Comparative proteomic investigation of metastatic and non-metastatic osteosarcoma cells of human and canine origin.

PubMed

Roy, Jahnabi; Wycislo, Kathryn L; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2017-01-01

Osteosarcoma is the most common bone cancer in dogs and people. In order to improve clinical outcomes, it is necessary to identify proteins that are differentially expressed by metastatic cells. Membrane bound proteins are responsible for multiple pro-metastatic functions. Therefore characterizing the differential expression of membranous proteins between metastatic and non-metastatic clonal variants will allow the discovery of druggable targets and consequently improve treatment methodology. The objective of this investigation was to systemically identify the membrane-associated proteomics of metastatic and non-metastatic variants of human and canine origin. Two clonal variants of divergent in vivo metastatic potential from human and canine origins were used. The plasma membranes were isolated and peptide fingerprinting was used to identify differentially expressed proteins. Selected proteins were further validated using western blotting, flow cytometry, confocal microscopy and immunohistochemistry. Over 500 proteins were identified for each cell line with nearly 40% of the proteins differentially regulated. Conserved between both species, metastatic variants demonstrated significant differences in expression of membrane proteins that are responsible for pro-metastatic functions. Additionally, CD147, CD44 and vimentin were validated using various biochemical techniques. Taken together, through a comparative proteomic approach we have identified several differentially expressed cell membrane proteins that will help in the development of future therapeutics.

Decreased expression of the stress protein HSP70 is an early event in murine erythroleukemic cell differentiation.

PubMed Central

Hensold, J O; Housman, D E

1988-01-01

Two-dimensional protein gels were used to systematically assess changes in gene expression in Friend erythroleukemia cells after exposure to inducers of differentiation. A rapid decrease in expression of the stress protein HSP70 was observed after exposure to inducers. The kinetics of this change suggest that it may be related to the cellular events that regulate the onset of differentiation. Images PMID:3164440

Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales

PubMed Central

Margres, Mark J.; Wray, Kenneth P.; Seavy, Margaret; McGivern, James J.; Herrera, Nathanael D.; Rokyta, Darin R.

2016-01-01

Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression proteins, enabling low-expression proteins to evolve and potentially lead to more rapid adaptation. PMID:26546003

Subcellular proteomic analysis of host-pathogen interactions using human monocytes exposed to Yersinia pestis and Yersinia pseudotuberculosis

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

Zhang, C G; Gonzales, A D; Choi, M W

2004-05-20

Yersinia pestis, the etiological agent of plague, is of concern to human health both from an infectious disease and a civilian biodefense perspective. While Y. pestis and Y. pseudotuberculosis share more than 90% DNA homology, they have significantly different clinical manifestations. Plague is often fatal if untreated, yet Y. pseudotuberculosis causes severe intestinal distress and is rarely fatal. A better understanding of host response to these closely related pathogens may help explain the different mechanisms of virulence and pathogenesis that result in such different clinical outcomes. The aim of this study was to characterize host protein expression changes in humanmore » monocyte-like U937 cells after exposure to Y. pestis and Y. pseudotuberculosis. In order to gain global proteomic coverage of host response, proteins from cytoplasmic, nuclear and membrane fractions of host cells were studied by 2-dimensional differential gel electrophoresis (2-D DIGE) and relative protein expression differences were quantitated. Differentially expressed proteins, with at least 1.5 fold expression changes and p values of 0.01 or less, were identified by MALDI-MS or LC/MS/MS. With these criteria, differential expression was detected in 16 human proteins after Y. pestis exposure and 13 human proteins after Y. pseudotuberculosis exposure, of which only two of the differentially expressed proteins identified were shared between the two exposures. Proteins identified in this study are reported to be involved in a wide spectrum of cellular functions and host defense mechanisms including apoptosis, cytoskeletal rearrangement, protein synthesis and degradation, DNA replication and transcription, metabolism, protein folding, and cell signaling. Notably, the differential expression patterns observed can distinguish the two pathogen exposures from each other and from unexposed host cells. The functions of the differentially expressed proteins identified provide insight on the different virulence and pathogenic mechanisms of Y. pestis and Y. pseudotuberculosis.« less

Proteins differentially expressed during limonene biotransformation by Penicillium digitatum DSM 62840 were examined using iTRAQ labeling coupled with 2D-LC-MS/MS.

PubMed

Zhang, Lu-Lu; Zhang, Yan; Ren, Jing-Nan; Liu, Yan-Long; Li, Jia-Jia; Tai, Ya-Nan; Yang, Shu-Zhen; Pan, Si-Yi; Fan, Gang

2016-10-01

This study focused on the differences in protein expression at various periods during limonene biotransformation by Penicillium digitatum DSM 62840. A total of 3644 protein-species were quantified by iTRAQ during limonene biotransformation (0 and 12 h). A total of 643 proteins were differentially expressed, 316 proteins were significantly up-regulated and 327 proteins were markedly down-regulated. GO, COG, and pathway enrichment analysis showed that the differentially expressed proteins possessed catalytic and binding functions and were involved in a variety of cellular and metabolic process. Furthermore, the enzymes involved in limonene transformation might be related to cytochrome P-450. This study provided a powerful platform for further exploration of biotransformation, and the identified proteins provided insight into the mechanism of limonene transformation.

Proteomic profiling of fetal esophageal epithelium, esophageal cancer, and tumor-adjacent esophageal epithelium and immunohistochemical characterization of a representative differential protein, PRX6

PubMed Central

Guo, Jun-Hui; Xing, Guo-Lan; Fang, Xin-Hui; Wu, Hui-Fang; Zhang, Bo; Yu, Jin-Zhong; Fan, Zong-Min; Wang, Li-Dong

2017-01-01

AIM To understand the molecular mechanism of esophageal cancer development and provide molecular markers for screening high-risk populations and early diagnosis. METHODS Two-dimensional electrophoresis combined with mass spectrometry were adopted to screen differentially expressed proteins in nine cases of fetal esophageal epithelium, eight cases of esophageal cancer, and eight cases of tumor-adjacent normal esophageal epithelium collected from fetuses of different gestational age, or esophageal cancer patients from a high-risk area of esophageal cancer in China. Immunohistochemistry (avidin-biotin-horseradish peroxidase complex method) was used to detect the expression of peroxiredoxin (PRX)6 in 91 cases of esophageal cancer, tumor-adjacent normal esophageal tissue, basal cell hyperplasia, dysplasia, and carcinoma in situ, as well as 65 cases of esophageal epithelium from fetuses at a gestational age of 3-9 mo. RESULTS After peptide mass fingerprint analysis and search of protein databases, 21 differential proteins were identified; some of which represent a protein isoform. Varying degrees of expression of PRX6 protein, which was localized mainly in the cytoplasm, were detected in adult and fetal normal esophageal tissues, precancerous lesions, and esophageal cancer. With the progression of esophageal lesions, PRX6 protein expression showed a declining trend (P < 0.05). In fetal epithelium from fetuses at gestational age 3-6 mo, PRX6 protein expression showed a declining trend with age (P < 0.05). PRX6 protein expression was significantly higher in well-differentiated esophageal cancer tissues than in poorly differentiated esophageal cancer tissues (P < 0.05). CONCLUSION Development and progression of esophageal cancer result from interactions of genetic changes (accumulation or superposition). PRX6 protein is associated with fetal esophageal development and cancer differentiation. PMID:28293090

Proteomic profiling of fetal esophageal epithelium, esophageal cancer, and tumor-adjacent esophageal epithelium and immunohistochemical characterization of a representative differential protein, PRX6.

PubMed

Guo, Jun-Hui; Xing, Guo-Lan; Fang, Xin-Hui; Wu, Hui-Fang; Zhang, Bo; Yu, Jin-Zhong; Fan, Zong-Min; Wang, Li-Dong

2017-02-28

To understand the molecular mechanism of esophageal cancer development and provide molecular markers for screening high-risk populations and early diagnosis. Two-dimensional electrophoresis combined with mass spectrometry were adopted to screen differentially expressed proteins in nine cases of fetal esophageal epithelium, eight cases of esophageal cancer, and eight cases of tumor-adjacent normal esophageal epithelium collected from fetuses of different gestational age, or esophageal cancer patients from a high-risk area of esophageal cancer in China. Immunohistochemistry (avidin-biotin-horseradish peroxidase complex method) was used to detect the expression of peroxiredoxin (PRX)6 in 91 cases of esophageal cancer, tumor-adjacent normal esophageal tissue, basal cell hyperplasia, dysplasia, and carcinoma in situ , as well as 65 cases of esophageal epithelium from fetuses at a gestational age of 3-9 mo. After peptide mass fingerprint analysis and search of protein databases, 21 differential proteins were identified; some of which represent a protein isoform. Varying degrees of expression of PRX6 protein, which was localized mainly in the cytoplasm, were detected in adult and fetal normal esophageal tissues, precancerous lesions, and esophageal cancer. With the progression of esophageal lesions, PRX6 protein expression showed a declining trend ( P < 0.05). In fetal epithelium from fetuses at gestational age 3-6 mo, PRX6 protein expression showed a declining trend with age ( P < 0.05). PRX6 protein expression was significantly higher in well-differentiated esophageal cancer tissues than in poorly differentiated esophageal cancer tissues ( P < 0.05). Development and progression of esophageal cancer result from interactions of genetic changes (accumulation or superposition). PRX6 protein is associated with fetal esophageal development and cancer differentiation.

Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

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

Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha

2008-02-15

There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPAR{gamma} agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA andmore » protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPAR{gamma}-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake.« less

bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.

PubMed

Li, Xiaosu; Chen, Rui; Zhu, Sijun

2017-11-15

Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of the differential gene and protein expression profile of the rolled leaf mutant of transgenic rice (Oryza sativa L.).

PubMed

Zhu, Qiuqiang; Yu, Shuguang; Chen, Guanshui; Ke, Lanlan; Pan, Daren

2017-01-01

The importance of leaf rolling in rice (Oryza sativa L.) has been widely recognized. Although several studies have investigated rice leaf rolling and identified some related genes, knowledge of the molecular mechanism underlying rice leaf rolling, especially outward leaf rolling, is limited. Therefore, in this study, differential proteomics and gene expression profiling were used to analyze rolled leaf mutant of transgenic rice in order to investigate differentially expressed genes and proteins related to rice leaf rolling. To this end, 28 differentially expressed proteins related to rolling leaf traits were isolated and identified. Digital expression profiling detected 10 genes related to rice leaf rolling. Some of the proteins and genes detected are involved in lipid metabolism, which is related to the development of bulliform cells, such as phosphoinositide phospholipase C, Mgll gene, and At4g26790 gene. The "omics"-level techniques were useful for simultaneously isolating several proteins and genes related to rice leaf rolling. In addition, the results of the analysis of differentially expressed proteins and genes were closely consistent with those from a corresponding functional analysis of cellular mechanisms; our study findings might form the basis for further research on the molecular mechanisms underlying rice leaf rolling.

Comparative proteomic study on Brassica hexaploid and its parents provides new insights into the effects of polyploidization.

PubMed

Shen, Yanyue; Zhang, Yu; Zou, Jun; Meng, Jinling; Wang, Jianbo

2015-01-01

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. Although genomic and transcriptomic changes have been observed in polyploids, the effects of polyploidization on proteomic divergence are poorly understood. In this study, we reported quantitative analysis of proteomic changes in leaves of Brassica hexaploid and its parents using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. A total of 2044 reproducible proteins were quantified by at least two unique peptides. We detected 452 proteins differentially expressed between Brassica hexaploid and its parents, and 100 proteins were non-additively expressed in Brassica hexaploid, which suggested a trend of non-additive protein regulation following genomic merger and doubling. Functional categories of cellular component biogenesis, immune system process, and response to stimulus, were significantly enriched in non-additive proteins, probably providing a driving force for variation and adaptation in allopolyploids. In particular, majority of the total 452 differentially expressed proteins showed expression level dominance of one parental expression, and there was an expression level dominance bias toward the tetraploid progenitor. In addition, the percentage of differentially expressed proteins that matched previously reported differentially genes were relatively low. This study aimed to get new insights into the effects of polyploidization on proteomic divergence. Using iTRAQ LC-MS/MS technology, we identified 452 differentially expressed proteins between allopolyploid and its parents which involved in response to stimulus, multi-organism process, and immune system process, much more than previous studies using 2-DE coupled with mass spectrometry technology. Therefore, our manuscript represents the most comprehensive analysis of protein profiles in allopolyploid and its parents, which will lead to a better understanding of novelty and plasticity of the allopolyploid genomes. Copyright © 2014 Elsevier B.V. All rights reserved.

Long Non-Coding RNAs Differentially Expressed between Normal versus Primary Breast Tumor Tissues Disclose Converse Changes to Breast Cancer-Related Protein-Coding Genes

PubMed Central

Reiche, Kristin; Kasack, Katharina; Schreiber, Stephan; Lüders, Torben; Due, Eldri U.; Naume, Bjørn; Riis, Margit; Kristensen, Vessela N.; Horn, Friedemann; Børresen-Dale, Anne-Lise; Hackermüller, Jörg; Baumbusch, Lars O.

2014-01-01

Breast cancer, the second leading cause of cancer death in women, is a highly heterogeneous disease, characterized by distinct genomic and transcriptomic profiles. Transcriptome analyses prevalently assessed protein-coding genes; however, the majority of the mammalian genome is expressed in numerous non-coding transcripts. Emerging evidence supports that many of these non-coding RNAs are specifically expressed during development, tumorigenesis, and metastasis. The focus of this study was to investigate the expression features and molecular characteristics of long non-coding RNAs (lncRNAs) in breast cancer. We investigated 26 breast tumor and 5 normal tissue samples utilizing a custom expression microarray enclosing probes for mRNAs as well as novel and previously identified lncRNAs. We identified more than 19,000 unique regions significantly differentially expressed between normal versus breast tumor tissue, half of these regions were non-coding without any evidence for functional open reading frames or sequence similarity to known proteins. The identified non-coding regions were primarily located in introns (53%) or in the intergenic space (33%), frequently orientated in antisense-direction of protein-coding genes (14%), and commonly distributed at promoter-, transcription factor binding-, or enhancer-sites. Analyzing the most diverse mRNA breast cancer subtypes Basal-like versus Luminal A and B resulted in 3,025 significantly differentially expressed unique loci, including 682 (23%) for non-coding transcripts. A notable number of differentially expressed protein-coding genes displayed non-synonymous expression changes compared to their nearest differentially expressed lncRNA, including an antisense lncRNA strongly anticorrelated to the mRNA coding for histone deacetylase 3 (HDAC3), which was investigated in more detail. Previously identified chromatin-associated lncRNAs (CARs) were predominantly downregulated in breast tumor samples, including CARs located in the protein-coding genes for CALD1, FTX, and HNRNPH1. In conclusion, a number of differentially expressed lncRNAs have been identified with relation to cancer-related protein-coding genes. PMID:25264628

Long non-coding RNAs differentially expressed between normal versus primary breast tumor tissues disclose converse changes to breast cancer-related protein-coding genes.

