBP can reduce the high glucose-induced retinal ganglion cell apoptosis and inhibit the delayed rectifier potassium current amplitude.

High level PHGDH expression in breast is predominantly associated with keratin 5-positive cell lineage independently of malignancy

DEFF Research Database (Denmark)

Gromova, Irina; Gromov, Pavel; Honma, Naoko

2015-01-01

in TNBC samples. One such protein was D-3-phosphoglycerate dehydrogenase (Phgdh), a candidate oncogene. We analysed expression of Phgdh in normal and TNBC mammary tissue samples by 2D gel-based proteomics and immunohistochemistry (IHC), and show here that high-level expression of Phgdh in mammary...

High-fat diet amplifies renal renin angiotensin system expression, blood pressure elevation, and renal dysfunction caused by Ceacam1 null deletion.

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Li, Caixia; Culver, Silas A; Quadri, Syed; Ledford, Kelly L; Al-Share, Qusai Y; Ghadieh, Hilda E; Najjar, Sonia M; Siragy, Helmy M

2015-11-01

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMl), a substrate of the insulin receptor tyrosine kinase, regulates insulin action by promoting insulin clearance. Global null mutation of Ceacam1 gene (Cc1(-/-)) results in features of the metabolic syndrome, including insulin resistance, hyperinsulinemia, visceral adiposity, elevated blood pressure, and albuminuria. It also causes activation of the renal renin-angiotensin system (RAS). In the current study, we tested the hypothesis that high-fat diet enhances the expression of RAS components. Three-month-old wild-type (Cc1(+/+)) and Cc1(-/-) mice were fed either a regular or a high-fat diet for 8 wk. At baseline under regular feeding conditions, Cc1(-/-) mice exhibited higher blood pressure, urine albumin-to-creatinine ratio (UACR), and renal expression of angiotensinogen, renin/prorenin, angiotensin-converting enzyme, (pro)renin receptor, angiotensin subtype AT1 receptor, angiotensin II, and elevated PI3K phosphorylation, as detected by p85α (Tyr(508)) immunostaining, inflammatory response, and the expression of collagen I and collagen III. In Cc1(+/+) mice, high-fat diet increased blood pressure, UACR, the expression of angiotensin-converting enzyme and angiotensin II, PI3K phosphorylation, inflammatory response, and the expression of collagen I and collagen III. In Cc1(-/-) mice, high-fat intake further amplified these parameters. Immunohistochemical staining showed increased p-PI3K p85α (Tyr(508)) expression in renal glomeruli, proximal, distal, and collecting tubules of Cc1(-/-) mice fed a high-fat diet. Together, this demonstrates that high-fat diet amplifies the permissive effect of Ceacam1 deletion on renal expression of all RAS components, PI3K phosphorylation, inflammation, and fibrosis. Copyright © 2015 the American Physiological Society.

Revealing complex function, process and pathway interactions with high-throughput expression and biological annotation data.

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Singh, Nitesh Kumar; Ernst, Mathias; Liebscher, Volkmar; Fuellen, Georg; Taher, Leila

2016-10-20

The biological relationships both between and within the functions, processes and pathways that operate within complex biological systems are only poorly characterized, making the interpretation of large scale gene expression datasets extremely challenging. Here, we present an approach that integrates gene expression and biological annotation data to identify and describe the interactions between biological functions, processes and pathways that govern a phenotype of interest. The product is a global, interconnected network, not of genes but of functions, processes and pathways, that represents the biological relationships within the system. We validated our approach on two high-throughput expression datasets describing organismal and organ development. Our findings are well supported by the available literature, confirming that developmental processes and apoptosis play key roles in cell differentiation. Furthermore, our results suggest that processes related to pluripotency and lineage commitment, which are known to be critical for development, interact mainly indirectly, through genes implicated in more general biological processes. Moreover, we provide evidence that supports the relevance of cell spatial organization in the developing liver for proper liver function. Our strategy can be viewed as an abstraction that is useful to interpret high-throughput data and devise further experiments.

High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains

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Masaru Nakata

2017-12-01

Full Text Available Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by

Influence of high-frequency electromagnetic fields on different modes of cell death and gene expression.

Science.gov (United States)

Port, M; Abend, M; Römer, B; Van Beuningen, D

2003-09-01

International thresholds for exposure to non-ionizing radiation leading to non-thermal effects were conservatively set by the International Commission on Non-Ionizing Radiation Protection (ICNIRP). The aim of this study was to examine whether biological effects such as different modes of cell death and gene expression modifications related to tumorgenesis are detectable above the threshold defined. Human leukaemia cells (HL-60) grown in vitro were exposed to electromagnetic fields (EMF; t 1/2(r) about 1 ns; field strength about 25 times higher than the ICNIRP reference levels for occupational exposure) leading to non-thermal effects using a high-voltage-improved GTEM cell 5302 (EMCO) connected to a pulse generator NP20 (C = 1 nF, U(Load) = 20kV). HL-60 cells were harvested at 0, 24, 48 and 72 h after radiation exposure. Micronuclei, apoptosis and abnormal cells (e.g. necrosis) were determined using morphological criteria. In parallel, the expression of 1176 genes was measured using Atlas Human 1.2. Array. Based on high data reproducibility calculated from two independent experiments (> 99%), array analysis was performed. No significant change in apoptosis, micronucleation, abnormal cells and differential gene expression was found. Exposure of HL-60 cells to EMFs 25 times higher than the ICNIRP reference levels for occupational exposure failed to induce any changes in apoptosis, micronucleation, abnormal morphologies and gene expression. Further experiments using EMFs above the conservatively defined reference level set by the ICNIRP may be desirable.

High-Throughput Screening to Identify Regulators of Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

Science.gov (United States)

Kassir, Yona

2017-01-01

Meiosis and gamete formation are processes that are essential for sexual reproduction in all eukaryotic organisms. Multiple intracellular and extracellular signals feed into pathways that converge on transcription factors that induce the expression of meiosis-specific genes. Once triggered the meiosis-specific gene expression program proceeds in a cascade that drives progress through the events of meiosis and gamete formation. Meiosis-specific gene expression is tightly controlled by a balance of positive and negative regulatory factors that respond to a plethora of signaling pathways. The budding yeast Saccharomyces cerevisiae has proven to be an outstanding model for the dissection of gametogenesis owing to the sophisticated genetic manipulations that can be performed with the cells. It is possible to use a variety selection and screening methods to identify genes and their functions. High-throughput screening technology has been developed to allow an array of all viable yeast gene deletion mutants to be screened for phenotypes and for regulators of gene expression. This chapter describes a protocol that has been used to screen a library of homozygous diploid yeast deletion strains to identify regulators of the meiosis-specific IME1 gene.

Copepod swimming behavior, respiration, and expression of stress-related genes in response to high stocking densities

DEFF Research Database (Denmark)

Nilsson, Birgitte; Jakobsen, Hans Henrik; Stief, Peter

2017-01-01

is problematic for calanoid copepod species like Acartia tonsa. In the present study, we evaluated the response of copepods experiencing stress under high-density conditions by assessing the acute stress level of A. tonsa. Control density was at 100 ind. L−1 while the treatments were increased stepwise up to 10......,000 ind. L−1. Three biological/physiological end-points were studied: swimming behavior, respiration rate and expression level of stress-related genes. None of the elevated densities caused any significant change in swimming behavior, respiration rate or gene expression level. This study suggests...... that adults of A. tonsa do not exhibit any measurable acute stress response when exposed to high culture densities for 12 h....

Tissue Specific Expression Of Sprouty1 In Mice Protects Against High Fat Diet Induced Fat Accumulation, Bone Loss, And Metabolic Dysfunction

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Urs, Sumithra; Henderson, Terry; Le, Phuong; Rosen, Clifford J.; Liaw, Lucy

2012-01-01

We recently characterized Sprouty1 (Spry1), a growth factor signaling inhibitor as a regulator of marrow progenitor cells promoting osteoblast differentiation at the expense of adipocytes. Adipose tissue specific Spry1 expression in mice resulted in increased bone mass and reduced body fat while conditional knockout of Spry1 had the opposite effect with decreased bone and increased body fat. Because Spry1 suppresses normal fat development, we tested the hypothesis that Spry1 expression prevents high fat diet-induced obesity, bone loss, and associated lipid abnormalities and demonstrate that Spry1 has a long-term protective effect on mice fed a high caloric diet. We studied diet-induced obesity in mice with fatty acid binding promoter (aP2)-driven expression or conditional knockout of Spry1 in adipocytes. Phenotyping was performed by whole body dual-energy X-ray absorptiometry, microCT, histology and blood analysis. In conditional Spry1 null mice, high fat diet increased body fat by 40%, impaired glucose regulation, and led to liver steatosis. However, over-expression of Spry1 led to 35% lower body fat, reduced bone loss, and normal metabolic function compared to single transgenics. This protective phenotype was associated with decreased circulating insulin (70%) and leptin (54%) compared to controls on a high fat diet. Additionally, Spry1 expression decreased adipose tissue inflammation by 45%. We show that conditional Spry1 expression in adipose tissue protects against high fat diet-induced obesity and associated bone loss. PMID:22142492

DNA methylation alters transcriptional rates of differentially expressed genes and contributes to pathophysiology in mice fed a high fat diet

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Pili Zhang

2017-04-01

Full Text Available Objective: Overnutrition can alter gene expression patterns through epigenetic mechanisms that may persist through generations. However, it is less clear if overnutrition, for example a high fat diet, modifies epigenetic control of gene expression in adults, or by what molecular mechanisms, or if such mechanisms contribute to the pathology of the metabolic syndrome. Here we test the hypothesis that a high fat diet alters hepatic DNA methylation, transcription and gene expression patterns, and explore the contribution of such changes to the pathophysiology of obesity. Methods: RNA-seq and targeted high-throughput bisulfite DNA sequencing were used to undertake a systematic analysis of the hepatic response to a high fat diet. RT-PCR, chromatin immunoprecipitation and in vivo knockdown of an identified driver gene, Phlda1, were used to validate the results. Results: A high fat diet resulted in the hypermethylation and decreased transcription and expression of Phlda1 and several other genes. A subnetwork of genes associated with Phlda1 was identified from an existing Bayesian gene network that contained numerous hepatic regulatory genes involved in lipid and body weight homeostasis. Hepatic-specific depletion of Phlda1 in mice decreased expression of the genes in the subnetwork, and led to increased oil droplet size in standard chow-fed mice, an early indicator of steatosis, validating the contribution of this gene to the phenotype. Conclusions: We conclude that a high fat diet alters the epigenetics and transcriptional activity of key hepatic genes controlling lipid homeostasis, contributing to the pathophysiology of obesity. Author Video: Author Video Watch what authors say about their articles Keywords: DNA methylation, RNA-seq, Transcription, High fat diet, Liver, Phlda1

High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

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Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

2016-03-01

An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

Genetic requirements for high constitutive SOS expression in recA730 mutants of Escherichia coli.

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Vlašić, Ignacija; Šimatović, Ana; Brčić-Kostić, Krunoslav

2011-09-01

The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5'-3' exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5'-3' exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

High E6 Gene Expression Predicts for Distant Metastasis and Poor Survival in Patients With HPV-Positive Oropharyngeal Squamous Cell Carcinoma

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Khwaja, Shariq S.; Baker, Callie; Haynes, Wesley; Spencer, Christopher R.; Gay, Hiram; Thorstad, Wade [Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri (United States); Adkins, Douglas R. [Division of Medical Oncology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri (United States); Nussenbaum, Brian [Department of Otolaryngology – Head and Neck Surgery, Washington University School of Medicine, St. Louis, Missouri (United States); Chernock, Rebecca D. [Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri (United States); Lewis, James S. [Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee (United States); Wang, Xiaowei, E-mail: xwang@radonc.wustl.edu [Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri (United States)

2016-07-15

Purpose: Patients with human papillomavirus (HPV)–positive oropharyngeal squamous cell carcinoma (OPSCC) have a favorable prognosis. As a result, de-escalation clinical trials are under way. However, approximately 10% of patients will experience distant recurrence even with standard-of-care treatment. Here, we sought to identify novel biomarkers to better risk-stratify HPV-positive patients with OPSCC. Methods and Materials: Gene expression profiling by RNA sequencing (RNA-seq) and quantitative polymerase chain reaction was performed on HPV-positive OPSCC primary tumor specimens from patients with and without distant metastasis (DM). Results: RNA-seq analysis of 39 HPV-positive OPSCC specimens revealed that patients with DM had 2-fold higher E6 gene expression levels than did patients without DM (P=.029). This observation was confirmed in a validation cohort comprising 93 patients with HPV-positive OPSCC. The mean normalized E6 expression level in the 17 recurring primary specimens was 13 ± 2 compared with 8 ± 1 in the remaining 76 nonrecurring primaries (P=.001). Receiver operating characteristic analysis established an E6 expression level of 7.3 as a cutoff for worse recurrence-free survival (RFS). Patients from this cohort with high E6 gene expression (E6-high) (n=51, 55%) had more cancer-related deaths (23% vs 2%, P<.001) and DM (26% vs 5%, P<.001) than did patients with low E6 gene expression (E6-low) (n=42, 45%). Kaplan-Meier survival analysis revealed that E6-high had worse RFS (95% vs 69%, P=.004) and cancer-specific survival (97% vs 79%, P=.007). E6-high maintained statistical significance in multivariate regression models balancing surgery, chemotherapy, nodal stage, and smoking status. Gene set enrichment analysis demonstrated that tumors with high E6 expression were associated with P53, epidermal growth factor receptor, activating transcription factor-2, and transforming growth factor-β signaling pathways. Conclusion: High E6 gene expression

High RBM3 expression in prostate cancer independently predicts a reduced risk of biochemical recurrence and disease progression

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Bjartell Anders

2011-09-01

Full Text Available Abstract Background High expression of the RNA-binding protein RBM3 has previously been found to be associated with good prognosis in breast cancer, ovarian cancer, malignant melanoma and colorectal cancer. The aim of this study was to examine the prognostic impact of immunohistochemical RBM3 expression in prostate cancer. Findings Immunohistochemical RBM3 expression was examined in a tissue microarray with malignant and benign prostatic specimens from 88 patients treated with radical prostatectomy for localized disease. While rarely expressed in benign prostate gland epithelium, RBM3 was found to be up-regulated in prostate intraepithelial neoplasia and present in various fractions and intensities in invasive prostate cancer. High nuclear RBM3 expression was significantly associated with a prolonged time to biochemical recurrence (BCR (HR 0.56, 95% CI: 0.34-0.93, p = 0.024 and clinical progression (HR 0.09, 95% CI: 0.01-0.71, p = 0.021. These associations remained significant in multivariate analysis, adjusted for preoperative PSA level in blood, pathological Gleason score and presence or absence of extracapsular extension, seminal vesicle invasion and positive surgical margin (HR 0.41, 95% CI: 0.19-0.89, p = 0.024 for BCR and HR 0.06, 95% CI: 0.01-0.50, p = 0.009 for clinical progression. Conclusion Our results demonstrate that high nuclear expression of RBM3 in prostate cancer is associated with a prolonged time to disease progression and, thus, a potential biomarker of favourable prognosis. The value of RBM3 for prognostication, treatment stratification and follow-up of prostate cancer patients should be further validated in larger studies.

Decreased expression of CD36 in circumvallate taste buds of high-fat diet induced obese rats.

Science.gov (United States)

Zhang, Xiao-Juan; Zhou, Li-Hong; Ban, Xiang; Liu, Dian-Xin; Jiang, Wei; Liu, Xiao-Min

2011-10-01

Mammals spontaneously prefer lipid rich foods. Overconsumption of high-fat diet leads to obesity and related diseases. Recent findings indicate that taste may participate in the orosensory perception of dietary lipids and the fatty taste may contribute to a preference for and excessive consumption of dietary fat. CD36, a trans-membrane glycoprotein, which is located in the taste buds of circumvallate papillae of rodents, appears to be a plausible receptor for this fatty taste. Obese subjects present a stronger preference for fatty foods, though the mechanisms involved are complex and are not fully investigated. Our data from immunofluorescence and real-time RT-PCR showed that the expression levels of CD36 in circumvallate taste buds were significantly lower in high-fat diet induced obese rats as compared with that of control rats fed a normal diet. These results suggest that decreased expression of CD36 in circumvallate taste buds of high-fat diet induced obese rats may be associated with diminished fatty taste sensitivity and in order to compensate the preference for dietary fat, rats consume more fatty foods. Therapeutic strategies designed to alter or manipulate CD36 expression or function in taste buds may have important implications in treating obesity and related diseases. Copyright © 2010 Elsevier GmbH. All rights reserved.

High Expressed Emotion and Schizophrenia: A Study of Illness ...

African Journals Online (AJOL)

Background: The prevention of relapse is one of the major aims of treatment of emotional disorders. Expressed emotion (EE) is one concept that has been associated with relapse. The study is aimed at studying the relationship between expressed emotion and the clinical characteristics of patients with schizophrenia.

High Laccase Expression by Trametes versicolor in a Simulated Textile Effluent with Different Carbon Sources and PHs

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Cristiane Ottoni

2016-08-01

Full Text Available Textile effluents are highly polluting and have variable and complex compositions. They can be extremely complex, with high salt concentrations and alkaline pHs. A fixed-bed bioreactor was used in the present study to simulate a textile effluent treatment, where the white-rot fungus, Trametes versicolor, efficiently decolourised the azo dye Reactive Black 5 over 28 days. This occurred under high alkaline conditions, which is unusual, but advantageous, for successful decolourisation processes. Active dye decolourisation was maintained by operation in continuous culture. Colour was eliminated during the course of operation and maximum laccase (Lcc activity (80.2 U∙L−1 was detected after glycerol addition to the bioreactor. Lcc2 gene expression was evaluated with different carbon sources and pH values based on reverse transcriptase-PCR (polymerase chain reaction. Glycerol was shown to promote the highest lcc2 expression at pH 5.5, followed by sucrose and then glucose. The highest levels of expression occurred between three and four days, which corroborate the maximum Lcc activity observed for sucrose and glycerol on the bioreactor. These results give new insights into the use of T. versicolor in textile dye wastewater treatment with high pHs.

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues.

Science.gov (United States)

Viana, Antonio A B; Fragoso, Rodrigo R; Guimarães, Luciane M; Pontes, Naiara; Oliveira-Neto, Osmundo B; Artico, Sinara; Nardeli, Sarah M; Alves-Ferreira, Marcio; Batista, João A N; Silva, Maria C M; Grossi-de-Sa, Maria F

2011-11-24

Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

High-level expression, purification and antibacterial activity of bovine lactoferricin and lactoferrampin in Photorhabdus luminescens.

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Tang, Zhiru; Zhang, Youming; Stewart, Adrian Francis; Geng, Meimei; Tang, Xiangsha; Tu, Qiang; Yin, Yulong

2010-10-01

Bovine lactoferricin (LFC) and bovine lactoferrampin (LFA) are two active fragments located in the N(1)-domain of bovine lactoferrin. Recent studies suggested that LFC and LFA have broad-spectrum activity against Gram-positive and Gram-negative bacteria. To date, LFC and LFA have usually been produced from milk. We report here the high-level expression, purification and characterization of LFC and LFA using the Photorhabdus luminescens expression system. After the cipA and cipB genes were deleted by ET recombination, the expression host P. luminescens TZR(001) was constructed. A synthetic LFC-LFA gene containing LFC and LFA was fused with the cipB gene to form a cipB-LFC-LFA gene. To obtain the expression vector pBAD-cipB-LFC-LFA, the cipB-LFC-LFA gene was cloned on the L-arabinose-inducible expression vector pBAD24. pBAD-cipB-LFC-LFA was transformed into P. luminescens TZR(001). The cipB-LFC-LFA fusion protein was expressed under the induction of L-arabinose and its yield reached 12 mg L(-1) bacterial culture. Recombinant LFC-LFA was released from cipB by pepsin. The MIC of recombinant LFC-LFA toward E. coli 0149, 0141 and 020 was 6.25, 12.5 and 3.175 microg ml(-1), respectively. Copyright 2010 Elsevier Inc. All rights reserved.

How African-American Elementary Students in High-Poverty Schools Experience Creative Expression: A Case Study Approach

Science.gov (United States)

Willis, Belinda F.

2016-01-01

Literature that addresses how the arts enhance student learning through creative expression is minimal. This is especially true for African-American elementary students from high-poverty backgrounds. The purpose of this study was to employ a case study design to explore how African-American elementary students in high-poverty schools experience…

The primary study of the radiosensitive effect on the H460 cell line by human lactotransferrin high expression in vitro

International Nuclear Information System (INIS)

Wang Yong; Wang Yan; Du Liqing; Yang Qingshan; Wang Yueying; Fan Feiyue

2009-01-01

Objective: To investigate the possible radiosensitive effect of human Lactotransferrin (hLF) high expression in lung cancer cell, we hereby constructed and transfected a recombinant vector pBC1 containing exogenous hLF gene. Methods: Firstly the recombinant plasmid pBC1-hLF was transferred into H460 and the hLF expression was evaluated by western blotting. Then we test the cell viability, apoptosis and clone formation after radiation along with the standard hLF treatment control. Results: The results showed that the hLF gene had been successfully transfect into H460 cells and hLF high expression can reduce the clone formation after radiation (P<0.01), and inhibit the cell proliferation with induced apoptosis (P<0.01). And the sensitization ratio of hLF treated and pBC1-hLF transfected were 1.29, 1.59. Conclusion: Our primary data shows that hLF high expression can radio sensitize the H460 cell in vitro. (authors)

Expression of phosphorylated extracellular signal-regulated kinase in rat kidneys exposed to high +Gz

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Hyun-Soo Kim

2012-11-01

Full Text Available Exposure to high gravitational acceleration forces acting along the body axis from the head to the feet (+Gz severely reduces blood flow to the visceral organs, including the kidneys. Extracellular signal-regulated kinase (ERK figures predominantly in mediating kidney cell responses to a wide variety of stress-related stimuli. Though previous studies have shown the activation of ERK in some experimental models, the regulation of ERK associated with +Gz exposure has not yet been investigated. The aim of this study was to examine the effect of high +Gz exposure on ERK activation in the kidneys. Using a small animal centrifuge, eight male Sprague-Dawley rats were exposed to +10Gz or +13Gz three times for 3 minutes each. The bilateral kidneys were obtained from each rat, and the expression levels of phosphorylated ERK (p-ERK were evaluated using immunohistochemistry. In the control group, the collecting duct epithelium displayed faint cytoplasmic staining with no nuclear staining of p-ERK. By contrast, rats exposed to +10Gz showed strong nuclear staining intensity for p-ERK. In the renal papilla, the epithelial cells of collecting ducts and thin segments of the loop of Henle exhibited strong nuclear immunoreactivity for p-ERK. Rats exposed to +13Gz also showed the same staining intensity and distribution of p-ERK expression as that of rats exposed to +10Gz. This study is the first to describe +Gz exposure-induced alteration in the expression of p-ERK in the kidneys. Our finding suggests that high +Gz exposure leads to the activation of ERK in the renal papilla.

Highly expressed genes within hippocampal sector CA1: implications for the physiology of memory

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Michael A. Meyer

2014-06-01

Full Text Available As the CA1 sector has been implicated to play a key role in memory formation, a dedicated search for highly expressed genes within this region was made from an on-line atlas of gene expression within the mouse brain (GENSAT. From a data base of 1013 genes, 16 were identified that had selective localization of gene expression within the CA1 region, and included Angpt2, ARHGEF6, CCK, Cntnap1, DRD3, EMP1, Epha2, Itm2b, Lrrtm2, Mdk, PNMT, Ppm1e, Ppp2r2d, RASGRP1, Slitrk5, and Sstr4. Of the 16 identified, the most selective and intense localization for both adult and post-natal day 7 was noted for ARHGEF6, which is known to be linked to non-syndromic mental retardation, and has also been localized to dendritic spines. Further research on the role played by ARHGEF6 in memory formation is strongly advocated.

Highly Expressed Genes within Hippocampal Sector CA1: Implications for the Physiology of Memory.

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Meyer, Michael A

2014-04-22

As the CA1 sector has been implicated to play a key role in memory formation, a dedicated search for highly expressed genes within this region was made from an on-line atlas of gene expression within the mouse brain (GENSAT). From a data base of 1013 genes, 16 were identified that had selective localization of gene expression within the CA1 region, and included Angpt2, ARHGEF6, CCK, Cntnap1, DRD3, EMP1, Epha2, Itm2b, Lrrtm2, Mdk, PNMT, Ppm1e, Ppp2r2d, RASGRP1, Slitrk5, and Sstr4. Of the 16 identified, the most selective and intense localization for both adult and post-natal day 7 was noted for ARHGEF6, which is known to be linked to non-syndromic mental retardation, and has also been localized to dendritic spines. Further research on the role played by ARHGEF6 in memory formation is strongly advocated.

High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

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Bing Xu

Full Text Available G protein-coupled receptors (GPCRs exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(--TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

Hydrogen sulfide inhibits high glucose-induced NADPH oxidase 4 expression and matrix increase by recruiting inducible nitric oxide synthase in kidney proximal tubular epithelial cells.

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Lee, Hak Joo; Lee, Doug Yoon; Mariappan, Meenalakshmi M; Feliers, Denis; Ghosh-Choudhury, Goutam; Abboud, Hanna E; Gorin, Yves; Kasinath, Balakuntalam S

2017-04-07

High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (H 2 S) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether H 2 S inhibits high glucose-induced expression of NOX4 and matrix protein and whether H 2 S and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of H 2 S. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by N (ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, H 2 S recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters H 2 S and NO and their interaction may serve as therapeutic targets in diabetic kidney disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Gene Expression Commons: an open platform for absolute gene expression profiling.

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Jun Seita

Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

Asymmetric Uncertainty Expression for High Gradient Aerodynamics

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Pinier, Jeremy T

2012-01-01

When the physics of the flow around an aircraft changes very abruptly either in time or space (e.g., flow separation/reattachment, boundary layer transition, unsteadiness, shocks, etc), the measurements that are performed in a simulated environment like a wind tunnel test or a computational simulation will most likely incorrectly predict the exact location of where (or when) the change in physics happens. There are many reasons for this, includ- ing the error introduced by simulating a real system at a smaller scale and at non-ideal conditions, or the error due to turbulence models in a computational simulation. The un- certainty analysis principles that have been developed and are being implemented today do not fully account for uncertainty in the knowledge of the location of abrupt physics changes or sharp gradients, leading to a potentially underestimated uncertainty in those areas. To address this problem, a new asymmetric aerodynamic uncertainty expression containing an extra term to account for a phase-uncertainty, the magnitude of which is emphasized in the high-gradient aerodynamic regions is proposed in this paper. Additionally, based on previous work, a method for dispersing aerodynamic data within asymmetric uncer- tainty bounds in a more realistic way has been developed for use within Monte Carlo-type analyses.

High-frequency deregulated expression of Wnt signaling pathway members in breast carcinomas.

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Khan, Zahid; Arafah, Maha; Shaik, Jilani Purusottapatnam; Mahale, Alka; Alanazi, Mohammad Saud

2018-01-01

Breast carcinoma is the most common malignancy and leading cause of cancer-related deaths in women worldwide including Saudi Arabia. Breast cancer in Saudi women develops at a much early age with median age of onset of 49 years compared to 62 years observed in patients from USA. Aberrations in wingless and integration site growth factor (Wnt) signaling pathway have been pathologically implicated in development of breast cancers and hence its role was examined in Saudi patients. We immunohistochemically examined various components of Wnt signaling pathway including β-catenin, tumor suppressor proteins, adenomatous polyposis coli (APC), and Axin, expression of naturally occurring pathway antagonists such as Dickkopf Wnt signaling pathway inhibitor 3 (DKK3), FRP2, and WIF1, as well as Wnt target cyclin D1 and c-Myc to establish if the pathway is constitutively activated in breast cancers arising in Saudi women. Cytoplasmic β-catenin, indicative of activation of the pathway, was observed in 24% of cases. Expression of APC and Axin, which are components of β-catenin destruction complex, was lost in 5% and 10% of tumors, respectively. Additionally, Wnt signaling inhibitors DKK3, FRP2, and Wnt inhibitory factor 1 (WIF1) were not expressed in 8%, 14%, and 5% breast tumors, respectively. Overall, accumulation of cytoplasmic β-catenin and downregulation of other Wnt pathway proteins (APC/Axin/DKK3/FRP2/WIF1) were found in approximately half of the breast cancers (47%) in our cohort. Consistent with this, analysis of Wnt target genes demonstrated moderate-to-strong expression of c-Myc in 58% and cyclin D1 in 50% of breast cancers. Deregulation of Wnt pathway was not associated with age of onset of the disease, tumor grade, and triple-negative status of breast cancers. High level of deregulated expression of Wnt pathway proteins suggests its important role in pathogenesis of breast cancers arising in Saudi women who may benefit from development of therapeutic drugs

High-level production of α-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis.

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He, Penghui; Zhang, Zeying; Cai, Dongbo; Chen, Yaozhong; Wang, Hao; Wei, Xuetuan; Li, Shunyi; Chen, Shouwen

2017-09-01

To improve target protein production by manipulating expression levels of alanine racemase in Bacillus licheniformis. The gene of dal was identified to be responsible for alanine racemase function. Based on the selection marker of dal, a food-grade expression system was constructed in B. licheniformis, and effects of different dal expression levels mediated by promoters on α-amylase production were investigated. The highest α-amylase activity (155 U/ml) was obtained in BL10D/pP43SAT-PtetDal, increased by 27% compared with that of the control strain BL10/pP43SAT in tetracycline-based system (123 U/ml). Moreover, the dal transcriptional level was not correlated positively with that of amyL. A food-grade system for high-level production of α-amylase was constructed in B. licheniformis, revealing that expression levels of selection marker significantly affected target protein production.