PubMed

Reiche, Kristin; Kasack, Katharina; Schreiber, Stephan; Lüders, Torben; Due, Eldri U; Naume, Bjørn; Riis, Margit; Kristensen, Vessela N; Horn, Friedemann; Børresen-Dale, Anne-Lise; Hackermüller, Jörg; Baumbusch, Lars O

2014-01-01

Breast cancer, the second leading cause of cancer death in women, is a highly heterogeneous disease, characterized by distinct genomic and transcriptomic profiles. Transcriptome analyses prevalently assessed protein-coding genes; however, the majority of the mammalian genome is expressed in numerous non-coding transcripts. Emerging evidence supports that many of these non-coding RNAs are specifically expressed during development, tumorigenesis, and metastasis. The focus of this study was to investigate the expression features and molecular characteristics of long non-coding RNAs (lncRNAs) in breast cancer. We investigated 26 breast tumor and 5 normal tissue samples utilizing a custom expression microarray enclosing probes for mRNAs as well as novel and previously identified lncRNAs. We identified more than 19,000 unique regions significantly differentially expressed between normal versus breast tumor tissue, half of these regions were non-coding without any evidence for functional open reading frames or sequence similarity to known proteins. The identified non-coding regions were primarily located in introns (53%) or in the intergenic space (33%), frequently orientated in antisense-direction of protein-coding genes (14%), and commonly distributed at promoter-, transcription factor binding-, or enhancer-sites. Analyzing the most diverse mRNA breast cancer subtypes Basal-like versus Luminal A and B resulted in 3,025 significantly differentially expressed unique loci, including 682 (23%) for non-coding transcripts. A notable number of differentially expressed protein-coding genes displayed non-synonymous expression changes compared to their nearest differentially expressed lncRNA, including an antisense lncRNA strongly anticorrelated to the mRNA coding for histone deacetylase 3 (HDAC3), which was investigated in more detail. Previously identified chromatin-associated lncRNAs (CARs) were predominantly downregulated in breast tumor samples, including CARs located in the protein-coding genes for CALD1, FTX, and HNRNPH1. In conclusion, a number of differentially expressed lncRNAs have been identified with relation to cancer-related protein-coding genes.

[Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells].

PubMed

Li, Pao; Gao, Shang; Zhou, Mei; Tang, Hong; Mu, Miduo; Zhang, Jiqiang; Tang, Kanglai

2017-04-01

To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation. TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot. The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch ( P <0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch ( P <0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch ( P <0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch ( P <0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch ( P <0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E ( P <0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch ( P <0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E ( P <0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E ( P >0.05). The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.

Interpretation of biological and mechanical variations between the Lowry versus Bradford method for protein quantification.

PubMed

Lu, Tzong-Shi; Yiao, Szu-Yu; Lim, Kenneth; Jensen, Roderick V; Hsiao, Li-Li

2010-07-01

The identification of differences in protein expression resulting from methodical variations is an essential component to the interpretation of true, biologically significant results. We used the Lowry and Bradford methods- two most commonly used methods for protein quantification, to assess whether differential protein expressions are a result of true biological or methodical variations. MATERIAL #ENTITYSTARTX00026; Differential protein expression patterns was assessed by western blot following protein quantification by the Lowry and Bradford methods. We have observed significant variations in protein concentrations following assessment with the Lowry versus Bradford methods, using identical samples. Greater variations in protein concentration readings were observed over time and in samples with higher concentrations, with the Bradford method. Identical samples quantified using both methods yielded significantly different expression patterns on Western blot. We show for the first time that methodical variations observed in these protein assay techniques, can potentially translate into differential protein expression patterns, that can be falsely taken to be biologically significant. Our study therefore highlights the pivotal need to carefully consider methodical approaches to protein quantification in techniques that report quantitative differences.

miR-764-5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression.

PubMed

Guo, Junwei; Ren, Fangli; Wang, Yinyin; Li, Shan; Gao, Zhengrong; Wang, Xiaoyan; Ning, Hongxiu; Wu, Jianguo; Li, Yi; Wang, Zhao; Chim, Shek Man; Xu, Jiake; Chang, Zhijie

2012-07-01

Differentiation of committed precursor cells into the osteoblast lineage is tightly regulated by several factors, including Runx2 and BMP2. We previously reported that C terminus of Hsc70-interacting protein/STIP1 homology and U-Box containing protein 1 (CHIP/STUB1) negatively regulated osteoblast differentiation through promoting Runx2 protein degradation. However, how CHIP is regulated during osteoblast differentiation remains unknown. In this study, we found that miR-764-5p is up-expressed during the osteoblast differentiation in calvarial and osteoblast progenitor cells, coupled with down-expression of CHIP protein. We observed that forced expression or inhibition of miR-764-5p decreased or increased the CHIP protein level through affecting its translation by targeting the 3'-UTR region. Perturbation of miR-764-5p resulted in altered differentiation fate of osteoblast progenitor cells and the role of miR-764-5p was reversed by overexpression of CHIP, whereas depletion of CHIP impaired the effect of miR-764-5p. Our data showed that miR-764-5p positively regulates osteoblast differentiation from osteoblast progenitor cells by repressing the translation of CHIP protein. Copyright © 2012 American Society for Bone and Mineral Research.

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

PubMed

Janjanam, Jagadeesh; Singh, Surender; Jena, Manoj K; Varshney, Nishant; Kola, Srujana; Kumar, Sudarshan; Kaushik, Jai K; Grover, Sunita; Dang, Ajay K; Mukesh, Manishi; Prakash, B S; Mohanty, Ashok K

2014-01-01

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

PubMed

Kawai, R; Ozeki, N; Yamaguchi, H; Tanaka, T; Nakata, K; Mogi, M; Nakamura, H

2014-05-01

We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Lineage specific expression of Polycomb Group Proteins in human embryonic stem cells in vitro.

PubMed

Pethe, Prasad; Pursani, Varsha; Bhartiya, Deepa

2015-05-01

Human embryonic (hES) stem cells are an excellent model to study lineage specification and differentiation into various cell types. Differentiation necessitates repression of specific genes not required for a particular lineage. Polycomb Group (PcG) proteins are key histone modifiers, whose primary function is gene repression. PcG proteins form complexes called Polycomb Repressive Complexes (PRCs), which catalyze histone modifications such as H2AK119ub1, H3K27me3, and H3K9me3. PcG proteins play a crucial role during differentiation of stem cells. The expression of PcG transcripts during differentiation of hES cells into endoderm, mesoderm, and ectoderm lineage is yet to be shown. In-house derived hES cell line KIND1 was differentiated into endoderm, mesoderm, and ectoderm lineages; followed by characterization using RT-PCR for HNF4A, CDX2, MEF2C, TBX5, SOX1, and MAP2. qRT-PCR and western blotting was performed to compare expression of PcG transcripts and proteins across all the three lineages. We observed that cells differentiated into endoderm showed upregulation of RING1B, BMI1, EZH2, and EED transcripts. Mesoderm differentiation was characterized by significant downregulation of all PcG transcripts during later stages. BMI1 and RING1B were upregulated while EZH2, SUZ12, and EED remained low during ectoderm differentiation. Western blotting also showed distinct expression of BMI1 and EZH2 during differentiation into three germ layers. Our study shows that hES cells differentiating into endoderm, mesoderm, and ectoderm lineages show distinct PcG expression profile at transcript and protein level. © 2015 International Federation for Cell Biology.

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

PubMed

Goppelt-Struebe, M; Reiser, C O; Schneider, N; Grell, M

1996-10-01

Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

PubMed

Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

2017-09-01

Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence.

PubMed

Amatngalim, Gimano D; Schrumpf, Jasmijn A; Dishchekenian, Fernanda; Mertens, Tinne C J; Ninaber, Dennis K; van der Linden, Abraham C; Pilette, Charles; Taube, Christian; Hiemstra, Pieter S; van der Does, Anne M

2018-04-01

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections. Copyright ©ERS 2018.

BAG3 is involved in neuronal differentiation and migration.

PubMed

Santoro, Antonietta; Nicolin, Vanessa; Florenzano, Fulvio; Rosati, Alessandra; Capunzo, Mario; Nori, Stefania L

2017-05-01

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

Proteomics Analysis of Tissue Samples Reveals Changes in Mitochondrial Protein Levels in Parathyroid Hyperplasia over Adenoma

PubMed Central

AKPINAR, GURLER; KASAP, MURAT; CANTURK, NUH ZAFER; ZULFIGAROVA, MEHIN; ISLEK, EYLÜL ECE; GULER, SERTAC ATA; SIMSEK, TURGAY; CANTURK, ZEYNEP

2017-01-01

Background/Aim: To unveil the pathophysiology of primary hyperparathyroidism, molecular details of parathyroid hyperplasia and adenoma have to be revealed. Such details will provide the tools necessary for differentiation of these two look-alike diseases. Therefore, in the present study, a comparative proteomic study using postoperative tissue samples from the parathyroid adenoma and parathyroid hyperplasia patients was performed. Materials and Methods: Protein extracts were prepared from tissue samples (n=8 per group). Protein pools were created for each group and subjected to DIGE and conventional 2DE. Following image analysis, spots representing the differentially regulated proteins were excised from the and used for identification via MALDI-TOF/TOF analysis. Results: The identities of 40 differentially-expressed proteins were revealed. Fourteen of these proteins were over-expressed in the hyperplasia while 26 of them were over-expressed in the adenoma. Conclusion: Most proteins found to be over-expressed in the hyperplasia samples were mitochondrial, underlying the importance of the mitochondrial activity as a potential biomarker for differentiation of parathyroid hyperplasia from adenoma. PMID:28446534

Comparative proteomic expression profile in all-trans retinoic acid differentiated neuroblastoma cell line.

PubMed

Cimmino, Flora; Spano, Daniela; Capasso, Mario; Zambrano, Nicola; Russo, Roberta; Zollo, Massimo; Iolascon, Achille

2007-07-01

Neuroblastoma (NB) is an infant tumor which frequently differentiates into neurons. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) to analyze the cytosolic and nuclear protein expression patterns of LAN-5 cells following neuronal differentiating agent all-trans-retinoic acid treatment. We identified several candidate proteins, from which G beta2 and Prefoldin 3 may have a role on NB development. These results strength the use of proteomics to discover new putative protein targets in cancer.

[Plasma proteomic analysis in children with infectious mononucleosis].

PubMed

Ran, Zhi-Ling; Xiao, Bin; Liu, Hong-Rui; Liu, You-Ping; Sheng, Qiao-Ni

2015-03-01

To explore the abnormal expression of plasma proteins by analysis of proteomic expression profile in children with infectious mononucleosis (IM). Two dimensional gel electrophoresis (2-DE) followed by the mass spectrometry was used to examine important protein spots with different expression levels between children with IM and normal controls. Seven differential proteins were obtained: hemopexin, vitamin D binding protein, fetuin A, C-reactive protein, apolipoprotein A, haptoglobin and transthyretin. Compared with the control group, haptoglobin showed a higher expression level in children with IM, and the expression levels of the other proteins were obviously down-regulated. The expression changes of differential proteins identified in this study are all related with the liver acute injury, suggesting that children with IM are associated with acute liver injury. Further studies on the characteristics of above proteins will contribute to the diagnosis and treatment of pediatric IM.

Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice.

PubMed

Quispe-Salcedo, Angela; Ida-Yonemochi, Hiroko; Nakatomi, Mitsushiro; Ohshima, Hayato

2012-04-01

Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.

Differential Expression of Zinc Transporters in Prostate Epithelia of Racial Groups

DTIC Science & Technology

2012-09-01

of significant genes or proteins in the prostate cancers taken from AAs versus those from European Americans (EAs)?” Because there is a well...may be differentially expressed in AAs and EAs. A study of the genes and proteins which influence the expression of any gene confirmed to be...type of TMA, based on long term clinical follow up was used to address the question of whether hZIP gene and protein expression is associated with

A zinc finger protein Zfp521 directs neural differentiation and beyond

PubMed Central

2011-01-01

Neural induction is largely considered a default process, whereas little is known about intrinsic factors that drive neural differentiation. Kamiya and colleagues now demonstrate that a transcription factor, Zfp521, is capable of directing embryonic stem (ES) cells into neural progenitors. They discovered that Zfp521 transcripts were enriched in early neural lineage of ES cell differentiation. Forced expression of Zfp521 turned ES cells into neural progenitors in culture conditions that would normally inhibit neural differentiation. Zfp521 was expressed in mouse embryos during gastrulation. The protein was shown to associate with a co-activator p300 and directly induce expression of early neural genes. Knockdown of the Zfp521 by shRNA halted cells at the epiblast stage and suppressed neural differentiation. Zfp521 is a nuclear protein with 30 Krüppel-like zinc fingers mediating multiple protein-protein interactions, and regulates transcription in diverse tissues and organs. The protein promotes proliferation, delays differentiation and reduces apoptosis. The findings by Kamiya and colleagues that Zfp521 directs and sustains early neural differentiation now opens up a series of studies to investigate roles of Zfp521 in stem cells and brain development of mice and men. PMID:21539723

Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

PubMed Central

Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

2014-01-01

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability. PMID:25050624

miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

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

McKenna, Declan J., E-mail: dj.mckenna@ulster.ac.uk; Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast BT9 7BL; Patel, Daksha, E-mail: d.patel@qub.ac.uk

2014-01-05

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in themore » cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.« less

RNA-binding Protein Quaking Stabilizes Sirt2 mRNA during Oligodendroglial Differentiation*

PubMed Central

Thangaraj, Merlin P.; Furber, Kendra L.; Gan, Jotham K.; Ji, Shaoping; Sobchishin, Larhonda; Doucette, J. Ronald; Nazarali, Adil J.

2017-01-01

Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3′ untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation. PMID:28188285

Comparative 2D-DIGE Proteomic Analysis of Bovine Mammary Epithelial Cells during Lactation Reveals Protein Signatures for Lactation Persistency and Milk Yield

PubMed Central

Janjanam, Jagadeesh; Singh, Surender; Jena, Manoj K.; Varshney, Nishant; Kola, Srujana; Kumar, Sudarshan; Kaushik, Jai K.; Grover, Sunita; Dang, Ajay K.; Mukesh, Manishi; Prakash, B. S.; Mohanty, Ashok K.

2014-01-01

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling. PMID:25111801

Proteomic changes during intestinal cell maturation in vivo

PubMed Central

Chang, Jinsook; Chance, Mark R.; Nicholas, Courtney; Ahmed, Naseem; Guilmeau, Sandra; Flandez, Marta; Wang, Donghai; Byun, Do-Sun; Nasser, Shannon; Albanese, Joseph M.; Corner, Georgia A.; Heerdt, Barbara G.; Wilson, Andrew J.; Augenlicht, Leonard H.; Mariadason, John M.

2008-01-01

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated. PMID:18824147

Soluble soy protein peptic hydrolysate stimulates adipocyte differentiation in 3T3-L1 cells.

PubMed

Goto, Tsuyoshi; Mori, Ayaka; Nagaoka, Satoshi

2013-08-01

The molecular mechanisms underlying the potential health benefit effects of soybean proteins on obesity-associated metabolic disorders have not been fully clarified. In this study, we investigated the effects of soluble soybean protein peptic hydrolysate (SPH) on adipocyte differentiation by using 3T3-L1 murine preadipocytes. The addition of SPH increased lipid accumulation during adipocyte differentiation. SPH increased the mRNA expression levels of an adipogenic marker gene and decreased that of a preadipocyte marker gene, suggesting that SPH promotes adipocyte differentiation. SPH induced antidiabetic and antiatherogenic adiponectin mRNA expression and secretion. Moreover, SPH increased the mRNA expression levels of insulin-responsive glucose transporter 4 and insulin-stimulated glucose uptake. The expression levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, during adipocyte differentiation were up-regulated in 3T3-L1 cells treated with SPH, and lipid accumulation during adipocyte differentiation induced by SPH was inhibited in the presence of a PPARγ antagonist. However, SPH did not exhibit PPARγ ligand activity. These findings indicate that SPH stimulates adipocyte differentiation, at least in part, via the up-regulation of PPARγ expression levels. These effects of SPH might be important for the health benefit effects of soybean proteins on obesity-associated metabolic disorders. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential proteome analysis of the cell differentiation regulated by BCC, CRH, CXCR4, GnRH, GPCR, IL1 signaling pathways in Chinese fire-bellied newt limb regeneration.