High expression of hexokinase domain containing 1 is associated with poor prognosis and aggressive phenotype in hepatocarcinoma

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Zhang, Zijian; Huang, Shanzhou [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Wang, Huanyu [Department of Thyroid and Breast Surgery, Nanshan District People’s Hospital, Shenzhen, 518000 (China); Wu, Jian [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Chen, Dong [Department of Biliopancreatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Peng, Baogang [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Zhou, Qi, E-mail: hnzhouqi@163.com [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China)

2016-06-10

Rapid progress and metastasis remain the major treatment failure modes of hepatocarcinoma (HCC). Unfortunately, the underlying molecular mechanisms of hepatoma cell proliferation and migration are poorly understood. Metabolic abnormalities play critical roles in tumorigenesis and progression. Hexokinase domain containing 1 (HKDC1) catalyzes the phosphorylation of glucose. However, the functions and mechanisms of HKDC1 in cancer remain unknown. In this study, real-time RT-PCR and Western blotting assays were used to detect the HKDC1 expression levels in HCC tissues and cell lines. The Oncomine™ Cancer Microarray Database was applied to analysis the correlations between HKDC1 expression and HCC clinical characteristics. MTT and Transwell migration assays were performed to determine the functions of HKDC1 in HCC cells. The effect of HKDC1 on Wnt/β-catenin signaling pathway was assessed using Western blotting assay. In this study, we found that HKDC1 expression levels were elevated in HCC tissues compared with the adjacent tissues. HCC patients with high expression levels of HKDC1 had poor overall survival (OS). Furthermore, higher HKDC1 levels also predicted a worse OS of patients within solitary, elevated pre-operated serum alpha fetoprotein (AFP) level and higher tumor diameter. Moreover, silencing HKDC1 suppressed HCC cells proliferation and migration in vitro. Downregulated HKDC1 expression repressed β-Catenin and c-Myc expression, which indicates that silencing HKDC1 may reduce proliferation and migration via inhibiting the Wnt/β-catenin signaling pathway in HCC. In summary, HKDC1 provides further insight into HCC tumor progression and may provide a novel prognostic biomarker and therapeutic target for HCC treatment. -- Highlights: •HKDC1 is upregulated in HCC. •Patients with high HKDC1 expressions perform worse OS. •Silencing HKDC1 suppresses proliferation and migration. •Silencing HKDC1 represses Wnt/β-catenin signaling pathway.

High expression of hexokinase domain containing 1 is associated with poor prognosis and aggressive phenotype in hepatocarcinoma

International Nuclear Information System (INIS)

Zhang, Zijian; Huang, Shanzhou; Wang, Huanyu; Wu, Jian; Chen, Dong; Peng, Baogang; Zhou, Qi

2016-01-01

Rapid progress and metastasis remain the major treatment failure modes of hepatocarcinoma (HCC). Unfortunately, the underlying molecular mechanisms of hepatoma cell proliferation and migration are poorly understood. Metabolic abnormalities play critical roles in tumorigenesis and progression. Hexokinase domain containing 1 (HKDC1) catalyzes the phosphorylation of glucose. However, the functions and mechanisms of HKDC1 in cancer remain unknown. In this study, real-time RT-PCR and Western blotting assays were used to detect the HKDC1 expression levels in HCC tissues and cell lines. The Oncomine™ Cancer Microarray Database was applied to analysis the correlations between HKDC1 expression and HCC clinical characteristics. MTT and Transwell migration assays were performed to determine the functions of HKDC1 in HCC cells. The effect of HKDC1 on Wnt/β-catenin signaling pathway was assessed using Western blotting assay. In this study, we found that HKDC1 expression levels were elevated in HCC tissues compared with the adjacent tissues. HCC patients with high expression levels of HKDC1 had poor overall survival (OS). Furthermore, higher HKDC1 levels also predicted a worse OS of patients within solitary, elevated pre-operated serum alpha fetoprotein (AFP) level and higher tumor diameter. Moreover, silencing HKDC1 suppressed HCC cells proliferation and migration in vitro. Downregulated HKDC1 expression repressed β-Catenin and c-Myc expression, which indicates that silencing HKDC1 may reduce proliferation and migration via inhibiting the Wnt/β-catenin signaling pathway in HCC. In summary, HKDC1 provides further insight into HCC tumor progression and may provide a novel prognostic biomarker and therapeutic target for HCC treatment. -- Highlights: •HKDC1 is upregulated in HCC. •Patients with high HKDC1 expressions perform worse OS. •Silencing HKDC1 suppresses proliferation and migration. •Silencing HKDC1 represses Wnt/β-catenin signaling pathway.

Low expression of Mda-7/IL-24 and high expression of C-myb in tumour tissues are predictors of poor prognosis for Burkitt lymphoma patients.

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Ma, Ming; Zhao, Riyang; Yang, Xingxiao; Zhao, Lianmei; Liu, Lihua; Zhang, Cong; Wang, Xuexiao; Shan, Baoen

2018-02-08

Burkitt lymphoma is one of the most common types of haematopoietic malignancy in children and adolescents. Mda-7/IL-24 had been identified as a differentiation inducer of Burkitt lymphoma cells. Previous studies have revealed that knockdown of C-myb can also lead to the terminal differentiation of Burkitt lymphoma cells. The aim of the present study was to investigate the correlation between the expression of Mda-7/IL-24 and C-myb, as well as their prognostic significance, for Burkitt lymphoma patients. The tumour tissues were collected from 59 cases of Burkitt lymphoma patients and detected with Western blotting and immunohistochemistry. The results showed that the expression of Mda-7/IL-24 was lower, whereas the expression of C-myb was higher in Burkitt lymphoma tissues compared to specimens of normal lymph node tissues. Furthermore, C-myb expression was negatively correlated with Mda-7/IL-24 expression at the protein level in Burkitt lymphoma tissues and cell lines. Both the expression of Mda-7/IL-24 and C-myb in Burkitt lymphoma tissues was associated with some clinicopathological parameters, such as clinical stage, infiltration in the bone marrow, Ki67 and overall survival rates. These results indicated that low expression of Mda-7/IL-24 along with high expression of C-myb are predictors for poor prognosis of Burkitt lymphoma patients; this outcome suggests that Mda-7/IL-24 and C-myb might be potential targets for clinical treatment of Burkitt lymphoma. Mda-7/IL-24: melanoma differentiation associated gene7/interleukin 24; FCM: flow cytometry; Ecog: Eastern Cooperative Oncology Group; IPI: International lymphoma prognosis index.

Multiple Sclerosis Risk Variant HLA-DRB1*1501 Associates with High Expression of DRB1 Gene in Different Human Populations

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Abad-Grau, María del Mar; Fedetz, María; Izquierdo, Guillermo; Lucas, Miguel; Fernández, Óscar; Ndagire, Dorothy; Catalá-Rabasa, Antonio; Ruiz, Agustín; Gayán, Javier; Delgado, Concepción; Arnal, Carmen

2012-01-01

The human leukocyte antigen (HLA) DRB1*1501 has been consistently associated with multiple sclerosis (MS) in nearly all populations tested. This points to a specific antigen presentation as the pathogenic mechanism though this does not fully explain the disease association. The identification of expression quantitative trait loci (eQTL) for genes in the HLA locus poses the question of the role of gene expression in MS susceptibility. We analyzed the eQTLs in the HLA region with respect to MS-associated HLA-variants obtained from genome-wide association studies (GWAS). We found that the Tag of DRB1*1501, rs3135388 A allele, correlated with high expression of DRB1, DRB5 and DQB1 genes in a Caucasian population. In quantitative terms, the MS-risk AA genotype carriers of rs3135388 were associated with 15.7-, 5.2- and 8.3-fold higher expression of DQB1, DRB5 and DRB1, respectively, than the non-risk GG carriers. The haplotype analysis of expression-associated variants in a Spanish MS cohort revealed that high expression of DRB1 and DQB1 alone did not contribute to the disease. However, in Caucasian, Asian and African American populations, the DRB1*1501 allele was always highly expressed. In other immune related diseases such as type 1 diabetes, inflammatory bowel disease, ulcerative colitis, asthma and IgA deficiency, the best GWAS-associated HLA SNPs were also eQTLs for different HLA Class II genes. Our data suggest that the DR/DQ expression levels, together with specific structural properties of alleles, seem to be the causal effect in MS and in other immunopathologies rather than specific antigen presentation alone. PMID:22253788

Melatonin counteracts changes in hypothalamic gene expression of signals regulating feeding behavior in high-fat fed rats.

Science.gov (United States)

Ríos-Lugo, María J; Jiménez-Ortega, Vanesa; Cano-Barquilla, Pilar; Mateos, Pilar Fernández; Spinedi, Eduardo J; Cardinali, Daniel P; Esquifino, Ana I

2015-03-01

Previous studies indicate that the administration of melatonin caused body weight and abdominal visceral fat reductions in rodent models of hyperadiposity. The objective of the present study performed in high-fat fed rats was to evaluate the activity of melatonin on gene expression of some medial basal hypothalamus (MBH) signals involved in feeding behavior regulation, including neuropeptide Y (NPY), proopiomelanocortin (POMC), prolactin-releasing peptide (PrRP), leptin- and insulin-receptors (R) and insulin-R substrate (IRS)-1 and -2. Blood levels of leptin and adiponectin were also measured. Adult Wistar male rats were divided into four groups (n=16 per group): (i) control diet (3% fat); (ii) high-fat (35%) diet; (iii) high-fat diet+melatonin; (iv) control diet+melatonin. Rats had free access to high-fat or control chow and one of the following drinking solutions: (a) tap water; (b) 25 μg/mL of melatonin. After 10 weeks, the high-fat fed rats showed augmented MBH mRNA levels of NPY, leptin-R, PrRP, insulin-R, IRS-1 and IRS-2. The concomitant administration of melatonin counteracted this increase. Feeding of rats with a high-fat diet augmented expression of the MBH POMC gene through an effect insensitive to melatonin treatment. The augmented levels of circulating leptin and adiponectin seen in high-fat fed rats were counteracted by melatonin as was the augmented body weight: melatonin significantly attenuated a body weight increase in high-fat fed rats without affecting chow or water consumption. Melatonin augmented plasma leptin and adiponectin in control rats. The results indicate that an effect on gene expression of feeding behavior signals at the central nervous system (CNS) may complement a peripheral rise of the energy expenditure produced by melatonin to decrease body weight in high-fat fed rats.

Incorporation of gene-specific variability improves expression analysis using high-density DNA microarrays

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Spitznagel Edward

2003-11-01

Full Text Available Abstract Background The assessment of data reproducibility is essential for application of microarray technology to exploration of biological pathways and disease states. Technical variability in data analysis largely depends on signal intensity. Within that context, the reproducibility of individual probe sets has not been hitherto addressed. Results We used an extraordinarily large replicate data set derived from human placental trophoblast to analyze probe-specific contribution to variability of gene expression. We found that signal variability, in addition to being signal-intensity dependant, is probe set-specific. Importantly, we developed a novel method to quantify the contribution of this probe set-specific variability. Furthermore, we devised a formula that incorporates a priori-computed, replicate-based information on probe set- and intensity-specific variability in determination of expression changes even without technical replicates. Conclusion The strategy of incorporating probe set-specific variability is superior to analysis based on arbitrary fold-change thresholds. We recommend its incorporation to any computation of gene expression changes using high-density DNA microarrays. A Java application implementing our T-score is available at http://www.sadovsky.wustl.edu/tscore.html.

The European Expression Of Interest For High Purity U-233 Materials

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Giaquinto, Joseph M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Younkin, James R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

2017-04-01

The purpose of this letter report is to document the response for an Expression of Interest (EOI) sent to the European Safeguards and research and development (R&D) scientific communities for the distribution of small amounts of high purity ²³³U materials for use in safeguards, nonproliferation, and basic R&D in the nuclear disciplines. The intent for the EOI was to gauge the level of international interest for these materials from government and research institutions with programmatic missions in the nuclear security or nuclear R&D arena. The information contained herein is intended to provide information to assist key decision makers in DOE as to the ultimate disposition path for the high purity materials currently being recovered at Oak Ridge National Laboratory (ORNL) and only those items for which there is no United States (U.S.) sponsor identified.

Requirements for growth and IL-10 expression of highly purified human T regulatory cells

OpenAIRE

Bonacci, Benedetta; Edwards, Brandon; Jia, Shuang; Williams, Calvin; Hessner, Martin J.; Gauld, Stephen; Verbsky, James

2012-01-01

Human regulatory T cells (TR) cells have potential for the treatment of a variety of immune mediated diseases but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human TR cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimu...

Pathway-based factor analysis of gene expression data produces highly heritable phenotypes that associate with age.

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Anand Brown, Andrew; Ding, Zhihao; Viñuela, Ana; Glass, Dan; Parts, Leopold; Spector, Tim; Winn, John; Durbin, Richard

2015-03-09

Statistical factor analysis methods have previously been used to remove noise components from high-dimensional data prior to genetic association mapping and, in a guided fashion, to summarize biologically relevant sources of variation. Here, we show how the derived factors summarizing pathway expression can be used to analyze the relationships between expression, heritability, and aging. We used skin gene expression data from 647 twins from the MuTHER Consortium and applied factor analysis to concisely summarize patterns of gene expression to remove broad confounding influences and to produce concise pathway-level phenotypes. We derived 930 "pathway phenotypes" that summarized patterns of variation across 186 KEGG pathways (five phenotypes per pathway). We identified 69 significant associations of age with phenotype from 57 distinct KEGG pathways at a stringent Bonferroni threshold ([Formula: see text]). These phenotypes are more heritable ([Formula: see text]) than gene expression levels. On average, expression levels of 16% of genes within these pathways are associated with age. Several significant pathways relate to metabolizing sugars and fatty acids; others relate to insulin signaling. We have demonstrated that factor analysis methods combined with biological knowledge can produce more reliable phenotypes with less stochastic noise than the individual gene expression levels, which increases our power to discover biologically relevant associations. These phenotypes could also be applied to discover associations with other environmental factors. Copyright © 2015 Brown et al.

Gene expression differences between PAXgene and Tempus blood RNA tubes are highly reproducible between independent samples and biobanks.

Science.gov (United States)

Skogholt, Anne Heidi; Ryeng, Einar; Erlandsen, Sten Even; Skorpen, Frank; Schønberg, Svanhild A; Sætrom, Pål

2017-03-23

Gene expression profiling from blood is sensitive to technology choices. For example, the main blood RNA collection systems-the PAXgene and Tempus tubes-differently influence RNA expression signatures. The aim of this study was to establish a common RNA isolation protocol for these two systems and investigate if it could reduce the differences in gene expression between them. We collected identical blood samples on the PAXgene and Tempus systems and retrieved blood samples from two independent biobanks-NOWAC and HUNT3-which are based on PAXgene and Tempus, respectively. High-quality RNA was extracted from both sampling systems by using their original protocols and our common modified protocol, and were profiled on Illumina microarrays. Regardless of the protocol used, we found most of the measured transcripts to be differently affected by the two sampling systems. However, our modified protocol reduced the number of transcripts that were significantly differentially expressed between PAXgene and Tempus by approximately 50%. Expression differences between PAXgene and Tempus were highly reproducible both between protocols and between different independent sample sets (Pearson correlation 0.563-0.854 across 47323 probes). Moreover, the modified protocol increased the microRNA output of the system with lowest microRNA yield, the PAXgene system. Most transcripts are affected by the choice of sampling system, but these effects are highly reproducible between independent samples. We propose that by running a control experiment with samples on both systems in parallel with biologically relevant samples, researchers may adjust for technical differences between the sampling systems.

Sparse Bayesian classification and feature selection for biological expression data with high correlations.

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Xian Yang

Full Text Available Classification models built on biological expression data are increasingly used to predict distinct disease subtypes. Selected features that separate sample groups can be the candidates of biomarkers, helping us to discover biological functions/pathways. However, three challenges are associated with building a robust classification and feature selection model: 1 the number of significant biomarkers is much smaller than that of measured features for which the search will be exhaustive; 2 current biological expression data are big in both sample size and feature size which will worsen the scalability of any search algorithms; and 3 expression profiles of certain features are typically highly correlated which may prevent to distinguish the predominant features. Unfortunately, most of the existing algorithms are partially addressing part of these challenges but not as a whole. In this paper, we propose a unified framework to address the above challenges. The classification and feature selection problem is first formulated as a nonconvex optimisation problem. Then the problem is relaxed and solved iteratively by a sequence of convex optimisation procedures which can be distributed computed and therefore allows the efficient implementation on advanced infrastructures. To illustrate the competence of our method over others, we first analyse a randomly generated simulation dataset under various conditions. We then analyse a real gene expression dataset on embryonal tumour. Further downstream analysis, such as functional annotation and pathway analysis, are performed on the selected features which elucidate several biological findings.

High Frequency of Programmed Death-ligand 1 Expression in Emphysematous Bullae-associated Lung Adenocarcinomas.

Science.gov (United States)

Toyokawa, Gouji; Takada, Kazuki; Okamoto, Tatsuro; Kozuma, Yuka; Matsubara, Taichi; Haratake, Naoki; Takamori, Shinkichi; Akamine, Takaki; Katsura, Masakazu; Shoji, Fumihiro; Oda, Yoshinao; Maehara, Yoshihiko

2017-09-01

Emphysematous bullae (EB) are known to be associated with a high incidence of lung cancer; however, the reason for this has yet to be elucidated. The objective of the present study was to clarify the prevalence of programmed death-ligand-1 (PD-L1) expression in EB-associated lung adenocarcinomas. A total of 369 patients with resected lung adenocarcinoma whose preoperative computed tomography findings were available for the examination of EB were analyzed for PD-L1 expression by immunohistochemistry and evaluated to determine the association between PD-L1 expression and EB-related adenocarcinomas. Among 369 patients, EB and cancer adjoining EB (Ca-ADJ) were identified in 81 (22.0%) and 50 (13.6%) patients, respectively. EB and Ca-ADJ were significantly associated with male gender, a smoking habit, a decreased forced expiratory volume in 1 second, a relatively higher tumor grade, advanced T status and stage, the presence of pleural and vessel invasion, invasive pathologic subtypes, and wild-type epidermal growth factor receptor. Seventy patients (19.0%) were positive for PD-L1 expression, whereas the remaining 299 patients (81.0%) were negative. Thirty-six (44.4%) and 29 (58.0%) of 81 and 50 patients with EB and Ca-ADJ, respectively, were positive for PD-L1 expression, which was shown to be significant by the Fisher exact test (P < .001 and P < .001, respectively). Among the 81 lung adenocarcinomas with EB, Ca-ADJ was significantly associated with PD-L1 expression (P = .021). In a multivariate analysis, the presence of Ca-ADJ was found to be an independent predictor of PD-L1 expression. EB-associated lung adenocarcinomas express PD-L1 protein more frequently than those without EB. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression profiling analysis: Uncoupling protein 2 deficiency improves hepatic glucose, lipid profiles and insulin sensitivity in high-fat diet-fed mice by modulating expression of genes in peroxisome proliferator-activated receptor signaling pathway.

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Zhou, Mei-Cen; Yu, Ping; Sun, Qi; Li, Yu-Xiu

2016-03-01

Uncoupling protein 2 (UCP2), which was an important mitochondrial inner membrane protein associated with glucose and lipid metabolism, widely expresses in all kinds of tissues including hepatocytes. The present study aimed to explore the impact of UCP2 deficiency on glucose and lipid metabolism, insulin sensitivity and its effect on the liver-associated signaling pathway by expression profiling analysis. Four-week-old male UCP2-/- mice and UCP2+/+ mice were randomly assigned to four groups: UCP2-/- on a high-fat diet, UCP2-/- on a normal chow diet, UCP2+/+ on a high-fat diet and UCP2+/+ on a normal chow diet. The differentially expressed genes in the four groups on the 16th week were identified by Affymetrix gene array. The results of intraperitoneal glucose tolerance test and insulin tolerance showed that blood glucose and β-cell function were improved in the UCP2-/- group on high-fat diet. Enhanced insulin sensitivity was observed in the UCP2-/- group. The differentially expressed genes were mapped to 23 pathways (P high-fat diet. The upregulation of genes in the PPAR signaling pathway could explain our finding that UCP2 deficiency ameliorated insulin sensitivity. The manipulation of UCP2 protein expression could represent a new strategy for the prevention and treatment of diabetes.

Selection of Highly Expressed Gene Variants in Escherichia coli Using Translationally Coupled Antibiotic Selection Markers

DEFF Research Database (Denmark)

Rennig, Maja; Daley, Daniel O.; Nørholm, Morten H. H.

2018-01-01

Strategies to select highly expressed variants of a protein coding sequence are usually based on trial-and-error approaches, which are time-consuming and expensive. We address this problem using translationally coupled antibiotic resistance markers. The system requires that the target gene can...

POMC and NPY mRNA expression during development is increased in rat offspring brain from mothers fed with a high fat diet.

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Klein, Marianne Orlandini; MacKay, Harry; Edwards, Alexander; Park, Su-Bin; Kiss, Ana Carolina Inhasz; Felicio, Luciano Freitas; Abizaid, Alfonso

2018-02-01

Developmental programing is influenced by perinatal nutrition and it has long-lasting impacts on adult metabolism in the offspring. In particular, maternal high fat diet has been associated with increased risk of obesity and metabolic disorders during adulthood in the descendants. These effects may be due to the effects of the high fat diet on the development of the systems that regulate food intake and energy balance in the offspring hypothalamus. The arcuate nucleus (ARC) may be a particularly sensitive region to it as this nucleus contains the POMC and AgRP/NPY neurons that integrate the melanocortin system. Thus, the aim of this study was to investigate the effects of maternal high fat diet during pregnancy on the transcription factors that regulate hypothalamic development in the offspring as a potential mechanism that may result in altered neuronal expression of POMC, NPY and/or AgRP. To this end, pregnant females exposed to high fat diet (60% fat diet since day 0 of pregnancy) or standard rat chow were sacrificed on days 12, 14, 16 and 18 of gestation to obtain brains from their developing fetuses and examine the mRNA expression of transcription factors associated with the development of cells in the ARC. Results show that, while no changes in transcription factor expression between groups were observed, POMC and NPY mRNA expression were higher on embryonic day 18 in the high fat group. These results suggest that POMC and NPY expression are altered by in utero exposure to a high fat diet, but these changes in gene expression are not associated with changes in the expression of transcription factors known to determine the fate of ARC cells. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing.

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Vannette, Rachel L; Mohamed, Abbas; Johnson, Brian R

2015-11-09

Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging.

Expression profiles of miRNA-122 and its target CAT1 in minipigs (Sus scrofa) fed a high-cholesterol diet

DEFF Research Database (Denmark)

Cirera Salicio, Susanna; Birck, Malene Muusfeldt; Busk, Peter Kamp

2010-01-01

The Göttingen minipig is an excellent model for studying effects of dietary high-fat intake on obesity. In this study, we analyzed the expression level of microRNA-122 (miRNA-122) and its target mRNA, CAT1, in intact young male minipigs fed either high-cholesterol or standard diet for 11 wk. Mi...... with a decrease in the expression of miRNA-122, confirming the implication of this microRNA in obesity. Gene expression levels of CAT1 did not differ between groups.......RNA-122 and CAT1 are known to be important regulators of lipid metabolism. The weight of the young minipigs was monitored once a week during the feeding period; measurements of total cholesterol, triglycerides, high-density lipoproteins, and low-density lipoproteins were recorded at 4 time points (8, 14...

High Nuclear Hypoxia-Inducible Factor 1 Alpha Expression Is a Predictor of Distant Recurrence in Patients With Resected Pancreatic Adenocarcinoma

International Nuclear Information System (INIS)

Colbert, Lauren E.; Fisher, Sarah B.; Balci, Serdar; Saka, Burcu; Chen, Zhengjia; Kim, Sungjin; El-Rayes, Bassel F.; Adsay, N. Volkan; Maithel, Shishir K.; Landry, Jerome C.

2015-01-01

Purpose: To evaluate nuclear hypoxia-inducible factor 1α (HIF-1α) expression as a prognostic factor for distant recurrence (DR) and local recurrence (LR) after pancreatic adenocarcinoma resection. Methods and Materials: Tissue specimens were collected from 98 patients with pancreatic adenocarcinoma who underwent resection without neoadjuvant therapy between January 2000 and December 2011. Local recurrence was defined as radiographic or pathologic evidence of progressive disease in the pancreas, pancreatic bed, or associated nodal regions. Distant recurrence was defined as radiographically or pathologically confirmed recurrent disease in other sites. Immunohistochemical staining was performed and scored by an independent pathologist blinded to patient outcomes. High HIF-1α overall expression score was defined as high percentage and intensity staining and thus score >1.33. Univariate analysis was performed for HIF-1α score with LR alone and with DR. Multivariate logistic regression was used to determine predictors of LR and DR. Results: Median follow-up time for all patients was 16.3 months. Eight patients (8%) demonstrated isolated LR, 26 patients (26.5%) had isolated DR, and 13 patients had both LR and DR. Fifty-three patients (54%) had high HIF-1α expression, and 45 patients (46%) had low HIF-1α expression. High HIF-1α expression was significantly associated with DR (P=.03), and low HIF-1α expression was significantly associated with isolated LR (P=.03). On multivariate logistic regression analysis, high HIF-1α was the only significant predictor of DR (odds ratio 2.46 [95% confidence interval 1.06-5.72]; P=.03). In patients with a known recurrence, an HIF-1α score ≥2.5 demonstrated a specificity of 100% for DR. Conclusions: High HIF-1α expression is a significant predictor of distant failure versus isolated local failure in patients undergoing resection of pancreatic adenocarcinoma. Expression of HIF-1α may have utility in determining candidates for

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues

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Viana Antonio AB

2011-11-01

Full Text Available Abstract Background Cotton (Gossypium spp. is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2 family member with no previous characterization. Results Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2 and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. Conclusions uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2 and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

Gender Expression, Violence, and Bullying Victimization: Findings from Probability Samples of High School Students in 4 US School Districts

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Gordon, Allegra R.; Conron, Kerith J.; Calzo, Jerel P.; White, Matthew T.; Reisner, Sari L.; Austin, S. Bryn

2018-01-01

Background: Young people may experience school-based violence and bullying victimization related to their gender expression, independent of sexual orientation identity. However, the associations between gender expression and bullying and violence have not been examined in racially and ethnically diverse population-based samples of high school…

High-fat diet feeding alters metabolic response to fasting/non fasting conditions. Effect on caveolin expression and insulin signalling.

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Gómez-Ruiz, Ana; Milagro, Fermín I; Campión, Javier; Martínez, J Alfredo; de Miguel, Carlos

2011-04-13

The effect of food intake on caveolin expression in relation to insulin signalling was studied in skeletal muscle and adipocytes from retroperitoneal (RP) and subcutaneous (SC) adipose tissue, comparing fasted (F) to not fasted (NF) rats that had been fed a control or high-fat (HF) diet for 72 days. Serum glucose was analysed enzymatically and insulin and leptin by ELISA. Caveolins and insulin signalling intermediaries (IR, IRS-1 and 2 and GLUT4) were determined by RT-PCR and western blotting. Caveolin and IR phosphorylation was measured by immunoprecipitation. Data were analysed with Mann-Whitney U test. High-fat fed animals showed metabolic alterations and developed obesity and insulin resistance. In skeletal muscle, food intake (NF) induced activation of IR and increased expression of IRS-2 in control animals with normal metabolic response. HF animals became overweight, hyperglycaemic, hyperinsulinemic, hyperleptinemic and showed insulin resistance. In skeletal muscle of these animals, food intake (NF) also induced IRS-2 expression together with IR, although this was not active. Caveolin 3 expression in this tissue was increased by food intake (NF) in animals fed either diet. In RP adipocytes of control animals, food intake (NF) decreased IR and IRS-2 expression but increased that of GLUT4. A similar but less intense response was found in SC adipocytes. Food intake (NF) did not change caveolin expression in RP adipocytes with either diet, but in SC adipocytes of HF animals a reduction was observed. Food intake (NF) decreased caveolin-1 phosphorylation in RP but increased it in SC adipocytes of control animals, whereas it increased caveolin-2 phosphorylation in both types of adipocytes independently of the diet. Animals fed a control-diet show a normal response to food intake (NF), with activation of the insulin signalling pathway but without appreciable changes in caveolin expression, except a small increase of caveolin-3 in muscle. Animals fed a high-fat diet

The study on highly expressed proteins as a function of an elevated ultraviolet radiation in the copepod, Tigriopus japonicus

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Zubrzycki, Igor Z.; Lee, Seunghan; Lee, Kanghyun; Wiacek, Magdalena; Lee, Wonchoel

2012-06-01

The objective of the study was to analyze constantlyhighly expressed proteins as a function of elevated midultraviolet (UVB, 280-315 nm) radiation in Tigriopus japonicus sensu lato ( T. japonicus s.l). We also analyzed associations between kinetics of radiation avoidance, measured as a covered distance per time unit, and highly expressed proteins. The obtained results indicate an increase in T. japonicus s.l. mobility between the control (no radiation) and mild UV radiation levels (15 kJ·m-2). Two-dimensional gel electrophoresis combined with MALDI-MS-MS resulted in 2D protein map comprising of 686 protein spots, of which 19 were identified as highly expressed proteins across all experimental conditions. Obtained results indicate that calpain, vitellogenin, and collagenase are housekeeping protein that are expressed at a constant level independently of environmental changes and that adoption of a locomotive system for the avoidance of a UV source may be, at least partially, supported by hepatopancreas-driven metabolism.

Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

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Zhang, Xiuren; Mason, Hugh

2006-02-05

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

International Nuclear Information System (INIS)

Wang, Yifan; Li, Shu Jie; Pan, Juncheng; Che, Yongzhe; Yin, Jian; Zhao, Qing

2011-01-01

Highlights: → Hv1 is specifically expressed in highly metastatic human breast tumor tissues. → Hv1 regulates breast cancer cytosolic pH. → Hv1 acidifies extracellular milieu. → Hv1 exacerbates the migratory ability of metastatic cells. -- Abstract: The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.

Multiple sclerosis risk variant HLA-DRB1*1501 associates with high expression of DRB1 gene in different human populations.

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Antonio Alcina

Full Text Available The human leukocyte antigen (HLA DRB1*1501 has been consistently associated with multiple sclerosis (MS in nearly all populations tested. This points to a specific antigen presentation as the pathogenic mechanism though this does not fully explain the disease association. The identification of expression quantitative trait loci (eQTL for genes in the HLA locus poses the question of the role of gene expression in MS susceptibility. We analyzed the eQTLs in the HLA region with respect to MS-associated HLA-variants obtained from genome-wide association studies (GWAS. We found that the Tag of DRB1*1501, rs3135388 A allele, correlated with high expression of DRB1, DRB5 and DQB1 genes in a Caucasian population. In quantitative terms, the MS-risk AA genotype carriers of rs3135388 were associated with 15.7-, 5.2- and 8.3-fold higher expression of DQB1, DRB5 and DRB1, respectively, than the non-risk GG carriers. The haplotype analysis of expression-associated variants in a Spanish MS cohort revealed that high expression of DRB1 and DQB1 alone did not contribute to the disease. However, in Caucasian, Asian and African American populations, the DRB1*1501 allele was always highly expressed. In other immune related diseases such as type 1 diabetes, inflammatory bowel disease, ulcerative colitis, asthma and IgA deficiency, the best GWAS-associated HLA SNPs were also eQTLs for different HLA Class II genes. Our data suggest that the DR/DQ expression levels, together with specific structural properties of alleles, seem to be the causal effect in MS and in other immunopathologies rather than specific antigen presentation alone.