PubMed

Geng, Xiaofang; Xu, Tiantian; Niu, Zhipeng; Zhou, Xiaochun; Zhao, Lijun; Xie, Zhaohui; Xue, Deming; Zhang, Fuchun; Xu, Cunshuan

2014-01-01

Following amputation, the newt has the remarkable ability to regenerate its limb, and this process involves dedifferentiation, proliferation and differentiation. To investigate the potential proteome during a dynamic network of Chinese fire-bellied newt limb regeneration (CNLR), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrum (MS) were applied to examine changes in the proteome that occurred at 11 time points after amputation. Meanwhile, several proteins were selected to validate their expression levels by Western blot. The results revealed that 1476 proteins had significantly changed as compared to the control group. Gene Ontology annotation and protein network analysis by Ingenuity Pathway Analysis 9.0 (IPA) software suggested that the differentially expressed proteins were involved in 33 kinds of physiological activities including signal transduction, cell proliferation, cell differentiation, etc. Among these proteins, 407 proteins participated in cell differentiation with 212 proteins in the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte, and 37 proteins participated in signaling pathways of BCC, CRH, CXCR4, GnRH, GPCR and IL1 which regulated cell differentiation and redifferentiation. On the other hand, the signal transduction activity and cell differentiation activity were analyzed by IPA based on the changes in the expression of these proteins. The results showed that BCC, CRH, CXCR4, GnRH, GPCR and IL1 signaling pathways played an important role in regulating the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte during CNLR. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Quantitative proteomic analysis of whey proteins in the colostrum and mature milk of yak (Bos grunniens).

PubMed

Yang, Yongxin; Zhao, Xiaowei; Yu, Shumin; Cao, Suizhong

2015-02-01

Yak (Bos grunniens) is an important natural resource in mountainous regions. To date, few studies have addressed the differences in the protein profiles of yak colostrum and milk. We used quantitative proteomics to compare the protein profiles of whey from yak colostrum and milk. Milk samples were collected from 21 yaks after calving (1 and 28 d). Whey protein profiles were generated through isobaric tag for relative and absolute quantification (iTRAQ)-labelled proteomics. We identified 183 proteins in milk whey; of these, the expression levels of 86 proteins differed significantly between the whey from colostrum and milk. Haemoglobin expression showed the greatest change; its levels were significantly higher in the whey from colostrum than in mature milk whey. Functional analysis revealed that many of the differentially expressed proteins were associated with biological regulation and response to stimuli. Further, eight differentially expressed proteins involved in the complement and coagulation cascade pathway were enriched in milk whey. These findings add to the general understanding of the protein composition of yak milk, suggest potential functions of the differentially expressed proteins, and provide novel information on the role of colostral components in calf survival. © 2014 Society of Chemical Industry.

Gene expression during ovarian differentiation in parasitic and non-parasitic lampreys: implications for fecundity and life history types.

PubMed

Spice, Erin K; Whyard, Steven; Docker, Margaret F

2014-11-01

Lampreys diverged from the jawed vertebrate lineage approximately 500million years ago. Lampreys undergo sex differentiation much later than most other vertebrates, and ovarian differentiation occurs several years before testicular differentiation. The genetic basis of lamprey sex differentiation is of particular interest both because of the phylogenetic importance of lampreys and because of their unusual pattern of sex differentiation. As well, differences between parasitic and non-parasitic lampreys may first become evident at ovarian differentiation. However, nothing is known about the genetic basis of ovarian differentiation in lampreys. This study examined potential differences in gene expression before, during, and after ovarian differentiation in parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey Ichthyomyzonfossor. Eight target genes (17β-hydroxysteroid dehydrogenase, germ cell-less, estrogen receptor β, insulin-like growth factor 1 receptor, daz-associated protein 1, cytochrome c oxidase subunit III, Wilms' tumour suppressor protein 1, and dehydrocholesterol reductase 7) were examined. Northern brook lamprey displayed higher expression of cytochrome c oxidase subunit III, whereas chestnut lamprey displayed higher expression of insulin-like growth factor 1 receptor; these genes may be involved in apoptosis and oocyte growth, respectively. Presumptive male larvae had higher expression of Wilms' tumour suppressor protein 1, which may be involved in the undifferentiated gonad and/or later testicular development. Differentiated females had higher expression of 17β hydroxysteroid dehydrogenase and daz-associated protein 1, which may be involved in female development. This study is the first to identify genes that may be involved in ovarian differentiation and fecundity in lampreys. Copyright © 2014 Elsevier Inc. All rights reserved.

Infection of epithelial cells with dengue virus promotes the expression of proteins favoring the replication of certain viral strains.

PubMed

Martínez-Betancur, Viviana; Marín-Villa, Marcel; Martínez-Gutierrez, Marlén

2014-08-01

Dengue virus (DENV) is the causative agent of dengue and severe dengue. To understand better the dengue virus-host interaction, it is important to determine how the expression of cellular proteins is modified due to infection. Therefore, a comparison of protein expression was conducted in Vero cells infected with two different DENV strains, both serotype 2: DENV-2/NG (associated with dengue) and DENV-2/16681 (associated with severe dengue). The viability of the infected cells was determined, and neither strain induced cell death at 48 hr. In addition, the viral genomes and infectious viral particles were quantified, and the genome of the DENV-2/16681 strain was determined to have a higher replication rate compared with the DENV-2/NG strain. Finally, the proteins from infected and uninfected cultures were separated using two-dimensional gel electrophoresis, and the differentially expressed proteins were identified by mass spectrometry. Compared with the uninfected controls, the DENV-2/NG- and DENV-2/16681-infected cultures had five and six differentially expressed proteins, respectively. The most important results were observed when the infected cultures were compared to each other (DENV-2/NG vs. DENV-2/16681), and 18 differentially expressed proteins were identified. Based on their cellular functions, many of these proteins were linked to the increase in the replication efficiency of DENV. Among the proteins were calreticulin, acetyl coenzyme A, acetyl transferase, and fatty acid-binding protein. It was concluded that the infection of Vero cells with DENV-2/NG or DENV-2/16681 differentially modifies the expression of certain proteins, which can, in turn, facilitate infection. © 2013 Wiley Periodicals, Inc.

Proteomics identification of differentially expressed proteins in the muscle of dysferlin myopathy patients.

PubMed

De la Torre, Carolina; Illa, Isabel; Faulkner, Georgine; Soria, Laura; Robles-Cedeño, Rene; Dominguez-Perles, Raul; De Luna, Noemí; Gallardo, Eduard

2009-04-01

The muscular dystrophies are a large and heterogeneous group of neuromuscular disorders that can be classified according to the mode of inheritance, the clinical phenotype and the molecular defect. To better understand the pathological mechanisms of dysferlin myopathy we compared the protein-expression pattern in the muscle biopsies of six patients with this disease with six patients with limb girdle muscular dystrophy 2A, five with facioscapulohumeral dystrophy and six normal control subjects. To investigate differences in the expression levels of skeletal muscle proteins we used 2-DE and MS. Western blot or immunohistochemistry confirmed relevant results. The study showed specific increase expression of proteins involved in fast-to-slow fiber type conversion (ankyrin repeat protein 2), type I predominance (phosphorylated forms of slow troponin T), sarcomere stabilization (actinin-associated LIM protein), protein ubiquitination (TRIM 72) and skeletal muscle differentiation (Rho-GDP-dissociation inhibitor ly-GDI) in dysferlin myopathy. As anticipated, we also found differential expression of proteins common to all the muscular dystrophies studied. This comparative proteomic analysis suggests that in dysferlin myopathy (i) the type I fiber predominance is an active process of fiber type conversion rather than a selective loss of type II fibers and (ii) the dysregulation of proteins involved in muscle differentiation further confirms the role of dysferlin in this process. Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling.

PubMed

Kandasamy, Saveetha; Loganathan, Karthiba; Muthuraj, Raveendran; Duraisamy, Saravanakumar; Seetharaman, Suresh; Thiruvengadam, Raguchander; Ponnusamy, Balasubramanian; Ramasamy, Samiyappan

2009-12-24

Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

PubMed Central

Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina

2015-01-01

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651

Chronic fluoxetine treatment directs energy metabolism towards the citric acid cycle and oxidative phosphorylation in rat hippocampal nonsynaptic mitochondria.

PubMed

Filipović, Dragana; Costina, Victor; Perić, Ivana; Stanisavljević, Andrijana; Findeisen, Peter

2017-03-15

Fluoxetine (Flx) is the principal treatment for depression; however, the precise mechanisms of its actions remain elusive. Our aim was to identify protein expression changes within rat hippocampus regulated by chronic Flx treatment versus vehicle-controls using proteomics. Fluoxetine-hydrohloride (15mg/kg) was administered daily to adult male Wistar rats for 3weeks, and cytosolic and nonsynaptic mitochondrial hippocampal proteomes were analyzed. All differentially expressed proteins were functionally annotated according to biological process and molecular function using Uniprot and Blast2GO. Our comparative study revealed that in cytosolic and nonsynaptic mitochondrial fractions, 60 and 3 proteins respectively, were down-regulated, and 23 and 60 proteins, respectively, were up-regulated. Proteins differentially regulated in cytosolic and nonsynaptic mitochondrial fractions were primarily related to cellular and metabolic processes. Of the identified proteins, the expressions of calretinin and parvalbumine were confirmed. The predominant molecular functions of differentially expressed proteins in both cell hippocampal fractions were binding and catalytic activity. Most differentially expressed proteins in nonsynaptic mitochondria were catalytic enzymes involved in the pyruvate metabolism, citric acid cycle, oxidative phosphorylation, ATP synthesis, ATP transduction and glutamate metabolism. Results indicate that chronic Flx treatment may influence proteins involved in calcium signaling, cytoskeletal structure, chaperone system and stimulates energy metabolism via the upregulation of GAPDH expression in cytoplasm, as well as directing energy metabolism toward the citric acid cycle and oxidative phosphorylation in nonsynaptic mitochondria. This approach provides new insight into the chronic effects of Flx treatment on protein expression in a key brain region associated with stress response and memory. Copyright © 2017 Elsevier B.V. All rights reserved.

IBT-based quantitative proteomics identifies potential regulatory proteins involved in pigmentation of purple sea cucumber, Apostichopus japonicus.

PubMed

Xing, Lili; Sun, Lina; Liu, Shilin; Li, Xiaoni; Zhang, Libin; Yang, Hongsheng

2017-09-01

Sea cucumbers are an important economic species and exhibit high yield value among aquaculture animals. Purple sea cucumbers are very rare and beautiful and have stable hereditary patterns. In this study, isobaric tags (IBT) were first used to reveal the molecular mechanism of pigmentation in the body wall of the purple sea cucumber. We analyzed the proteomes of purple sea cucumber in early pigmentation stage (Pa), mid pigmentation stage (Pb) and late pigmentation stage (Pc), resulting in the identification of 5580 proteins, including 1099 differentially expressed proteins in Pb: Pa and 339 differentially expressed proteins in Pc: Pb. GO and KEGG analyses revealed possible differentially expressed proteins, including"melanogenesis", "melanosome", "melanoma", "pigment-biosynthetic process", "Epidermis development", "Ras-signaling pathway", "Wnt-signaling pathway", "response to UV light", and "tyrosine metabolism", involved in pigment synthesis and regulation in purple sea cucumbers. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying pigmentation in sea cucumbers. Furthermore, these results may also provide the base for further identification of proteins involved in resistance mechanisms against melanoma, albinism, UV damage, and other diseases in sea cucumbers. Copyright © 2017. Published by Elsevier Inc.

Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells.

PubMed

Zogovic, Nevena; Tovilovic-Kovacevic, Gordana; Misirkic-Marjanovic, Maja; Vucicevic, Ljubica; Janjetovic, Kristina; Harhaji-Trajkovic, Ljubica; Trajkovic, Vladimir

2015-04-01

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, β-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and β-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process. © 2014 International Society for Neurochemistry.

Identification of Proteins Differentially Expressed by Quercetin Treatment in a Middle Cerebral Artery Occlusion Model: A Proteomics Approach.

PubMed

Shah, Fawad-Ali; Park, Dong-Ju; Koh, Phil-Ok

2018-06-20

Cerebral ischemia is a major cause of death and neurological disability. It also leads to severe brain tissue damage by excessive generation of oxidative stress. Quercetin is a bioflavonoid substance that acts an antioxidant agent and exerts a neuroprotective effect against cerebral ischemia. The aim of this study was to detect specific proteins that are differentially expressed in response to quercetin treatment in focal cerebral ischemia. Adult male rats were intraperitoneally injected with vehicle or quercetin (10 mg/kg) 30 min prior to right middle cerebral artery occlusion (MCAO). Brain tissues were collected 24 h after MCAO surgery and right cerebral cortices proteins were identified by two-dimensional gel electrophoresis and mass spectrometry. MCAO leads to neurological behavior disorders, infarction, and histopathological change. However, quercetin treatment alleviated MCAO-induced neuronal deficits and damages. We identified specific proteins differentially expressed between vehicle- and quercetin-treated animals. Among these detected proteins, isocitrate dehydrogenase [NAD + ], adenosylhomocysteinase, pyruvate kinase, and ubiquitin carboxy terminal hydrolase L1 were decreased in vehicle-treated animals, while quercetin administration alleviated the MCAO-induced decreases in these proteins. However, 60 kDa heat shock protein and collapsin response mediator protein 2 were increased in the vehicle-treated animals, and quercetin treatment attenuated increases in these proteins. The expression changes in these proteins were confirmed by Western blot and reverse transcription-PCR analyses. These proteins are associated with cellular differentiation, metabolism, and oxidative stress related proteins. These results suggest that quercetin reduces ischemic injury by modulating the expression of various proteins in focal cerebral ischemia.

Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

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

Walsh, Erica M.; Niu, MengMeng; Bergholz, Johann

The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification.more » In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.« less

Proteomic Analysis of Apis cerana and Apis mellifera Larvae Fed with Heterospecific Royal Jelly and by CSBV Challenge

PubMed Central

Huang, Xiu; Han, Richou

2014-01-01

Chinese honeybee Apis cerana (Ac) is one of the major Asian honeybee species for local apiculture. However, Ac is frequently damaged by Chinese sacbrood virus (CSBV), whereas Apis mellifera (Am) is usually resistant to it. Heterospecific royal jelly (RJ) breeding in two honeybee species may result in morphological and genetic modification. Nevertheless, knowledge on the resistant mechanism of Am to this deadly disease is still unknown. In the present study, heterospecific RJ breeding was conducted to determine the effects of food change on the larval mortality after CSBV infection at early larval stage. 2-DE and MALDI-TOF/TOF MS proteomic technology was employed to unravel the molecular event of the bees under heterospecific RJ breeding and CSBV challenge. The change of Ac larval food from RJC to RJM could enhance the bee resistance to CSBV. The mortality rate of Ac larvae after CSBV infection was much higher when the larvae were fed with RJC compared with the larvae fed with RJM. There were 101 proteins with altered expressions after heterospecific RJ breeding and viral infection. In Ac larvae, 6 differential expression proteins were identified from heterospecific RJ breeding only, 21 differential expression proteins from CSBV challenge only and 7 differential expression proteins from heterospecific RJ breeding plus CSBV challenge. In Am larvae, 17 differential expression proteins were identified from heterospecific RJ breeding only, 26 differential expression proteins from CSBV challenge only and 24 differential expression proteins from heterospecific RJ breeding plus CSBV challenge. The RJM may protect Ac larvae from CSBV infection, probably by activating the genes in energy metabolism pathways, antioxidation and ubiquitin-proteasome system. The present results, for the first time, comprehensively descript the molecular events of the viral infection of Ac and Am after heterospecific RJ breeding and are potentially useful for establishing CSBV resistant populations of Ac for apiculture. PMID:25102167

Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation.

PubMed

Liu, Chang; Tong, Huili; Li, Shufeng; Yan, Yunqin

2018-05-01

Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern in bovine MDSCs during differentiation in vitro. CRISPR/Cas9 technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation of bovine MDSCs. ECM2 expression was shown to increase gradually during bovine MDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression during bovine MDSC differentiation; these results could lead to treatments that help to increase beef cattle muscularity. © 2018 International Federation for Cell Biology.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

PubMed

Cordeiro, Fabio Aparecido; Tadra-Sfeir, Michelle Zibetti; Huergo, Luciano Fernandes; de Oliveira Pedrosa, Fábio; Monteiro, Rose Adele; de Souza, Emanuel Maltempi

2013-03-01

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.

The altered expression of perineuronal net elements during neural differentiation.

PubMed

Eskici, Nazli F; Erdem-Ozdamar, Sevim; Dayangac-Erden, Didem

2018-01-01

Perineuronal nets (PNNs), which are localized around neurons during development, are specialized forms of neural extracellular matrix with neuroprotective and plasticity-regulating roles. Hyaluronan and proteoglycan link protein 1 (HAPLN1), tenascin-R (TNR) and aggrecan (ACAN) are key elements of PNNs. In diseases characterized by neuritogenesis defects, the expression of these proteins is known to be downregulated, suggesting that PNNs may have a role in neural differentiation. In this study, the mRNA and protein levels of HAPLN1, TNR and ACAN were determined and compared at specific time points of neural differentiation. We used PC12 cells as the in vitro model because they reflect this developmental process. On day 7, the HAPLN1 mRNA level showed a 2.9-fold increase compared to the non-differentiated state. However, the cellular HAPLN1 protein level showed a decrease, indicating that the protein may have roles in neural differentiation, and may be secreted during the early period of differentiation. By contrast, TNR mRNA and protein levels remained unchanged, and the amount of cellular ACAN protein showed a 3.7-fold increase at day 7. These results suggest that ACAN may be secreted after day 7, possibly due to its large amount of post-translational modifications. Our results provide preliminary data on the expression of PNN elements during neural differentiation. Further investigations will be performed on the role of these elements in neurological disease models.

Genome-wide methylation and gene expression changes in newborn rats following maternal protein restriction and reversal by folic acid.

PubMed

Altobelli, Gioia; Bogdarina, Irina G; Stupka, Elia; Clark, Adrian J L; Langley-Evans, Simon

2013-01-01

A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures.

Exocyst Complex Protein Expression in the Human Placenta

PubMed Central

Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

2014-01-01

Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

Proteomic and Transcriptomic Analysis of Aspergillus fumigatus on Exposure to Amphotericin B▿ †

PubMed Central

Gautam, Poonam; Shankar, Jata; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Gade, Wasudev Namdeo; Basir, Seemi Farhat; Sarma, Puranam Usha

2008-01-01

Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development. PMID:18838595

Proteomic and transcriptomic analysis of Aspergillus fumigatus on exposure to amphotericin B.

PubMed

Gautam, Poonam; Shankar, Jata; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Gade, Wasudev Namdeo; Basir, Seemi Farhat; Sarma, Puranam Usha

2008-12-01

Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development.

Proteomic changes during adult stage in pre-optic, hypothalamus, hippocampus and pituitary regions of female rat brain following neonatal exposure to estradiol-17β.

PubMed

Govindaraj, Vijayakumar; Shridharan, Radhika Nagamangalam; Rao, Addicam Jagannadha

2018-05-16

Although neonatal exposure to estrogen or estrogenic compounds results in irreversible changes in the brain function and reproductive abnormalities during adulthood but the underlying mechanisms are still largely unknown. The present study has attempted to compare the protein profiles of sexually dimorphic brain regions of adult female rats which were exposed to estradiol- 17β during neonatal period. The total proteins extracted from pre-optic area (POA), hypothalamus, hippocampus and pituitary of control and neonatally E2 treated female rats was subjected to 2D-SDS-PAGE and differentially expressed proteins were identified by MALDI TOF/TOF-MS. Our results revealed that a total of 21 protein spots which were identified as differentially expressed in all the four regions analyzed; the differential expression was further validated by RT-PCR and western blotting. The differentially expressed proteins such as 14-3-3 zeta/delta (POA), LMNA (hippocampus), Axin2 (hypothalamus), Syntaxin-7 (hippocampus), prolactin and somatotropin (pituitary) which have very important functions in the process of neuronal differentiation, migration, axon outgrowth, formation of dendritic spine density and synaptic plasticity and memory have not been previously reported in association with neonatal estrogen exposure. The affected brain functions are very important for the establishment of sex specific brain morphology and behavior. Our results suggest that the differentially expressed proteins may play an important role in irreversible changes in the brain function as well as reproductive abnormalities observed in the female rats during adulthood. Copyright © 2018 Elsevier Inc. All rights reserved.

Proteomic Analysis and qRT-PCR Verification of Temperature Response to Arthrospira (Spirulina) platensis

PubMed Central

Huili, Wang; Xiaokai, Zhao; Meili, Lin; Dahlgren, Randy A.; Wei, Chen; Jaiopeng, Zhou; Chengyang, Xu; Chunlei, Jin; Yi, Xu; Xuedong, Wang; Li, Ding; Qiyu, Bao

2013-01-01

Arthrospira (Spirulina) platensis (ASP) is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C) using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328). The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones), energy metabolism (photosynthesis, respiratory electron transport), translation (ribosomal structure and biogenesis) and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and translation levels. The remaining 12 genes showed inconsistent protein expression with transcription level and accounted for 31.6% of the total target genes. PMID:24349519

Proteomic Analysis of Saliva Identifies Potential Biomarkers for Orthodontic Tooth Movement

PubMed Central

Ellias, Mohd Faiz; Zainal Ariffin, Shahrul Hisham; Karsani, Saiful Anuar; Abdul Rahman, Mariati; Senafi, Shahidan; Megat Abdul Wahab, Rohaya

2012-01-01

Orthodontic treatment has been shown to induce inflammation, followed by bone remodelling in the periodontium. These processes trigger the secretion of various proteins and enzymes into the saliva. This study aims to identify salivary proteins that change in expression during orthodontic tooth movement. These differentially expressed proteins can potentially serve as protein biomarkers for the monitoring of orthodontic treatment and tooth movement. Whole saliva from three healthy female subjects were collected before force application using fixed appliance and at 14 days after 0.014′′ Niti wire was applied. Salivary proteins were resolved using two-dimensional gel electrophoresis (2DE) over a pH range of 3–10, and the resulting proteome profiles were compared. Differentially expressed protein spots were then identified by MALDI-TOF/TOF tandem mass spectrometry. Nine proteins were found to be differentially expressed; however, only eight were identified by MALDI-TOF/TOF. Four of these proteins—Protein S100-A9, immunoglobulin J chain, Ig alpha-1 chain C region, and CRISP-3—have known roles in inflammation and bone resorption. PMID:22919344

ITRAQ-based quantitative proteomic analysis of Cynops orientalis limb regeneration.

PubMed

Tang, Jie; Yu, Yuan; Zheng, Hanxue; Yin, Lu; Sun, Mei; Wang, Wenjun; Cui, Jihong; Liu, Wenguang; Xie, Xin; Chen, Fulin

2017-09-22

Salamanders regenerate their limbs after amputation. However, the molecular mechanism of this unique regeneration remains unclear. In this study, isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to quantitatively identify differentially expressed proteins in regenerating limbs 3, 7, 14, 30 and 42 days post amputation (dpa). Of 2636 proteins detected in total, 253 proteins were differentially expressed during different regeneration stages. Among these proteins, Asporin, Cadherin-13, Keratin, Collagen alpha-1(XI) and Titin were down-regulated. CAPG, Coronin-1A, AnnexinA1, Cathepsin B were up-regulated compared with the control. The identified proteins were further analyzed to obtain information about their expression patterns and functions in limb regeneration. Functional analysis indicated that the differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding. This work indicated that significant proteome alternations occurred during salamander limb regeneration. The results may provide fundamental knowledge to understand the mechanism of limb regeneration.

Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes.

PubMed

Lechner, Stefan; Mitterberger, Maria C; Mattesich, Monika; Zwerschke, Werner

2013-01-01

We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes. Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling

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

Liu, Chang; Chen, Lin; Zeng, Jing

Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observedmore » that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling. - Highlights: • CBDL-rat serum promotes the myogenic differentiaton and expression of BMP2 in PMVECs. • CBDL-rat serum activates the BMP2/smad signaling pathway. • The downregulation of Smurf1 stimulates the accumulation of Smad1/5 in PMVECs. • Noggin reverses partially the myogenic differentiaton in PMVECs.« less

[Role of CD2-associated protein in podocyte differentiation.].

PubMed

Jiang, Hua-Jun; Chang, Ying; Zhu, Zhong-Hua; Liu, Jian-She; Deng, An-Guo; Zhang, Chun

2008-02-25

To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.

Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.).

PubMed

Wang, Shuzhen; Chen, Wenyue; Xiao, Wenfei; Yang, Changdeng; Xin, Ya; Qiu, Jieren; Hu, Weimin; Ying, Wu; Fu, Yaping; Tong, Jianxin; Hu, Guocheng; Chen, Zhongzhong; Fang, Xianping; Yu, Hong; Lai, Wenguo; Ruan, Songlin; Ma, Huasheng

2015-01-01

Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesium-chelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various growth stages and chlorophyll biosynthesis pathway related proteins, especially magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), may provide new insights into the molecular mechanisms of rice hull development and chlorophyll associated regulation.

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation.

PubMed

Tapia, Olga; Gerace, Larry

2016-01-01

We describe straightforward methodology for structure-function mapping of nuclear lamina proteins in myoblast differentiation, using populations of C2C12 myoblasts in which the endogenous lamina components are replaced with ectopically expressed mutant versions of the proteins. The procedure involves bulk isolation of C2C12 cell populations expressing the ectopic proteins by lentiviral transduction, followed by depletion of the endogenous proteins using siRNA, and incubation of cells under myoblast differentiation conditions. Similar methodology may be applied to mouse embryo fibroblasts or to other cell types as well, for the identification and characterization of sequences of lamina proteins involved in functions that can be measured biochemically or cytologically.

Identification of differentially expressed genes in childhood asthma.

PubMed

Zhang, Nian-Zhen; Chen, Xiu-Juan; Mu, Yu-Hua; Wang, Hewen

2018-05-01

Asthma has been the most common chronic disease in children that places a major burden for affected people and their families.An integrated analysis of microarrays studies was performed to identify differentially expressed genes (DEGs) in childhood asthma compared with normal control. We also obtained the differentially methylated genes (DMGs) in childhood asthma according to GEO. The genes that were both differentially expressed and differentially methylated were identified. Functional annotation and protein-protein interaction network construction were performed to interpret biological functions of DEGs. We performed q-RT-PCR to verify the expression of selected DEGs.One DNA methylation and 3 gene expression datasets were obtained. Four hundred forty-one DEGs and 1209 DMGs in childhood asthma were identified. Among which, 16 genes were both differentially expressed and differentially methylated in childhood asthma. Natural killer cell mediated cytotoxicity pathway, Jak-STAT signaling pathway, and Wnt signaling pathway were 3 significantly enriched pathways in childhood asthma according to our KEGG enrichment analysis. The PPI network of top 20 up- and downregulated DEGs consisted of 822 nodes and 904 edges and 2 hub proteins (UBQLN4 and MID2) were identified. The expression of 8 DEGs (GZMB, FGFBP2, CLC, TBX21, ALOX15, IL12RB2, UBQLN4) was verified by qRT-PCR and only the expression of GZMB and FGFBP2 was inconsistent with our integrated analysis.Our finding was helpful to elucidate the underlying mechanism of childhood asthma and develop new potential diagnostic biomarker and provide clues for drug design.

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

PubMed Central

2009-01-01

Background Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion. PMID:20034395

Understanding mechanism of sea cucumber Apostichopus japonicus aestivation: Insights from TMT-based proteomic study.

PubMed

Chen, Muyan; Li, Xingke; Zhu, Aijun; Storey, Kenneth B; Sun, Lina; Gao, Tianxiang; Wang, Tianming

2016-09-01

Marine invertebrate aestivation is a unique strategy for summer survival in response to hot marine conditions. The sea cucumber, Apostichopus japonicus, is an excellent model marine invertebrate for studies of environmentally-induced aestivation. In the present study, we used a tandem mass tag (TMT)-coupled LC-MS/MS approach to identify and quantify the global proteome expression profile over the aestivation-arousal cycle of A. japonicus. A total of 3920 proteins were identified from the intestine of sea cucumber. Among them, 630 proteins showed significant differential expression when comparing three conditions of sea cucumbers: non-aestivating (active), deep-aestivation (at least 15days of continuous aestivation), and arousal after aestivation (renewed moving and feeding). Sea cucumbers in deep aestivation showed substantial differentially expressed proteins (143 up-regulated and 267 down-regulated proteins compared with non-aestivating controls). These differentially expressed proteins suggested that protein and phospholipid probably are major fuel sources during hypometabolism and a general attenuation of carbohydrate metabolism was observed during deep aestivation. Differentially expressed proteins also provided the first global picture of a shift in protein synthesis, protein folding, DNA binding, apoptosis, cellular transport and signaling, and cytoskeletal proteins during deep aestivation in sea cucumbers. A comparison of arousal from aestivation with deep aestivation, revealed a general reversal of the changes that occurred in aestivation for most proteins. Western blot detection further validated the significant up-regulation of HSP70 and down-regulation of methyltransferase-like protein 7A-like in deep-aestivation. Our results suggest that there is substantial post-transcriptional regulation of proteins during the aestivation-arousal cycle in sea cucumbers. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein Profiles Reveal Diverse Responsive Signaling Pathways in Kernels of Two Maize Inbred Lines with Contrasting Drought Sensitivity

PubMed Central

Yang, Liming; Jiang, Tingbo; Fountain, Jake C.; Scully, Brian T.; Lee, Robert D.; Kemerait, Robert C.; Chen, Sixue; Guo, Baozhu

2014-01-01

Drought stress is a major factor that contributes to disease susceptibility and yield loss in agricultural crops. To identify drought responsive proteins and explore metabolic pathways involved in maize tolerance to drought stress, two maize lines (B73 and Lo964) with contrasting drought sensitivity were examined. The treatments of drought and well water were applied at 14 days after pollination (DAP), and protein profiles were investigated in developing kernels (35 DAP) using iTRAQ (isobaric tags for relative and absolute quantitation). Proteomic analysis showed that 70 and 36 proteins were significantly altered in their expression under drought treatments in B73 and Lo964, respectively. The numbers and levels of differentially expressed proteins were generally higher in the sensitive genotype, B73, implying an increased sensitivity to drought given the function of the observed differentially expressed proteins, such as redox homeostasis, cell rescue/defense, hormone regulation and protein biosynthesis and degradation. Lo964 possessed a more stable status with fewer differentially expressed proteins. However, B73 seems to rapidly initiate signaling pathways in response to drought through adjusting diverse defense pathways. These changes in protein expression allow for the production of a drought stress-responsive network in maize kernels. PMID:25334062

Orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) protein negatively regulates bone morphogenetic protein 2-induced osteoblast differentiation through suppressing runt-related gene 2 (Runx2) activity.

PubMed

Lee, Kkot-Nim; Jang, Won-Gu; Kim, Eun-Jung; Oh, Sin-Hye; Son, Hye-Ju; Kim, Sun-Hun; Franceschi, Renny; Zhang, Xiao-Kun; Lee, Shee-Eun; Koh, Jeong-Tae

2012-06-01

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

Differentially expressed proteins in nitric oxide-stimulated NIH/3T3 fibroblasts: implications for inhibiting cancer development.

PubMed

Shim, Dong Hwi; Lim, Joo Weon; Kim, Hyeyoung

2015-03-01

Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. The cells were treated with 300 μM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (α1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (ε subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (ε subunit) are unknown in relation to carcinogenesis. Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

Transcriptional profiling of murine osteoblast differentiation based on RNA-seq expression analyses.