Pre-amplification in the context of high-throughput qPCR gene expression experiment

Czech Academy of Sciences Publication Activity Database

Korenková, Vlasta; Scott, J.; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjoback, R.

2015-01-01

Roč. 16, č. 5 (2015) ISSN 1471-2199 R&D Projects: GA ČR(CZ) GAP304/12/1585; GA ČR(CZ) GA15-08239S; GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : High-throughput qPCR * Gene expression * Exponential pre-amplification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.500, year: 2015

High calcium concentration in bones promotes bone metastasis in renal cell carcinomas expressing calcium-sensing receptor.

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Joeckel, Elke; Haber, Tobias; Prawitt, Dirk; Junker, Kerstin; Hampel, Christian; Thüroff, Joachim W; Roos, Frederik C; Brenner, Walburgis

2014-02-28

The prognosis for renal cell carcinoma (RCC) is related to a high rate of metastasis, including 30% of bone metastasis. Characteristic for bone tissue is a high concentration of calcium ions. In this study, we show a promoting effect of an enhanced extracellular calcium concentration on mechanisms of bone metastasis via the calcium-sensing receptor (CaSR) and its downstream signaling molecules. Our analyses were performed using 33 (11/category) matched specimens of normal and tumor tissue and 9 (3/category) primary cells derived from RCC patients of the 3 categories: non-metastasized, metastasized into the lung and metastasized into bones during a five-year period after nephrectomy. Expression of CaSR was determined by RT-PCR, Western blot analyses and flow cytometry, respectively. Cells were treated by calcium and the CaSR inhibitor NPS 2143. Cell migration was measured in a Boyden chamber with calcium (10 μM) as chemotaxin and proliferation by BrdU incorporation. The activity of intracellular signaling mediators was quantified by a phospho-kinase array and Western blot. The expression of CaSR was highest in specimens and cells of patients with bone metastases. Calcium treatment induced an increased migration (19-fold) and proliferation (2.3-fold) exclusively in RCC cells from patients with bone metastases. The CaSR inhibitor NPS 2143 elucidated the role of CaSR on the calcium-dependent effects. After treatment with calcium, the activity of AKT, PLCγ-1, p38α and JNK was clearly enhanced and PTEN expression was almost completely abolished in bone metastasizing RCC cells. Our results indicate a promoting effect of extracellular calcium on cell migration and proliferation of bone metastasizing RCC cells via highly expressed CaSR and its downstream signaling pathways. Consequently, CaSR may be regarded as a new prognostic marker predicting RCC bone metastasis.

The expression profile of phosphatidylinositol in high spatial resolution imaging mass spectrometry as a potential biomarker for prostate cancer.

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Takayuki Goto

Full Text Available High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS is an emerging application for the comprehensive and detailed analysis of the spatial distribution of ionized molecules in situ on tissue slides. HR-MALDI-IMS in negative mode in a mass range of m/z 500-1000 was performed on optimal cutting temperature (OCT compound-embedded human prostate tissue samples obtained from patients with prostate cancer at the time of radical prostatectomy. HR-MALDI-IMS analysis of the 14 samples in the discovery set identified 26 molecules as highly expressed in the prostate. Tandem mass spectrometry (MS/MS showed that these molecules included 14 phosphatidylinositols (PIs, 3 phosphatidylethanolamines (PEs and 3 phosphatidic acids (PAs. Among the PIs, the expression of PI(18:0/18:1, PI(18:0/20:3 and PI(18:0/20:2 were significantly higher in cancer tissue than in benign epithelium. A biomarker algorithm for prostate cancer was formulated by analyzing the expression profiles of PIs in cancer tissue and benign epithelium of the discovery set using orthogonal partial least squares discriminant analysis (OPLS-DA. The sensitivity and specificity of this algorithm for prostate cancer diagnosis in the 24 validation set samples were 87.5 and 91.7%, respectively. In conclusion, HR-MALDI-IMS identified several PIs as being more highly expressed in prostate cancer than benign prostate epithelium. These differences in PI expression profiles may serve as a novel diagnostic tool for prostate cancer.

EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

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Zhu Yuelin

2008-01-01

Full Text Available Abstract Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from http://www.ezarray.com/.

Highly tissue specific expression of Sphinx supports its male courtship related role in Drosophila melanogaster.

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Chen, Ying; Dai, Hongzheng; Chen, Sidi; Zhang, Luoying; Long, Manyuan

2011-04-26

Sphinx is a lineage-specific non-coding RNA gene involved in regulating courtship behavior in Drosophila melanogaster. The 5' flanking region of the gene is conserved across Drosophila species, with the proximal 300 bp being conserved out to D. virilis and a further 600 bp region being conserved amongst the melanogaster subgroup (D. melanogaster, D. simulans, D. sechellia, D. yakuba, and D. erecta). Using a green fluorescence protein transformation system, we demonstrated that a 253 bp region of the highly conserved segment was sufficient to drive sphinx expression in male accessory gland. GFP signals were also observed in brain, wing hairs and leg bristles. An additional ∼800 bp upstream region was able to enhance expression specifically in proboscis, suggesting the existence of enhancer elements. Using anti-GFP staining, we identified putative sphinx expression signal in the brain antennal lobe and inner antennocerebral tract, suggesting that sphinx might be involved in olfactory neuron mediated regulation of male courtship behavior. Whole genome expression profiling of the sphinx knockout mutation identified significant up-regulated gene categories related to accessory gland protein function and odor perception, suggesting sphinx might be a negative regulator of its target genes.

Highly tissue specific expression of Sphinx supports its male courtship related role in Drosophila melanogaster.

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Ying Chen

2011-04-01

Full Text Available Sphinx is a lineage-specific non-coding RNA gene involved in regulating courtship behavior in Drosophila melanogaster. The 5' flanking region of the gene is conserved across Drosophila species, with the proximal 300 bp being conserved out to D. virilis and a further 600 bp region being conserved amongst the melanogaster subgroup (D. melanogaster, D. simulans, D. sechellia, D. yakuba, and D. erecta. Using a green fluorescence protein transformation system, we demonstrated that a 253 bp region of the highly conserved segment was sufficient to drive sphinx expression in male accessory gland. GFP signals were also observed in brain, wing hairs and leg bristles. An additional ∼800 bp upstream region was able to enhance expression specifically in proboscis, suggesting the existence of enhancer elements. Using anti-GFP staining, we identified putative sphinx expression signal in the brain antennal lobe and inner antennocerebral tract, suggesting that sphinx might be involved in olfactory neuron mediated regulation of male courtship behavior. Whole genome expression profiling of the sphinx knockout mutation identified significant up-regulated gene categories related to accessory gland protein function and odor perception, suggesting sphinx might be a negative regulator of its target genes.

Acute exposure to high-peak-power pulsed microwaves affecting the histamine H3 receptor expression in rat hippocampus

International Nuclear Information System (INIS)

Yu Xiaodong; Li Bo; Li Dehua; He Qiyi; Yu Zhengping

2006-01-01

In the Morris Water test, high-peak-power pulsed microwave (MW)-exposed rats displayed some learning and memory behavior dysfunctions, and their escape time and swimming distance to the submerged platform were longer than those of the sham-exposed rats. to understand the molecular mechanism involved, the reverse transcription-polymerase chain reation (RT-PCR) and the Western-blotting technique were used for investigating the mRNA and protein expression patterns of the histamine H 3 receptor (H 3 R) in rat hippocampus. High-peak-power pulsed microwave-exposure did not remarkably lead to the change in expression of H 3 R mRNA in rat hippocampi; however, it promoted the up-regulatory expression of the H 3 R protein, which was possibly triggered through the mitogen-activated protein kinase (MAPK) pathways. Therefore, further investigation of the molecular mechanism of the MW effects on the learning and memory behaviors is required. (authors)

Acute exposure to high-peak-power pulsed microwaves affecting the histamine H3 receptor expression in rat hippocampus

Institute of Scientific and Technical Information of China (English)

无

2006-01-01

In the Morris Water Maze test, high-peak-power pulsed microwave (MW)-exposed rats displayed some learning and memory behavior dysfunctions, and their escape time and swimming distance to the submerged platform were longer than those of the sham-exposed rats. To understand the molecular mechanism involved, the reverse transcription-polymerase chain reaction (RT-PCR) and the Western-blotting technique were used for investigating the mRNA and protein expression patterns of the histamine H3 receptor (H3R) in rat hippocampus. High-peak-power pulsed microwave-exposure did not remarkably lead to the change in expression of H3R mRNA in rat hippocampi;however, it promoted the up-regulatory expression of the H3R protein, which was possibly triggered through the mitogen-activated protein kinase (MAPK) pathways. Therefore, further investigation of the molecular mechanism of the MW effects on the learning and memory behaviors is required.

Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.

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Constance Ciaudo

2009-08-01

Full Text Available Embryonic stem (ES cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19-30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription

High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.

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Zhang, Kang; Su, Lingqia; Duan, Xuguo; Liu, Lina; Wu, Jing

2017-02-20

We recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, P amyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536. Using the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the P amyQ' promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter P HpaII -P amyQ' produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter P HpaII -P amyQ' also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter P HpaII -P amyQ' system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications. The dual-promoter P HpaII -P amyQ' system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

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Thorgersen, Michael P. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Lancaster, W. Andrew [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Rajeev, Lara [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Ge, Xiaoxuan [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Vaccaro, Brian J. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Poole, Farris L. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Arkin, Adam P. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Mukhopadhyay, Aindrila [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Adams, Michael W. W. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology

2016-12-02

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by

Gene Expression Analysis of Escherichia Coli Grown in Miniaturized Bioreactor Platforms for High-Throughput Analysis of Growth and genomic Data

DEFF Research Database (Denmark)

Boccazzi, P.; Zanzotto, A.; Szita, Nicolas

2005-01-01

Combining high-throughput growth physiology and global gene expression data analysis is of significant value for integrating metabolism and genomics. We compared global gene expression using 500 ng of total RNA from Escherichia coli cultures grown in rich or defined minimal media in a miniaturize...... cultures using just 500 ng of total RNA indicate that high-throughput integration of growth physiology and genomics will be possible with novel biochemical platforms and improved detection technologies....

Vocational Preference Inventory High Point Codes Versus Expressed Choices as Predictors of College Major and Career Entry

Science.gov (United States)

Gade, Eldon M.; Soliah, David

1975-01-01

For 151 male graduates of the University of North Dakota, expressed choices measured by preferences made as high school seniors on the ACT Student Profile Section were significantly more accurate predictors of graduating college major and of career entry occupation than were their Vocational Preference Inventory high point codes. (Author)

High Rab27A expression indicates favorable prognosis in CRC.

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Shi, Chuanbing; Yang, Xiaojun; Ni, Yijiang; Hou, Ning; Xu, Li; Zhan, Feng; Zhu, Huijun; Xiong, Lin; Chen, Pingsheng

2015-06-13

Rab27A is a peculiar member in Rab family and has been suggested to play essential roles in the development of human cancers. However, the association between Rab27A expression and clinicopathological characteristics of colorectal cancer (CRC) has not been elucidated yet. One-step quantitative real-time polymerase chain reaction (qPCR) test with 18 fresh-frozen CRC samples and immunohistochemistry (IHC) analysis in 112 CRC cases were executed to evaluate the relationship between Rab27A expression and the clinicopathological features of CRC. Cox regression and Kaplan-Meier survival analyses were performed to identify the prognostic factors for 112 CRC patients. The results specified that the expression levels of Rab27A mRNA and protein were significantly higher in CRC tissues than that in matched non-cancerous tissues, in both qPCR test (p = 0.029) and IHC analysis (p = 0.020). The IHC data indicated that the Rab27A protein expression in CRC was statistically correlated with lymph node metastasis (p = 0.022) and TNM stage (p = 0.026). Cox multi-factor analysis and Kaplan-Meier method suggested Rab27A protein expression (p = 0.012) and tumor differentiation (p = 0.004) were significantly associated with the overall survival of CRC patients. The data indicated the differentiate expression of Rab27A in CRC tissues and matched non-cancerous tissues. Rab27A may be used as a valuable prognostic biomarker for CRC patients.

Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells.

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Prashanthi Karyala

Full Text Available BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B, β-galactosidase (β-gal and green fluorescent protein (GFP from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to

Tumor Classification Using High-Order Gene Expression Profiles Based on Multilinear ICA

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Ming-gang Du

2009-01-01

Full Text Available Motivation. Independent Components Analysis (ICA maximizes the statistical independence of the representational components of a training gene expression profiles (GEP ensemble, but it cannot distinguish relations between the different factors, or different modes, and it is not available to high-order GEP Data Mining. In order to generalize ICA, we introduce Multilinear-ICA and apply it to tumor classification using high order GEP. Firstly, we introduce the basis conceptions and operations of tensor and recommend Support Vector Machine (SVM classifier and Multilinear-ICA. Secondly, the higher score genes of original high order GEP are selected by using t-statistics and tabulate tensors. Thirdly, the tensors are performed by Multilinear-ICA. Finally, the SVM is used to classify the tumor subtypes. Results. To show the validity of the proposed method, we apply it to tumor classification using high order GEP. Though we only use three datasets, the experimental results show that the method is effective and feasible. Through this survey, we hope to gain some insight into the problem of high order GEP tumor classification, in aid of further developing more effective tumor classification algorithms.

A novel mean-centering method for normalizing microRNA expression from high-throughput RT-qPCR data

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Wylie Dennis

2011-12-01

Full Text Available Abstract Background Normalization is critical for accurate gene expression analysis. A significant challenge in the quantitation of gene expression from biofluids samples is the inability to quantify RNA concentration prior to analysis, underscoring the need for robust normalization tools for this sample type. In this investigation, we evaluated various methods of normalization to determine the optimal approach for quantifying microRNA (miRNA expression from biofluids and tissue samples when using the TaqMan® Megaplex™ high-throughput RT-qPCR platform with low RNA inputs. Findings We compared seven normalization methods in the analysis of variation of miRNA expression from biofluid and tissue samples. We developed a novel variant of the common mean-centering normalization strategy, herein referred to as mean-centering restricted (MCR normalization, which is adapted to the TaqMan Megaplex RT-qPCR platform, but is likely applicable to other high-throughput RT-qPCR-based platforms. Our results indicate that MCR normalization performs comparable to or better than both standard mean-centering and other normalization methods. We also propose an extension of this method to be used when migrating biomarker signatures from Megaplex to singleplex RT-qPCR platforms, based on the identification of a small number of normalizer miRNAs that closely track the mean of expressed miRNAs. Conclusions We developed the MCR method for normalizing miRNA expression from biofluids samples when using the TaqMan Megaplex RT-qPCR platform. Our results suggest that normalization based on the mean of all fully observed (fully detected miRNAs minimizes technical variance in normalized expression values, and that a small number of normalizer miRNAs can be selected when migrating from Megaplex to singleplex assays. In our study, we find that normalization methods that focus on a restricted set of miRNAs tend to perform better than methods that focus on all miRNAs, including

Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

DEFF Research Database (Denmark)

Moe, Maren; Meuwissen, Theo; Lien, Sigbjørn

2007-01-01

Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low...

High-Methionine Diet Attenuates Severity of Arthritis and Modulates IGF-I Related Gene Expressions in an Adjuvant Arthritis Rats Model

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Mingxin Li

2016-01-01

Full Text Available Rheumatoid arthritis, a synthesized form of adjuvant arthritis exhibited throughout many animal species, inhibits liver function and circulation of IGF-I and contributes to the degradation of skeletal muscle mass. One of the primary goals of the present study is determining whether a high-Methionine (high-Met diet is capable of reducing the adverse effects of arthritis, namely, loss of body mass. Following adjuvant injection, forty arthritic rats were randomly assigned to either a control group with a basal diet or a high-Met group with the same basal diet + 0.5% Methionine. After 14 days all rats were terminated. The high-Met group exhibited an increase in body weight and food intake in comparison with the control group (P<0.05. High-Met diet debilitated arthritis-induced surges in the gastrocnemius in both atrogin-1 and the MuRF1 expressions; however, it was observed to have little to no effect on atrogin-1 and MuRF1 gene expression in soleus. At the same time, high-Met diet rats experienced a rise in IGF-I, with lowering of IGFBP-3 gene expression in the gastrocnemius and the soleus. These data suggest that arthritis severity can be partly attenuated by high-Met diet.

HER-2 immunohistochemical expression as prognostic marker in high-grade T1 bladder cancer (T1G3

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Luca Bongiovanni

2013-06-01

Full Text Available Objectives: To evaluate if the Human epidermal growth factor receptor 2 (HER-2 expression levels may be used as potential prognostic marker in high grade T1 blad- der cancer (T1G3 Methods: Specimens from transurethral resection of bladder tumour (TURBT of 103 patients with high-grade T1 bladder cancer were collected. This pathologic database was reviewed. Four-year follow-up data were matched with pathologic data. Eighty-three patients entered the study. HER-2 staining was performed. Patients were grouped for HER-2 status. Statistical analysis included Kaplan Meier survival analysis and Log-rank test. Results: Pathological review of TURBT specimens confirmed high-grade T1 transitional cell bladder cancer in all patients. Median follow-up was 12 months (mean 23,5; range 3-48. Twenty-one patients (25.4% present strong HER-2 expression (3+, 28 (33.7% moderate expression (2+, 26 (33.7% weak staining (1+ and 8 (9.6% negative expression (0. Thirty- one patients of 83 (37.4% had not evidence of disease, 41 (49.4% recurred, 11 (13.2% had a progression of disease. Forty-one patients had high grade T1 recurrence. Patients with HER-2 status 0 did not showed progression of disease. Patients with HER-2 status 3+, undergoing cys- tectomy because progression of disease, had a pathological stage > pT2 and a nodal involve- ment. Median Disease-Free Survival (DFS for all patients was 12 months (DFS probability (pDFS = 49.3%; 95% CI, -11.1/+10.1. Median DFS in HER-2 groups was 8 (pDFS 37.5%; 95% CI,-28.8/+29.9, 24 (pDFS 46.1%; 95% CI,-19.5/+17.5, 20 (pDFS 46.4%; 95% CI,-18.8/+16.9 and 10 months (pDFS 47.6%; 95% CI,-21.9/+19.1 respectively in HER-2 status 0,1+,2+,3+. Log-Rank test is not statistically significant (p = 0,39. Conclusions: This study showed that HER-2 expression does not represent a prognostic mark- er of recurrence/progression of disease in high-grade T1 bladder cancer.

Enhancement of high glucose-induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2 /Akt/NF-κB pathway.

Science.gov (United States)

Onphachanh, Xaykham; Lee, Hyun Jik; Lim, Jae Ryong; Jung, Young Hyun; Kim, Jun Sung; Chae, Chang Woo; Lee, Sei-Jung; Gabr, Amr Ahmed; Han, Ho Jae

2017-09-01

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM-associated neuronal cell death. Previous investigators reported on a genome-wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose-induced neuronal cell death and the effect of melatonin against high glucose-induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN-induced putative kinase 1 (PINK1) and LC-3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker™ fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V-positive cells. In addition, high glucose-stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N-acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT 2 receptor-specific inhibitor 4-P-PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin-regulated mitochondrial ROS production, cleaved caspase-3 and caspase-9 expressions, and the number of annexin V-positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT 2 /Akt/NF-κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions. © 2017 The Authors. Journal of Pineal Research

Tissue-specific expression of Sprouty1 in mice protects against high-fat diet-induced fat accumulation, bone loss and metabolic dysfunction.

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Urs, Sumithra; Henderson, Terry; Le, Phuong; Rosen, Clifford J; Liaw, Lucy

2012-09-28

We recently characterised Sprouty1 (Spry1), a growth factor signalling inhibitor as a regulator of marrow progenitor cells promoting osteoblast differentiation at the expense of adipocytes. Adipose tissue-specific Spry1 expression in mice resulted in increased bone mass and reduced body fat, while conditional knockout of Spry1 had the opposite effect with decreased bone mass and increased body fat. Because Spry1 suppresses normal fat development, we tested the hypothesis that Spry1 expression prevents high-fat diet-induced obesity, bone loss and associated lipid abnormalities, and demonstrate that Spry1 has a long-term protective effect on mice fed a high-energy diet. We studied diet-induced obesity in mice with fatty acid binding promoter-driven expression or conditional knockout of Spry1 in adipocytes. Phenotyping was performed by whole-body dual-energy X-ray absorptiometry, microCT, histology and blood analysis. In conditional Spry1-null mice, a high-fat diet increased body fat by 40 %, impaired glucose regulation and led to liver steatosis. However, overexpression of Spry1 led to 35 % (P < 0·05) lower body fat, reduced bone loss and normal metabolic function compared with single transgenics. This protective phenotype was associated with decreased circulating insulin (70 %) and leptin (54 %; P < 0·005) compared with controls on a high-fat diet. Additionally, Spry1 expression decreased adipose tissue inflammation by 45 %. We show that conditional Spry1 expression in adipose tissue protects against high-fat diet-induced obesity and associated bone loss.

High expression of SDF-1 and VEGF is associated with poor prognosis in patients with synovial sarcomas.

Science.gov (United States)

Feng, Qi; Guo, Peng; Wang, Jin; Zhang, Xiaoyu; Yang, Hui-Chai; Feng, Jian-Gang

2018-03-01

Stromal cell-derived factor-1 (SDF-1) predicts poor clinical outcomes of certain types of cancer. Furthermore, vascular endothelial growth factor (VEGF) promotes the growth and metastasis of solid tumors. The aim of the present study was to examine the expression of SDF-1 and VEGF in patients with synovial sarcoma and to determine their expression is correlated with unfavorable outcomes. Levels of SDF-1 and VEGF proteins were evaluated in 54 patients with synovial sarcoma using immunohistochemical and immunofluorescence staining. Potential associations between the expression of SDF-1 and VEGF and various clinical parameters were analyzed using Pearson's χ 2 test and the Spearman-rho test. Additionally, univariate and multivariate Cox regression analyses were used to identify potential prognostic factors, and the Kaplan-Meier method was used to analyze the overall survival rates of patients. Low SDF-1 and VEGF expression was detected in 20.4% (11/54) and 22.2% (12/54) of patients with synovial sarcoma; moderate expression was detected in 35.2% (19/54) and 37.0% (20/54) of patients and high expression was detected in 44.4% (24 of 54) and 40.7% (22 of 54) of patients, respectively. Levels of SDF-1 and VEGF proteins were significantly associated with histological grade (P<0.05), metastasis (P<0.05) and American Joint Committee on Cancer staging (P<0.05). In addition, levels of SDF-1 and VEGF expression were positively correlated with each other (P<0.001). Univariate analysis also indicated that VEGF expression was associated with shorter overall survival rates in (P<0.05), whereas multivariate analysis demonstrated that SDF-1 expression was associated with shorter patient survival rates (P<0.05). Finally, both SDF-1 and VEGF expression were associated with various characteristics of synovial sarcoma. Therefore, SDF-1 expression may be a potential independent prognostic indicator in patients with synovial sarcomas.

Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum

International Nuclear Information System (INIS)

Marcel, Y.L.; Jewer, D.; Vezina, C.; Milthorp, P.; Weech, P.K.

1987-01-01

The expression and immunoreactivity of apolipoprotein (apo) A-I epitopes in high density lipoproteins (HDL) and serum has been investigated using two series of monoclonal antibodies (Mabs) which have been described elsewhere. Series 1 Mabs, identified as 3D4, 6B8, and 5G6, were obtained by immunization and screening with apoA-I, and series 2 Mabs, identified as 2F1, 4H1, 3G10, 4F7, and 5F6, were obtained by immunization and screening with HDL. These Mabs were characterized with respect to their binding to HDL particles in solution. In series 2 Mabs, 2F1, 3G10, and 4F7, which react with apoA-I CNBr-fragments 1 and 2, could precipitate 100% of 125 I-labeled HDL, while 4H1 and 5F6, which react with CNBr fragments 1 and 3, precipitated 90 and 60% of 125 I-labeled HDL, respectively. Therefore, three distinct epitopes mapped to CNBr fragments 1 and 2 have been identified which are expressed on all HDL particles, indicating that several antigenic do mains exist on apoA-I which have the same conformation on all apoA-I-containing lipoproteins. The Mabs reacting at these sites have significantly higher affinity constants for 125 I-labeled HDL than those that failed to precipitate 100% of HDL. This suggests that the high affinity Mabs react with apoA-I epitopes that are both expressed on all lipoproteins and located in thermo-dynamically stable regions of the molecules. All Mabs from series 1 precipitated 35% or less of 125 I-labeled HDL prepared from freshly collected serum, but the proportion of HDL particles expressing the epitopes for these Mabs doubled or more upon serum storage at 4 degrees C. The time course of the alteration of apoA-I antigen in vitro was measured in three normolipemic donors

High expression of markers of apoptosis in Langerhans cell histiocytosis

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Petersen, Bodil Laub; Lundegaard, Pia Rengtved; Bank, M I

2003-01-01

53 and the number of cells in apoptosis detected with TUNEL. Langerhans cell histiocytosis cells showed strong expression of p53 and in some cases co-expression of Fas and Fas-L. The expression of Fas-L was significantly higher in infiltrates from patients with single-system disease. The actual...... number of pathological Langerhans cells in apoptosis as estimated by TUNEL was low. CONCLUSIONS: The low number of TUNEL-reactive cells can be explained by the rapid turnover of apoptotic cells in the tissue, not leaving the apoptotic cells long enough in the tissue to be detected. The co......-expression of Fas and Fas-L in some Langerhans cells can lead to an autocrine apoptotic shortcut, mediating the death of the double-positive cells. Our findings suggest that apoptosis mediated through the Fas/Fas-L pathway may contribute to the spontaneous regression of lesions in single-system disease. A delicate...

Differential gene expression at different stages of mesocarp development in high- and low-yielding oil palm.

Science.gov (United States)

Wong, Yick Ching; Teh, Huey Fang; Mebus, Katharina; Ooi, Tony Eng Keong; Kwong, Qi Bin; Koo, Ka Loo; Ong, Chuang Kee; Mayes, Sean; Chew, Fook Tim; Appleton, David R; Kulaveerasingam, Harikrishna

2017-06-21

The oil yield trait of oil palm is expected to involve multiple genes, environmental influences and interactions. Many of the underlying mechanisms that contribute to oil yield are still poorly understood. In this study, we used a microarray approach to study the gene expression profiles of mesocarp tissue at different developmental stages, comparing genetically related high- and low- oil yielding palms to identify genes that contributed to the higher oil-yielding palm and might contribute to the wider genetic improvement of oil palm breeding populations. A total of 3412 (2001 annotated) gene candidates were found to be significantly differentially expressed between high- and low-yielding palms at at least one of the different stages of mesocarp development evaluated. Gene Ontologies (GO) enrichment analysis identified 28 significantly enriched GO terms, including regulation of transcription, fatty acid biosynthesis and metabolic processes. These differentially expressed genes comprise several transcription factors, such as, bHLH, Dof zinc finger proteins and MADS box proteins. Several genes involved in glycolysis, TCA, and fatty acid biosynthesis pathways were also found up-regulated in high-yielding oil palm, among them; pyruvate dehydrogenase E1 component Subunit Beta (PDH), ATP-citrate lyase, β- ketoacyl-ACP synthases I (KAS I), β- ketoacyl-ACP synthases III (KAS III) and ketoacyl-ACP reductase (KAR). Sucrose metabolism-related genes such as Invertase, Sucrose Synthase 2 and Sucrose Phosphatase 2 were found to be down-regulated in high-yielding oil palms, compared to the lower yield palms. Our findings indicate that a higher carbon flux (channeled through down-regulation of the Sucrose Synthase 2 pathway) was being utilized by up-regulated genes involved in glycolysis, TCA and fatty acid biosynthesis leading to enhanced oil production in the high-yielding oil palm. These findings are an important stepping stone to understand the processes that lead to

Comparative profiling of miRNA expression in developing seeds of high linoleic and high oleic safflower (Carthamus tinctorius L. plants

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Shijiang eCao

2013-12-01

Full Text Available Vegetable oils high in oleic acid are considered to be advantageous because of their better nutritional value and potential industrial applications. The oleic acid content in the classic safflower oil is normally 10-15% while a natural mutant (ol accumulates elevated oleic acid up to 70% in seed oil. As a part of our investigation into the molecular features of the high oleic (HO trait in safflower we have profiled the microRNA (miRNA populations in developing safflower seeds expressing the ol allele in comparison to the wild type high linoleic (HL safflower using deep sequencing technology. The small RNA populations of the mid-maturity developing embryos of homozygous ol HO and wild type HL safflower had a very similar size distribution pattern, however, only ~16.5% of the unique small RNAs were overlapping in these two genotypes. From these two small RNA populations we have found 55 known miRNAs and identified two candidate novel miRNA families to be likely unique to the developing safflower seeds. Target genes with conserved as well as novel functions were predicted for the conserved miRNAs. We have also identified 13 miRNAs differentially expressed between the HO and HL safflower genotypes. The results may lay a foundation for unravelling the miRNA-mediated molecular processes that regulate oleic acid accumulation in the HO safflower mutant and developmental processes in safflower embryos in general.

Association of high proliferation marker Ki-67 expression with DCEMR imaging features of breast: a large scale evaluation

Science.gov (United States)

Saha, Ashirbani; Harowicz, Michael R.; Grimm, Lars J.; Kim, Connie E.; Ghate, Sujata V.; Walsh, Ruth; Mazurowski, Maciej A.

2018-02-01

One of the methods widely used to measure the proliferative activity of cells in breast cancer patients is the immunohistochemical (IHC) measurement of the percentage of cells stained for nuclear antigen Ki-67. Use of Ki-67 expression as a prognostic marker is still under investigation. However, numerous clinical studies have reported an association between a high Ki-67 and overall survival (OS) and disease free survival (DFS). On the other hand, to offer non-invasive alternative in determining Ki-67 expression, researchers have made recent attempts to study the association of Ki-67 expression with magnetic resonance (MR) imaging features of breast cancer in small cohorts (AUC) of the values predicted. Our model was able to predict high versus low Ki-67 in the test set with an AUC of 0.67 (95% CI: 0.58-0.75, p<1.1e-04). Thus, a moderate strength of association of Ki-67 values and MRextracted imaging features was demonstrated in our experiments.

Gender-specific reduction of hepatic Mrp2 expression by high-fat diet protects female mice from ANIT toxicity

International Nuclear Information System (INIS)

Kong, Bo; Csanaky, Iván L.; Aleksunes, Lauren M.; Patni, Meghan; Chen, Qi; Ma, Xiaochao; Jaeschke, Hartmut; Weir, Scott; Broward, Melinda; Klaassen, Curtis D.; Guo, Grace L.

2012-01-01

Emerging evidence suggests that feeding a high-fat diet (HFD) to rodents affects the expression of genes involved in drug transport. However, gender-specific effects of HFD on drug transport are not known. The multidrug resistance-associated protein 2 (Mrp2, Abcc2) is a transporter highly expressed in the hepatocyte canalicular membrane and is important for biliary excretion of glutathione-conjugated chemicals. The current study showed that hepatic Mrp2 expression was reduced by HFD feeding only in female, but not male, C57BL/6J mice. In order to determine whether down-regulation of Mrp2 in female mice altered chemical disposition and toxicity, the biliary excretion and hepatotoxicity of the Mrp2 substrate, α-naphthylisothiocyanate (ANIT), were assessed in male and female mice fed control diet or HFD for 4 weeks. ANIT-induced biliary injury is a commonly used model of experimental cholestasis and has been shown to be dependent upon Mrp2-mediated efflux of an ANIT glutathione conjugate that selectively injures biliary epithelial cells. Interestingly, HFD feeding significantly reduced early-phase biliary ANIT excretion in female mice and largely protected against ANIT-induced liver injury. In summary, the current study showed that, at least in mice, HFD feeding can differentially regulate Mrp2 expression and function and depending upon the chemical exposure may enhance or reduce susceptibility to toxicity. Taken together, these data provide a novel interaction between diet and gender in regulating hepatobiliary excretion and susceptibility to injury. -- Highlights: ► High-fat diet decreases hepatic Mrp2 expression only in female but not in male mice. ► HFD significantly reduces early-phase biliary ANIT excretion in female mice. ► HFD protects female mice against ANIT-induced liver injury.

Expressive writing for high-risk drug dependent patients in a primary care clinic: A pilot study

Directory of Open Access Journals (Sweden)

Wedgwood Lucinda

2006-11-01

Full Text Available Abstract Background Previous research has shown that expressive writing is beneficial in terms of both physical and emotional health outcomes. This study aimed to investigate the effectiveness and acceptability of a brief expressive writing intervention for high-risk drug dependent patients in a primary care clinic, and to determine the relationship between linguistic features of writing and health outcomes. Methods Participants completed four 15-minute expressive writing tasks over a week, in which they described their thoughts and feelings about a recent stressful event. Self-report measures of physical (SF-12 and psychological health (DASS-21 were administered at baseline and at a two-week follow-up. Fifty-three participants were recruited and 14 (26% completed all measures. Results No statistically significant benefits in physical or psychological health were found, although all outcomes changed in the direction of improvement. The intervention was well-received and was rated as beneficial by participants. The use of more positive emotion words in writing was associated with improvements in depression and stress, and flexibility in first person pronoun use was associated with improvements in anxiety. Increasing use of cognitive process words was associated with worsening depressive mood. Conclusion Although no significant benefits in physical and psychological health were found, improvements in psychological wellbeing were associated with certain writing styles and expressive writing was deemed acceptable by high-risk drug dependent patients. Given the difficulties in implementing psychosocial interventions in this population, further research using a larger sample is warranted.

High-level expression, purification, polyclonal antibody preparation ...

African Journals Online (AJOL)

OprD is a specific porin which can binds imipenem and carbapenems in Pseudomonas aeruginosa. OprD loss plays a central role in mediating carbapenem resistance. Therefore, purification of oprD protein lays a pavement for the study in vivo and in vitro. In our study, the oprD gene was cloned into pQE30 expression ...

Expression of NgBR Is Highly Associated with Estrogen Receptor Alpha and Survivin in Breast Cancer

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North, Paula; Kong, Amanda; Huang, Jian; Miao, Qing Robert

2013-01-01

NgBR is a type I receptor with a single transmembrane domain and was identified as a specific receptor for Nogo-B. Our recent findings demonstrated that NgBR binds farnesylated Ras and recruits Ras to the plasma membrane, which is a critical step required for the activation of Ras signaling in human breast cancer cells and tumorigenesis. Here, we first use immunohistochemistry and real-time PCR approaches to examine the expression patterns of Nogo-B and NgBR in both normal and breast tumor tissues. Then, we examine the relationship between NgBR expression and molecular subtypes of breast cancer, and the roles of NgBR in estrogen-dependent survivin signaling pathway. Results showed that NgBR and Nogo-B protein were detected in both normal and breast tumor tissues. However, the expression of Nogo-B and NgBR in breast tumor tissue was much stronger than in normal breast tissue. The statistical analysis demonstrated that NgBR is highly associated with ER-positive/HER2-negative breast cancer. We also found that the expression of NgBR has a strong correlation with the expression of survivin, which is a well-known apoptosis inhibitor. The correlation between NgBR and survivin gene expression was further confirmed by real-time PCR. In vitro results also demonstrated that estradiol induces the expression of survivin in ER-positive T47D breast tumor cells but not in ER-negative MDA-MB-468 breast tumor cells. NgBR knockdown with siRNA abolishes estradiol-induced survivin expression in ER-positive T47D cells but not in ER-negative MDA-MB-468 cells. In addition, estradiol increases the expression of survivin and cell growth in ER-positive MCF-7 and T47D cells whereas knockdown of NgBR with siRNA reduces estradiol-induced survivin expression and cell growth. In summary, these results indicate that NgBR is a new molecular marker for breast cancer. The data suggest that the expression of NgBR may be essential in promoting ER-positive tumor cell proliferation via survivin induction

High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

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Chang, L.; Liu, Y.Y.; Zhu, B.; Li, Y.; Hua, H.; Wang, Y.H.; Zhang, J.; Jiang, Z.; Wang, Z.R. [Sichuan University, Chengdu (China). West China Medical Center. Health Ministry Key Lab. of Chronobiology], e-mail: wangzhengrong@126.com

2009-10-15

Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to {sup 60}Co-{gamma}-rays. NIH 3T3 cells were treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with {sup 6}0Co-{gamma}-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 {+-} 6.51 vs 66.0 {+-} 3.51 and 67.7 {+-} 7.37; transfection: 121.7 {+-} 6.50 vs 65.3 {+-} 3.51 and 69.0 {+-} 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, cmyc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes. (author)

High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

International Nuclear Information System (INIS)

Chang, L.; Liu, Y.Y.; Zhu, B.; Li, Y.; Hua, H.; Wang, Y.H.; Zhang, J.; Jiang, Z.; Wang, Z.R.

2009-01-01

Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to 60 Co-γ-rays. NIH 3T3 cells were treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with 6 0Co-γ-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 ± 6.51 vs 66.0 ± 3.51 and 67.7 ± 7.37; transfection: 121.7 ± 6.50 vs 65.3 ± 3.51 and 69.0 ± 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, cmyc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes. (author)

Wilms’ Tumor 1 Gene Mutations Independently Predict Poor Outcome in Adults With Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

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Paschka, Peter; Marcucci, Guido; Ruppert, Amy S.; Whitman, Susan P.; Mrózek, Krzysztof; Maharry, Kati; Langer, Christian; Baldus, Claudia D.; Zhao, Weiqiang; Powell, Bayard L.; Baer, Maria R.; Carroll, Andrew J.; Caligiuri, Michael A.; Kolitz, Jonathan E.; Larson, Richard A.; Bloomfield, Clara D.

2008-01-01

Purpose To analyze the prognostic impact of Wilms’ tumor 1 (WT1) gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML). Patients and Methods We studied 196 adults younger than 60 years with newly diagnosed primary CN-AML, who were treated similarly on Cancer and Leukemia Group B (CALGB) protocols 9621 and 19808, for WT1 mutations in exons 7 and 9. The patients also were assessed for the presence of FLT3 internal tandem duplications (FLT3-ITD), FLT3 tyrosine kinase domain mutations (FLT3-TKD), MLL partial tandem duplications (MLL-PTD), NPM1 and CEBPA mutations, and for the expression levels of ERG and BAALC. Results Twenty-one patients (10.7%) harbored WT1 mutations. Complete remission rates were not significantly different between patients with WT1 mutations and those with unmutated WT1 (P = .36; 76% v 84%). Patients with WT1 mutations had worse disease-free survival (DFS; P < .001; 3-year rates, 13% v 50%) and overall survival (OS; P < .001; 3-year rates, 10% v 56%) than patients with unmutated WT1. In multivariable analyses, WT1 mutations independently predicted worse DFS (P = .009; hazard ratio [HR] = 2.7) when controlling for CEBPA mutational status, ERG expression level, and FLT3-ITD/NPM1 molecular-risk group (ie, FLT3-ITDnegative/NPM1mutated as low risk v FLT3-ITDpositive and/or NPM1wild-type as high risk). WT1 mutations also independently predicted worse OS (P < .001; HR = 3.2) when controlling for CEBPA mutational status, FLT3-ITD/NPM1 molecular-risk group, and white blood cell count. Conclusion We report the first evidence that WT1 mutations independently predict extremely poor outcome in intensively treated, younger patients with CN-AML. Future trials should include testing for WT1 mutations as part of molecularly based risk assessment and risk-adapted treatment stratification of patients with CN-AML. PMID:18559874

The Effect of Silicon on Photosynthesis and Expression of Its Relevant Genes in Rice (Oryza sativa L.) under High-Zinc Stress

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Song, Alin; Li, Ping; Fan, Fenliang; Li, Zhaojun; Liang, Yongchao

2014-01-01

The main objectives of this study were to elucidate the roles of silicon (Si) in alleviating the effects of 2 mM zinc (high Zn) stress on photosynthesis and its related gene expression levels in leaves of rice (Oryza sativa L.) grown hydroponically with high-Zn stress. The results showed that photosynthetic parameters, including net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration, chlorophyll concentration and the chlorophyll fluorescence, were decreased in rice exposed to high-Zn treatment. The leaf chloroplast structure was disordered under high-Zn stress, including uneven swelling, disintegrated and missing thylakoid membranes, and decreased starch granule size and number, which, however, were all counteracted by the addition of 1.5 mM Si. Furthermore, the expression levels of Os08g02630 (PsbY), Os05g48630 (PsaH), Os07g37030 (PetC), Os03g57120 (PetH), Os09g26810 and Os04g38410 decreased in Si-deprived plants under high-Zn stress. Nevertheless, the addition of 1.5 mM Si increased the expression levels of these genes in plants under high-Zn stress at 72 h, and the expression levels were higher in Si-treated plants than in Si-deprived plants. Therefore, we conclude that Si alleviates the Zn-induced damage to photosynthesis in rice. The decline of photosynthesis in Zn-stressed rice was attributed to stomatal limitation, and Si activated and regulated some photosynthesis-related genes in response to high-Zn stress, consequently increasing photosynthesis. PMID:25426937

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

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Ribeiro, Thuanne Pires; Arraes, Fabricio Barbosa Monteiro; Lourenço-Tessutti, Isabela Tristan; Silva, Marilia Santos; Lisei-de-Sá, Maria Eugênia; Lucena, Wagner Alexandre; Macedo, Leonardo Lima Pepino; Lima, Janaina Nascimento; Santos Amorim, Regina Maria; Artico, Sinara; Alves-Ferreira, Márcio; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

2017-08-01

Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T 0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2 -ΔΔCt analyses revealed that T 0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T 0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g -1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T 0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T 1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g -1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Comparative Genomics in Switchgrass Using 61,585 High-Quality Expressed Sequence Tags

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Christian M. Tobias

2008-11-01

Full Text Available The development of genomic resources for switchgrass ( L., a perennial NAD-malic enzyme type C grass, is required to enable molecular breeding and biotechnological approaches for improving its value as a forage and bioenergy crop. Expressed sequence tag (EST sequencing is one method that can quickly sample gene inventories and produce data suitable for marker development or analysis of tissue-specific patterns of expression. Toward this goal, three cDNA libraries from callus, crown, and seedling tissues of ‘Kanlow’ switchgrass were end-sequenced to generate a total of 61,585 high-quality ESTs from 36,565 separate clones. Seventy-three percent of the assembled consensus sequences could be aligned with the sorghum [ (L. Moench] genome at a -value of <1 × 10, indicating a high degree of similarity. Sixty-five percent of the ESTs matched with gene ontology molecular terms, and 3.3% of the sequences were matched with genes that play potential roles in cell-wall biogenesis. The representation in the three libraries of gene families known to be associated with C photosynthesis, cellulose and β-glucan synthesis, phenylpropanoid biosynthesis, and peroxidase activity indicated likely roles for individual family members. Pairwise comparisons of synonymous codon substitutions were used to assess genome sequence diversity and indicated an overall similarity between the two genome copies present in the tetraploid. Identification of EST–simple sequence repeat markers and amplification on two individual parents of a mapping population yielded an average of 2.18 amplicons per individual, and 35% of the markers produced fragment length polymorphisms.

Both high expression of pyruvate kinase M2 and vascular endothelial growth factor-C predicts poorer prognosis in human breast cancer.

Science.gov (United States)

Lin, Yang; Liu, Fangfang; Fan, Yu; Qian, Xiaolong; Lang, Ronggang; Gu, Feng; Gu, Jun; Fu, Li

2015-01-01

Pyruvate kinase M2 (PKM2) and vascular endothelial growth factor-C (VEGF-C) have been known to play an important role in tumorigenesis and tumor progression in breast cancer. However, the association between PKM2 and VEGF-C in breast cancer remains unclear. In the present study, a total of 218 specimens from breast cancer patients and 26 paired breast tumors with adjacent normal tissues as well as two breast cancer cell lines were enrolled to investigate the correlation between PKM2 and VEGF-C. We found that PKM2 and VEGF-C mRNA levels were both significantly increasing in breast tumors compared with adjacent normal tissues. Knockdown of PKM2 mRNA expression resulted in VEGF-C mRNA and protein down-regulated as well as cell proliferation inhibited. A positive correlation between PKM2 and VEGF-C expression was identified by immunohistochemical analyses of 218 specimens of patients with breast cancer (P=0.023). PKM2 high expression was significantly correlated with histological grade (P=0.030), lymph node stage (P=0.001), besides VEGF-C high expression was significantly associated with lymphovascular invasion (P=0.012). While combined high expression of PKM2 and VEGF-C was found to be associated with worse histological grade, more lymph node metastasis, more lymphovascular invasion, shorter progression free survival (PFS), and poorer overall survival (OS) in human breast cancer. The results of the present study suggested that PKM2 expression was correlated with VEGF-C expression, and combination of PKM2 and VEGF-C levels had the better prognostic significance in predicting the poor outcome of patients with breast cancer.

Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

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Bendixen Christian

2007-11-01

Full Text Available Abstract Background: Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels, which is essential for recognising potential molecular markers for breeding purposes. Results: Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26,877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p Conclusion: This study contributes to the understanding of the complex genetic system controlling and responding to androstenone levels in pig testis. The identification of new pathways and genes involved in the biosynthesis and metabolism of androstenone is an important first step towards finding molecular markers to reduce boar taint.

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

Science.gov (United States)

Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

2018-02-01

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris.

Science.gov (United States)

Kittl, Roman; Mueangtoom, Kitti; Gonaus, Christoph; Khazaneh, Shima Tahvilda; Sygmund, Christoph; Haltrich, Dietmar; Ludwig, Roland

2012-01-20

Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L(-1) of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K(M) values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I(50)=1.4M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research. Copyright © 2011 Elsevier B.V. All rights reserved.

Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages.

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Nan Li

Full Text Available Non-coding small RNAs (sRNAs are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.

High level over-expression of different NCX isoforms in HEK293 cell lines and primary neuronal cultures is protective following oxygen glucose deprivation.

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Cross, Jane L; Boulos, Sherif; Shepherd, Kate L; Craig, Amanda J; Lee, Sharon; Bakker, Anthony J; Knuckey, Neville W; Meloni, Bruno P

2012-07-01

In this study we have assessed sodium-calcium exchanger (NCX) protein over-expression on cell viability in primary rat cortical neuronal and HEK293 cell cultures when subjected to oxygen-glucose deprivation (OGD). In cortical neuronal cultures, NCX2 and NCX3 over-expression was achieved using adenoviral vectors, and following OGD increased neuronal survival from ≈20% for control vector treated cultures to ≈80% for both NCX isoforms. In addition, we demonstrated that NCX2 and NCX3 over-expression in cortical neuronal cultures enables neurons to maintain intracellular calcium at significantly lower levels than control vector treated cultures when exposed to high (9mM) extracellular calcium challenge. Further assessment of NCX activity during OGD was performed using HEK293 cell lines generated to over-express NCX1, NCX2 or NCX3 isoforms. While it was shown that NCX isoform expression differed considerably in the different HEK293 cell lines, high levels of NCX over-expression was associated with increased resistance to OGD. Taken together, our findings show that high levels of NCX over-expression increases neuronal and HEK293 cell survival following OGD, improves calcium management in neuronal cultures and provides additional support for NCX as a therapeutic target to reduce ischemic brain injury. Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Lewis x is highly expressed in normal tissues: a comparative immunohistochemical study and literature revision.

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Croce, María V; Isla-Larrain, Marina; Rabassa, Martín E; Demichelis, Sandra; Colussi, Andrea G; Crespo, Marina; Lacunza, Ezequiel; Segal-Eiras, Amada

2007-01-01

An immunohistochemical analysis was employed to determine the expression of carbohydrate antigens associated to mucins in normal epithelia. Tissue samples were obtained as biopsies from normal breast (18), colon (35) and oral cavity mucosa (8). The following carbohydrate epitopes were studied: sialyl-Lewis x, Lewis x, Lewis y, Tn hapten, sialyl-Tn and Thomsen-Friedenreich antigen. Mucins were also studied employing antibodies against MUC1, MUC2, MUC4, MUC5AC, MUC6 and also normal colonic glycolipid. Statistical analysis was performed and Kendall correlations were obtained. Lewis x showed an apical pattern mainly at plasma membrane, although cytoplasmic staining was also found in most samples. TF, Tn and sTn haptens were detected in few specimens, while sLewis x was found in oral mucosa and breast tissue. Also, normal breast expressed MUC1 at a high percentage, whereas MUC4 was observed in a small number of samples. Colon specimens mainly expressed MUC2 and MUC1, while most oral mucosa samples expressed MUC4 and MUC1. A positive correlation between MUC1VNTR and TF epitope (r=0.396) was found in breast samples, while in colon specimens MUC2 and colonic glycolipid versus Lewis x were statistically significantly correlated (r=0.28 and r=0.29, respectively). As a conclusion, a defined carbohydrate epitope expression is not exclusive of normal tissue or a determined localization, and it is possible to assume that different glycoproteins and glycolipids may be carriers of carbohydrate antigens depending on the tissue localization considered.

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

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A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

DEFF Research Database (Denmark)

Christensen, Søren Tvorup; Voss, Jesper W.; Teilmann, Stefan C.

2005-01-01

Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na+-dependent taurine transporter...... TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium....

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

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Craveiro R.B.

2006-01-01

Full Text Available Carboxypeptidase M (CPM is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1. This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa, suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

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R.B. Craveiro

2006-02-01

Full Text Available Carboxypeptidase M (CPM is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1. This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa, suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

Gene promoter methylation and protein expression of BRMS1 in uterine cervix in relation to high-risk human papilloma virus infection and cancer.

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Panagopoulou, Maria; Lambropoulou, Maria; Balgkouranidou, Ioanna; Nena, Evangelia; Karaglani, Makrina; Nicolaidou, Christina; Asimaki, Anthi; Konstantinidis, Theocharis; Constantinidis, Theodoros C; Kolios, George; Kakolyris, Stylianos; Agorastos, Theodoros; Chatzaki, Ekaterini

2017-04-01

Cervical cancer is strongly related to certain high-risk types of human papilloma virus infection. Breast cancer metastasis suppressor 1 (BRMS1) is a tumor suppressor gene, its expression being regulated by DNA promoter methylation in several types of cancers. This study aims to evaluate the methylation status of BRMS1 promoter in relation to high-risk types of human papilloma virus infection and the development of pre-cancerous lesions and describe the pattern of BRMS1 protein expression in normal, high-risk types of human papilloma virus-infected pre-cancerous and malignant cervical epithelium. We compared the methylation status of BRMS1 in cervical smears of 64 women with no infection by high-risk types of human papilloma virus to 70 women with proven high-risk types of human papilloma virus infection, using real-time methylation-specific polymerase chain reaction. The expression of BRMS1 protein was described by immunohistochemistry in biopsies from cervical cancer, pre-cancerous lesions, and normal cervices. Methylation of BRMS1 promoter was detected in 37.5% of women with no high-risk types of human papilloma virus infection and was less frequent in smears with high-risk types of human papilloma virus (11.4%) and in women with pathological histology (cervical intraepithelial neoplasia) (11.9%). Methylation was detected also in HeLa cervical cancer cells. Immunohistochemistry revealed nuclear BRMS1 protein staining in normal high-risk types of human papilloma virus-free cervix, in cervical intraepithelial neoplasias, and in malignant tissues, where staining was occasionally also cytoplasmic. In cancer, expression was stronger in the more differentiated cancer blasts. In conclusion, BRMS1 promoter methylation and aberrant protein expression seem to be related to high-risk types of human papilloma virus-induced carcinogenesis in uterine cervix and is worthy of further investigation.

Ectopic High Expression of E2-EPF Ubiquitin Carrier Protein Indicates a More Unfavorable Prognosis in Brain Glioma.

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Zhang, Xiaohui; Zhao, Fangbo; Zhang, Shujun; Song, Yichun

2017-04-01

Ubiquitination of proteins meant for elimination is a primary method of eukaryotic cellular protein degradation. The ubiquitin carrier protein E2-EPF is a key degradation enzyme that is highly expressed in many tumors. However, its expression and prognostic significance in brain glioma are still unclear. The aim of this study was to reveal how the level of E2-EPF relates to prognosis in brain glioma. Thirty low-grade and 30 high-grade brain glioma samples were divided into two tissue microarrays each. Levels of E2-EPF protein were examined by immunohistochemistry and immunofluorescence. Quantitative real-time polymerase chain reaction was used to analyze the level of E2-EPF in 60 glioma and 3 normal brain tissue samples. The relationship between E2-EPF levels and prognosis was analyzed by Kaplan-Meier survival curves. E2-EPF levels were low in normal brain tissue samples but high in glioma nuclei. E2-EPF levels gradually increased as glioma grade increased (p EPF levels in high-grade glioma were significantly higher than in low-grade glioma (p EPF levels was shorter than in patients with low expression (p EPF was significantly shorter than patients with only nuclear E2-EPF (p EPF levels, especially ectopic, are associated with higher grade glioma and shorter survival. E2-EPF levels may play a key role in predicting the prognosis for patients with brain glioma.

Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish.

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Cristina Santoriello

2010-12-01

Full Text Available Melanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.Using the combinatorial Gal4-UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.

High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells.

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Fu, J; Tay, S S W; Ling, E A; Dheen, S T

2006-05-01

Maternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs. Cell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2'-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT-PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically. High glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells. The changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the

Evidence for gene-environment interaction in a genome wide study of nonsyndromic cleft palate

DEFF Research Database (Denmark)

Beaty, Terri H; Ruczinski, Ingo; Murray, Jeffrey C

2011-01-01

Nonsyndromic cleft palate (CP) is a common birth defect with a complex and heterogeneous etiology involving both genetic and environmental risk factors. We conducted a genome-wide association study (GWAS) using 550 case-parent trios, ascertained through a CP case collected in an international...... consortium. Family-based association tests of single nucleotide polymorphisms (SNP) and three common maternal exposures (maternal smoking, alcohol consumption, and multivitamin supplementation) were used in a combined 2 df test for gene (G) and gene-environment (G × E) interaction simultaneously, plus...... multiple SNPs associated with higher risk of CP in the presence of maternal smoking. Additional evidence of reduced risk due to G × E interaction in the presence of multivitamin supplementation was observed for SNPs in BAALC on chr. 8. These results emphasize the need to consider G × E interaction when...

High expression of high-mobility group box 1 in the blood and lungs is associated with the development of chronic obstructive pulmonary disease in smokers.

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Ko, Hsin-Kuo; Hsu, Wen-Hu; Hsieh, Chih-Cheng; Lien, Te-Cheng; Lee, Tzong-Shyuan; Kou, Yu Ru

2014-02-01

High-mobility group box 1 (HMGB1) is an important mediator in multiple pathological conditions, but the expression of HMGB1 in chronic obstructive pulmonary disease (COPD) has not yet been completely investigated. We aimed to analyze the relationship between HMGB1 expression in blood and lung tissue and the development of COPD. Twenty-eight patients admitted for single pulmonary surgical intervention were enrolled. The expression of HMGB1 in blood and lung tissue was evaluated by enzyme-linked immunosorbent assay analysis and immunohistochemistry stain, respectively. The study patients were divided into smokers with COPD (n = 11), smokers without COPD (n = 8) and non-smoker healthy controls (n = 9). Smokers with COPD compared with smokers without COPD and healthy controls were older in age, with lower post-bronchodilator forced expiratory volume in 1 s/forced vital capacity (FEV1 /FVC) ratio (63.1 ± 5.5 vs 77.6 ± 3.6 and 84.5 ± 5.8, P vs 7.3 ± 4.8 and 17.0 ± 19.6 ng/mL, P = 0.016 and P = 0.021, respectively). In smokers with COPD, the numbers and portion of HMGB1-expressing cells in epithelium and submucosal areas were significantly increased. Notably, plasma HMGB1 levels negatively correlated with post-bronchodilator FEV1 /FVC ratio (r = -0.585, P = 0.008) in smokers, but not in non-smokers. In smokers, high expression of HMGB1 in the blood and lungs is related to the lung function impairment and appears to be associated with the development of COPD. © 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.

Molecular and functional expression of high conductance Ca 2+ activated K+ channels in the eel intestinal epithelium

DEFF Research Database (Denmark)

Lionetto, Maria G; Rizzello, Antonia; Giordano, Maria E

2008-01-01

Several types of K(+) channels have been identified in epithelial cells. Among them high conductance Ca(2+)-activated K(+) channels (BK channels) are of relevant importance for their involvement in regulatory volume decrease (RVD) response following hypotonic stress. The aim of the present work...... was to investigate the functional and molecular expression of BK in the eel intestine, which is a useful experimental model for cell volume regulation research. In the present paper using rat BK channel-specific primer, a RT-PCR signal of 696 pb cDNA was detected in eel intestine, whole nucleotide sequence showed...... high similarity (83%) to the alpha subunit of BK channel family. BK channel protein expression was verified by immunoblotting and confocal microscopy, while the functional role of BK channels in epithelial ion transport mechanisms and cell volume regulation was examined by electrophysiological...

Rapamycin-ameliorated diabetic symptoms involved in increasing adiponectin expression in diabetic mice on a high-fat diet.

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Gong, Fang-Hua; Ye, Yan-Na; Li, Jin-Meng; Zhao, Hai-Yang; Li, Xiao-Kun

2017-07-01

Recent studies showed that rapamycin improved diabetic complications. Here, we investigated the metabolic effects of rapamycin in type 2 diabetes model (T2DM) mice. Mice were treated with a daily intraperitoneal injection of rapamycin at 2 mg/kg or vehicle only for 3 weeks and were maintained on a high-fat diet. The treated diabetic mice exhibited decreased body weight, blood glucose levels, and fat mass. FGF21 expression was suppressed in C57B/L6 mice, but adiponectin expression increased both in FGF21 KO and C57B/L6 mice. These results suggest that rapamycin may alleviate FGF21 resistance in mice on a high-fat diet. The reduction of adipose tissue mass of the diabetic mice may be due to the increased adiponectin. Copyright © 2017. Published by Elsevier Taiwan.

Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

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Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj

2006-01-01

Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells....

Effect of high-intensity training on exercise-induced gene expression specific to ion homeostasis and metabolism

DEFF Research Database (Denmark)

Nordsborg, Nikolai; Bangsbo, Jens; Pilegaard, Henriette

2003-01-01

Changes in gene expression during recovery from high-intensity, intermittent, one-legged exercise were studied before and after 5.5 wk of training. Genes related to metabolism, as well as Na+, K+, and pH homeostasis, were selected for analyses. After the same work was performed before and after...

High-Throughput Agrobacterium-mediated Transformation of Medicago Truncatula in Comparison to Two Expression Vectors

International Nuclear Information System (INIS)

Sultana, T.; Deeba, F.; Naqvi, S. M. S.

2016-01-01

Legumes have been turbulent to efficient Agrobacterium-mediated transformation for a long time. The selection of Medicago truncatula as a model legume plant for molecular analysis resulted in the development of efficient Agrobacterium-mediated transformation protocols. In current study, M. truncatula transformed plants expressing OsRGLP1 were obtained through GATEWAY technology using pGOsRGLP1 (pH7WG2.0=OsRGLP1). The transformation efficiency of this vector was compared with expression vector from pCAMBIA series over-expressing same gene (pCOsRGLP1). A lower number of explants generated hygromycin resistant plantlet for instance, 18.3 with pGOsRGLP1 vector as compared to 35.5 percent with pCOsRGLP1 vector. Transformation efficiency of PCR positive plants generated was 9.4 percent for pGOsRGLP1 while 21.6 percent for pCOsRGLP1. Furthermore 24.4 percent of explants generated antibiotic resistant plantlet on 20 mgl/sup -1/ of hygromycin which was higher than on 15 mgl/sup -1/ of hygromycin such as 12.2 percent. T/sub 1/ progeny analysis indicated that the transgene was inherited in Mendelian manner. The functionally active status of transgene was monitored by high level of Superoxide dismutase (SOD) activity in transformed progeny. (author)

Expression

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Wang-Xia Wang

2014-02-01

Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

The impact of high trait social anxiety on neural processing of facial emotion expressions in females.