PubMed

Khayal, Layal Abo; Grünhagen, Johannes; Provazník, Ivo; Mundlos, Stefan; Kornak, Uwe; Robinson, Peter N; Ott, Claus-Eric

2018-04-11

Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology. Copyright © 2018 Elsevier Inc. All rights reserved.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

PubMed

Gai, Yingping; Yuan, Shuo-Shuo; Liu, Zhao-Yang; Zhao, Huai-Ning; Liu, Qi; Qin, Yong-Li; Fang, Li-Jing; Ji, Xian-Ling

2018-05-30

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signalling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 ( MuMLPL329 ) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 ( Pst. DC3000 ) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative proteomic analysis in children with idiopathic short stature (ISS) before and after short-term recombinant human growth hormone (rhGH) therapy.

PubMed

Heo, Sun Hee; Choi, Jin-Ho; Kim, Yoo-Mi; Jung, Chang-Woo; Lee, Jin; Jin, Hye Young; Kim, Gu-Hwan; Lee, Beom Hee; Shin, Choong Ho; Yoo, Han-Wook

2013-04-01

This study was undertaken to identify growth hormone (GH) responsive proteins and protein expression patterns by short-term recombinant human growth hormone (rhGH) therapy in patients with idiopathic short stature (ISS) using proteomic analysis. Seventeen children (14 males and three females) with ISS were included. They were treated with rhGH at a dose of 0.31 ± 0.078 mg/kg/week for 3 months. Immunodepletion of six highly-abundant serum proteins followed by 2D DIGE analysis, and subsequent MALDI TOF MS, were employed to generate a panel of proteins differentially expressed after short-term rhGH therapy and verify the differences in serum levels of specific proteins by rhGH therapy. Fourteen spots were differentially expressed after rhGH treatment. Among them, apo E and apo L-1 expression were consistently enhanced, whereas serum amyloid A was reduced after rhGH therapy. The differential expressions of these proteins were subsequently verified by Western blot analysis using sera of the before and after rhGH treatment. This study suggests that rhGH therapy influences lipoprotein metabolism and enhances apo L-1 protein expression in ISS patients. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

PubMed Central

Zhang, Xianghui; Fu, Jianming; Hiromasa, Yasuaki; Pan, Hongyu; Bai, Guihua

2013-01-01

Background Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1. Methods The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1+NIL were also compared to identify pathogen-responsive proteins. Results Eight proteins were either induced or upregulated in inoculated Fhb1+NIL when compared with mock-inoculated Fhb1+NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1+NIL when compared with Fusarium-inoculated Fhb1−NIL. Proteins that were differentially expressed in the Fhb1+NIL, not in the Fhb1−NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification. Conclusions Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1+NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance. PMID:24376514

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

He, Yonghan; Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3; State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223

Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 daysmore » of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.« less

Aberrant Expression of Retinoic Acid Signaling Molecules Influences Patient Survival in Astrocytic Gliomas

PubMed Central

Campos, Benito; Centner, Franz-Simon; Bermejo, Justo Lorenzo; Ali, Ramadan; Dorsch, Katharina; Wan, Feng; Felsberg, Jörg; Ahmadi, Rezvan; Grabe, Niels; Reifenberger, Guido; Unterberg, Andreas; Burhenne, Jürgen; Herold-Mende, Christel

2011-01-01

Undifferentiated cell populations may influence tumor growth in malignant glioma. We investigated potential disruptions in the retinoic acid (RA) differentiation pathway that could lead to a loss of differentiation capacity, influencing patient prognosis. Expression of key molecules belonging to the RA differentiation pathway was analyzed in 283 astrocytic gliomas and was correlated with tumor proliferation, tumor differentiation, and patient survival. In addition, in situ concentrations of retinoids were measured in tumors, and RA signaling events were studied in vitro. Unlike other tumors, in gliomas expression of most RA signaling molecules increased with malignancy and was associated with augmented intratumoral retinoid levels in high-grade gliomas. Aberrantly expressed RA signaling molecules included i) the retinol-binding protein CRBP1, which facilitates cellular retinoid uptake; ii) ALDH1A1, capable of activating RA precursors; iii) the RA-degrading enzyme CYP26B1; and iv) the RA-binding protein FABP5, which can inhibit RA-induced differentiation. In contrast, expression of the RA-binding protein CRABP2, which fosters differentiation, was decreased in high-grade tumors. Moreover, expression of CRBP1 correlated with tumor proliferation, and FABP5 expression correlated with an undifferentiated tumor phenotype. CRBP1 and ALDH1A1 were independent prognostic markers for adverse patient survival. Our data indicate a complex and clinically relevant deregulation of RA signaling, which seems to be a central event in glioma pathogenesis. PMID:21514413

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

PubMed Central

Christmann, Martin; Friesenhagen, Judith; Westphal, Andreas; Pietsch, Daniel; Brand, Korbinian

2015-01-01

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation. PMID:26646662

A novel protein expression signature differentiates benign lipomas from well-differentiated liposarcomas.

PubMed

Mather, Quang; Priego, Jonathon; Ward, Kristi; Kundan, Verma; Tran, Dat; Dwivedi, Alok; Bryan, Brad A

2017-09-01

Benign lipomas and well-differentiated liposarcomas share many histological and molecular features. Due to their similarities, patients with these lipomatous tumors are misdiagnosed up to 40% of the time following radiological detection, up to 17% of the time following histological examination, and in as many as 15% of cases following fluorescent in situ hybridization for chromosomal anomalies. Incorrect classification of these two tumor types leads to increased costs to the patient and delayed accurate diagnoses. In this study, we used genomics analysis to identify several genes whose mRNA expression patterns were significantly altered between lipomas and well-differentiated liposarcomas. We confirmed our findings at the protein level using a panel of 30 human lipomatous tumors, revealing that C4BPB, class II, major histocompatibility complex, CIITA, EPHB2, HOXB7, GLS2, RBBP5, and regulator of RGS2 protein levels were increased in well-differentiated liposarcomas compared to lipomas. We developed a multi-protein model of these markers to increase discriminatory ability, finding the combined expression model with CIITA and RGS2 provided a high ability (AUC=0.93) to differentiate between lipomas and well-differentiated liposarcomas with sensitivity at 83.3% and specificity at 90.9%.

Specificity protein 1 regulates topoisomerase IIβ expression in SH-SY5Y cells during neuronal differentiation.

PubMed

Guo, Hui; Cao, Cuili; Chi, Xueqian; Zhao, Junxia; Liu, Xia; Zhou, Najing; Han, Shuo; Yan, Yongxin; Wang, Yanling; Xu, Yannan; Yan, Yunli; Cui, Huixian; Sun, Hongxia

2014-10-01

Topoisomerase IIβ (top IIβ) is a nuclear enzyme with an essential role in neural development. The regulation of top IIβ gene expression during neural differentiation is poorly understood. Functional analysis of top IIβ gene structure displayed a GC box sequence in its transcription promoter, which binds the nuclear transcription factor specificity protein 1 (Sp1). Sp1 regulates gene expression via multiple mechanisms and is essential for early embryonic development. This study seeks to determine whether Sp1 regulates top IIβ gene expression during neuronal differentiation. For this purpose, human neuroblastoma SH-SY5Y cells were induced to neuronal differentiation in the presence of all-trans retinoic acid (RA) for 5 days. After incubation with 10 μM RA for 3-5 days, a majority of the cells exited the cell cycle to become postmitotic neurons, characterized by the presence of longer neurite outgrowths and expression of the neuronal marker microtubule-associated protein-2 (MAP2). Elevated Sp1 and top IIβ mRNA and protein levels were detected and found to be positively correlated with the differentiation stage. Chromatin immunoprecipitation assay demonstrated an increased recruitment of Sp1 to the top IIβ promoter after RA treatment. Mithramycin A, a compound that interferes with Sp1 binding to GC-rich DNA sequences, downregulated the expression of top IIβ, resulting in reduced expression of MAP2 and decreased neurite length compared with the control group. Our results indicate that Sp1 regulates top IIβ expression by binding to the GC box of the gene promoter during neuronal differentiation in SH-SY5Y cells. © 2014 Wiley Periodicals, Inc.

Transcriptome interrogation of human myometrium identifies differentially expressed sense-antisense pairs of protein-coding and long non-coding RNA genes in spontaneous labor at term.

PubMed

Romero, Roberto; Tarca, Adi L; Chaemsaithong, Piya; Miranda, Jezid; Chaiworapongsa, Tinnakorn; Jia, Hui; Hassan, Sonia S; Kalita, Cynthia A; Cai, Juan; Yeo, Lami; Lipovich, Leonard

2014-09-01

To identify differentially expressed long non-coding RNA (lncRNA) genes in human myometrium in women with spontaneous labor at term. Myometrium was obtained from women undergoing cesarean deliveries who were not in labor (n = 19) and women in spontaneous labor at term (n = 20). RNA was extracted and profiled using an Illumina® microarray platform. We have used computational approaches to bound the extent of long non-coding RNA representation on this platform, and to identify co-differentially expressed and correlated pairs of long non-coding RNA genes and protein-coding genes sharing the same genomic loci. We identified co-differential expression and correlation at two genomic loci that contain coding-lncRNA gene pairs: SOCS2-AK054607 and LMCD1-NR_024065 in women in spontaneous labor at term. This co-differential expression and correlation was validated by qRT-PCR, an experimental method completely independent of the microarray analysis. Intriguingly, one of the two lncRNA genes differentially expressed in term labor had a key genomic structure element, a splice site, that lacked evolutionary conservation beyond primates. We provide, for the first time, evidence for coordinated differential expression and correlation of cis-encoded antisense lncRNAs and protein-coding genes with known as well as novel roles in pregnancy in the myometrium of women in spontaneous labor at term.

Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization.

PubMed

Wu, Yun H; Wu, Tsung M; Hong, Chwan Y; Wang, Yei S; Yen, Jui H

2014-01-01

Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger-containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism.

Identification of inflammation-related proteins in a murine colitis model by 2D fluorescence difference gel electrophoresis and mass spectrometry.

PubMed

Naito, Yuji; Takagi, Tomohisa; Okada, Hitomi; Omatsu, Tatsushi; Mizushima, Katsura; Handa, Osamu; Kokura, Satoshi; Ichikawa, Hiroshi; Fujiwake, Hideshi; Yoshikawa, Toshikazu

2010-05-01

The aim of this study was to identify new intestinal proteins potentially associated with acute inflammation using proteomic profiling of an in vivo mice model of ulcerative colitis. 2D fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight spectrometer (MALDI-TOF) peptide mass fingerprinting were used to determine differentially expressed proteins between normal and inflamed intestinal mucosa. Acute colitis was induced by 8.0% dextran sodium sulfate (DSS) given p.o. for 7 days. Among a total of seven protein spots showing differential expression, we identified five different proteins, of which two were upregulated and three downregulated in colitis in comparison to normal mucosa, using the MASCOT search engine. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase 2 and serpin b1a were upregulated proteins, and protein disulfide-isomerase A3, peroxiredoxin-6 and vimentin were identified as downregulated proteins. These identified proteins may be responsible for the development of the intestinal inflammation. 2D-DIGE and MALDI-TOF mass spectrometry are useful in the search for the differentially expressed proteins.

Liraglutide attenuates the osteoblastic differentiation of MC3T3-E1 cells by modulating AMPK/mTOR signaling

PubMed Central

Hu, Xiong-Ke; Yin, Xin-Hua; Zhang, Hong-Qi; Guo, Chao-Feng; Tang, Ming-Xing

2016-01-01

Liraglutide, a synthetic analogue of glucagon-like peptide-1, is utilized in the treatment of type 2 diabetes and obesity. Liraglutide has been previously demonstrated to prevent osteoblastic differentiation of human vascular smooth muscle cells, resulting in the slowing of arterial calcification, however, its effect on bone formation remains unclear. The present study investigated the effect of liraglutide on osteoblastic differentiation using Alizarin Red S staining, and examined the molecular mechanisms underlying the regulatory effect by western blot analysis. The present study demonstrated that protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) were downregulated in MC3T3-E1 cells during osteoblastic differentiation in commercial osteogenic differentiation medium, whereas protein expression levels of transforming growth factor-β (TGF-β) and phosphorylated mammalian target of rapamycin (p-mTOR) increased. Liraglutide was subsequently demonstrated to dose-dependently attenuate the osteoblastic differentiation of MC3T3-E1 cells, to upregulate p-AMPK, and downregulate p-mTOR and TGF-β protein expression levels. Treatment with an AMPK-specific inhibitor, Compound C, eradicated the effect of liraglutide on osteoblastic differentiation, and p-mTOR and TGF-β downregulation. An mTOR activator, MHY1485, also abolished the inhibitory effect of liraglutide on osteoblastic differentiation, and resulted in p-mTOR and TGF-β downregulation, but did not attenuate the liraglutide-induced increase in p-AMPK protein expression levels. The results of the present study demonstrate that liraglutide attenuates osteoblastic differentiation of MC3T3-E1 cells via modulation of AMPK/mTOR signaling. The present study revealed a novel function of liraglutide, which contributes to the understanding of its pharmacological and physiological effects in clinical settings. PMID:27600753

Novel insights into the effect of nitrogen on storage protein biosynthesis and protein body development in wheat caryopsis.

PubMed

Yu, Xurun; Chen, Xinyu; Wang, Leilei; Yang, Yang; Zhu, Xiaowei; Shao, Shanshan; Cui, Wenxue; Xiong, Fei

2017-04-01

Molecular and cytological mechanisms concerning the effects of nitrogen on wheat (Triticum aestivum L.) storage protein biosynthesis and protein body development remain largely elusive. We used transcriptome sequencing, proteomics techniques, and light microscopy to investigate these issues. In total, 2585 differentially expressed genes (DEGs) and 57 differentially expressed proteins (DEPs) were found 7 days after anthesis (DAA), and 2456 DEGs and 64 DEPs were detected 18 DAA after nitrogen treatment. Gene ontology terms related to protein biosynthesis processes enriched these numbers by 678 and 582 DEGs at 7 and 18 DAA, respectively. Further, 25 Kyoto Encyclopedia of Genes and Genomes pathways were involved in protein biosynthesis at both 7 and 18 DAA. DEPs related to storage protein biosynthesis contained gliadin and glutenin subunits, most of which were up-regulated after nitrogen treatment. Quantitative real-time PCR analysis indicated that some gliadin and glutenin subunit encoding genes were differentially expressed at 18 DAA. Structural observation revealed that wheat endosperm accumulated more and larger protein bodies after nitrogen treatment. Collectively, our findings suggest that nitrogen treatment enhances storage protein content, endosperm protein body quantity, and partial processing quality by altering the expression levels of certain genes involved in protein biosynthesis pathways and storage protein expression at the proteomics level. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

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

Reis Monteiro dos-Santos, Guilherme Rodrigo; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida

The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

Identification of differentially expressed proteins in Ostrinia furnacalis adults after exposure to ultraviolet A.

PubMed

Zhang, Changyu; Meng, Jianyu

2018-06-23

Ultraviolet A (UVA), the major component of solar UV irradiation, is an important environmental factor inducing damage to insects including cell death, photoreceptor damage, and oxidative stress. In order to improve understanding of the adaptation mechanisms of insect after UVA exposure, a comparative proteomic analysis was carried out to reveal differential protein expression in Ostrinia furnacalis. Three-day-old adults were treated with UVA for 1 h. Total proteins of control and UVA-treated insects were examined using two-dimensional electrophoresis (2-DE). 2-DE analysis demonstrated that 19 proteins were increased and 18 proteins were decreased significantly in O. furnacalis after UVA exposure, respectively. Thirty differentially expressed proteins were successfully identified by mass spectrometry. The identified proteins were involved in diverse biological processes, such as signal transduction, transport processing, cellular stress, metabolisms, and cytoskeleton organization. Our results reveal that the response patterns of O. furnacalis to UVA irradiation are complex and provide novel insights into the adaptation response to UVA irradiation stress.