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Felmingham, Kim L; Stewart, Laura F; Kemp, Andrew H; Carr, Andrea R

2016-05-01

A cognitive model of social anxiety predicts that an early attentional bias leads to greater cognitive processing of social threat signals, whereas the vigilance-avoidance model predicts there will be subsequent reduction in cognitive processing. This study tests these models by examining neural responses to social threat stimuli using Event-related potentials (ERP). 19 women with high trait social anxiety and 19 women with low trait social anxiety viewed emotional expressions (angry, disgusted, happy and neutral) in a passive viewing task whilst ERP responses were recorded. The HSA group revealed greater automatic attention, or hypervigilance, to all facial expressions, as indexed by greater N1 amplitude compared to the LSA group. They also showed greater sustained attention and elaborative processing of all facial expressions, indexed by significantly increased P2 and P3 amplitudes compared to the LSA group. These results support cognitive models of social anxiety, but are not consistent with predictions of the vigilance-avoidance model. Copyright © 2016. Published by Elsevier B.V.

High natural gene expression variation in the reef-building coral Acropora millepora: potential for acclimative and adaptive plasticity.

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Granados-Cifuentes, Camila; Bellantuono, Anthony J; Ridgway, Tyrone; Hoegh-Guldberg, Ove; Rodriguez-Lanetty, Mauricio

2013-04-08

Ecosystems worldwide are suffering the consequences of anthropogenic impact. The diverse ecosystem of coral reefs, for example, are globally threatened by increases in sea surface temperatures due to global warming. Studies to date have focused on determining genetic diversity, the sequence variability of genes in a species, as a proxy to estimate and predict the potential adaptive response of coral populations to environmental changes linked to climate changes. However, the examination of natural gene expression variation has received less attention. This variation has been implicated as an important factor in evolutionary processes, upon which natural selection can act. We acclimatized coral nubbins from six colonies of the reef-building coral Acropora millepora to a common garden in Heron Island (Great Barrier Reef, GBR) for a period of four weeks to remove any site-specific environmental effects on the physiology of the coral nubbins. By using a cDNA microarray platform, we detected a high level of gene expression variation, with 17% (488) of the unigenes differentially expressed across coral nubbins of the six colonies (jsFDR-corrected, p natural variation between reef corals when assessing experimental gene expression differences. The high transcriptional variation detected in this study is interpreted and discussed within the context of adaptive potential and phenotypic plasticity of reef corals. Whether this variation will allow coral reefs to survive to current challenges remains unknown.

Identification of distal regulatory regions in the human alpha IIb gene locus necessary for consistent, high-level megakaryocyte expression.

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Thornton, Michael A; Zhang, Chunyan; Kowalska, Maria A; Poncz, Mortimer

2002-11-15

The alphaIIb/beta3-integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of alphaIIb, and studies of the alphaIIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the alphaIIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) alphaIIb loci revealed high levels of conservation at intergenic regions both 5' and 3' to the alphaIIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive (HS) sites that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the h(alpha)IIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue specificity, but that a region 2.5 to 7.1 kb upstream of the h(alpha)IIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold and led to levels of h(alpha)IIb mRNA that were about 30% of the native m(alpha)IIb mRNA level. These constructs also resulted in detectable h(alpha)IIb/m(beta)3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the alphaIIb gene for tissue specificity, but also characterizes the distal organization of the alphaIIb gene locus and provides an initial localization of 2 important regulatory regions needed for the expression of the alphaIIb gene at high levels during megakaryopoiesis.

Placenta-specific novel splice variants of Rho GDP dissociation inhibitor β are highly expressed in cancerous cells

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Hatakeyama Keiichi

2012-12-01

Full Text Available Abstract Background Alternative splicing of pre-mRNA transcripts not only plays a role in normal molecular processes but is also associated with cancer development. While normal transcripts are ubiquitously expressed in normal tissues, splice variants created through abnormal alternative splicing events are often expressed in cancer cells. Although the Rho GDP dissociation inhibitor β (ARHGDIB gene has been found to be ubiquitously expressed in normal tissues and involved in cancer development, the presence of splice variants of ARHGDIB has not yet been investigated. Results Validation analysis for the presence of and exon structures of splice variants of ARHGDIB, performed using reverse-transcriptase polymerase chain reaction and DNA sequencing, successfully identified novel splice variants of ARHGDIB, that is, 6a, 6b, and 6c, in colon, pancreas, stomach, and breast cancer cell lines. Quantitative real-time polymerase chain reaction analysis showed that these variants were also highly expressed in normal placental tissue but not in other types of normal tissue. Conclusions Expression of ARHGDIB variants 6a, 6b, and 6c appears to be restricted to cancer cells and normal placental tissue, suggesting that these variants possess cancer-specific functions and, as such, are potential cancer-related biomarkers.

Impaired detection of happy facial expressions in autism.

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Sato, Wataru; Sawada, Reiko; Uono, Shota; Yoshimura, Sayaka; Kochiyama, Takanori; Kubota, Yasutaka; Sakihama, Morimitsu; Toichi, Motomi

2017-10-17

The detection of emotional facial expressions plays an indispensable role in social interaction. Psychological studies have shown that typically developing (TD) individuals more rapidly detect emotional expressions than neutral expressions. However, it remains unclear whether individuals with autistic phenotypes, such as autism spectrum disorder (ASD) and high levels of autistic traits (ATs), are impaired in this ability. We examined this by comparing TD and ASD individuals in Experiment 1 and individuals with low and high ATs in Experiment 2 using the visual search paradigm. Participants detected normal facial expressions of anger and happiness and their anti-expressions within crowds of neutral expressions. In Experiment 1, reaction times were shorter for normal angry expressions than for anti-expressions in both TD and ASD groups. This was also the case for normal happy expressions vs. anti-expressions in the TD group but not in the ASD group. Similarly, in Experiment 2, the detection of normal vs. anti-expressions was faster for angry expressions in both groups and for happy expressions in the low, but not high, ATs group. These results suggest that the detection of happy facial expressions is impaired in individuals with ASD and high ATs, which may contribute to their difficulty in creating and maintaining affiliative social relationships.

Unwinding after high salinity stress: Pea DNA helicase 45 over- expression in tobacco confers high salinity tolerance without affecting yield (abstract)

International Nuclear Information System (INIS)

Tuteja, N.

2005-01-01

Soil salinity is an increasing threat for agriculture and is a major factor in reducing plant productivity; therefore, it is necessary to obtain salinity-tolerant varieties. A typical characteristic of soil salinity is the induction of multiple stress- inducible genes. Some of the genes encoding osmolytes, ion channels or enzymes are able to confer salinity-tolerant phenotypes when transferred to sensitive plants. As salinity stress affects the cellular gene-expression machinery, it is evident that molecules involved in nucleic acid processing including helicases, are likely to be affected as well. DNA helicases unwind duplex DNA and are involved in replication, repair, recombination and transcription while RNA helicases unfold the secondary structures in RNA and are involved in transcription, ribosome biogenesis and translation initiation. We have earlier reported the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with eIF-4A (Plant J. 24:219-230,2000). Here we report that PDH45 mRNA is induced in pea seedlings in response to high salt and its over- expression driven by a constitutive CAMV-355-promoter in tobacco plants confers salinity tolerance, thus suggesting a new pathway for manipulating stress tolerance in crop plants. The T0 transgenic plants showed high-levels of PDH45 protein in normal and stress conditions, as compared to wild type (WT) plants. The T0 transgenics also showed tolerance to high salinity as tested by a leaf disc senescence assay. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress, without any reduction in plant yield, in terms of seed weight. Measurement of Na/sup +/ ions in different parts of the plant showed higher accumulation in the old leaves and negligible in seeds of T1 transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance will be discussed. Over-expression of PDH45 provides a possible example of the

AICAR Protects against High Palmitate/High Insulin-Induced Intramyocellular Lipid Accumulation and Insulin Resistance in HL-1 Cardiac Cells by Inducing PPAR-Target Gene Expression

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Ricardo Rodríguez-Calvo

2015-01-01

Full Text Available Here we studied the impact of 5-aminoimidazole-4-carboxamide riboside (AICAR, a well-known AMPK activator, on cardiac metabolic adaptation. AMPK activation by AICAR was confirmed by increased phospho-Thr172-AMPK and phospho-Ser79-ACC protein levels in HL-1 cardiomyocytes. Then, cells were exposed to AICAR stimulation for 24 h in the presence or absence of the AMPK inhibitor Compound C, and the mRNA levels of the three PPARs were analyzed by real-time RT-PCR. Treatment with AICAR induced gene expression of all three PPARs, but only the Ppara and Pparg regulation were dependent on AMPK. Next, we exposed HL-1 cells to high palmitate/high insulin (HP/HI conditions either in presence or in absence of AICAR, and we evaluated the expression of selected PPAR-targets genes. HP/HI induced insulin resistance and lipid storage was accompanied by increased Cd36, Acot1, and Ucp3 mRNA levels. AICAR treatment induced the expression of Acadvl and Glut4, which correlated to prevention of the HP/HI-induced intramyocellular lipid build-up, and attenuation of the HP/HI-induced impairment of glucose uptake. These data support the hypothesis that AICAR contributes to cardiac metabolic adaptation via regulation of transcriptional mechanisms.

A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity.

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Mateusz G Adamski

Full Text Available Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA permits the normalization of results between different batches and between different instruments--regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1 the achievement of absolute quantification and (2 a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

DAP1 high expression increases risk of lymph node metastases in squamous cell carcinoma of the oral cavity.

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Santos, M; Maia, L L; Silva, C V M; Peterle, G T; Mercante, A M C; Nunes, F D; Carvalho, M B; Tajara, E H; Louro, I D; Silva-Conforti, A M A

2015-09-08

Death-associated protein 1 (DAP1) is a member of the DAP family. Its expression is associated with cell growth and normal death of the neoplastic cells, regulated by the mammalian target of the rapamycin protein. Activated DAP1 negatively regulates autophagy, which has been associated with the development and progression of several diseases, such as cancer, and with prognosis and survival of diverse tumor types. Therefore, in this study we analyzed DAP1 expression in 54 oral squamous cell carcinoma tumor samples and in 20 non-tumoral margins by immunohistochemistry. The results showed that DAP1 is more frequently expressed in tumor tissues compared with marginal non-tumoral cells. Additionally, high DAP1 expression is associated with a 4-fold increase in the risk of lymph node metastases. Our results suggest that the DAP1 protein can be used as a potential marker of lymph node metastases predisposition, helping define the best therapy for each patient to minimize risk of developing metastases.

Expression and mechanism of high mobility group box protein-1 in retinal tissue of diabetic rats

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Shuang Jiang

2016-05-01

Full Text Available AIM:To investigate the expression and mechanism of high mobility group box protein-1(HMGB1in the retina of diabetic rats. METHODS:Sixty SD rats were randomly divided into diabetic group and control group. Diabetic rat model was produced by intraperitioneal injection of 1% STZ with 60mg/Kg weight. The rats in control group received intraperitioneal injection of normal saline with same dosage. After injection, the rats were sacrificed and eyeballs were enucleated for HE staining, the retina fluorescence angiography, TUNEL and Western Blot detection at 1, 2 and 4mo for the expressions of HMGB1 and NF-κB. RESULTS:Compared with the control group, the retinal cells disorder, cell densities decreases, microvasculars occlusion were founded with inner and outer nuclear layer thinning and ganglion cell apoptosis. The fluorescence angiography showed that peripheral capillaries became circuitous and vascular occlusion and non-perfusion area could be seen. The expressions of HMGB1 and NF-κB were higher than those of control with time dependence and they had significant positive correlations(PCONCLUSION:The expression of HMGB1 increases in diabetic rat retina, which may involve in the occurrence of diabetic retinopathy through the NF- κB pathway.

Nuclear Kaiso expression is associated with high grade and triple-negative invasive breast cancer.

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Jeroen F Vermeulen

Full Text Available Kaiso is a BTB/POZ transcription factor that is ubiquitously expressed in multiple cell types and functions as a transcriptional repressor and activator. Little is known about Kaiso expression and localization in breast cancer. Here, we have related pathological features and molecular subtypes to Kaiso expression in 477 cases of human invasive breast cancer. Nuclear Kaiso was predominantly found in invasive ductal carcinoma (IDC (p = 0.007, while cytoplasmic Kaiso expression was linked to invasive lobular carcinoma (ILC (p = 0.006. Although cytoplasmic Kaiso did not correlate to clinicopathological features, we found a significant correlation between nuclear Kaiso, high histological grade (p = 0.023, ERα negativity (p = 0.001, and the HER2-driven and basal/triple-negative breast cancers (p = 0.018. Interestingly, nuclear Kaiso was also abundant in BRCA1-associated breast cancer (p<0.001 and invasive breast cancer overexpressing EGFR (p = 0.019. We observed a correlation between nuclear Kaiso and membrane-localized E-cadherin and p120-catenin (p120 (p<0.01. In contrast, cytoplasmic p120 strongly correlated with loss of E-cadherin and low nuclear Kaiso (p = 0.005. We could confirm these findings in human ILC cells and cell lines derived from conditional mouse models of ILC. Moreover, we present functional data that substantiate a mechanism whereby E-cadherin controls p120-mediated relief of Kaiso-dependent gene repression. In conclusion, our data indicate that nuclear Kaiso is common in clinically aggressive ductal breast cancer, while cytoplasmic Kaiso and a p120-mediated relief of Kaiso-dependent transcriptional repression characterize ILC.

Contemporary and Traditional : the identity of the Swedish folk high school as expressed in its vocational orientation

OpenAIRE

Landström, Inger

2004-01-01

In this thesis the vocational orientation of the courses offered - as an expression of the folk high school's self-identity- is analysed. The point of departure was that what the folk high school do, is a way to show what it wants to be and, therefore, reflects what it is. Focus is on how the folk high school meets with contemporary tasks and demands in different areas of society. The research objects are, the folk high school as type of school (a total of 147 schools) and a sample of ten ind...

A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria

DEFF Research Database (Denmark)

Ferro, Roberto; Rennig, Maja; Hernández Rollán, Cristina

2018-01-01

with a long history in food fermentation. We have developed a synbio approach for increasing gene expression in two Gram-positive bacteria. First of all, the gene of interest was coupled to an antibiotic resistance gene to create a growth-based selection system. We then randomised the translation initiation...... region (TIR) preceding the gene of interest and selected clones that produced high protein titres, as judged by their ability to survive on high concentrations of antibiotic. Using this approach, we were able to significantly increase production of two industrially relevant proteins; sialidase in B....... subtilis and tyrosine ammonia lyase in L. lactis. Gram-positive bacteria are widely used to produce industrial enzymes. High titres are necessary to make the production economically feasible. The synbio approach presented here is a simple and inexpensive way to increase protein titres, which can be carried...

Effects of a free-choice high-fat high-sugar diet on brain PER2 and BMAL1 protein expression in mice.

Science.gov (United States)

Blancas-Velazquez, Aurea; la Fleur, Susanne E; Mendoza, Jorge

2017-10-01

The suprachiasmatic nucleus (SCN) times the daily rhythms of behavioral processes including feeding. Beyond the SCN, the hypothalamic arcuate nucleus (ARC), involved in feeding regulation and metabolism, and the epithalamic lateral habenula (LHb), implicated in reward processing, show circadian rhythmic activity. These brain oscillators are functionally coupled to coordinate the daily rhythm of food intake. In rats, a free choice high-fat high-sugar (fcHFHS) diet leads to a rapid increase of calorie intake and body weight gain. Interestingly, under a fcHFHS condition, rats ingest a similar amount of sugar during day time (rest phase) as during night time (active phase), but keep the rhythmic intake of regular chow-food. The out of phase between feeding patterns of regular (chow) and highly rewarding food (sugar) may involve alterations of brain circadian oscillators regulating feeding. Here, we report that the fcHFHS diet is a successful model to induce calorie intake, body weight gain and fat tissue accumulation in mice, extending its effectiveness as previously reported in rats. Moreover, we observed that whereas in the SCN the day-night difference in the PER2 clock protein expression was similar between chow-fed and fcHFHS-fed animals, in the LHb, this day-night difference was altered in fcHFHS-exposed animals compared to control chow mice. These findings confirm previous observations in rats showing disrupted daily patterns of feeding behavior under a fcHFHS diet exposure, and extend our insights on the effects of the diet on circadian gene expression in brain clocks. Copyright © 2017 Elsevier Ltd. All rights reserved.

Insight into small RNA abundance and expression in high- and low-temperature stress response using deep sequencing in Arabidopsis.

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Baev, Vesselin; Milev, Ivan; Naydenov, Mladen; Vachev, Tihomir; Apostolova, Elena; Mehterov, Nikolay; Gozmanva, Mariyana; Minkov, Georgi; Sablok, Gaurav; Yahubyan, Galina

2014-11-01

Small RNA profiling and assessing its dependence on changing environmental factors have expanded our understanding of the transcriptional and post-transcriptional regulation of plant stress responses. Insufficient data have been documented earlier to depict the profiling of small RNA classes in temperature-associated stress which has a wide implication for climate change biology. In the present study, we report a comparative assessment of the genome-wide profiling of small RNAs in Arabidopsis thaliana using two conditional responses, induced by high- and low-temperature. Genome-wide profiling of small RNAs revealed an abundance of 21 nt small RNAs at low temperature, while high temperature showed an abundance of 21 nt and 24 nt small RNAs. The two temperature treatments altered the expression of a specific subset of mature miRNAs and displayed differential expression of a number of miRNA isoforms (isomiRs). Comparative analysis demonstrated that a large number of protein-coding genes can give rise to differentially expressed small RNAs following temperature shifts. Low temperature caused accumulation of small RNAs, corresponding to the sense strand of a number of cold-responsive genes. In contrast, high temperature stimulated the production of small RNAs of both polarities from genes encoding functionally diverse proteins. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Proton-pumping rhodopsins are abundantly expressed by microbial eukaryotes in a high-Arctic fjord.

Science.gov (United States)

Vader, Anna; Laughinghouse, Haywood D; Griffiths, Colin; Jakobsen, Kjetill S; Gabrielsen, Tove M

2018-02-01

Proton-pumping rhodopsins provide an alternative pathway to photosynthesis by which solar energy can enter the marine food web. Rhodopsin genes are widely found in marine bacteria, also in the Arctic, and were recently reported from several eukaryotic lineages. So far, little is known about rhodopsin expression in Arctic eukaryotes. In this study, we used metatranscriptomics and 18S rDNA tag sequencing to examine the mid-summer function and composition of marine protists (size 0.45-10 µm) in the high-Arctic Billefjorden (Spitsbergen), especially focussing on the expression of microbial proton-pumping rhodopsins. Rhodopsin transcripts were highly abundant, at a level similar to that of genes involved in photosynthesis. Phylogenetic analyses placed the environmental rhodopsins within disparate eukaryotic lineages, including dinoflagellates, stramenopiles, haptophytes and cryptophytes. Sequence comparison indicated the presence of several functional types, including xanthorhodopsins and a eukaryotic clade of proteorhodopsin. Transcripts belonging to the proteorhodopsin clade were also abundant in published metatranscriptomes from other oceanic regions, suggesting a global distribution. The diversity and abundance of rhodopsins show that these light-driven proton pumps play an important role in Arctic microbial eukaryotes. Understanding this role is imperative to predicting the future of the Arctic marine ecosystem faced by a changing light climate due to diminishing sea-ice. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

OpenAIRE

Ezer, Daphne; Moignard, Victoria; G?ttgens, Berthold; Adryan, Boris

2016-01-01

Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete ...

High Expression of HULC Is Associated with Poor Prognosis in Osteosarcoma Patients.

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Vanessa Regina Maciel Uzan

Full Text Available Osteosarcoma (OS is the most common primary bone cancer in childhood. OS is an aggressive disease, and metastatic patients evolve with very poor clinical outcomes. Genetically, OSs are extremely complex tumors, and the related metastatic process is not well understood in terms of the biology of the disease. In this context, long non-coding RNAs (lncRNAs have emerged as an important class of gene expression regulators that play key roles in the invasion and metastasis of several human tumors. Here, we evaluated the expression of HULC, which is an lncRNA that is associated with the tumor metastatic process, and assessed its potential role as a prognostic marker in OS. HULC expression was evaluated in primary OS samples using real-time RT-PCR. HULC expression status was determined by receiver operating characteristic (ROC analysis, and its association with survival was assessed using the Kaplan-Meier method. The HULC expression level was not significantly associated with the clinicopathological characteristics of the OS patients. However, our data demonstrated that higher levels of expression of HULC were associated with lower survival rates in OS patients, both in terms of overall and event-free survival. Elevated HULC expression was associated with poor clinical outcomes among the OS patients, which suggests that HULC could be a potential prognostic biomarker in OS.

Influence of high glucose and advanced glycation end-products (ages) levels in human osteoblast-like cells gene expression.

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Miranda, Cristina; Giner, Mercè; Montoya, M José; Vázquez, M Angeles; Miranda, M José; Pérez-Cano, Ramón

2016-08-31

Type 2 diabetes mellitus (T2DM) is associated with an increased risk of osteoporotic fracture. Several factors have been identified as being potentially responsible for this risk, such as alterations in bone remodelling that may have been induced by changes in circulating glucose or/and by the presence of non-oxidative end products of glycosylation (AGEs). The aim of this study is to assess whether such variations generate a change in the gene expression related to the differentiation and osteoblast activity (OPG, RANKL, RUNX2, OSTERIX, and AGE receptor) in primary cultures of human osteoblast-like cells (hOB). We recruited 32 patients; 10 patients had osteoporotic hip fractures (OP group), 12 patients had osteoporotic hip fractures with T2DM (T2DM group), and 10 patients had hip osteoarthritis (OA group) with no osteoporotic fractures and no T2DM. The gene expression was analyzed in hOB cultures treated with physiological glucose concentration (4.5 mM) as control, high glucose (25 mM), and high glucose plus AGEs (2 μg/ml) for 24 h. The hOB cultures from patients with hip fractures presented slower proliferation. Additionally, the hOB cultures from the T2DM group were the most negatively affected with respect to RUNX2 and OSX gene expression when treated solely with high glucose or with high glucose plus AGEs. Moreover, high levels of glucose induced a major decrease in the RANKL/OPG ratio when comparing the OP and the T2DM groups to the OA group. Our data indicates an altered bone remodelling rate in the T2DM group, which may, at least partially, explain the reduced bone strength and increased incidence of non-traumatic fractures in diabetic patients.

Over-expression of β-catenin is associated with high grade of ...

African Journals Online (AJOL)

The aim of this study was to evaluate abnormalities of β-catenin protein expression, subcellular ... Patients and methods: Forty prostate cancer specimens, obtained from patients with different stages ... Conclusions: We concluded that changes in expression and cell distribution of β-catenin correlated ... HOW TO USE AJOL.

High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

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Amarjeet Kumar Singh

Full Text Available Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp, which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

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Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

2015-09-01

A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver. Copyright © 2015 Elsevier Inc. All rights reserved.

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

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

2014-04-25

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

About using of the high-speed modular computing structures in the systems of radiation express analysis

Energy Technology Data Exchange (ETDEWEB)

Ermalitski, F A

1996-12-31

In accordance with parallel processing conception the original method for definition of the stream intensity of Poisson group is proposed, which is able to use for radiation express analysis. Within the framework of this method the parallel measure-calculating procedure is created and the high-speed modular computing system is designed for its implementation. 5 refs.

Phenotypic and gene expression changes between low (glucose-responsive) and High (glucose non-responsive) MIN-6 beta cells

DEFF Research Database (Denmark)

O´Driscoll, L.; Gammell, p.; McKierman, E.

2006-01-01

The long-term potential to routinely use replacement beta cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous beta cell studies, by ourselves and other researchers, have indicated...... that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H......, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies...

[Genetic cloning and expression of hypoxia inducible factor 1 alpha in high altitude hypoxic adaptation species Tibetan antelope (Pantholops hodgsonii)].

Science.gov (United States)

Liu, Fang; Wuren, Tana; Ma, Lan; Yang, Ying-Zhong; Ge, Ri-Li

2011-12-25

In order to investigate the role of the hypoxia inducible factor 1 alpha (HIF-1α) in the adaptation mechanism to high altitude hypoxia, the cloning of the HIF-1α gene cDNA of Tibetan antelope (Pantholops hodgsonii), using RT-PCR and RACE, was applied, and the comparative analysis of the tissue-specific expressions of HIF-1α among Tibetan antelope, Tibetan sheep and plain sheep was performed using real-time PCR and Western blot. The sequence analysis indicated that the cDNA sequences acquired by cloning from the HIF-1α gene of Tibetan antelope comprised a 2 471-bp open reading frame (ORF) and a 1 911-bp 3'UTR. The similarity between its coding sequence, predicted amino acid sequence and HIF-1α of other mammals exceeded 87%, in which the similarity with cow was up to more than 98%, which showed that this sequence was the cDNA of HIF-1α of Tibetan antelope. The results of real-time PCR and Western blot showed that expressions of HIF-1α mRNA and protein appeared in Tibetan antelope's lung, cardiac muscle and skeletal muscle, with the highest expression in lung. HIF-1α mRNA and protein had obvious differential expression in these tissues. Further research showed that Tibetan antelope and Tibetan sheep possessed higher expressions of HIF-1α protein in the three tissues above-mentioned compared with plain sheep, and the expressions of HIF-1α mRNA and protein in Tibetan antelope's lung, cardiac muscle and skeletal muscle were higher than those of Tibetan sheep. It illustrates that the hypoxic HIF-1α-specific expression is one of the molecular bases of high altitude hypoxia adaptation in Tibetan antelope.

Survivin Expression as a Predictive Marker for Local Control in Patients With High-Risk T1 Bladder Cancer Treated With Transurethral Resection and Radiochemotherapy

International Nuclear Information System (INIS)

Weiss, Christian; Roemer, Felix von; Capalbo, Gianni; Ott, Oliver J.; Wittlinger, Michael; Krause, Steffen F.; Sauer, Rolf; Roedel, Claus; Roedel, Franz

2009-01-01

Purpose: The objectives of this study were to investigate the expression of survivin in tumor samples from patients with high-risk T1 bladder cancer and to correlate its expression with clinicopathologic features as well as clinical outcomes after initial transurethral resection (TURBT) followed by radiotherapy (RT) or radiochemotherapy (RCT). Methods and Materials: Survivin protein expression was evaluated by immunohistochemistry on tumor specimen (n = 48) from the initial TURBT, and was correlated with clinical and histopathologic characteristics as well as with 5-year rates of local failure, tumor progression, and death from urothelial cancer after primary bladder sparring treatment with RT/RCT. Results: Survivin was not expressed in normal bladder urothelium but was overexpressed in 67% of T1 tumors. No association between survivin expression and clinicopathologic factors (age, gender, grading, multifocality, associated carcinoma in situ) could be shown. With a median follow-up of 27 months (range, 3-140 months), elevated survivin expression was significantly associated with an increased probability of local failure after TURBT and RCT/RT (p = 0.003). There was also a clear trend toward a higher risk of tumor progression (p = 0.07) and lower disease-specific survival (p = 0.10). Conclusions: High survivin expression is a marker of tumor aggressiveness and may help to identify a subgroup of patients with T1 bladder cancer at a high risk for recurrence when treated with primary organ-sparing approaches such as TURBT and RCT.

Effect of berberine on the ratio of high-molecular weight adiponectin to total adiponectin and adiponectin receptors expressions in high-fat diet fed rats.

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Wu, Yue-Yue; Zha, Ying; Liu, Jun; Wang, Fang; Xu, Jiong; Chen, Zao-Ping; Ding, He-Yuan; Sheng, Li; Han, Xiao-Jie

2016-11-17

To assess the effects of berberine (BBR) on high-molecular weight (HMW) adiponectin and adiponectin receptors (adipoR1/adipoR2) expressions in high-fat (HF) diet fed rats. Forty Wistar male rats were randomly assigned into a normal diet fed group and three HF diet (fat for 45% calories) fed groups (n=10 for each group). All rats underwent 12 weeks of feeding. After 4 weeks feeding, rats in the two of three HF diet fed groups were treated with 150 mg·kg -1 ·day -1 BBR (HF+LBBR group) and 380 mg·kg -1 ·day -1 BBR (HF+HBBR group) by gavage once a day respectively for the next 8 weeks while the rats in other groups treated with vehicle (NF+Veh and HF+Veh). Body weight and food intake were observed and recorded on daily basis. At the end of 12 weeks, the blood, liver, epididymal fat tissues and quadriceps femoris muscles were collected. Fasting insulin, plasma fasting glucose, serum free fatty acid (FFA), total adiponectin and HMW adiponectin levels were measured by enzyme linked immunosorbent assay method. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed to determine the insulinsensitizing. Meanwhile the homeostasis model assessment (HOMA) method was used to determine insulin resistance (HOMA-IR). The expressions of adipoR1, adipoR2 and adenosine monophophate activated protein kinase (AMPK) phosphorylation level in skeletal muscle and liver tissue were detected by Western blot. Liver and kidney toxicity were evaluated during treatment. The body weight of rats in high- or low-dose BBR group reduced as well as HOMA-IR, FFA concentrations and fasting insulin levels decreased compared with HF+Veh group (Pinsulin resistance by increasing the expression of adiponectin receptors and the ratio of HMW to total adiponectin.

Effects of Persian leek (Allium ampeloprasum) on hepatic lipids and the expression of proinflammatory gene in hamsters fed a high-fat/ high-cholesterol diet.

Science.gov (United States)

Fatoorechi, Vahideh; Rismanchi, Marjan; Nasrollahzadeh, Javad

2016-01-01

Persian leek is one of the most widely used herbal foods among Iranians. In this study, effects of oral administration of Persian leek on plasma and liver lipids were examined in hamster. Male Syrian hamsters were randomly divided into three groups: control (standard diet), high fat control (high-fat/high-cholesterol diet), Persian leek (high-fat/high-cholesterol diet + 1% per weight of diet from dried powdered Persian leek) for 14 weeks. High fat diet increased plasma and liver lipids as compared to standard diet. Adding Persian leek to the high-fat/high-cholesterol diet resulted in no significant changes in the concentration of the plasma lipids or liver cholesterol. However, liver triglycerides (TG), plasma Alanine aminotransferase and gene expression of tumor necrosis factor- α were decreased in hamsters fed high-fat diet containing Persian leek as compared to high-fat diet only. Persian leek might be considered as a herbal food that can reduce liver TG accumulation induced by high fat diets.

Positive expression of LSD1 and negative expression of E-cadherin correlate with metastasis and poor prognosis of colon cancer.

Science.gov (United States)

Jie, Ding; Zhongmin, Zhang; Guoqing, Liao; Sheng, Liu; Yi, Zhang; Jing, Wen; Liang, Zeng

2013-06-01

The first identified lysine-specific demethylase, LSD1, plays an important role in the metastatic progression of several types of cancer. The aim of this study was to investigate LSD1, E-cadherin, and N-cadherin expression in colon cancer specimens and their clinical significance. The expression of LSD1, E-cadherin, and N-cadherin in colon cancer specimens was determined by immunohistochemistry, and the relationship between the expression of the respective molecules and clinicopathological characteristics was analyzed. The positive expression rates of LSD1, E-cadherin, and N-cadherin in colon cancer specimens were 66.7 % (72/108), 85.2 % (92/108), and 41.7 % (45/108), respectively. LSD1 was significantly more highly expressed in colon cancer specimens classified as high TNM stage lesions and with distant metastasis (P colon cancer specimens classified as high TNM stage lesions and with distant metastasis (P clinical and pathological characteristics (P > 0.05). Correlation analysis revealed that LSD1 expression was negatively correlated with E-cadherin expression (r s = -0.318, P = 0.001), but not evidently correlated with N-cadherin expression (r s = 0.182, P = 0.06). Colon cancer specimens with positive LSD1 expression and negative E-cadherin expression were correlated with significantly lower overall survival. LSD1 showed a significantly higher expression, in contrast to the significantly lower expression of E-cadherin, in colon cancer specimens classified as high TNM stage lesions and with distant metastasis. Positive expression of LSD1 and negative expression of E-cadherin may be predictors of a worse colon cancer prognosis.

High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

OpenAIRE

Bonvini, P; Zin, A; Alaggio, R; Pawel, B; Bisogno, G; Rosolen, A

2013-01-01

Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clin...

High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli

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Kuang-Ting Cheng

2018-03-01

Full Text Available P-113, which was originally derived from the human saliva protein histatin 5, is a histidine-rich antimicrobial peptide with the sequence AKRHHGYKRKFH. P-113 is currently undergoing phase II clinical trial as a pharmaceutical agent to fight against fungal infections in HIV patients with oral candidiasis. Previously, we developed a new procedure for the high-yield expression and purification of hG31P, an analogue and antagonist of human CXCL8. Moreover, we have successfully removed lipopolysaccharide (LPS, endotoxin associated with hG31P in the expression with Escherichia coli. In this paper, we have used hG31P as a novel fusion protein for the expression and purification of P-113. The purity of the expressed P-113 is more than 95% and the yield is 4 mg P-113 per liter of E. coli cell culture in Luria-Bertani (LB medium. The antimicrobial activity of the purified P-113 was tested. Furthermore, we used circular dichroism (CD and nuclear magnetic resonance (NMR spectroscopy to study the structural properties of P-113. Our results indicate that using hG31P as a fusion protein to obtain large quantities of P-113 is feasible and is easy to scale up for commercial production. An effective way of producing enough P-113 for future clinical studies is evident in this study.

Combination of Bcl-2 and MYC protein expression improves high-risk stratification in diffuse large B-cell lymphoma

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Wang J

2015-09-01

Full Text Available Jing Wang,* Min Zhou,* Jing-Yan Xu,* Bing Chen, Jian OuyangDepartment of Hematology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, People’s Republic of China*These authors contributed equally to this work and should be considered as cofirst authorsPurpose: To evaluate whether the addition of two biological markers (MYC and BCL-2 protein overexpression improves the stratification of high-risk patients with diffuse large B-cell lymphoma (DLBCL.Method: Seven risk factors were identified at diagnosis, and a maximum of 7 points were assigned to each patient. The patients were classified according to four risk groups: low (0–1, low-intermediate (2–3, high-intermediate (4, and high (5–7. Only high-risk patients with DLBCL were included in this analysis. We retrospectively examined 20 cases from 2008 to 2013 at the Nanjing Drum Tower Hospital.Results: The median expression of MYC protein was 60%, and 17 of 20 (65% evaluable cases overexpressed MYC. The median expression of BCL-2 protein was also 60%. Eighteen of 20 (90% evaluable cases showed BCL-2 overexpression. Additionally, 12 out of 20 cases (60% demonstrated coexpression of MYC and BCL-2 proteins. The percentages of overall survival and progression-free survival at the median follow-up time (36 months were 33.3%±16.1% and 16.9%±13.5%, respectively. By comparison, nine, four, and 20 patients were classified as high risk based on the International Prognostic Index (IPI, National Comprehensive Cancer Network(NCCN-IPI, and revised IPI criteria, respectively. According to the IPI and NCCN-IPI stratification, the risk groups demonstrated closely overlapping survival curves. In addition, four out of 20 cases were identified as low-intermediate risk according to the NCCN-IPI criteria.Conclusion: The addition of MYC and BCL-2 protein expression to the IPI could identify a subset of DLBCL patients with high-risk clinicopathological characteristics and

High expression of liver histone deacetylase 3 contributes to high-fat-diet-induced metabolic syndrome by suppressing the PPAR-γ and LXR-α-pathways in E3 rats.

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Li, Dongmin; Wang, Xuan; Ren, Wuchao; Ren, Juan; Lan, Xi; Wang, Feimiao; Li, Hongmin; Zhang, Fujun; Han, Yan; Song, Tianbao; Holmdahl, Rikard; Lu, Shemin

2011-09-15

In the previous experiment, we found that there was a different response between E3 rats and DA.1U rats to high-fat-diet-induced metabolic syndrome (HFD-MetS). The aim of this study was to explore the cause and molecular mechanism of the genetic difference in susceptibility to metabolic syndrome in E3 rats as compared with DA.1U rats. Firstly, a 12-week HFD-MetS model in E3 and DA.1U rats was carried out and assessed. Then, the expression of key insulin signaling molecules, metabolic nuclear receptors, metabolic key enzymes and histone deacetylases (Hdacs) was determined by different methods. Finally, the effects of overexpression and disruption of Hdac3 on metabolic nuclear receptors were analyzed in CBRH-7919 cells and primarily-hepatic cells from DA.1U and E3 rats. We found that E3 rats were susceptible, while DA.1U rats were resisted to HFD-MetS. The expression of liver X receptor α,β (LXR-α,β), farnesoid X receptor (FXR), peroxisome proliferator activated receptor γ (PPAR-γ) and cholesterol 7α-hydroxylase (CYP7A1) increased markedly in DA.1U rat liver, whereas they decreased significantly in E3 rats. The expression of Hdac3 increased by HFD treatment in both E3 and DA.1U rat livers, but the constitutive Hdac3 expression was lower in DA.IU rat liver than in E3 rat liver. Importantly, overexpression of Hdac3 could downregulate the expression of LXR-α, PPAR-γ and CYP7A1 in both CBRH-7919 cells and primarily cultured hepatic cells from DA.IU rats. On the contrary, disruption of Hdac3 by shRNA upregulated the expression of LXR-α, PPAR-γ and CYP7A1 in both CBRH-7919 cells and primarily cultured hepatic cells from E3 rats. The results suggested that a high constitutive expression of Hdac3 inhibiting the expression of PPAR-γ, LXR-α and CYP7A1 in liver contributes to HFD-MetS in E3 rats. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Increased COX-2 expression in epithelial and stromal cells of high mammographic density tissues and in a xenograft model of mammographic density.

Science.gov (United States)

Chew, G L; Huo, C W; Huang, D; Hill, P; Cawson, J; Frazer, H; Hopper, J L; Haviv, I; Henderson, M A; Britt, K; Thompson, E W

2015-08-01

Mammographic density (MD) adjusted for age and body mass index is one of the strongest known risk factors for breast cancer. Given the high attributable risk of MD for breast cancer, chemoprevention with a safe and available agent that reduces MD and breast cancer risk would be beneficial. Cox-2 has been implicated in MD-related breast cancer risk, and was increased in stromal cells in high MD tissues in one study. Our study assessed differential Cox-2 expression in epithelial and stromal cells in paired samples of high and low MD human breast tissue, and in a validated xenograft biochamber model of MD. We also examined the effects of endocrine treatment upon Cox-2 expression in high and low MD tissues in the MD xenograft model. Paired high and low MD human breast tissue samples were immunostained for Cox-2, then assessed for differential expression and staining intensity in epithelial and stromal cells. High and low MD human breast tissues were separately maintained in biochambers in mice treated with Tamoxifen, oestrogen or placebo implants, then assessed for percentage Cox-2 staining in epithelial and stromal cells. Percentage Cox-2 staining was greater for both epithelial (p = 0.01) and stromal cells (p tissues. In high MD biochamber tissues, percentage Cox-2 staining was greater in stromal cells of oestrogen-treated versus placebo-treated tissues (p = 0.05).

High Nrf2 expression in alveolar type I pneumocytes is associated with low recurrences in primary spontaneous pneumothorax.

Science.gov (United States)

Chen, Yu-Wen; Chiu, Wen-Chin; Chou, Shah-Hwa; Su, Yu-Han; Huang, Ying-Fong; Lee, Yen-Lung; Yuan, Shyng-Shiou F; Lee, Yi-Chen

2017-10-01

Recurrent primary spontaneous pneumothorax (PSP) is a troublesome problem and a major concern for the patients. This study examined whether nuclear factor erythroid 2-related factor 2 (Nrf2) expression in alveolar type I pneumocytes was associated with the clinical manifestations of PSP patients including disease recurrence. Eighty-eight PSP patients who were managed with needlescopic video-assisted thoracoscopic surgery (NVATS) were included in this study. Immunohistochemistry (IHC) was assessed to determine Nrf2 expression in resected lung tissues and the results were correlated with clinicopathological characteristics by the chi-square or the Fisher's exact test. The prognostic value of Nrf2 for overall recurrence was evaluated by univariate and multivariable Cox regression model. The expression of Nrf2 was observed in type I pneumocytes of lung tissues from PSP patients by IHC. We found that low Nrf2 expression in PSP patients, especially in young (age ≤ 20, p = 0.033) and body mass index (BMI) ≥18 kg/m 2 (p = 0.019) groups, was significantly correlated with PSP recurrence. In the univariate and multivariate analyses, high Nrf2 expression was a significant protective factor for overall recurrence in PSP patients (univariate: p = 0.026; multivariate: p = 0.004). The expression level of Nrf2 in alveolar type I pneumocytes was a potential factor involved in PSP recurrence. Our findings suggest that elevated Nrf2 expression in PSP patients may be a promising way for reducing PSP recurrence. Copyright © 2017. Published by Elsevier Taiwan.

Diets high in corn oil or extra-virgin olive oil differentially modify the gene expression profile of the mammary gland and influence experimental breast cancer susceptibility.

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Moral, Raquel; Escrich, Raquel; Solanas, Montserrat; Vela, Elena; Ruiz de Villa, M Carme; Escrich, Eduard

2016-06-01

Nutritional factors, especially dietary lipids, may have a role in the etiology of breast cancer. We aimed to analyze the effects of high-fat diets on the susceptibility of the mammary gland to experimental malignant transformation. Female Sprague-Dawley rats were fed a low-fat, high-corn-oil, or high-extra-virgin olive oil (EVOO) diet from weaning or from induction. Animals were induced with 7,12-dimethylbenz[a]anthracene at 53 days and euthanized at 36, 51, 100 and 246 days. Gene expression profiles of mammary glands were determined by microarrays. Further molecular analyses were performed by real-time PCR, TUNEL and immunohistochemistry. Carcinogenesis parameters were determined at 105 and 246 days. High-corn-oil diet increased body weight and mass when administered from weaning. The EVOO diet did not modify these parameters and increased the hepatic expression of UCP2, suggesting a decrease in intake/expenditure balance. Both diets differentially modified the gene expression profile of the mammary gland, especially after short dietary intervention. Corn oil down-regulated the expression of genes related to immune system and apoptosis, whereas EVOO modified the expression of metabolism genes. Further analysis suggested an increase in proliferation and lower apoptosis in the mammary glands by effect of the high-corn-oil diet, which may be one of the mechanisms of its clear stimulating effect on carcinogenesis. The high-corn-oil diet strongly stimulates mammary tumorigenesis in association with modifications in the expression profile and an increased proliferation/apoptosis balance of the mammary gland.

[Effect of resveratrol on expression of TLR4 and inflammatory factors in gingival epithelial cells under high glucose environment].

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Lv, Jia-Shu; Jiang, Xue-Wei; Zhang, Yan; Zhen, Lei

2017-02-01

Through a study of the molecular mechanism of the effect of resveratrol(RSV) on expression of TLR4 and inflammatory factors in gingival epithelial cells under high glucose environment, the therapeutic effect and molecular mechanism of resveratrol on periodontitis in patients with diabetes mellitus was investigated. Gingival epithelial cells were cultured in vitro; according to the way of action, the cultured cells were divided into control group, high glucose group(HG) and HG+RSV group. The mRNA expression of TLR4 was detected by PCR; The third generation of gingival epithelial cells were pre-treated with or without RSV for 24 h under high glucose conditions, and subsequently treated with LPS at 100 ng/mL for 2 h. ELISA was used to detect the secretion of IL-1 beta, IL-6, IL-8 and TNF- alpha; the activation of TLR4 downstream signaling molecules NF-κB p65, p38 MAPK, and STAT3 was determined by Western blot. SPSS17.0 software package was used for statistical analysis. RSV could reverse the increase of TLR4 level in gingival epithelial cells in high glucose medium.LPS markedly increased the expression and secretion of IL-1β, IL-6, IL-8, and TNF-α in GECs cultured in high glucose medium, which was partly blocked in the presence of RSV. Furthermore, Western blot results showed that RSV significantly suppressed the phosphorylation of TLR4 downstream factors NF-κB p65, p38MAPK, and STAT3. RSV reduces inflammatory cytokine secretion in gingival epithelial cells, through negative regulation of TLR4 signaling pathway.

Parallel Sequencing of Expressed Sequence Tags from Two Complementary DNA Libraries for High and Low Phosphorus Adaptation in Common Beans

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Matthew W. Blair

2011-11-01

Full Text Available Expressed sequence tags (ESTs have proven useful for gene discovery in many crops. In this work, our objective was to construct complementary DNA (cDNA libraries from root tissues of common beans ( L. grown under low and high P hydroponic conditions and to conduct EST sequencing and comparative analyses of the libraries. Expressed sequence tag analysis of 3648 clones identified 2372 unigenes, of which 1591 were annotated as known genes while a total of 465 unigenes were not associated with any known gene. Unigenes with hits were categorized according to biological processes, molecular function, and cellular compartmentalization. Given the young tissue used to make the root libraries, genes for catalytic activity and binding were highly expressed. Comparisons with previous root EST sequencing and between the two libraries made here resulted in a set of genes to study further for differential gene expression and adaptation to low P, such as a 14 kDa praline-rich protein, a metallopeptidase, tonoplast intrinsic protein, adenosine triphosphate (ATP citrate synthase, and cell proliferation genes expressed in the low P treated plants. Given that common beans are often grown on acid soils of the tropics and subtropics that are usually low in P these genes and the two parallel libraries will be useful for selection for better uptake of this essential macronutrient. The importance of EST generation for common bean root tissues under low P and other abiotic soil stresses is also discussed.

Identification of genes preferentially expressed by highly virulent piscine Streptococcus agalactiae upon interaction with macrophages.

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Chang-Ming Guo

Full Text Available Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood-brain barrier (BBB. The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB.

Identification of Genes Preferentially Expressed by Highly Virulent Piscine Streptococcus agalactiae upon Interaction with Macrophages

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Guo, Chang-Ming; Chen, Rong-Rong; Kalhoro, Dildar Hussain; Wang, Zhao-Fei; Liu, Guang-Jin; Lu, Cheng-Ping; Liu, Yong-Jie

2014-01-01

Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood–brain barrier (BBB). The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB. PMID:24498419

Bile salt receptor TGR5 is highly expressed in esophageal adenocarcinoma and precancerous lesions with significantly worse overall survival and gender differences

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Pang CH

2017-02-01

Full Text Available Chunhong Pang,1,2 Amy LaLonde,3 Tony E Godfrey,4 Jianwen Que,5,6 Jun Sun,7 Tong Tong Wu,3 Zhongren Zhou2 1Department of Pathology, China-Japan Friendship Hospital, 2Department of Pathology and Laboratory Medicine, 3Department of Biostatistics and Computational Biology, University of Rochester, Rochester, NY, 4Department of Surgery, Boston University Medical Center, Boston, MA, 5Center for Human Development, 6Division of Digestive and Liver Diseases, Columbia University, New York, NY, 7Division of Gastroenterology and Hepatology, University of Illinois College of Medicine, Chicago, IL, USA Abstract: Bile acid reflux in the esophagus plays an important role in the carcinogenesis of esophageal adenocarcinoma (EAC. The G-protein coupled bile acid receptor (TGR5 has been associated with the development of gastrointestinal cancer. However, little is known regarding the role of TGR5 in esophageal carcinoma and precancerous lesions. We analyzed genomic DNA from 116 EACs for copy number aberrations via Affymetrix SNP6.0 microarrays. The TGR5 gene locus was amplified in 12.7% (14/116 of the EACs. The TGR5 protein expression was also assessed using immunohistochemistry from tissue microarrays, including Barrett’s esophagus (BE, low- (LGD and high-grade dysplasia (HGD, columnar cell metaplasia (CM, squamous epithelium (SE, EAC and squamous cell carcinoma. The TGR5 protein was highly expressed in 71% of EAC (75/106, 100% of HGD (11/11, 72% of LGD (13/18, 66% of BE (23/35, 84% of CM (52/62, and 36% of SE (30/83. The patients with high expression of TGR5 exhibited significantly worse overall survival compared to the patients with nonhigh expression. TGR5 high expression was significantly increased in the males compared to the females in all cases with an odds ratio of 1.9 times. The vitamin D receptor (VDR was significantly correlated with TGR5 expression. Our findings indicated that TGR5 may play an important role in the development and prognosis of EAC

Suppression of endothelial t-PA expression by prolonged high laminar shear stress

International Nuclear Information System (INIS)

Ulfhammer, Erik; Carlstroem, Maria; Bergh, Niklas; Larsson, Pia; Karlsson, Lena; Jern, Sverker

2009-01-01

Primary hypertension is associated with an impaired capacity for acute release of endothelial tissue-type plasminogen activator (t-PA), which is an important local protective response to prevent thrombus extension. As hypertensive vascular remodeling potentially results in increased vascular wall shear stress, we investigated the impact of shear on regulation of t-PA. Cultured human endothelial cells were exposed to low (≤1.5 dyn/cm 2 ) or high (25 dyn/cm 2 ) laminar shear stress for up to 48 h in two different experimental models. Using real-time RT-PCR and ELISA, shear stress was observed to time and magnitude-dependently suppress t-PA transcript and protein secretion to approximately 30% of basal levels. Mechanistic experiments revealed reduced nuclear protein binding to the t-PA specific CRE element (EMSA) and an almost completely abrogated shear response with pharmacologic JNK inhibition. We conclude that prolonged high laminar shear stress suppresses endothelial t-PA expression and may therefore contribute to the enhanced risk of arterial thrombosis in hypertensive disease.

Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography.

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Kim, Soung Min; Jeong, Dasul; Kim, Min Keun; Lee, Sang Shin; Lee, Suk Keun

2017-08-08

Oral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods. The present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC). Two types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin. The increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction

The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon

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Fernández de Marco, María del Mar; Alejo, Alí; Hudson, Paul; Damon, Inger K.; Alcami, Antonio

2010-01-01

Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.—Fernández de Marco, M. M., Alejo, A., Hudson, P., Damon, I. K., Alcami, A. The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon. PMID:20019241

Expression of Mitochondrial Non-coding RNAs (ncRNAs) Is Modulated by High Risk Human Papillomavirus (HPV) Oncogenes*

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Villota, Claudio; Campos, América; Vidaurre, Soledad; Oliveira-Cruz, Luciana; Boccardo, Enrique; Burzio, Verónica A.; Varas, Manuel; Villegas, Jaime; Villa, Luisa L.; Valenzuela, Pablo D. T.; Socías, Miguel; Roberts, Sally; Burzio, Luis O.

2012-01-01

The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis. PMID:22539350

Expression of mitochondrial non-coding RNAs (ncRNAs) is modulated by high risk human papillomavirus (HPV) oncogenes.

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Villota, Claudio; Campos, América; Vidaurre, Soledad; Oliveira-Cruz, Luciana; Boccardo, Enrique; Burzio, Verónica A; Varas, Manuel; Villegas, Jaime; Villa, Luisa L; Valenzuela, Pablo D T; Socías, Miguel; Roberts, Sally; Burzio, Luis O

2012-06-15

The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

Effect of endurance and high intensity interval swimming training on cardiac structure and Hand2 expression of rats

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Mohammad Ali Gharaat

2017-12-01

Conclusion: In the situation of the present study, both of Endurance and Interval training regimens increase Hand2 gene expression, heart weight and left ventricle weight. Because of the shorter training time, high intensity interval training can be more beneficial to be executed.

High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

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Yao Nie

successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli.

Time perception and dynamics of facial expressions of emotions.

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Sophie L Fayolle

Full Text Available Two experiments were run to examine the effects of dynamic displays of facial expressions of emotions on time judgments. The participants were given a temporal bisection task with emotional facial expressions presented in a dynamic or a static display. Two emotional facial expressions and a neutral expression were tested and compared. Each of the emotional expressions had the same affective valence (unpleasant, but one was high-arousing (expressing anger and the other low-arousing (expressing sadness. Our results showed that time judgments are highly sensitive to movements in facial expressions and the emotions expressed. Indeed, longer perceived durations were found in response to the dynamic faces and the high-arousing emotional expressions compared to the static faces and low-arousing expressions. In addition, the facial movements amplified the effect of emotions on time perception. Dynamic facial expressions are thus interesting tools for examining variations in temporal judgments in different social contexts.

miR-375 is highly expressed and possibly transactivated by achaete-scute complex homolog 1 in small-cell lung cancer cells

Institute of Scientific and Technical Information of China (English)

Huijie Zhao; Lei Zhu; Yujuan Jin; Hongbin Ji; Xiumin Yan; Xueliang Zhu

2012-01-01

In this study,we identified five miRNAs highly expressed in the small-cell lung cancer (SCLC) cell line NCI-H209.Among them,the expression levels of miR-375 were dramatically elevated in all SCLC cell lines examined,coincident with the expression of the transcription factor achaete-scute complex homolog 1 (ASCL1).Moreover,miR-375 was upregulated and correlated with ASCL1 in the cell lines generated from mouse SCLC-like tumors as well.Dual-luciferase assays further showed that ASCL1 activated the expression of miR-375 by binding to the three E-box elements in the miR-375 promoter.These results imply a role of ASCL1 in SCLC via the upregulation of miR-375.

Press Law and Press Freedom for High School Publications: Court Cases and Related Decisions Discussing Free Expression Guarantees and Limitations for High School Students and Journalists. Contributions to the Study of Mass Media and Communications, Number 6.

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Ingelhart, Louis E.

According to this reference manual, the nation's courts offer public high school journalists the same constitutional protection for expression, free speech, and free press as adults. Part 1 traces the development of the First and Fourth Amendments and explains how these provisions apply to high school publications. Part 2 examines expression that…

MLH1 expression predicts the response to preoperative therapy and is associated with PD-L1 expression in esophageal cancer.

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Momose, Kota; Yamasaki, Makoto; Tanaka, Koji; Miyazaki, Yasuhiro; Makino, Tomoki; Takahashi, Tsuyoshi; Kurokawa, Yukinori; Nakajima, Kiyokazu; Takiguchi, Shuji; Mori, Masaki; Doki, Yuichiro

2017-07-01

Programmed death-ligand 1 (PD-1/PD-L1) inhibition therapy demonstrates potential as a future treatment for esophageal cancer. Mismatch repair status and tumor PD-L1 expression are the candidate predictive biomarkers for response to this therapy. In colorectal cancer, mismatch repair-deficient tumors are associated with improved survival, although they are not sensitive to 5-fluorouracil-based chemotherapy. The purpose of the present study was to investigate the association between MutL homolog 1 (MLH1) expression and prognosis, response to therapy and PD-L1 expression in esophageal cancer. Immunohistochemistry was used to evaluate MLH1 and PD-L1 expression in 251 resected specimens. Of the specimens, 30.3% exhibited low MLH1 expression and 15.5% exhibited high PD-L1 expression. The 5-year overall survival rates for the high MLH1 expression group and the low MLH1 expression group were 51.3 and 55.6%, respectively (P=0.5260). The responder ratio was 45.7% in the high MLH1 expression group and 15.4% in the low MLH1 expression group (PMLH1 expression group (P=0.0064) and 25.0% in the low MLH1 expression group. MLH1 expression may be a predictive factor for the response to preoperative therapy in esophageal cancer, and esophageal cancer with low MLH1 expression may have a mechanism that assists in promoting tumor PD-L1 expression.

Continuous high expression of XBP1 and GRP78 is important for the survival of bone marrow cells in CCl4-treated cirrhotic liver

International Nuclear Information System (INIS)

Marumoto, Yoshio; Terai, Shuji; Urata, Yohei; Matsumoto, Toshihiko; Mizunaga, Yuko; Yamamoto, Naoki; Jin, Haiyan; Fujisawa, Koichi; Murata, Tomoaki; Shinoda, Koh; Nishina, Hiroshi; Sakaida, Isao

2008-01-01

We have previously shown that infusion of bone marrow cells (BMC) improves CCl 4 -induced cirrhosis. However, it is unclear why the injected BMC are resistant to CCl 4 damage and subsequently improve the local microenvironment in damaged liver. To analyze the cellular phenomena involved in this process, we studied the damaged liver using electron microscopy. We found that CCl 4 caused rough endoplasmic reticula to swell in hepatocytes. To analyze the gene expression patterns associated with this process, we conducted PCR-selected suppressive subtractive hybridization. We found that expression levels of HSP84, HSP40, and XBP1 differed markedly between control liver and liver infused with BMC. Immunohistochemical staining revealed that expression levels of HSP84 and HSP40 were markedly higher in the early phase of differentiation immediately after BMC infusion, but decreased over time. XBP1 expression remained high during the late phase, and GRP78 expression increased with XBP1 activation. We also found that GFP-positive BMC expressed XBP1 and GRP78. XBP1 and GRP78 are associated with ER stress. Thus, continuous high XBP1 and GRP78 expression might be essential for the survival and proliferation of BMC in a CCl 4 -induced persistent liver damage environment

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease.

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Palacios, Florencia; Moreno, Pilar; Morande, Pablo; Abreu, Cecilia; Correa, Agustín; Porro, Valentina; Landoni, Ana Ines; Gabus, Raul; Giordano, Mirta; Dighiero, Guillermo; Pritsch, Otto; Oppezzo, Pablo

2010-06-03

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia

DEFF Research Database (Denmark)

Kristensen, Louise; Kielsgaard Kristensen, Thomas; Abildgaard, Niels

2015-01-01

included in the study. All three genes were observed to be independent markers of prognosis in CLL with high expression being associated with more aggressive disease. With this clear association with outcome in CLL, these genes thereby represent promising candidates for future functional studies...

CYP2W1 is highly expressed in adrenal glands and is positively associated with the response to mitotane in adrenocortical carcinoma.

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Cristina L Ronchi

Full Text Available Adrenocortical tumors comprise frequent adenomas (ACA and rare carcinomas (ACC. Human cytochrome P450 2W1 (CYP2W1 is highly expressed in some cancers holding the potential to activate certain drugs into tumor cytotoxins.To investigate the CYP2W1 expression in adrenal samples and its relationship with clinical outcome in ACC.CYP2W1 expression was investigated by qRT-PCR in 13 normal adrenal glands, 32 ACA, 25 ACC, and 9 different non-adrenal normal tissue samples and by immunohistochemistry in 352 specimens (23 normal adrenal glands, 33 ACA, 239 ACC, 67 non-adrenal normal or neoplastic samples.CYP2W1 mRNA expression was absent/low in normal non-adrenal tissues, but high in normal and neoplastic adrenal glands (all P<0.01 vs non-adrenal normal tissues. Accordingly, CYP2W1 immunoreactivity was absent/low (H-score 0-1 in 72% of non-adrenal normal tissues, but high (H-score 2-3 in 44% of non-adrenal cancers, in 65% of normal adrenal glands, in 62% of ACAs and in 50% of ACCs (all P<0.001 vs non-adrenal normal tissues, being significantly increased in steroid-secreting compared to non-secreting tumors. In ACC patients treated with mitotane only, high CYP2W1 immunoreactivity adjusted for ENSAT stage was associated with longer overall survival and time to progression (P<0.05 and P<0.01, respectively, and with a better response to therapy both as palliative (response/stable disease in 42% vs 6%, P<0.01 or adjuvant option (absence of disease recurrence in 69% vs 45%, P<0.01.CYP2W1 is highly expressed in both normal and neoplastic adrenal glands making it a promising tool for targeted therapy in ACC. Furthermore, CYP2W1 may represent a new predictive marker for the response to mitotane treatment.

Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

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Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

2016-06-01

Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris.

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Cardoso-Júnior, Carlos Antônio Mendes; Silva, Renato Pereira; Borges, Naiara Araújo; de Carvalho, Washington João; Walter, S Leal; Simões, Zilá Luz Paulino; Bitondi, Marcia Maria Gentile; Ueira Vieira, Carlos; Bonetti, Ana Maria; Hartfelder, Klaus

2017-08-01

In social insects, juvenile hormone (JH) has acquired novel functions related to caste determination and division of labor among workers, and this is best evidenced in the honey bee. In contrast to honey bees, stingless bees are a much more diverse group of highly eusocial bees, and the genus Melipona has long called special attention due to a proposed genetic mechanism of caste determination. Here, we examined methyl farnesoate epoxidase (mfe) gene expression, encoding an enzyme relevant for the final step in JH biosynthesis, and measured the hemolymph JH titers for all life cycle stages of Melipona scutellaris queens and workers. We confirmed that mfe is exclusively expressed in the corpora allata. The JH titer is high in the second larval instar, drops in the third, and rises again as the larvae enter metamorphosis. During the pupal stage, mfe expression is initialy elevated, but then gradually drops to low levels before adult emergence. No variation was, however, seen in the JH titer. In adult virgin queens, mfe expression and the JH titer are significantly elevated, possibly associated with their reproductive potential. For workers we found that JH titers are lower in foragers than in nurse bees, while mfe expression did not differ. Stingless bees are, thus, distinct from honey bee workers, suggesting that they have maintained the ancestral gonadotropic function for JH. Hence, the physiological circuitries underlying a highly eusocial life style may be variable, even within a monophyletic clade such as the corbiculate bees. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression of p53 protein in high-grade gastroenteropancreatic neuroendocrine carcinoma

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Ali, Abir Salwa; Grönberg, Malin; Federspiel, Birgitte

2017-01-01

of immunoreactive p53 protein in GEP-NEC. Materials and methods Tumor tissues from 124 GEP-NEC patients with locally advanced or metastatic disease treated with platinum-based chemotherapy were collected from Nordic centers and clinical data were obtained from the Nordic NEC register. Tumor proliferation rate...... In this cohort of GEP-NEC patients, p53 expression could not be correlated with clinical outcome. However, in patients with colorectal NECs, p53 expression was correlated with shorter PFS and OS. Further studies are needed to establish the role of immunoreactive p53 as a prognostic marker for GEP-NEC patients.......Background Gastroenteropancreatic neuroendocrine carcinomas (GEP-NECs) are aggressive, rapidly proliferating tumors. Therapeutic response to current chemotherapy regimens is usually short lasting. The aim of this study was to examine the expression and potential clinical importance...