Proteome analysis of human substantia nigra in Parkinson's disease

PubMed Central

Werner, Cornelius J; Heyny-von Haussen, Roland; Mall, Gerhard; Wolf, Sabine

2008-01-01

Background Parkinson's disease (PD) is the most common neurodegenerative disorder involving the motor system. Although not being the only region involved in PD, affection of the substantia nigra and its projections is responsible for some of the most debilitating features of the disease. To further advance a comprehensive understanding of nigral pathology, we conducted a tissue based comparative proteome study of healthy and diseased human substantia nigra. Results The gross number of differentially regulated proteins in PD was 221. In total, we identified 37 proteins, of which 16 were differentially expressed. Identified differential proteins comprised elements of iron metabolism (H-ferritin) and glutathione-related redox metabolism (GST M3, GST P1, GST O1), including novel redox proteins (SH3BGRL). Additionally, many glial or related proteins were found to be differentially regulated in PD (GFAP, GMFB, galectin-1, sorcin), as well as proteins belonging to metabolic pathways sparsely described in PD, such as adenosyl homocysteinase (methylation), aldehyde dehydrogenase 1 and cellular retinol-binding protein 1 (aldehyde metabolism). Further differentially regulated proteins included annexin V, beta-tubulin cofactor A, coactosin-like protein and V-type ATPase subunit 1. Proteins that were similarly expressed in healthy or diseased substantia nigra comprised housekeeping proteins such as COX5A, Rho GDI alpha, actin gamma 1, creatin-kinase B, lactate dehydrogenase B, disulfide isomerase ER-60, Rab GDI beta, methyl glyoxalase 1 (AGE metabolism) and glutamine synthetase. Interestingly, also DJ-1 and UCH-L1 were expressed similarly. Furthermore, proteins believed to serve as internal standards were found to be expressed in a constant manner, such as 14-3-3 epsilon and hCRMP-2, thus lending further validity to our results. Conclusion Using an approach encompassing high sensitivity and high resolution, we show that alterations of SN in PD include many more proteins than previously thought. The results point towards a heterogeneous aetiopathogenesis of the disease, including alterations of GSH-related proteins as well as alterations of proteins involved in retinoid metabolism, and they indicate that proteins involved in familial PD may not be differentially regulated in idiopathic Parkinson's disease. PMID:18275612

Expression of Antisense Long Noncoding RNAs as Potential Regulators in Rainbow Trout with Different Tolerance to Plant-Based Diets.

PubMed

Abernathy, Jason; Overturf, Ken

2018-01-04

Reformulation of aquafeeds in salmonid diets to include more plant proteins is critical for sustainable aquaculture. However, increasing plant proteins can lead to stunted growth and enteritis. Toward an understanding of the regulatory mechanisms behind plant protein utilization, directional RNA sequencing of liver tissues from a rainbow trout strain selected for growth on an all plant-protein diet and a control strain, both fed a plant diet for 12 weeks, were utilized to construct long noncoding RNAs. Antisense long noncoding RNAs were selected for differential expression and functional analyses since they have been shown to have regulatory actions within a genome. A total of 142 unique antisense long noncoding RNAs were differentially expressed between strains, 60 of which could be mapped to a gene. Genes underlying these noncoding RNAs are indicated in lipid metabolism and immunity. Six noncoding transcripts were also found to overlap with differentially expressed protein-coding genes, all of which were co-expressed. Associating variation in regulatory elements between rainbow trout strains with differing tolerance to plant-protein diets will assist in future studies toward increased gains throughout carnivorous aquaculture.

Regulation of Silk Genes by Hox and Homeodomain Proteins in the Terminal Differentiated Silk Gland of the Silkworm Bombyx mori

PubMed Central

Takiya, Shigeharu; Tsubota, Takuya; Kimoto, Mai

2016-01-01

The silk gland of the silkworm Bombyx mori is a long tubular organ that is divided into several subparts along its anteroposterior (AP) axis. As a trait of terminal differentiation of the silk gland, several silk protein genes are expressed with unique regional specificities. Most of the Hox and some of the homeobox genes are also expressed in the differentiated silk gland with regional specificities. The expression patterns of Hox genes in the silk gland roughly correspond to those in embryogenesis showing “colinearity”. The central Hox class protein Antennapedia (Antp) directly regulates the expression of several middle silk gland–specific silk genes, whereas the Lin-1/Isl-1/Mec3 (LIM)-homeodomain transcriptional factor Arrowhead (Awh) regulates the expression of posterior silk gland–specific genes for silk fiber proteins. We summarize our results and discuss the usefulness of the silk gland of Bombyx mori for analyzing the function of Hox genes. Further analyses of the regulatory mechanisms underlying the region-specific expression of silk genes will provide novel insights into the molecular bases for target-gene selection and regulation by Hox and homeodomain proteins. PMID:29615585

PodNet, a protein-protein interaction network of the podocyte.

PubMed

Warsow, Gregor; Endlich, Nicole; Schordan, Eric; Schordan, Sandra; Chilukoti, Ravi K; Homuth, Georg; Moeller, Marcus J; Fuellen, Georg; Endlich, Karlhans

2013-07-01

Interactions between proteins crucially determine cellular structure and function. Differential analysis of the interactome may help elucidate molecular mechanisms during disease development; however, this analysis necessitates mapping of expression data on protein-protein interaction networks. These networks do not exist for the podocyte; therefore, we built PodNet, a literature-based mouse podocyte network in Cytoscape format. Using database protein-protein interactions, we expanded PodNet to XPodNet with enhanced connectivity. In order to test the performance of XPodNet in differential interactome analysis, we examined podocyte developmental differentiation and the effect of cell culture. Transcriptomes of podocytes in 10 different states were mapped on XPodNet and analyzed with the Cytoscape plugin ExprEssence, based on the law of mass action. Interactions between slit diaphragm proteins are most significantly upregulated during podocyte development and most significantly downregulated in culture. On the other hand, our analysis revealed that interactions lost during podocyte differentiation are not regained in culture, suggesting a loss rather than a reversal of differentiation for podocytes in culture. Thus, we have developed PodNet as a valuable tool for differential interactome analysis in podocytes, and we have identified established and unexplored regulated interactions in developing and cultured podocytes.

Differentially expressed proteins of soybean (Glycine max) pulvinus in light and dark conditions

USDA-ARS?s Scientific Manuscript database

Soybean leaves (Glycine max) both track and avoid the sun through turgor changes of the pulvinus tissue at the base of leaves. Pulvinar response is known to be affected by both diurnally varying environmental factors and circadian patterns. Differential expression of the proteins between light and d...

The Network Organization of Cancer-associated Protein Complexes in Human Tissues

PubMed Central

Zhao, Jing; Lee, Sang Hoon; Huss, Mikael; Holme, Petter

2013-01-01

Differential gene expression profiles for detecting disease genes have been studied intensively in systems biology. However, it is known that various biological functions achieved by proteins follow from the ability of the protein to form complexes by physically binding to each other. In other words, the functional units are often protein complexes rather than individual proteins. Thus, we seek to replace the perspective of disease-related genes by disease-related complexes, exemplifying with data on 39 human solid tissue cancers and their original normal tissues. To obtain the differential abundance levels of protein complexes, we apply an optimization algorithm to genome-wide differential expression data. From the differential abundance of complexes, we extract tissue- and cancer-selective complexes, and investigate their relevance to cancer. The method is supported by a clustering tendency of bipartite cancer-complex relationships, as well as a more concrete and realistic approach to disease-related proteomics. PMID:23567845

The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma.

PubMed

Leone, A; Mitsiades, N; Ward, Y; Spinelli, B; Poulaki, V; Tsokos, M; Kelly, K

2001-05-31

Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.

Comparative proteomics of umbilical vein blood plasma from normal and gestational diabetes mellitus patients reveals differentially expressed proteins associated with childhood obesity.

PubMed

Miao, Zhijing; Wang, Jianqing; Wang, Fuqiang; Liu, Lan; Ding, Hongjuan; Shi, Zhonghua

2016-11-01

Offspring obesity is one of long-term complications of gestational diabetes mellitus (GDM). The aim of this study is to identify proteins differentially expressed in the umbilical vein blood plasma, which could become markers for early diagnosis of childhood obesity. Umbilical vein plasma samples were collected from 30 control and 30 GDM patients in 2007-2008 whose offspring were suffering from obesity at 6-7 years old. Multiplexed isobaric tandem mass tag labeling combined with LC-MS/MS was used to identify differentially expressed proteins. Ingenuity pathway analysis was performed to identify canonical pathways, biological functions, and networks of interacting proteins. Western blotting was used to verify the expression of three selected proteins. A total of 318 proteins were identified, of which 12 proteins were upregulated in GDM group while 24 downregulated. Lipid metabolism was the top category identified by ingenuity pathway analysis. Three randomly chosen proteins were validated by Western blotting, which were consistent with LC-MS. There are significant differences of protein profile in the umbilical vein blood plasma between normal and GDM patients with obese offspring. The results indicate that a variety of proteins and biological mechanisms may contribute to childhood obesity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integrated analysis of long noncoding RNA and mRNA expression profile in children with obesity by microarray analysis.

PubMed

Liu, Yuesheng; Ji, Yuqiang; Li, Min; Wang, Min; Yi, Xiaoqing; Yin, Chunyan; Wang, Sisi; Zhang, Meizhen; Zhao, Zhao; Xiao, Yanfeng

2018-06-08

Long noncoding RNAs (lncRNAs) have an important role in adipose tissue function and energy metabolism homeostasis, and abnormalities may lead to obesity. To investigate whether lncRNAs are involved in childhood obesity, we investigated the differential expression profile of lncRNAs in obese children compared with non-obese children. A total number of 1268 differentially expressed lncRNAs and 1085 differentially expressed mRNAs were identified. Gene Ontology (GO) and pathway analysis revealed that these lncRNAs were involved in varied biological processes, including the inflammatory response, lipid metabolic process, osteoclast differentiation and fatty acid metabolism. In addition, the lncRNA-mRNA co-expression network and the protein-protein interaction (PPI) network were constructed to identify hub regulatory lncRNAs and genes based on the microarray expression profiles. This study for the first time identifies an expression profile of differentially expressed lncRNAs in obese children and indicated hub lncRNA RP11-20G13.3 attenuated adipogenesis of preadipocytes, which is conducive to the search for new diagnostic and therapeutic strategies of childhood obesity.

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells.

PubMed

Gregorio-King, Claudia C; McLeod, Janet L; Collier, Fiona McL; Collier, Gregory R; Bolton, Karyn A; Van Der Meer, Gavin J; Apostolopoulos, Jim; Kirkland, Mark A

2002-03-20

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.

Protein expression profiling in head fragments during planarian regeneration after amputation.

PubMed

Chen, Xiaoguang; Xu, Cunshuan

2015-04-01

Following amputation, a planarian tail fragment can regrow into a complete organism including a well-organized brain within about 2-3 weeks, thus restoring the structure and function to presurgical levels. Despite the enormous potential of these animals for regenerative medicine, our understanding of the exact mechanism of planarian regeneration is incomplete. To better understand the molecular nature of planarian head regeneration, we applied two-dimensional electrophoresis (2-DE)/matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/time-of-flight mass spectrometry (TOF MS) technique to analyze the dynamic proteomic expression profiles over the course of 6 to 168 h post-decapitation. This approach identified a total of 141 differentially expressed proteins, 47 of which exhibited exceptionally high fold changes (≥3-fold change). Of these, Rx protein, an important regulator of head and brain development, was considered to be closely related to planarian head regeneration because of its exceptional high expression almost throughout the time course of regeneration process. Functional annotation analysis classified the 141 proteins into eight categories: (1) signaling, (2) Ca(2+) binding and translocation, (3) transcription and translation, (4) cytoskeleton, (5) metabolism, (6) cell protection, (7) tissue differentiation, and (8) cell cycle. Signaling pathway analysis indicated that Wnt1/Ca(2+) signaling pathway was activated during head regeneration. Integrating the analyses of proteome expression profiling, functional annotation, and signaling pathway, amputation-induced head reformation requires some mechanisms to promote cell proliferation and differentiation, including differential regulation of proapoptotic and antiapoptotic proteins, and the regulation of proliferation and differentiation-related proteins. Importantly, Wnt1/Ca(2+) signaling pathway upregulates Rx expression, finally facilitating the differentiation of neoblasts into various cell types. Taken together, our study demonstrated that proteomic analysis approach used by us is a powerful tool in understanding molecular process related to head regeneration of planarian.

Protein expression differs between neural progenitor cells from the adult rat brain subventricular zone and olfactory bulb.

PubMed

Maurer, Martin H; Feldmann, Robert E; Bürgers, Heinrich F; Kuschinsky, Wolfgang

2008-01-16

Neural progenitor cells can be isolated from various regions of the adult mammalian brain, including the forebrain structures of the subventricular zone and the olfactory bulb. Currently it is unknown whether functional differences in these progenitor cell populations can already be found on the molecular level. Therefore, we compared protein expression profiles between progenitor cells isolated from the subventricular zone and the olfactory bulb using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry. The subventricular zone and the olfactory bulb are connected by the Rostral Migratory Stream (RMS), in which glial fibrillary acidic protein (GFAP)-positive cells guide neuroblasts. Recent literature suggested that these GFAP-positive cells possess neurogenic potential themselves. In the current study, we therefore compared the cultured neurospheres for the fraction of GFAP-positive cells and their morphology of over a prolonged period of time. We found significant differences in the protein expression patterns between subventricular zone and olfactory bulb neural progenitor cells. Of the differentially expressed protein spots, 105 were exclusively expressed in the subventricular zone, 23 showed a lower expression and 51 a higher expression in the olfactory bulb. The proteomic data showed that more proteins are differentially expressed in olfactory bulb progenitors with regard to proteins involved in differentiation and microenvironmental integration, as compared to the subventricular zone progenitors. Compared to 94% of all progenitors of the subventricular zone expressed GFAP, nearly none in the olfactory bulb cultures expressed GFAP. Both GFAP-positive subpopulations differed also in morphology, with the olfactory bulb cells showing more branching. No differences in growth characteristics such as doubling time, and passage lengths could be found over 26 consecutive passages in the two cultures. In this study, we describe differences in protein expression of neural progenitor populations isolated from two forebrain regions, the subventricular zone and the olfactory bulb. These subpopulations can be characterized by differential expression of marker proteins. We isolated fractions of progenitor cells with GFAP expression from both regions, but the GFAP-positive cells differed in number and morphology. Whereas in vitro growth characteristics of neural progenitors are preserved in both regions, our proteomic and immunohistochemical data suggest that progenitor cells from the two regions differ in morphology and functionality, but not in their proliferative capacity.

Proteomic analysis of cell cycle arrest and differentiation induction caused by ATPR, a derivative of all-trans retinoic acid, in human gastric cancer SGC-7901 cells.

PubMed

Xia, Quan; Zhao, Yingli; Wang, Jiali; Qiao, Wenhao; Zhang, Dongling; Yin, Hao; Xu, Dujuan; Chen, Feihu

2017-07-01

4-amino-2-trifluoromethyl-phenyl retinate (ATPR) was reported to potentially inhibit proliferation and induce differentiation activity in some tumor cells. In this study, a proteomics approach was used to investigate the possible mechanism by screening the differentially expressed protein profiles of SGC-7901 cells before and after ATPR-treatment in vitro. Peptides digested from the total cellular proteins were analyzed by reverse phase LC-MS/MS followed by a label-free quantification analysis. The SEQUEST search engine was used to identify proteins and bioinformatics resources were used to investigate the involved pathways for the differentially expressed proteins. Thirteen down-regulated proteins were identified in the ATPR-treated group. Bioinformatics analysis showed that the effects of ATPR on 14-3-3ε might potentially involve the PI3K-AKT-FOXO pathway and P27Kip1 expression. Western blot and RT-PCR analysis showed that ATPR could inhibit AKT phosphorylation, up-regulate the expression of FOXO1A and P27Kip1 at both the protein and mRNA levels, and down-regulate the cytoplasmic expression of cyclin E and CDK2. ATPR-induced G0/G1 phase arrest and differentiation can be ablated if the P27kip1 gene is silenced with sequence-specific siRNA or in 14-3-3ε overexpression of SGC-7901 cells. ATPR might cause cell cycle arrest and differentiation in SGC-7901 cells by simultaneously inhibiting the phosphorylation of AKT and down-regulating 14-3-3ε. This change would then enhance the inhibition of cyclin E/CDK2 by up-regulating FOXO1A and P27Kip1. Our findings could be of value for finding new drug targets and for developing more effective differentiation inducer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ectopic expression of necdin induces differentiation of mouse neuroblastoma cells.