Widespread ectopic expression of olfactory receptor genes

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Yanai Itai

2006-05-01

Full Text Available Abstract Background Olfactory receptors (ORs are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information.

Diet-dependent gene expression in honey bees: honey vs. sucrose or high fructose corn syrup.

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Wheeler, Marsha M; Robinson, Gene E

2014-07-17

Severe declines in honey bee populations have made it imperative to understand key factors impacting honey bee health. Of major concern is nutrition, as malnutrition in honey bees is associated with immune system impairment and increased pesticide susceptibility. Beekeepers often feed high fructose corn syrup (HFCS) or sucrose after harvesting honey or during periods of nectar dearth. We report that, relative to honey, chronic feeding of either of these two alternative carbohydrate sources elicited hundreds of differences in gene expression in the fat body, a peripheral nutrient-sensing tissue analogous to vertebrate liver and adipose tissues. These expression differences included genes involved in protein metabolism and oxidation-reduction, including some involved in tyrosine and phenylalanine metabolism. Differences between HFCS and sucrose diets were much more subtle and included a few genes involved in carbohydrate and lipid metabolism. Our results suggest that bees receive nutritional components from honey that are not provided by alternative food sources widely used in apiculture.

Significant differences in gene expression and key genetic components associated with high growth vigor in populus section tacamahaca as revealed by comparative transcriptome analysis

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Cheng, S.; Chen, M.; Li, Y.; Wang, J.; Sun, X.; Wang, J.

2017-01-01

To identify genetic components involved in high growth vigor in F1 Populus section Tacamahaca hybrid plants, high and low vigor plants showing significant differences in apical dominance during a rapid growth period were selected. Apical bud transcriptomes of high and low-growth-vigor hybrids and their parents were analyzed using high-throughput RNA sequencing on an Illumina HiSeq 2000 platform. A total of 5,542 genes were differently expressed between high growth vigor hybrid and its parents, the genes were significantly enriched in pathways related to processes such as photosynthesis, pyrimidine ribonucleotide biosynthetic processes and nucleoside metabolic processes. There were 1410 differentially expressed genes between high and low growth vigor hybrid, the genes were mainly involved in photosynthesis, chlorophyll biosynthetic process, carbon fixation in photosynthetic organisms, porphyrin and chlorophyll metabolism and nitrogen metabolism. Moreover, a k-core of a gene co-expression network analysis was performed to identify the potential functions of genes related to high growth vigor. The functions of 8 selected candidate genes were associated mainly with circadian rhythm, water transport, cellulose catabolic processes, sucrose biosynthesis, pyrimidine ribonucleotide biosynthesis, purine nucleotide biosynthesis, meristem maintenance, and carbohydrate metabolism. Our results may contribute to a better understanding of the molecular basis of high growth vigor in hybrids and its regulation. (author)

Effects of Curcuma comosa on the expression of atherosclerosis-related cytokine genes in rabbits fed a high-cholesterol diet.

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Charoenwanthanang, Puttavee; Lawanprasert, Somsong; Phivthong-Ngam, Laddawal; Piyachaturawat, Pawinee; Sanvarinda, Yupin; Porntadavity, Sureerut

2011-04-12

Curcuma comosa has been known to have potential use in cardiovascular diseases, but its immunoregulatory role in atherosclerosis development and liver toxicity has not been well studied. We therefore investigated the effects of Curcuma comosa on the expression of atherosclerosis-related cytokine genes in rabbits fed a high-cholesterol diet. Twelve male New Zealand White rabbits were treated with 1.0% cholesterol for one month and were subsequently treated with 0.5% cholesterol either alone, or in combination with 5mg/kg/day of simvastatin or with 400mg/kg/day of Curcuma comosa powder for three months. The expression of IL-1, MCP-1, TNF-α, IL-10, and TGF-β in the isolated abdominal aorta and liver were determined by real-time RT-PCR. Liver toxicity was determined by hepatic enzyme activity. Curcuma comosa significantly decreased the expression of pro-inflammatory cytokines, leading to a stronger reduction in IL-1, MCP-1, and TNF-α expression compared to that was suppressed by simvastatin treatment. However, neither Curcuma comosa nor simvastatin affected the expression of anti-inflammation cytokines. In the liver, Curcuma comosa insignificantly decreased the expression of pro-inflammatory cytokines and significantly increased the expression of the anti-inflammatory cytokine IL-10 without altering the activity of hepatic enzymes. In contrast, simvastatin significantly increased the MCP-1 and TNF-α expressions and serum ALT level, without affecting the expression of anti-inflammatory cytokines. In this study, we demonstrated that Curcuma comosa exerts anti-inflammatory activity in the aorta and liver without causing liver toxicity, indicating that Curcuma comosa is a potential candidate as an alternative agent in cardiovascular disease therapy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Regulation of the Na+/K+-ATPase Ena1 Expression by Calcineurin/Crz1 under High pH Stress: A Quantitative Study.

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Silvia Petrezsélyová

Full Text Available Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase.

Endothelial lipase is highly expressed in macrophages in advanced human atherosclerotic lesions

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Bartels, Emil D; Nielsen, John E; Lindegaard, Marie Louise Skakkebæk

2007-01-01

Endothelial lipase (EL) is expressed in endothelial cells, and affects plasma lipoprotein metabolism by hydrolyzing phospholipids in HDL. To determine the cellular expression of EL mRNA and protein in human atherosclerotic lesions, we performed in situ hybridization and immunohistochemical studies...

High expression of PTBP1 promote invasion of colorectal cancer by alternative splicing of cortactin.

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Wang, Zhi-Na; Liu, Dan; Yin, Bin; Ju, Wen-Yi; Qiu, Hui-Zhong; Xiao, Yi; Chen, Yuan-Jia; Peng, Xiao-Zhong; Lu, Chong-Mei

2017-05-30

Polypyrimidine tract-binding protein 1 (PTBP1) involving in almost all steps of mRNA regulation including alternative splicing metabolism during tumorigenesis due to its RNA-binding activity. Initially, we found that high expressed PTBP1 and poor prognosis was interrelated in colorectal cancer (CRC) patients with stages II and III CRC, which widely different in prognosis and treatment, by immunohistochemistry. PTBP1 was also upregulated in colon cancer cell lines. In our study, knockdown of PTBP1 by siRNA transfection decreased cell proliferation and invasion in vitro. Denovirus shRNA knockdown of PTBP1 inhibited colorectal cancer growth in vivo. Furthermore, PTBP1 regulates alternative splicing of many target genes involving in tumorgenesis in colon cancer cells. We confirmed that the splicing of cortactin exon 11 which was only contained in cortactin isoform-a, as a PTBP1 target. Knockdown of PTBP1 decreased the expression of cortactin isoform-a by exclusion of exon 11. Also the mRNA levels of PTBP1 and cortactin isoform-a were cooperatively expressed in colorectal cancer tissues. Knocking down cortactin isoform-a significantly decreased cell migration and invasion in colorectal cancer cells. Overexpression of cortactin isoform-a could rescue PTBP1-knockdown effect of cell motility. In summary the study revealed that PTBP1 facilitates colorectal cancer migration and invasion activities by inclusion of cortactin exon 11.

Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

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Nakamura, Tsuyoshi; Omasa, Takeshi

2015-09-01

Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Differential Gene Expression and Concentration-Response Modeling Workflow for High-Throughput Transcriptomic (HTTr) Data: Results From MCF7 Cells

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Increasing efficiency and declining cost of generating whole transcriptome profiles has made high-throughput transcriptomics a practical option for chemical bioactivity screening. The resulting data output provides information on the expression of thousands of genes and is amenab...

Gene design, cloning and protein-expression methods for high-value targets at the Seattle Structural Genomics Center for Infectious Disease

International Nuclear Information System (INIS)

Raymond, Amy; Haffner, Taryn; Ng, Nathan; Lorimer, Don; Staker, Bart; Stewart, Lance

2011-01-01

An overview of one salvage strategy for high-value SSGCID targets is given. Any structural genomics endeavor, particularly ambitious ones such as the NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) and Center for Structural Genomics of Infectious Disease (CSGID), face technical challenges at all points of the production pipeline. One salvage strategy employed by SSGCID is combined gene engineering and structure-guided construct design to overcome challenges at the levels of protein expression and protein crystallization. Multiple constructs of each target are cloned in parallel using Polymerase Incomplete Primer Extension cloning and small-scale expressions of these are rapidly analyzed by capillary electrophoresis. Using the methods reported here, which have proven particularly useful for high-value targets, otherwise intractable targets can be resolved

Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2

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Sinilnikova Olga

2011-05-01

Full Text Available Abstract Background The gene CHEK2 encodes a checkpoint kinase playing a key role in the DNA damage pathway. Though CHEK2 has been identified as an intermediate breast cancer susceptibility gene, only a small proportion of high-risk families have been explained by genetic variants located in its coding region. Alteration in gene expression regulation provides a potential mechanism for generating disease susceptibility. The detection of differential allelic expression (DAE represents a sensitive assay to direct the search for a functional sequence variant within the transcriptional regulatory elements of a candidate gene. We aimed to assess whether CHEK2 was subject to DAE in lymphoblastoid cell lines (LCLs from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified. Methods We implemented an assay based on high-resolution melting (HRM curve analysis and developed an analysis tool for DAE assessment. Results We observed allelic expression imbalance in 4 of the 41 LCLs examined. All four were carriers of the truncating mutation 1100delC. We confirmed previous findings that this mutation induces non-sense mediated mRNA decay. In our series, we ruled out the possibility of a functional sequence variant located in the promoter region or in a regulatory element of CHEK2 that would lead to DAE in the transcriptional regulatory milieu of freely proliferating LCLs. Conclusions Our results support that HRM is a sensitive and accurate method for DAE assessment. This approach would be of great interest for high-throughput mutation screening projects aiming to identify genes carrying functional regulatory polymorphisms.

Hepatic expression of inflammatory genes and microRNAs in pigs with high “cholesteryl ester transfer protein” (CETP) activity

DEFF Research Database (Denmark)

Cirera, Susanna; Tørsleff, Benedicte C Juul; Ritz, Christian

2016-01-01

levels (designated as CETP-high and CETP-low, respectively). Furthermore, breed and gender differences were also investigated. We found significant difference (P hepatic expression levels of several mRNAs and microRNAs between the CETP-high and -low groups (C5, IL1RN, IL18, and miR-223-5p......) promoting the redistribution of cholesteryl esters, triglycerides, and phospholipids between plasma proteins. Moreover, obesity and ORD are often linked with chronic low-grade inflammation leading to insulin resistance and endothelial and microvascular dysfunctions. The aim of this study was to detect...... differences in the hepatic expression of genes involved in low-grade inflammation and of obesity- and cholesterol-related microRNAs in two mixed breed populations of pigs (Yorkshire-Göttingen minipig, YM and Duroc-Göttingen minipig, DM) including males and females, with extreme phenotypes for CETP activity...

Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

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Aznar-Moreno, Jose A; Venegas Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Mullen, Robert; Gidda, Satinder K; Salas, Joaquín J

2014-03-01

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis. © 2013 Scandinavian Plant Physiology Society.

Apolipoprotein E expression and behavioral toxicity of high charge, high energy (HZE) particle radiation

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Higuchi, Yoshinori; Nelson, Gregory A.; Vazquez, Marcelo; Laskowitz, Daniel T.; Slater, James M.; Pearlstein, Robert D.

2002-01-01

Apolipoprotein E (apoE) is a lipid binding protein that plays an important role in tissue repair following brain injury. In the present studies, we have investigated whether apoE affects the behavioral toxicity of high charge, high energy (HZE) particle radiation. METHODS: Sixteen male apoE knockout (KO) mice and sixteen genetically matched wild-type (WT) C57BL mice were used in this experiment. Half of the KO and half of the WT animals were irradiated with 600 MeV/amu iron particles (2 Gy whole body). The effect of irradiation on motor coordination and stamina (Rotarod test), exploratory behavior (open field test), and spatial working and reference memory (Morris water maze) was assessed. ROTAROD TEST: Performance was adversely affected by radiation exposure in both KO and WT groups at 30 d after irradiation. By 60 d after radiation, the radiation effect was lost in WT, but still apparent in irradiated KO mice. OPEN FIELD TEST: Radiation reduced open field exploratory activity 14, 28, 56, 84, and 168 d after irradiation of KO mice, but had no effect on WT mice. MORRIS WATER MAZE: Radiation adversely affected spatial working memory in the KO mice, but had no discernible effect in the WT mice as assessed 180 d after irradiation. In contrast, irradiated WT mice showed marked impairment of spatial reference memory in comparison to non-irradiated mice, while no effect of radiation was observed in KO mice. CONCLUSIONS: These studies show that apoE expression influences the behavioral toxicity of HZE particle radiation and suggest that apoE plays a role in the repair/recovery from radiation injury of the CNS. ApoE deficiency may exacerbate the previously reported effects of HZE particle radiation in accelerating the brain aging process.

Apolipoprotein E expression and behavioral toxicity of high charge, high energy (HZE) particle radiation

International Nuclear Information System (INIS)

Higuchi, Yoshinori; Nelson, G.A.; Slater, J.M.; Pearlstein, R.D.; Laskowitz, D.T.

2002-01-01

Apolipoprotein E (apoE) is a lipid binding protein that plays an important role in tissue repair following brain injury. In the present studies, we have investigated whether apoE affects the behavioral toxicity of high charge, high energy (HZE) particle radiation. Sixteen male apoE knockout (KO) mice and sixteen genetically matched wild-type (WT) C57BL mice were used in this experiment. Half of the KO and half of the WT animals were irradiated with 600 MeV/amu iron particles (2 Gy whole body). The effect of irradiation on motor coordination and stamina (Rotarod test), exploratory behavior (open field test), and spatial working and reference memory (Morris water maze) was assessed. Rotarod test: Performance was adversely affected by radiation exposure in both KO and WT groups at 30 d after irradiation. By 60 d after radiation, the radiation effect was lost in WT, but still apparent in irradiated KO mice. Open field test: Radiation reduced open field exploratory activity 14, 28, 56, 84, and 168 d after irradiation of KO mice, but had no effect on WT mice. Morris water maze: Radiation adversely affected spatial working memory in the KO mice, but had no discernible effect in the WT mice as assessed 180 d after irradiation. In contrast, irradiated WT mice showed marked impairment of spatial reference memory in comparison to non-irradiated mice, while no effect of radiation was observed in KO mice. These studies show that apoE expression influences the behavioral toxicity of HZE particle radiation and suggest that apoE plays a role in the repair/recovery from radiation injury of the central nervous system (CNS). ApoE deficiency may exacerbate the previously reported effects of HZE particle radiation in accelerating the brain aging process. (author)

Apolipoprotein E expression and behavioral toxicity of high charge, high energy (HZE) particle radiation

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Higuchi, Yoshinori; Nelson, G.A.; Slater, J.M.; Pearlstein, R.D. [Loma Linda Univ., CA (United States). Medical Center; Vazquez, M. [Brookhaven National Lab., Upton, NY (United States); Laskowitz, D.T. [Duke Univ., Durham, NC (United States). Medical Center

2002-12-01

Apolipoprotein E (apoE) is a lipid binding protein that plays an important role in tissue repair following brain injury. In the present studies, we have investigated whether apoE affects the behavioral toxicity of high charge, high energy (HZE) particle radiation. Sixteen male apoE knockout (KO) mice and sixteen genetically matched wild-type (WT) C57BL mice were used in this experiment. Half of the KO and half of the WT animals were irradiated with 600 MeV/amu iron particles (2 Gy whole body). The effect of irradiation on motor coordination and stamina (Rotarod test), exploratory behavior (open field test), and spatial working and reference memory (Morris water maze) was assessed. Rotarod test: Performance was adversely affected by radiation exposure in both KO and WT groups at 30 d after irradiation. By 60 d after radiation, the radiation effect was lost in WT, but still apparent in irradiated KO mice. Open field test: Radiation reduced open field exploratory activity 14, 28, 56, 84, and 168 d after irradiation of KO mice, but had no effect on WT mice. Morris water maze: Radiation adversely affected spatial working memory in the KO mice, but had no discernible effect in the WT mice as assessed 180 d after irradiation. In contrast, irradiated WT mice showed marked impairment of spatial reference memory in comparison to non-irradiated mice, while no effect of radiation was observed in KO mice. These studies show that apoE expression influences the behavioral toxicity of HZE particle radiation and suggest that apoE plays a role in the repair/recovery from radiation injury of the central nervous system (CNS). ApoE deficiency may exacerbate the previously reported effects of HZE particle radiation in accelerating the brain aging process. (author)

Macroscopic Expressions of Molecular Adiabatic Compressibility of Methyl and Ethyl Caprate under High Pressure and High Temperature

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Fuxi Shi

2014-01-01

Full Text Available The molecular compressibility, which is a macroscopic quantity to reveal the microcompressibility by additivity of molecular constitutions, is considered as a fixed value for specific organic liquids. In this study, we introduced two calculated expressions of molecular adiabatic compressibility to demonstrate its pressure and temperature dependency. The first one was developed from Wada’s constant expression based on experimental data of density and sound velocity. Secondly, by introducing the 2D fitting expressions and their partial derivative of pressure and temperature, molecular compressibility dependency was analyzed further, and a 3D fitting expression was obtained from the calculated data of the first one. The third was derived with introducing the pressure and temperature correction factors based on analogy to Lennard-Jones potential function and energy equipartition theorem. In wide range of temperatures (293

Combination of isocitrate dehydrogenase 1 (IDH1) mutation and podoplanin expression in brain tumors identifies patients at high or low risk of venous thromboembolism.

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Mir Seyed Nazari, Pegah; Riedl, Julia; Preusser, Matthias; Posch, Florian; Thaler, Johannes; Marosi, Christine; Birner, Peter; Ricken, Gerda; Hainfellner, Johannes A; Pabinger, Ingrid; Ay, Cihan

2018-04-19

Venous thromboembolism (VTE) is a frequent complication in primary brain tumor patients. Independent studies revealed that podoplanin expression in brain tumors is associated with increased VTE risk, while the isocitrate dehydrogenase 1 (IDH1) mutation is associated with very low VTE risk. To investigate the interrelation between intratumoral podoplanin expression and IDH1 mutation, and their mutual impact on VTE development. In a prospective cohort study, intratumoral IDH1 R132H mutation and podoplanin were determined in brain tumor specimens (mainly glioma) by immunohistochemistry. Primary endpoint of the study was symptomatic VTE during a 2-year follow-up. All brain tumors that expressed podoplanin to a medium-high extent showed also an IDH1 wildtype status. A score based on IDH1 status and podoplanin expression levels allowed predicting risk of VTE. Patients with wildtype IDH1 brain tumors and high podoplanin expression had a significantly increased VTE risk compared to those with mutant IDH1 tumors and no podoplanin expression (6-month risk 18.2% vs. 0%). IDH1 mutation and podoplanin overexpression seem to be exclusive. While brain tumor patients with IDH1 mutation are at very low VTE risk, the risk of VTE in patients with IDH1 wildtype tumors is strongly linked to podoplanin expression levels. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Different gene-expression profiles for the poorly differentiated carcinoma and the highly differentiated papillary adenocarcinoma in mammary glands support distinct metabolic pathways

International Nuclear Information System (INIS)

Eilon, Tali; Barash, Itamar

2008-01-01

Deregulation of Stat5 in the mammary gland of transgenic mice causes tumorigenesis. Poorly differentiated carcinoma and highly differentiated papillary adenocarcinoma tumors evolve. To distinguish the genes and elucidate the cellular processes and metabolic pathways utilized to preserve these phenotypes, gene-expression profiles were analyzed. Mammary tumors were excised from transgenic mice carrying a constitutively active variant of Stat5, or a Stat5 variant lacking s transactivation domain. These tumors displayed either the carcinoma or the papillary adenocarcinoma phenotypes. cRNAs, prepared from each tumor were hybridized to an Affymetrix GeneChip ® Mouse Genome 430A 2.0 array. Gene-ontology analysis, hierarchical clustering and biological-pathway analysis were performed to distinct the two types of tumors. Histopathology and immunofluorescence staining complemented the comparison between the tumor phenotypes. The nucleus-cytoskeleton-plasma membrane axis is a major target for differential gene expression between phenotypes. In the carcinoma, stronger expression of genes coding for specific integrins, cytoskeletal proteins and calcium-binding proteins highlight cell-adhesion and motility features of the tumor cells. This is supported by the higher expression of genes involved in O-glycan synthesis, TGF-β, activin, their receptors and Smad3, as well as the Notch ligands and members of the γ-secretase complex that enable Notch nuclear localization. The Wnt pathway was also a target for differential gene expression. Higher expression of genes encoding the degradation complex of the canonical pathway and limited TCF expression in the papillary adenocarcinoma result in membranal accumulation of β-catenin, in contrast to its nuclear translocation in the carcinoma. Genes involved in cell-cycle arrest at G1 and response to DNA damage were more highly expressed in the papillary adenocarcinomas, as opposed to favored G2/M regulation in the carcinoma tumors. At least

High yield of recombinant human Apolipoprotein A-I expressed in Pichia pastoris by using mixed-mode chromatography.

Science.gov (United States)

Narasimhan Janakiraman, Vignesh; Noubhani, Abdelmajid; Venkataraman, Krishnan; Vijayalakshmi, Mookambeswaran; Santarelli, Xavier

2016-01-01

A vast majority of the cardioprotective properties exhibited by High-Density Lipoprotein (HDL) is mediated by its major protein component Apolipoprotein A-I (ApoA1). In order to develop a simplified bioprocess for producing recombinant human Apolipoprotein A-I (rhApoA1) in its near-native form, rhApoA1was expressed without the use of an affinity tag in view of its potential therapeutic applications. Expressed in Pichia pastoris at expression levels of 58.2 mg ApoA1 per litre of culture in a reproducible manner, the target protein was purified by mixed-mode chromatography using Capto™ MMC ligand with a purity and recovery of 84% and 68%, respectively. ApoA1 purification was scaled up to Mixed-mode Expanded Bed Adsorption chromatography to establish an 'on-line' process for the efficient capture of rhApoA1 directly from the P. pastoris expression broth. A polishing step using anion exchange chromatography enabled the recovery of ApoA1 up to 96% purity. Purified ApoA1 was identified and verified by RPLC-ESI-Q-TOF mass spectrometry. This two-step process would reduce processing times and therefore costs in comparison to the twelve-step procedure currently used for recovering rhApoA1 from P. pastoris. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Change of SPARC expression after chemotherapy in gastric cancer

International Nuclear Information System (INIS)

Gao, Yong-Yin; Han, Ru-Bing; Wang, Xia; Ge, Shao-Hua; Li, Hong-Li; Deng, Ting; Liu, Rui; Bai, Ming; Zhou, Li-Kun; Zhang, Xin-Yuan; Ba, Yi; Huang, Ding-Zhi

2015-01-01

The expression of tumor biomarkers may change after chemotherapy. However, whether secreted protein acidic and rich in cysteine (SPARC) expression changes after chemotherapy in gastric cancer (GC) is unclear. This study investigated the influence of chemotherapy on SPARC expression in GC. Immunohistochemistry was used to analyze SPARC expression in 132 GC cases (including 54 cases with preoperative chemotherapy and 78 cases without preoperative chemotherapy). SPARC expression of postoperative specimens with and without preoperative chemotherapy was assessed to analyze the influence of chemotherapy on SPARC expression. SPARC was highly expressed in GC compared with the desmoplastic stroma surrounding tumor cells and noncancerous tissues. High SPARC expression was correlated with invasion depth, lymph node, and TNM stage. After chemotherapy, a lower proportion of high SPARC expression was observed in patients with preoperative chemotherapy than in the controls. For 54 patients with preoperative chemotherapy, gross type, histology, depth of invasion, lymph node, TNM stage, and SPARC expression were related to overall survival. Further multivariate analysis showed that lymph node, histology, and SPARC expression after chemotherapy were independent prognostic factors. SPARC expression may change after chemotherapy in GC. SPARC expression should be reassessed for patients with GC after chemotherapy

Myocardial Lipid Profiling During Time Course of High Fat Diet and its Relationship to the Expression of Fatty Acid Transporters

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Ewa Harasim

2015-09-01

Full Text Available Background/Aims: It is well documented that increased fatty acids (FA supply causes lipid accumulation and insulin resistance in skeletal muscles. Whether the same mechanism is present in the heart is still unclear. Therefore, the goal of our study was to determine the content of specific myocardial lipid fractions during feeding rats a high fat diet (HFD for 5 weeks. Moreover, the relation between changes in myocardial lipid content, whole body insulin resistance and the expression of fatty acid transporters in each week of HFD was established. Methods: Gas liquid chromatography and high performance liquid chromatography were used to determine the content of lipid fractions in the left ventricle. Expression of selected proteins was estimated by Western blot technique. All measurements were made after each week of HFD. Results: As expected, lipid profile in myocardium was altered by HFD in different weeks of the study with the most intense changes in triacylglycerols, long chain fatty acid-CoA and ceramide. Furthermore, there was a significant elevation of plasmalemmal (the 4th and the 5th week and mitochondrial expression (from the 3rd to the 5th week of fatty acid translocase. Conclusion: High fat diet affects myocardial lipid profile in each week of its duration and causes alternations in FA metabolism in cardiomyocytes.

Effect of Qingguang'an II on expression of PAX6, Ngn1 and Ngn2 mRNA of rats with chronic high intraocular pressure

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Ya-Sha Zhou

2017-09-01

Full Text Available AIM: To remark the effect of Qingguang'an II on expression of PAX6, Ngn1, and Ngn2 mRNA of rats with chronic high intraocular pressure. METHODS: Totally 40 male SD rats were randomly divided into 6 groups, that was: A: blank group, B: model group, C: Qingguang'an II low dose group, D: Qingguang'an II moderate dose group, E: Qingguang'an II high dose group, F: Yimaikang disket group. B, C, D, E, F groups of experimental rats were established the model of chronic high intraocular pressure(IOPby cauterizing of superficial scleral vein. Animal model was established successfully by using monitoring IOP consistently keep above 25mmHg for 8wk as cut-off criterion. Tissues of Eyes were obtained after intragastric administration for 2wk and 4wk. The expressions of PAX6, Ngn1, and Ngn2 mRNA were investigated by Real-time PCR. RESULTS: At the time-point of 2wk, PAX6, Ngn1, and Ngn2 mRNA in group B were statistically expressed in lower level comparing with other groups(PPPP>0.05. CONCLUSION: In summar, Qingguang'an II and Yimaikang disket can remarkably increase the expressions of PAX6, Ngn1, and Ngn2, which suggest protecting the optic nerve of rats caused by chronic high IOP. What's more, this study indicated that, in the protection of optic nerve of rats with chronic high IOP, the high dose of Qingguang'an II at the time-point of 4wk was the better choice.

High-level Gpr56 expression is dispensable for the maintenance and function of hematopoietic stem and progenitor cells in mice

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Tata Nageswara Rao

2015-05-01

Full Text Available Blood formation by hematopoietic stem cells (HSCs is regulated by a still incompletely defined network of general and HSC-specific regulators. In this study, we analyzed the role of G-protein coupled receptor 56 (Gpr56 as a candidate HSC regulator based on its differential expression in quiescent relative to proliferating HSCs and its common targeting by core HSC regulators. Detailed expression analysis revealed that Gpr56 is abundantly expressed by HSPCs during definitive hematopoiesis in the embryo and in the adult bone marrow, but its levels are reduced substantially as HSPCs differentiate. However, despite enriched expression in HSPCs, Gpr56-deficiency did not impair HSPC maintenance or function during steady-state or myeloablative stress-induced hematopoiesis. Gpr56-deficient HSCs also responded normally to physiological and pharmacological mobilization signals, despite the reported role of this GPCR as a regulator of cell adhesion and migration in neuronal cells. Moreover, Gpr56-deficient bone marrow engrafted with equivalent efficiency as wild-type HSCs in primary recipients; however, their reconstituting ability was reduced when subjected to serial transplantation. These data indicate that although GPR56 is abundantly and selectively expressed by primitive HSPCs, its high level expression is largely dispensable for steady-state and regenerative hematopoiesis.

Early adaptive response of the retina to a pro-diabetogenic diet: Impairment of cone response and gene expression changes in high-fructose fed rats.

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Thierry, Magalie; Pasquis, Bruno; Buteau, Bénédicte; Fourgeux, Cynthia; Dembele, Doulaye; Leclere, Laurent; Gambert-Nicot, Ségolène; Acar, Niyazi; Bron, Alain M; Creuzot-Garcher, Catherine P; Bretillon, Lionel

2015-06-01

The lack of plasticity of neurons to respond to dietary changes, such as high fat and high fructose diets, by modulating gene and protein expression has been associated with functional and behavioral impairments that can have detrimental consequences. The inhibition of high fat-induced rewiring of hypothalamic neurons induced obesity. Feeding rodents with high fructose is a recognized and widely used model to trigger obesity and metabolic syndrome. However the adaptive response of the retina to short term feeding with high fructose is poorly documented. We therefore aimed to characterize both the functional and gene expression changes in the neurosensory retina of Brown Norway rats fed during 3 and 8 days with a 60%-rich fructose diet (n = 16 per diet and per time point). Glucose, insulin, leptin, triacylglycerols, total cholesterol, HDL-cholesterol, LDL-cholesterol and fructosamine were quantified in plasma (n = 8 in each group). Functionality of the inner retina was studied using scotopic single flash electroretinography (n = 8 in each group) and the individual response of rod and cone photoreceptors was determined using 8.02 Hz Flicker electroretinography (n = 8 in each group). Analysis of gene expression in the neurosensory retina was performed by Affymetrix genechips, and confirmed by RT-qPCR (n = 6 in each group). Elevated glycemia (+13%), insulinemia (+83%), and leptinemia (+172%) was observed after 8 days of fructose feeding. The cone photoreceptor response was altered at day 8 in high fructose fed rats (Δ = 0.5 log unit of light stimulus intensity). Affymetrix analysis of gene expression highlighted significant modulation of the pathways of eIF2 signaling and endoplasmic reticulum stress, regulation of eIF4 and p70S6K signaling, as well as mTOR signaling and mitochondrial dysfunction. RT-qPCR analysis confirmed the down regulation of Crystallins, Npy, Nid1 and Optc genes after 3 days of fructose feeding, and up regulation of End2. Meanwhile, a trend

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

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Blanca Iglesias-Figueroa

2016-06-01

Full Text Available In this study, bovine lactoferrin (bLf, an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin demonstrated antibacterial activity against Escherichia coli (E. coli BL21DE3, Staphylococcus aureus (S. aureus FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly.