PubMed

Kobayashi, Masakatsu; Taniura, Hideo; Yoshikawa, Kazuaki

2002-11-01

Necdin is expressed predominantly in postmitotic neurons, and ectopic expression of this protein strongly suppresses cell growth. Necdin has been implicated in the pathogenesis of Prader-Willi syndrome, a human neurodevelopmental disorder associated with genomic imprinting. Here we demonstrate that ectopic expression of necdin induces a neuronal phenotype in neuroblastoma cells. Necdin was undetectable in mouse neuroblastoma N1E-115 cells under undifferentiated and differentiated conditions. N1E-115 cells transfected with necdin cDNA showed morphological differentiation such as neurite outgrowth and expression of the synaptic marker proteins synaptotagmin and synaptophysin. In addition, Western blot analysis of the retinoblastoma protein (Rb) family members Rb, p130, and p107 revealed that necdin cDNA transfectants contained an increased level of p130 and a reduced level of p107, a pattern seen in differentiated G(0) cells. The transcription factors E2F1 and E2F4 physically interacted with necdin via their carboxyl-terminal transactivation domains, but only E2F1 abrogated necdin-induced growth arrest and neurite outgrowth of neuroblastoma cells. Overexpression of E2F1 in differentiated N1E-115 cells induced apoptosis, which was antagonized by co-expression of necdin. These results suggest that necdin promotes the differentiation and survival of neurons through its antagonistic interactions with E2F1.

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

PubMed

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-02-15

To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

Mapping gene expression patterns during myeloid differentiation using the EML hematopoietic progenitor cell line.

PubMed

Du, Yang; Campbell, Janee L; Nalbant, Demet; Youn, Hyewon; Bass, Ann C Hughes; Cobos, Everardo; Tsai, Schickwann; Keller, Jonathan R; Williams, Simon C

2002-07-01

The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line. Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML. Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes. Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.

Proteomic analysis associated with coronary artery dilatation caused by Kawasaki disease using serum exosomes.

PubMed

Zhang, Li; Wang, Wei; Bai, Jun; Xu, Yu-Fen; Li, Lai-Qing; Hua, Liang; Deng, Li; Jia, Hong-Ling

2016-05-01

The aim of this study was to investigate the serum exosome proteome profile of coronary artery dilatation (CAD) caused by Kawasaki disease (KD). Two-dimensional electrophoresis was implemented on proteins of serum exosomes obtained from children with CAD caused by KD and from healthy controls. Differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis. We identified 38 differentially expressed proteins (13 up-regulated and 25 down-regulated) from serum exosomes of patients with CAD caused by KD compared with healthy controls. Expression levels of three differentially expressed proteins (leucine-rich alpha-2-glycoprotein, sex hormone-binding globulin, and serotransferrin) were validated using western blot analysis. Classification and protein-protein network analysis showed that they are associated with multiple functional groups involved in the acute inflammatory response, defense response, complement activation, humoral immune response, and response to wounding. The majority of the proteins are involved in the inflammation and coagulation cascades. These findings establish a comprehensive proteome profile of CAD caused by KD and increase our knowledge of scientific insight into its mechanisms. Copyright © 2016 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

A Pilot Proteogenomic Study with Data Integration Identifies MCT1 and GLUT1 as Prognostic Markers in Lung Adenocarcinoma.

PubMed

Stewart, Paul A; Parapatics, Katja; Welsh, Eric A; Müller, André C; Cao, Haoyun; Fang, Bin; Koomen, John M; Eschrich, Steven A; Bennett, Keiryn L; Haura, Eric B

2015-01-01

We performed a pilot proteogenomic study to compare lung adenocarcinoma to lung squamous cell carcinoma using quantitative proteomics (6-plex TMT) combined with a customized Affymetrix GeneChip. Using MaxQuant software, we identified 51,001 unique peptides that mapped to 7,241 unique proteins and from these identified 6,373 genes with matching protein expression for further analysis. We found a minor correlation between gene expression and protein expression; both datasets were able to independently recapitulate known differences between the adenocarcinoma and squamous cell carcinoma subtypes. We found 565 proteins and 629 genes to be differentially expressed between adenocarcinoma and squamous cell carcinoma, with 113 of these consistently differentially expressed at both the gene and protein levels. We then compared our results to published adenocarcinoma versus squamous cell carcinoma proteomic data that we also processed with MaxQuant. We selected two proteins consistently overexpressed in squamous cell carcinoma in all studies, MCT1 (SLC16A1) and GLUT1 (SLC2A1), for further investigation. We found differential expression of these same proteins at the gene level in our study as well as in other public gene expression datasets. These findings combined with survival analysis of public datasets suggest that MCT1 and GLUT1 may be potential prognostic markers in adenocarcinoma and druggable targets in squamous cell carcinoma. Data are available via ProteomeXchange with identifier PXD002622.

Proteomic Characterization of Inbreeding-Related Cold Sensitivity in Drosophila melanogaster

PubMed Central

Beck, Hans C.; Petersen, Jørgen; Gagalova, Kristina Kirilova; Loeschcke, Volker

2013-01-01

Inbreeding depression is a widespread phenomenon of central importance to agriculture, medicine, conservation biology and evolutionary biology. Although the population genetic principles of inbreeding depression are well understood, we know little about its functional genomic causes. To provide insight into the molecular interplay between intrinsic stress responses, inbreeding depression and temperature tolerance, we performed a proteomic characterization of a well-defined conditional inbreeding effect in a single line of Drosophila melanogaster, which suffers from extreme cold sensitivity and lethality. We identified 48 differentially expressed proteins in a conditional lethal line as compared to two control lines. These proteins were enriched for proteins involved in hexose metabolism, in particular pyruvate metabolism, and many were found to be associated with lipid particles. These processes can be linked to known cold tolerance mechanisms, such as the production of cryoprotectants, membrane remodeling and the build-up of energy reserves. We checked mRNA-expression of seven genes with large differential protein expression. Although protein expression poorly correlated with gene expression, we found a single gene (CG18067) that, after cold shock, was upregulated in the conditional lethal line both at the mRNA and protein level. Expression of CG18067 also increased in control flies after cold shock, and has previously been linked to cold exposure and chill coma recovery time. Many differentially expressed proteins in our study appear to be involved in cold tolerance in non-inbred individuals. This suggest the conditional inbreeding effect to be caused by misregulation of physiological cold tolerance mechanisms. PMID:23658762

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

Identification and expression of the protein ubiquitination system in Giardia intestinalis.

PubMed

Gallego, Eva; Alvarado, Magda; Wasserman, Moises

2007-06-01

Giardia intestinalis is a single-cell eukaryotic microorganism, regarded as one of the earliest divergent eukaryotes and thus an attractive model to study the evolution of regulatory systems. Giardia has two different forms throughout its life cycle, cyst and trophozoite, and changes from one to the other in response to environmental signals. The two differentiation processes involve a differential gene expression as well as a quick and specific protein turnover that may be mediated by the ubiquitin/proteasome system. The aim of this work was to search for unreported components of the ubiquitination system and to experimentally demonstrate their expression in the parasite and during the two differentiation processes. We found activity of protein ubiquitination in G. intestinalis trophozoites and analyzed the transcription of the ubiquitin gene, as well as that of the activating (E1), conjugating (E2), and ligase (E3) ubiquitin enzymes during encystation and excystation. A constant ubiquitin expression persisted during the parasite's differentiation processes, whereas variation in transcription was observed in the other genes under study.

The protein expression landscape of mitosis and meiosis in diploid budding yeast.

PubMed

Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

2017-03-06

Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We have integrated protein profiling data using mass spectrometry with tiling array RNA profiling data and information on double-stranded RNAs (dsRNAs) by overlapping sense/antisense transcripts from an RNA-Sequencing experiment. This work therefore provides quantitative insight into protein expression during cell division and development and associates changing protein levels with developmental stage specific induction of antisense transcripts and the formation of dsRNAs. Copyright © 2017 Elsevier B.V. All rights reserved.

PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway

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

Li, Haiying; Cui, Yazhou; Luan, Jing

Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit themore » level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.« less

Cloning, sequencing and expression in MEL cells of a cDNA encoding the mouse ribosomal protein S5.

PubMed

Vanegas, N; Castañeda, V; Santamaría, D; Hernández, P; Schvartzman, J B; Krimer, D B

1997-06-05

We describe the isolation and characterization of a cDNA encoding the mouse S5 ribosomal protein. It was isolated from a MEL (murine erythroleukemia) cell cDNA library by differential hybridization as a down regulated sequence during HMBA-induced differentiation. Northern series analysis showed that S5 mRNA expression is reduced 5-fold throughout the differentiation process. The mouse S5 mRNA is 760 bp long and encodes for a 204 amino acid protein with 94% homology with the human and rat S5.

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia

PubMed Central

Bakker, Astrid D.; Hogervorst, Jolanda M. A.; Nolte, Peter A.; Klein-Nulend, Jenneke

2017-01-01

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation. PMID:28166273

[Expression of ICAT and Wnt signaling-related proteins in the monocytic differentiation of HL-60 cells induced by a new steroidal drug NSC67657].

PubMed

Wang, J S; Wang, W J; Wang, T; Zhang, Y

2016-04-01

To investigate the expression of mRNA and proteins of β-catenin, TCF-4 (ICAT) and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new steroidal drug NSC67657. Wright's staining and α-NBE staining were used to observe the differentiation of HL-60 cells after 5 days of 10 μmol/L NSC67657 treatment. Flow cytometry (FCM) was used to detect the differentiation and cell cycles. The expressions of mRNA and proteins of ICAT and Wnt signaling pathway-related factors, including β-catenin, TCF-4, c-myc, cyclin D1 and TCF-1 before and after differentiation, were detected by RT-PCR and Western blot. Morphological observation showed that NSC67657 induced monocytic differentiation of HL-60 cells. At 5 days after 10 μmol/L NSC67657 treatment, the number of CD14(+) HL-60 cells was (94.37±2.84)%, significantly higher than the (1.31±0.09)% in control group (P<0.01). The flow cytometry assay revealed that NSC67657 induced (76.46±2.83)% of G1/G0 phase arrest, significantly higher than that of (59.40±5.42)% in the control group (P<0.05), while the S phase cells were of (18.76±0.98)%, significantly lower than that of (34.38±2.61) % in the control group (P<0.05). The NSC67657 treatment also up-regulated the expression of ICAT mRNA and protein, and down-regulated the expression of β-catenin mRNA and protin (P<0.01 for all). However, the nuclear expression of β-catenin was down-regulated (P<0.01). The NSC67657 treatment induced nonsignificant alterations of TCF-4 mRNA, total protein and nuclear protein in the HL-60 cells (P>0.05 for all). The target genes of Wnt signaling pathway, including c-myc, cyclinD1 and TCF-1 mRNA and proteins in the HL-60 cells were significantly down-regulated after NSC67657 treatment (P<0.05). The new steroidal drug NSC67657 induces monocytic differentiation of HL-60 cells, and down-regulates the expression of β-catenin and target genes of Wnt signaling pathway. These results indicate that Wnt signaling pathway may be directly or indirectly involved in the monocytic differentiation process of HL-60 cells.

Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

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

Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.

Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference mapmore » of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.« less

Transcription factors SOHLH1 and SOHLH2 coordinate oocyte differentiation without affecting meiosis I.

PubMed

Shin, Yong-Hyun; Ren, Yu; Suzuki, Hitomi; Golnoski, Kayla J; Ahn, Hyo Won; Mico, Vasil; Rajkovic, Aleksandar

2017-06-01

Following migration of primordial germ cells to the genital ridge, oogonia undergo several rounds of mitotic division and enter meiosis at approximately E13.5. Most oocytes arrest in the dictyate (diplotene) stage of meiosis circa E18.5. The genes necessary to drive oocyte differentiation in parallel with meiosis are unknown. Here, we have investigated whether expression of spermatogenesis and oogenesis bHLH transcription factor 1 (Sohlh1) and Sohlh2 coordinates oocyte differentiation within the embryonic ovary. We found that SOHLH2 protein was expressed in the mouse germline as early as E12.5 and preceded SOHLH1 protein expression, which occurred circa E15.5. SOHLH1 protein appearance at E15.5 correlated with SOHLH2 translocation from the cytoplasm into the nucleus and was dependent on SOHLH1 expression. NOBOX oogenesis homeobox (NOBOX) and LIM homeobox protein 8 (LHX8), two important regulators of postnatal oogenesis, were coexpressed with SOHLH1. Single deficiency of Sohlh1 or Sohlh2 disrupted the expression of LHX8 and NOBOX in the embryonic gonad without affecting meiosis. Sohlh1-KO infertility was rescued by conditional expression of the Sohlh1 transgene after the onset of meiosis. However, Sohlh1 or Sohlh2 transgene expression could not rescue Sohlh2-KO infertility due to a lack of Sohlh1 or Sohlh2 expression in rescued mice. Our results indicate that Sohlh1 and Sohlh2 are essential regulators of oocyte differentiation but do not affect meiosis I.