Investigation of hTERT gene expression levels in two cell lines infected by high-risk human papilloma virus

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Maryam Akhtari

2016-07-01

Full Text Available Background: Human papilloma virus (HPV is one of the most important factors in cervical cancer. Viral sequences are integrated into the host cell genome. In mild cases the virus causes skin damages, in severe cases it leads to cancer. Like many other cancers, telomerase gene expression was increased in cervical cancer. This enzyme is a reverse transcriptase that contains two common subunits: i catalytic protein called human telomerase reverse transcriptase (hTERT and, ii RNA sequence called hTR. hTERT expression is hardly found in any somatic tissues. Detection of high telomerase activity in human cells, lead to tumor genesis. So hTERT can be used as a diagnostic tool in cancer detection. Methods: This experimental study was carried out from May 2013 to April 2014 in Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences in Tehran, Iran. Caski and Hela cancer cell lines were used which contain HPV16 and HPV18 respectively. Cell lines were cultured and total RNA was extracted. Following normalization agent glyceraldehyde-3-phosphate dehydrogenase (GADPH, hTERT expression level was determining by real-time PCR method. For each sample, the expression level of hTERT and GAPDH were quantified as copy numbers (per reaction using the standard curve. Finally, hTERT levels in Hela and Caski cell lines were compared quantitatively by t-test using GraphPad statistic software version 5 (San Diego, CA, USA. Results: According to the charts real-time PCR, hTERT gene expression in Hela and Caski cancer cell lines is significantly different (t=0.0319. Conclusion: All results confirm that hTERT expression levels in Hela and Caski cell lines are significantly different and the level of hTERT expression in the Caski cell line was slightly higher than that of Hela cell line. The significant difference between hTERT mRNA expression levels reported here could be used as a tumor marker for HPV16 and HPV18 in cervical cancer.

Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans.

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Smith, Kate E; Kelly, Amy C; Min, Catherine G; Weber, Craig S; McCarthy, Fiona M; Steyn, Leah V; Badarinarayana, Vasudeo; Stanton, J Brett; Kitzmann, Jennifer P; Strop, Peter; Gruessner, Angelika C; Lynch, Ronald M; Limesand, Sean W; Papas, Klearchos K

2017-11-01

Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived β cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high β cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic β cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to β cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and β cell function and leads to increased inflammatory signaling.

Decaffeinated coffee consumption induces expression of tight junction proteins in high fat diet fed rats

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Mazzone G

2016-09-01

Full Text Available Background: Recent evidence indicates that gut microbiota plays a key role in the development of NAFLD through the gut-liver axis. An altered gut permeability induced by alterations of tight junction (TJ proteins allows the passage of bacteria and substances leading to liver inflammation, hepatocyte damage and fibrosis. This study aims to evaluate the influence of decaffeinated coffee on gut permeability in a rat model of fat liver damage induced by a high fat diet (HFD. Methods: Twelve male Wistar rats were assigned to 3 groups. The first group received HFD for 5 months and drank water. The second group received HFD for 5 months and drank water added with 1.2mL decaffeinated coffee/day starting from the 4th month. The third group received standard diet (SD and drank water. Protein and mRNA expression levels of Toll-Like Receptor- 4 (TLR-4, Occludin and Zonula occludens-1 (ZO-1 were assessed in rat intestines. Results: A significant reduction of Occludin and ZO-1 was observed in HFD fed rats (0.97±0.05 vs 0.15±0.08 p˂0.01, and 0.97±0.05 vs 0.57±0.14 p˂0.001 respectively. This reduction was reverted in HFD+COFFEE rats (0.15±0.08 vs 0.83±0.27 p˂0.01 and 0.57±0.14 vs 0.85±0.12 p˂0.01 respectively. The TLR-4 expression up-regulated by HFD was partially reduced by coffee administration. Conclusions: HFD impairs the intestinal TJ barrier integrity. Coffee increases the expression of TJ proteins, reverting the altered gut permeability and reducing TLR-4 expression.

High expression of GRP78/BiP as a novel predictor of favorable outcomes in patients with advanced thymic carcinoma.

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Miura, Yosuke; Kaira, Kyoichi; Sakurai, Reiko; Imai, Hisao; Tomizawa, Yoshio; Sunaga, Noriaki; Minato, Koichi; Hisada, Takeshi; Oyama, Tetsunari; Yamada, Masanobu

2017-10-01

Glucose-regulated protein (GRP) 78/immunoglobulin heavy chain binding protein (BiP) is a member of the endoplasmic reticulum chaperone family, and its role in various types of human malignancies has recently been investigated. However, the clinicopathological characteristics of GRP78/BiP in advanced thymic carcinoma (ATC) remain unknown. We aimed to examine the relationship between GRP78/BiP expression and the clinical outcomes of ATC patients. Thirty-four patients with ATC receiving combination chemotherapy at three institutions between April 1998 and April 2014 were enrolled in this study. We retrospectively collected patient characteristics such as therapeutic efficacy, pathological findings, and survival data from their medical records. We performed immunohistochemical analysis to evaluate the expression of GRP78/BiP in tumor specimens obtained from surgical resection or biopsy. This study included 21 men (68%) and 13 women (32%) with a median age of 62 years (range 36-75 years). GRP78/BiP overexpression was observed in 65% of the patients (22 of 34 patients). There was no correlation between GRP78/BiP expression and any patient characteristic. Patients with a high level of GRP78/BiP expression had significantly longer overall survival (OS) compared to those with a low level (46.2 vs. 16.8 months, p = 0.04). Multivariate analysis demonstrated that a high level of GRP78/BiP expression was an independent prognostic factor for prolonged OS. Our findings indicate that the overexpression of GRP78/BiP is a novel predictor of favorable outcomes in patients with ATC who receive combination chemotherapy.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

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Ming-Wai Poon

Full Text Available A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs. Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2 reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%. Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs. This cell type may also have advantages in retinal pigmented epithelial differentiation.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

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Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

2015-01-01

A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

High Frequency Electromagnetic Field Induces Lipocalin 2 Expression in

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Amaneh Mohammadi Roushandeh

2010-06-01

Full Text Available Objective(sNeutrophil gelatinase-associated lipocalin (NGAL/Lcn2, comprise a group of small extracellular proteins with a common β-sheet-dominated 3-dimensional structure. In the past, it was assumed that the predominant role of lipocalin was acting as transport proteins. Recently it has been found that oxidative stress induces Lcn2 expression. It has been also proved that electromagnetic field (EMF produces reactive oxygen species (ROS in different tissues. Expression of Lcn2 following exposure to electromagnetic field has been investigated in this study. Materials and MethodsBalb/c mice (8 weeks old were exposed to 3 mT, 50 HZ EMF for 2 months, 4 hr/day. Afterwards, the mice were sacrificed by cervical dislocation and livers were removed. The liver specimens were stained with Haematoxylin- Eosin (H&E and analyzed under an optical microscope. Total RNA was extracted from liver and reverse transcription was performed by SuperScript III reverse transcriptase with 1 µg of total RNA. Assessment of Lcn2 expression was performed by semiquantitative and real time- PCR.ResultsThe light microscopic studies revealed that the number of lymphocyte cells was increased compared to control and dilation of sinosoids was observed in the liver. Lcn2 was up-regulated in the mice exposed to EMF both in mRNA and protein levels.ConclusionTo the extent of our knowledge, this is the first report dealing with up-regulation of Lcn2 in liver after exposure to EMF. The up-regulation might be a compensatory response that involves cell defense pathways and protective effects against ROS. However, further and complementary studies are required in this regards.

A maternal high-fat, high-sucrose diet alters insulin sensitivity and expression of insulin signalling and lipid metabolism genes and proteins in male rat offspring: effect of folic acid supplementation.

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Cuthbert, Candace E; Foster, Jerome E; Ramdath, D Dan

2017-10-01

A maternal high-fat, high-sucrose (HFS) diet alters offspring glucose and lipid homoeostasis through unknown mechanisms and may be modulated by folic acid. We investigated the effect of a maternal HFS diet on glucose homoeostasis, expression of genes and proteins associated with insulin signalling and lipid metabolism and the effect of prenatal folic acid supplementation (HFS/F) in male rat offspring. Pregnant Sprague-Dawley rats were randomly fed control (CON), HFS or HFS/F diets. Offspring were weaned on CON; at postnatal day 70, fasting plasma insulin and glucose and liver and skeletal muscle gene and protein expression were measured. Treatment effects were assessed by one-way ANOVA. Maternal HFS diet induced higher fasting glucose in offspring v. HFS/F (P=0·027) and down-regulation (Pinsulin resistance v. CON (P=0·030) and HFS/F was associated with higher insulin (P=0·016) and lower glucose (P=0·025). Maternal HFS diet alters offspring insulin sensitivity and de novo hepatic lipogenesis via altered gene and protein expression, which appears to be potentiated by folate supplementation.

Lysine-specific demethylase 2A expression is associated with cell growth and cyclin D1 expression in colorectal adenocarcinoma.

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Cao, Lin-Lin; Du, Changzheng; Liu, Hangqi; Pei, Lin; Qin, Li; Jia, Mei; Wang, Hui

2018-04-01

Lysine-specific demethylase 2A (KDM2A), a specific H3K36me1/2 demethylase, has been reported to be closely associated with several types of cancer. In this study, we aimed to investigate the expression and function of KDM2A in colorectal adenocarcinoma. A total of 215 colorectal adenocarcinoma specimens were collected, and then subjected to immunohistochemistry assay to evaluate the expression levels of KDM2A, cyclin D1 and other proteins in colorectal adenocarcinoma tissues. Real-time polymerase chain reaction, Western blot, and other molecular biology methods were used to explore the role of KDM2A in colorectal adenocarcinoma cells. In this study, we report that the expression level of KDM2A is high in colorectal adenocarcinoma tissues, and this high expression promotes the proliferation and colony formation of colorectal adenocarcinoma cells, as demonstrated by KDM2A knockdown experiments. In addition, the expression of KDM2A is closely associated with cyclin D1 expression in colorectal adenocarcinoma tissues and cell lines. Our study reveals a novel role for high-expressed KDM2A in colorectal adenocarcinoma cell growth, and that the expression of KDM2A is associated with that of cyclin D1 in colorectal adenocarcinoma.

High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris

DEFF Research Database (Denmark)

Oledzka, G.; Dabrowski, Slawomir; Kur, J.

2003-01-01

Aqualysin I is a heat-stable subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. We report the high-level expression of an aqualysin I protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia...... to that of the native enzyme. We also explored the possibility of secreting the GAP expressed aqualysin I in P. pastoris by in-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal. However, the levels of secreted pro-aqualysin I particles were approximately 10 times lower, possibly...

Integrated analyses of microRNAs demonstrate their widespread influence on gene expression in high-grade serous ovarian carcinoma.

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Creighton, Chad J; Hernandez-Herrera, Anadulce; Jacobsen, Anders; Levine, Douglas A; Mankoo, Parminder; Schultz, Nikolaus; Du, Ying; Zhang, Yiqun; Larsson, Erik; Sheridan, Robert; Xiao, Weimin; Spellman, Paul T; Getz, Gad; Wheeler, David A; Perou, Charles M; Gibbs, Richard A; Sander, Chris; Hayes, D Neil; Gunaratne, Preethi H

2012-01-01

The Cancer Genome Atlas (TCGA) Network recently comprehensively catalogued the molecular aberrations in 487 high-grade serous ovarian cancers, with much remaining to be elucidated regarding the microRNAs (miRNAs). Here, using TCGA ovarian data, we surveyed the miRNAs, in the context of their predicted gene targets. Integration of miRNA and gene patterns yielded evidence that proximal pairs of miRNAs are processed from polycistronic primary transcripts, and that intronic miRNAs and their host gene mRNAs derive from common transcripts. Patterns of miRNA expression revealed multiple tumor subtypes and a set of 34 miRNAs predictive of overall patient survival. In a global analysis, miRNA:mRNA pairs anti-correlated in expression across tumors showed a higher frequency of in silico predicted target sites in the mRNA 3'-untranslated region (with less frequency observed for coding sequence and 5'-untranslated regions). The miR-29 family and predicted target genes were among the most strongly anti-correlated miRNA:mRNA pairs; over-expression of miR-29a in vitro repressed several anti-correlated genes (including DNMT3A and DNMT3B) and substantially decreased ovarian cancer cell viability. This study establishes miRNAs as having a widespread impact on gene expression programs in ovarian cancer, further strengthening our understanding of miRNA biology as it applies to human cancer. As with gene transcripts, miRNAs exhibit high diversity reflecting the genomic heterogeneity within a clinically homogeneous disease population. Putative miRNA:mRNA interactions, as identified using integrative analysis, can be validated. TCGA data are a valuable resource for the identification of novel tumor suppressive miRNAs in ovarian as well as other cancers.

High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor-binding domain of botulinum neurotoxin serotype D

International Nuclear Information System (INIS)

Zhang, Yanfeng; Gao, Xiaoli; Qin, Ling; Buchko, Garry W.; Robinson, Howard; Varnum, Susan M.

2010-01-01

The receptor-binding domain of botulinum neurotoxin serotype D was expressed in E. coli using a codon-optimized cDNA. The highly purified protein crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 Å, and the crystals diffracted to 1.65 Å resolution. Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D-HCR was expressed at a high level (150–200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D-HCR was obtained. The recombinant BoNT/D-HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 Å. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit

Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression

International Nuclear Information System (INIS)

Nguyen, Kim-Lien; Llano, Manuel; Akari, Hirofumi; Miyagi, Eri; Poeschla, Eric M.; Strebel, Klaus; Bour, Stephan

2004-01-01

Two HIV-1 accessory proteins, Vpu and Vif, are notoriously difficult to express autonomously in the absence of the viral Tat and Rev proteins. We examined whether the codon bias observed in the vpu and vif genes relative to highly expressed human genes contributes to the Rev dependence and low expression level outside the context of the viral genome. The entire vpu gene as well as the 5' half of the vif gene were codon optimized and the resulting open reading frames (ORFs) (vphu and hvif, respectively) were cloned in autonomous expression vectors under the transcriptional control of the CMV promoter. Codon optimization efficiently removed the expression block observed in the native genes and allowed high levels of Rev- and Tat-independent expression of Vpu and Vif. Most of the higher protein levels detected are accounted for by enhanced steady-state levels of the mRNA encoding the optimized species. Nuclear run-on experiments show for the first time that codon optimization has no effect on the rate of transcriptional initiation or elongation of the vphu mRNA. Instead, optimization of the vpu gene was found to stabilize the vphu mRNA in the nucleus and enhance its export to the cytoplasm. This was achieved by allowing the optimized mRNA to use a new CRM1-independent nuclear export pathway. This work provides a better understanding of the molecular mechanisms underlying the process of codon optimization and introduces novel tools to study the biological functions of the Vpu and Vif proteins independently of other viral proteins

Hypothyroidism leads to increased collagen-based stiffness and re-expression of large cardiac titin isoforms with high compliance.

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Wu, Yiming; Peng, Jun; Campbell, Kenneth B; Labeit, Siegfried; Granzier, Henk

2007-01-01

Because long-term hypothyroidism results in diastolic dysfunction, we investigated myocardial passive stiffness in hypothyroidism and focused on the possible role of titin, an important determinant of diastolic stiffness. A rat model of hypothyroidism was used, obtained by administering propylthiouracil (PTU) for times that varied from 1 month (short-term) to 4 months (long-term). Titin expression was determined by transcript analysis, gel electrophoresis and immunoelectron microscopy. Diastolic function was measured at the isolated heart, skinned muscle, and cardiac myocyte levels. We found that hypothyroidism resulted in expression of a large titin isoform, the abundance of which gradually increased with time to become the most dominant isoform in long-term hypothyroid rats. This isoform co-migrates on high-resolution gels with fetal cardiac titin. Transcript analysis on myocardium of long-term PTU rats, provided evidence for expression of additional PEVK and Ig domain exons, similar to what has been described in fetal myocardium. Consistent with the expression of a large titin isoform, titin-based restoring and passive forces were significantly reduced in single cardiac myocytes and muscle strips of long-term hypothyroid rats. Overall muscle stiffness and LV diastolic wall stiffness were increased, however, due to increased collagen-based stiffness. We conclude that long term hypothyroidism triggers expression of a large cardiac titin isoform and that the ensuing reduction in titin-based passive stiffness functions as a compensatory mechanism to reduce LV wall stiffness.

Gene-Expression Biomarkers for Application to High-Throughput Radiation Biodosimetry

National Research Council Canada - National Science Library

Grace, M. B; Salter, C. A; Bullard, J. R; Prasanna, P. G; Manglapus, G. L; Blakely, W. F

2005-01-01

.... Even with the delayed onset of symptoms, sometimes several days after exposure, gene-expression biomarkers can identify these exposed individuals very early after exposure, allowing for prompt medical intervention...

High expression of WWP1 predicts poor prognosis and associates with tumor progression in human colorectal cancer

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Chen, Jian-Jun; Zhang, Wei

2018-01-01

WWP1 (WW domain-containing E3 ubiquitin protein ligase 1), which is frequently up-regulated in multiple human malignancies, has been demonstrated to play a critical function in cell proliferation, apoptosis and invasion. However, limited knowledge is known about the expression pattern and prognostic value of WWP1 in colorectal cancer (CRC). In this study, we firstly observed that WWP1 mRNA and protein is commonly up-regulated in CRC tissues compared with normal counterparts. Furthermore, by immunohistochemical analysis in 348 cases of CRC specimens, we demonstrated that the WWP1 protein expression is up-regulated in 58.91% (205/348) samples and detected increasing WWP1 expression is closely correlated with enhanced tumor size (P=0.022), CEA level (P=0.021), T classification (P=0.010), distant metastasis (P=0.021) and TNM stage (P=0.005). Meanwhile, Kaplan-Meier survival analysis showed CRC patients with a high WWP1 expression have a poorer overall survival (P<0.001) and disease-free survival (P=0.001) than those with a low WWP1 expression. Multivariate Cox regression analysis revealed WWP1 is the independent prognostic factors for overall survival rate of CRC patients. What’s more, by CCK-8 assays and Transwell assays, we found WWP1 depletion markedly inhibited tumor proliferation and invasion in CRC cells, and cells with WWP1 overexpression had a prominently higher proliferative and invasive capacity. Most notably, we illuminated WWP1 downregulation inactivated PTEN/Akt pathway in CRC cells. Taken together, our studies revealed the prognostic value of WWP1 in CRC and support that WWP1 may act as a molecular target for CRC treatment. PMID:29511596

Hedgehog signaling pathway is active in GBM with GLI1 mRNA expression showing a single continuous distribution rather than discrete high/low clusters.

Science.gov (United States)

Chandra, Vikas; Das, Tapojyoti; Gulati, Puneet; Biswas, Nidhan K; Rote, Sarang; Chatterjee, Uttara; Ghosh, Samarendra N; Deb, Sumit; Saha, Suniti K; Chowdhury, Anup K; Ghosh, Subhashish; Rudin, Charles M; Mukherjee, Ankur; Basu, Analabha; Dhara, Surajit

2015-01-01

Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression--as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution-unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the "high-Hh" cluster of MB but 5.6 fold higher than that of the "low-Hh" cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

Human TMEM174 that is highly expressed in kidney tissue activates AP-1 and promotes cell proliferation

International Nuclear Information System (INIS)

Wang, Pingzhang; Sun, Bo; Hao, Dongxia; Zhang, Xiujun; Shi, Taiping; Ma, Dalong

2010-01-01

Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174ΔTM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.

Human TMEM174 that is highly expressed in kidney tissue activates AP-1 and promotes cell proliferation

Energy Technology Data Exchange (ETDEWEB)

Wang, Pingzhang [Chinese National Human Genome Center, 3-707 North YongChang Road BDA, Beijing 100191 (China); Laboratory of Medical Immunology, School of Basic Medical Science, Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191 (China); Peking University Center for Human Disease Genomics, No. 38 Xueyuan Road, Beijing 100191 (China); Sun, Bo; Hao, Dongxia [Department of Biology, Northchina Coal Medical College, No. 57 JianShe South Road, Tangshan 063000 (China); Zhang, Xiujun, E-mail: zhangxiujun66@yahoo.com.cn [Department of Biology, Northchina Coal Medical College, No. 57 JianShe South Road, Tangshan 063000 (China); Shi, Taiping, E-mail: taiping_shi@yahoo.com.cn [Chinese National Human Genome Center, 3-707 North YongChang Road BDA, Beijing 100191 (China); Laboratory of Medical Immunology, School of Basic Medical Science, Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191 (China); Peking University Center for Human Disease Genomics, No. 38 Xueyuan Road, Beijing 100191 (China); Ma, Dalong [Chinese National Human Genome Center, 3-707 North YongChang Road BDA, Beijing 100191 (China); Laboratory of Medical Immunology, School of Basic Medical Science, Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191 (China); Peking University Center for Human Disease Genomics, No. 38 Xueyuan Road, Beijing 100191 (China)

2010-04-16

Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174{Delta}TM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.

Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status.

Science.gov (United States)

Meiyalaghan, Sathiyamoorthy; Thomson, Susan J; Fiers, Mark W E J; Barrell, Philippa J; Latimer, Julie M; Mohan, Sara; Jones, E Eirian; Conner, Anthony J; Jacobs, Jeanne M E

2014-01-02

GSL1 and GSL2, Gibberellin Stimulated-Like proteins (also known as Snakin-1 and Snakin-2), are cysteine-rich peptides from potato (Solanum tuberosum L.) with antimicrobial properties. Similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. To help resolve the biological roles of GSL1 and GSL2 peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato. We have characterised the full length genes for both GSL1 (chromosome 4) and GSL2 (chromosome 1) from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. The frequency of SNPs in GSL1 and GSL2 were very low with only one SNP every 67 and 53 nucleotides in exon regions of GSL1 and GSL2, respectively. Analysis of comprehensive RNA-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. Expression analysis based on the frequency of next generation sequence reads established that GSL2 was expressed at a higher level than GSL1 in 30 out of 32 tissue and treatment libraries. Furthermore, both the GSL1 and GSL2 genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. Potato transformation with antisense knock-down expression cassettes failed to recover viable plants. The potato GSL1 and GSL2 genes are very highly conserved suggesting they contribute to an important biological function. The known antimicrobial activity of the GSL proteins, coupled with the FPKM analysis from RNA-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. The lethality of antisense knock

Adaptations to High Salt in a Halophilic Protist: Differential Expression and Gene Acquisitions through Duplications and Gene Transfers

Science.gov (United States)

Harding, Tommy; Roger, Andrew J.; Simpson, Alastair G. B.

2017-01-01

The capacity of halophiles to thrive in extreme hypersaline habitats derives partly from the tight regulation of ion homeostasis, the salt-dependent adjustment of plasma membrane fluidity, and the increased capability to manage oxidative stress. Halophilic bacteria, and archaea have been intensively studied, and substantial research has been conducted on halophilic fungi, and the green alga Dunaliella. By contrast, there have been very few investigations of halophiles that are phagotrophic protists, i.e., protozoa. To gather fundamental knowledge about salt adaptation in these organisms, we studied the transcriptome-level response of Halocafeteria seosinensis (Stramenopiles) grown under contrasting salinities. We provided further evolutionary context to our analysis by identifying genes that underwent recent duplications. Genes that were highly responsive to salinity variations were involved in stress response (e.g., chaperones), ion homeostasis (e.g., Na+/H+ transporter), metabolism and transport of lipids (e.g., sterol biosynthetic genes), carbohydrate metabolism (e.g., glycosidases), and signal transduction pathways (e.g., transcription factors). A significantly high proportion (43%) of duplicated genes were also differentially expressed, accentuating the importance of gene expansion in adaptation by H. seosinensis to high salt environments. Furthermore, we found two genes that were lateral acquisitions from bacteria, and were also highly up-regulated and highly expressed at high salt, suggesting that this evolutionary mechanism could also have facilitated adaptation to high salt. We propose that a transition toward high-salt adaptation in the ancestors of H. seosinensis required the acquisition of new genes via duplication, and some lateral gene transfers (LGTs), as well as the alteration of transcriptional programs, leading to increased stress resistance, proper establishment of ion gradients, and modification of cell structure properties like membrane

Adaptations to High Salt in a Halophilic Protist: Differential Expression and Gene Acquisitions through Duplications and Gene Transfers

Directory of Open Access Journals (Sweden)

Tommy Harding

2017-05-01

Full Text Available The capacity of halophiles to thrive in extreme hypersaline habitats derives partly from the tight regulation of ion homeostasis, the salt-dependent adjustment of plasma membrane fluidity, and the increased capability to manage oxidative stress. Halophilic bacteria, and archaea have been intensively studied, and substantial research has been conducted on halophilic fungi, and the green alga Dunaliella. By contrast, there have been very few investigations of halophiles that are phagotrophic protists, i.e., protozoa. To gather fundamental knowledge about salt adaptation in these organisms, we studied the transcriptome-level response of Halocafeteria seosinensis (Stramenopiles grown under contrasting salinities. We provided further evolutionary context to our analysis by identifying genes that underwent recent duplications. Genes that were highly responsive to salinity variations were involved in stress response (e.g., chaperones, ion homeostasis (e.g., Na+/H+ transporter, metabolism and transport of lipids (e.g., sterol biosynthetic genes, carbohydrate metabolism (e.g., glycosidases, and signal transduction pathways (e.g., transcription factors. A significantly high proportion (43% of duplicated genes were also differentially expressed, accentuating the importance of gene expansion in adaptation by H. seosinensis to high salt environments. Furthermore, we found two genes that were lateral acquisitions from bacteria, and were also highly up-regulated and highly expressed at high salt, suggesting that this evolutionary mechanism could also have facilitated adaptation to high salt. We propose that a transition toward high-salt adaptation in the ancestors of H. seosinensis required the acquisition of new genes via duplication, and some lateral gene transfers (LGTs, as well as the alteration of transcriptional programs, leading to increased stress resistance, proper establishment of ion gradients, and modification of cell structure properties like

Neurotensin neural mRNA expression correlates with vocal communication and other highly-motivated social behaviors in male European starlings.

Science.gov (United States)

Merullo, Devin P; Cordes, Melissa A; Susan DeVries, M; Stevenson, Sharon A; Riters, Lauren V

2015-11-01

Vocalizations coordinate social interactions in many species and often are important for behaviors such as mate attraction or territorial defense. Although the neural circuitry underlying vocal communication is well-known for some animal groups, such as songbirds, the motivational processes that regulate vocal signals are not as clearly understood. Neurotensin (NT) is a neuropeptide implicated in motivation that can modulate the activity of dopaminergic neurons. Dopaminergic projections from the ventral tegmental area (VTA) are key to mediating highly motivated, goal-directed behaviors, including sexually-motivated birdsong. However, the role of NT in modifying vocal communication or other social behaviors has not been well-studied. Here in European starlings (Sturnus vulgaris) we analyzed relationships between sexually-motivated song and NT and NT1 receptor (NTSR1) expression in VTA. Additionally, we examined NT and NTSR1 expression in four regions that receive dopaminergic projections from VTA and are involved in courtship song: the medial preoptic nucleus (POM), the lateral septum (LS), Area X, and HVC. Relationships between NT and NTSR1 expression and non-vocal courtship and agonistic behaviors were also examined. NT expression in Area X positively related to sexually-motivated song production. NT expression in POM positively correlated with non-vocal courtship behavior and agonistic behavior. NT expression in POM was greatest in males owning nesting sites, and the opposite pattern was observed for NTSR1 expression in LS. These results are the first to implicate NT in Area X in birdsong, and further highlight NT as a potential neuromodulator for the control of vocal communication and other social behaviors. Copyright © 2015 Elsevier Inc. All rights reserved.

Aryl hydrocarbon receptor expression is associated with a family history of upper gastrointestinal tract cancer in a high-risk population exposed to aromatic hydrocarbons

Energy Technology Data Exchange (ETDEWEB)

Roth, M.J.; Wei, W.Q.; Baer, J.; Abnet, C.C.; Wang, G.Q.; Sternberg, L.R.; Warner, A.C.; Johnson, L.L.; Lu, N.; Giffen, C.A.; Dawsey, S.M.; Qiao, Y.L.; Cherry, J. [NCI, Bethesda, MD (United States)

2009-09-15

Polycyclic aromatic hydrocarbon (PAH) exposure is a risk factor for esophageal squamous cell carcinoma, and PAHs are ligands of the aryl hydrocarbon receptor (AhR). This study measured the expression of AhR and related genes in frozen esophageal cell samples from patients exposed to different levels of indoor air pollution, who did or did not have high-grade squamous dysplasia and who did or did not have a family history of upper gastrointestinal tract (UGI) cancer. 147 samples were evaluated, including 23 (16%) from patients with high-grade dysplasia and 48 (33%) from patients without dysplasia who heated their homes with coal, without a chimney (a 'high' indoor air pollution group), and 27 (18%) from patients with high-grade dysplasia and 49 (33%) from patients without dysplasia who did not heat their homes at all (a 'low' indoor air pollution group). Sixty-four (44%) had a family history of UGI cancer. RNA was extracted and quantitative PCR analysis was done. AhR gene expression was detectable in 85 (58%) of the samples and was >9-fold higher in those with a family history of UGI cancer (median expression (interquartile range), -1,964 (-18,000, -610) versus -18,000 (-18,000, -1036); P = 0.02, Wilcoxon rank-sum test). Heating status, dysplasia category, age, gender, and smoking were not associated with AhR expression (linear regression; all P values {ge} 0.1). AhR expression was higher in patients with a family history of UGI cancer. Such individuals may be more susceptible to the deleterious effects of PAH exposure, including PAH-induced cancer.