Comparative proteomic analysis of differentially expressed proteins in the early milky stage of rice grains during high temperature stress

PubMed Central

Liao, Jiang-Lin; Zhou, Hui-Wen; Huang, Ying-Jin

2014-01-01

Rice yield and quality are adversely affected by high temperatures, and these effects are more pronounced at the ‘milky stage’ of the rice grain ripening phase. Identifying the functional proteins involved in the response of rice to high temperature stress may provide the basis for improving heat tolerance in rice. In the present study, a comparative proteomic analysis of paired, genetically similar heat-tolerant and heat-sensitive rice lines was conducted. Two-dimensional electrophoresis (2-DE) revealed a total of 27 differentially expressed proteins in rice grains, predominantly from the heat-tolerant lines. The protein profiles clearly indicated variations in protein expression between the heat-tolerant and heat-sensitive rice lines. Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis revealed that 25 of the 27 differentially displayed proteins were homologous to known functional proteins. These homologous proteins were involved in biosynthesis, energy metabolism, oxidation, heat shock metabolism, and the regulation of transcription. Seventeen of the 25 genes encoding the differentially displayed proteins were mapped to rice chromosomes according to the co-segregating conditions between the simple sequence repeat (SSR) markers and the target genes in recombinant inbred lines (RILs). The proteins identified in the present study provide a basis to elucidate further the molecular mechanisms underlying the adaptation of rice to high temperature stress. PMID:24376254

Quantitative proteomic analysis of the Salmonella-lettuce interaction

PubMed Central

Zhang, Yuping; Nandakumar, Renu; Bartelt-Hunt, Shannon L; Snow, Daniel D; Hodges, Laurie; Li, Xu

2014-01-01

Human pathogens can internalize food crops through root and surface uptake and persist inside crop plants. The goal of the study was to elucidate the global modulation of bacteria and plant protein expression after Salmonella internalizes lettuce. A quantitative proteomic approach was used to analyse the protein expression of Salmonella enterica serovar Infantis and lettuce cultivar Green Salad Bowl 24 h after infiltrating S. Infantis into lettuce leaves. Among the 50 differentially expressed proteins identified by comparing internalized S. Infantis against S. Infantis grown in Luria Broth, proteins involved in glycolysis were down-regulated, while one protein involved in ascorbate uptake was up-regulated. Stress response proteins, especially antioxidant proteins, were up-regulated. The modulation in protein expression suggested that internalized S. Infantis might utilize ascorbate as a carbon source and require multiple stress response proteins to cope with stresses encountered in plants. On the other hand, among the 20 differentially expressed lettuce proteins, proteins involved in defense response to bacteria were up-regulated. Moreover, the secreted effector PipB2 of S. Infantis and R proteins of lettuce were induced after bacterial internalization into lettuce leaves, indicating human pathogen S. Infantis triggered the defense mechanisms of lettuce, which normally responds to plant pathogens. PMID:24512637

Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells

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

Zhu, Yue; Tong, Hui-Li; Li, Shu-Feng

Bovine muscle-derived satellite cells (MDSCs) are important for animal growth. In this study, the effect of transcription elongation factor A3 (TCEA3) on bovine MDSC differentiation was investigated. Western blotting, immunofluorescence assays, and cytoplasmic and nuclear protein isolation and purification techniques were used to determine the expression pattern and protein localization of TCEA3 in bovine MDSCs during in vitro differentiation. TCEA3 expression was upregulated using the CRISPR/Cas9 technique to study its effects on MDSC differentiation in vitro. TCEA3 expression gradually increased during the in vitro differentiation of bovine MDSCs and peaked on the 5th day of differentiation. TCEA3 was mainly localized in the cytoplasmmore » of bovine MDSCs, and its expression was not detected in the nucleus. The level of TCEA3 was relatively higher in myotubes at a higher degree of differentiation than during early differentiation. After transfection with a TCEA3-activating plasmid vector (TCEA3 overexpression) for 24 h, the myotube fusion rate, number of myotubes, and expression levels of the muscle differentiation-related loci myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly during the in vitro differentiation of bovine MDSCs. After transfection with a TCEA3-inhibiting plasmid vector for 24 h, the myotube fusion rate, number of myotubes, and expression levels of MYOG and MYH3 decreased significantly. Our results indicated, for the first time, that TCEA3 promotes the differentiation of bovine MDSCs and have implications for meat production and animal rearing. - Highlights: • Muscle-derived satellite cell differentiation is promoted by TCEA3. • TCEA3 protein was localized in the cytoplasm, but not nuclei of bovine MDSCs. • TCEA3 levels increased as myotube differentiation increased. • TCEA3 affected myotube fusion, myotube counts, and MYOG and MYH3 levels.« less

Comparative differential gene expression analysis of nucleus-encoded proteins for Rafflesia cantleyi against Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Ng, Siuk-Mun; Lee, Xin-Wei; Wan, Kiew-Lian; Firdaus-Raih, Mohd

2015-09-01

Regulation of functional nucleus-encoded proteins targeting the plastidial functions was comparatively studied for a plant parasite, Rafflesia cantleyi versus a photosynthetic plant, Arabidopsis thaliana. This study involved two species of different feeding modes and different developmental stages. A total of 30 nucleus-encoded proteins were found to be differentially-regulated during two stages in the parasite; whereas 17 nucleus-encoded proteins were differentially-expressed during two developmental stages in Arabidopsis thaliana. One notable finding observed for the two plants was the identification of genes involved in the regulation of photosynthesis-related processes where these processes, as expected, seem to be present only in the autotroph.

Dual-compartmental transcriptomic + proteomic analysis of a marine endosymbiosis exposed to environmental change.

PubMed

Mayfield, Anderson B; Wang, Yu-Bin; Chen, Chii-Shiarng; Chen, Shu-Hwa; Lin, Chung-Yen

2016-12-01

As significant anthropogenic pressures are putting undue stress on the world's oceans, there has been a concerted effort to understand how marine organisms respond to environmental change. Transcriptomic approaches, in particular, have been readily employed to document the mRNA-level response of a plethora of marine invertebrates exposed to an array of simulated stress scenarios, with the tacit and untested assumption being that the respective proteins show a corresponding trend. To better understand the degree of congruency between mRNA and protein expression in an endosymbiotic marine invertebrate, mRNAs and proteins were sequenced from the same samples of the common, Indo-Pacific coral Seriatopora hystrix exposed to stable or upwelling-simulating conditions for 1 week. Of the 167 proteins downregulated at variable temperature, only two were associated with mRNAs that were also differentially expressed between treatments. Of the 378 differentially expressed genes, none were associated with a differentially expressed protein. Collectively, these results highlight the inherent risk of inferring cellular behaviour based on mRNA expression data alone and challenge the current, mRNA-focused approach taken by most marine and many molecular biologists. © 2016 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Differential expression of syndecan isoforms during mouse incisor amelogenesis.

PubMed

Muto, Taro; Miyoshi, Keiko; Munesue, Seiichi; Nakada, Hiroshi; Okayama, Minoru; Matsuo, Takashi; Noma, Takafumi

2007-08-01

Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells.

PubMed

Moulding, D A; Giles, R V; Spiller, D G; White, M R; Tidd, D M; Edwards, S W

2000-09-01

Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 beta-myristate 13 alpha-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of other BCL2 family members and PMA-induced tumor necrosis factor-alpha (TNF-alpha). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1. (Blood. 2000;96:1756-1763)

Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

PubMed

Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

2018-03-02

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells.

PubMed

Yan, Guokai; Lestari, Retno; Long, Baisheng; Fan, Qiwen; Wang, Zhichang; Guo, Xiaozhen; Yu, Jie; Hu, Jun; Yang, Xingya; Chen, Changqing; Liu, Lu; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

2016-03-17

L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells.

Electromagnetic Radiation Disturbed the Photosynthesis of Microcystis aeruginosa at the Proteomics Level.

PubMed

Tang, Chao; Yang, Chuanjun; Yu, Hui; Tian, Shen; Huang, Xiaomei; Wang, Weiyi; Cai, Peng

2018-01-11

Photosynthesis of Microcystis aeruginosa under Electromagnetic Radiation (1.8 GHz, 40 V/m) was studied by using the proteomics. A total of 30 differentially expressed proteins, including 15 up-regulated and 15 down-regulated proteins, were obtained in this study. The differentially expressed proteins were significantly enriched in the photosynthesis pathway, in which the protein expression levels of photosystems II cytochrome b559 α subunit, cytochrome C550, PsbY, and F-type ATP synthase (a, b) decreased. Our results indicated that electromagnetic radiation altered the photosynthesis-related protein expression levels, and aimed at the function of photosynthetic pigments, photosystems II potential activity, photosynthetic electron transport process, and photosynthetic phosphorylation process of M. aeruginosa. Based on the above evidence, that photoreaction system may be deduced as a target of electromagnetic radiation on the photosynthesis in cyanobacteria; the photoreaction system of cyanobacteria is a hypothetical "shared target effector" that responds to light and electromagnetic radiation; moreover, electromagnetic radiation does not act on the functional proteins themselves but their expression processes.

Neuroglian is expressed on cells destined to form the prothoracic glands of Manduca embryos as they segregate from surrounding cells and rearrange during morphogenesis.

PubMed

Chen, C L; Lampe, D J; Robertson, H M; Nardi, J B

1997-01-01

A cell surface protein (3B11) is differentially expressed in the embryonic labial segment of Manduca as two circular monolayers of epithelial cells invaginate and segregate from surrounding epithelial cells. The cells that invaginate and preferentially express 3B11 represent the presumptive prothoracic glands. These cells continue to express protein 3B11 as they rearrange to form first a three-dimensional aggregate and later anastomosing filaments of cells. In the differentiated prothoracic gland, expression of 3B11 is restricted to sites of cell-cell contact. Cloning and sequencing of the cDNA for protein 3B11 revealed that this protein is the Manduca counterpart of Drosophila neuroglian and mouse L1. These surface proteins are known to function as adhesion/recognition molecules during development. Manduca neuroglian shares 58 and 31% identity respectively with the Drosophila and mouse proteins and has a cytoplasmic domain of over 100 amino acids.

Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

PubMed

de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

2017-08-10

Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open reading frames were predicted for 7947 transcripts. This class represented 17% of the differentially expressed transcripts, suggesting a potential transcriptional regulatory function of long non-coding RNA in tick blood-feeding. The assembled sialotranscriptome greatly expands the sequence availability of R. zambeziensis, assists in our understanding of the transcription of secretory proteins during blood-feeding and will be a valuable resource for future vaccine candidate selection.

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

1997-09-03

Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis.

PubMed

Han, Xiang Hua; Jin, Yong-Ri; Seto, Marianne; Yoon, Jeong Kyo

2011-03-25

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.

A WNT/β-Catenin Signaling Activator, R-spondin, Plays Positive Regulatory Roles during Skeletal Myogenesis*

PubMed Central

Han, Xiang Hua; Jin, Yong-Ri; Seto, Marianne; Yoon, Jeong Kyo

2011-01-01

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway. PMID:21252233

Caste- and development-associated gene expression in a lower termite

PubMed Central

Scharf, Michael E; Wu-Scharf, Dancia; Pittendrigh, Barry R; Bennett, Gary W

2003-01-01

Background Social insects such as termites express dramatic polyphenism (the occurrence of multiple forms in a species on the basis of differential gene expression) both in association with caste differentiation and between castes after differentiation. We have used cDNA macroarrays to compare gene expression between polyphenic castes and intermediary developmental stages of the termite Reticulitermes flavipes. Results We identified differentially expressed genes from nine ontogenic categories. Quantitative PCR was used to quantify precise differences in gene expression between castes and between intermediary developmental stages. We found worker and nymph-biased expression of transcripts encoding termite and endosymbiont cellulases; presoldier-biased expression of transcripts encoding the storage/hormone-binding protein vitellogenin; and soldier-biased expression of gene transcripts encoding two transcription/translation factors, two signal transduction factors and four cytoskeletal/muscle proteins. The two transcription/translation factors showed significant homology to the bicaudal and bric-a-brac developmental genes of Drosophila. Conclusions Our results show differential expression of regulatory, structural and enzyme-coding genes in association with termite castes and their developmental precursor stages. They also provide the first glimpse into how insect endosymbiont cellulase gene expression can vary in association with the caste of a host. These findings shed light on molecular processes associated with termite biology, polyphenism, caste differentiation and development and highlight potentially interesting variations in developmental themes between termites, other insects, and higher animals. PMID:14519197

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

PubMed

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Protein Malnutrition Induces Bone Marrow Mesenchymal Stem Cells Commitment to Adipogenic Differentiation Leading to Hematopoietic Failure

PubMed Central

Cunha, Mayara Caldas Ramos; Lima, Fabiana da Silva; Vinolo, Marco Aurélio Ramirez; Hastreiter, Araceli; Curi, Rui; Borelli, Primavera; Fock, Ricardo Ambrósio

2013-01-01

Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states. PMID:23516566

In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.

PubMed

Uhl, P B; Szober, C M; Amann, B; Alge-Priglinger, C; Ueffing, M; Hauck, S M; Deeg, C A

2014-09-23

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication. Copyright © 2014 Elsevier B.V. All rights reserved.

QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.

PubMed

Choi, Hyungwon; Kim, Sinae; Fermin, Damian; Tsou, Chih-Chiang; Nesvizhskii, Alexey I

2015-11-03

We introduce QPROT, a statistical framework and computational tool for differential protein expression analysis using protein intensity data. QPROT is an extension of the QSPEC suite, originally developed for spectral count data, adapted for the analysis using continuously measured protein-level intensity data. QPROT offers a new intensity normalization procedure and model-based differential expression analysis, both of which account for missing data. Determination of differential expression of each protein is based on the standardized Z-statistic based on the posterior distribution of the log fold change parameter, guided by the false discovery rate estimated by a well-known Empirical Bayes method. We evaluated the classification performance of QPROT using the quantification calibration data from the clinical proteomic technology assessment for cancer (CPTAC) study and a recently published Escherichia coli benchmark dataset, with evaluation of FDR accuracy in the latter. QPROT is a statistical framework with computational software tool for comparative quantitative proteomics analysis. It features various extensions of QSPEC method originally built for spectral count data analysis, including probabilistic treatment of missing values in protein intensity data. With the increasing popularity of label-free quantitative proteomics data, the proposed method and accompanying software suite will be immediately useful for many proteomics laboratories. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative Temporal Transcriptome Profiling of Wheat near Isogenic Line Carrying Lr57 under Compatible and Incompatible Interactions

PubMed Central

Yadav, Inderjit S.; Sharma, Amandeep; Kaur, Satinder; Nahar, Natasha; Bhardwaj, Subhash C.; Sharma, Tilak R.; Chhuneja, Parveen

2016-01-01

Leaf rust caused by Puccinia triticina (Pt) is one of the most important diseases of bread wheat globally. Recent advances in sequencing technologies have provided opportunities to analyse the complete transcriptomes of the host as well as pathogen for studying differential gene expression during infection. Pathogen induced differential gene expression was characterized in a near isogenic line carrying leaf rust resistance gene Lr57 and susceptible recipient genotype WL711. RNA samples were collected at five different time points 0, 12, 24, 48, and 72 h post inoculation (HPI) with Pt 77-5. A total of 3020 transcripts were differentially expressed with 1458 and 2692 transcripts in WL711 and WL711+Lr57, respectively. The highest number of differentially expressed transcripts was detected at 12 HPI. Functional categorization using Blast2GO classified the genes into biological processes, molecular function and cellular components. WL711+Lr57 showed much higher number of differentially expressed nucleotide binding and leucine rich repeat genes and expressed more protein kinases and pathogenesis related proteins such as chitinases, glucanases and other PR proteins as compared to susceptible genotype. Pathway annotation with KEGG categorized genes into 13 major classes with carbohydrate metabolism being the most prominent followed by amino acid, secondary metabolites, and nucleotide metabolism. Gene co-expression network analysis identified four and eight clusters of highly correlated genes in WL711 and WL711+Lr57, respectively. Comparative analysis of the differentially expressed transcripts led to the identification of some transcripts which were specifically expressed only in WL711+Lr57. It was apparent from the whole transcriptome sequencing that the resistance gene Lr57 directed the expression of different genes involved in building the resistance response in the host to combat invading pathogen. The RNAseq data and differentially expressed transcripts identified in present study is a genomic resource which can be used for further studying the host pathogen interaction for Lr57 and wheat transcriptome in general. PMID:28066494

Development of sodium channel protein during chemically induced differentiation of neuroblastoma cells.

PubMed

Baumgold, J; Spector, I

1987-04-01

We have previously shown that the [3H]saxitoxin binding site of the sodium channel is expressed independently of the [125I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E-115 neuroblastoma cells, using [3H]saxitoxin binding, [125I]scorpion toxin binding, and 22Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferentiated, bind 90 +/- 10 fmol of [3H]saxitoxin/mg of protein and 112 +/- 14 fmol of [125I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 +/- 5 nmol of 22Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [3H]saxitoxin binding, the [125I]scorpion toxin binding, and the 22Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [3H]saxitoxin site and [125I]scorpion toxin site appear to be coregulated in N1E-115 cells.

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

PubMed Central

Cullingford, Timothy E; Markou, Thomais; Fuller, Stephen J; Giraldo, Alejandro; Pikkarainen, Sampsa; Zoumpoulidou, Georgia; Alsafi, Ali; Ekere, Collins; Kemp, Timothy J; Dennis, Jayne L; Game, Laurence; Sugden, Peter H; Clerk, Angela

2008-01-01

Background Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response. PMID:18275597

Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.

PubMed

Pastorelli, Roberta; Carpi, Donatella; Campagna, Roberta; Airoldi, Luisa; Pohjanvirta, Raimo; Viluksela, Matti; Hakansson, Helen; Boutros, Paul C; Moffat, Ivy D; Okey, Allan B; Fanelli, Roberto

2006-05-01

One characteristic feature of acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is dramatic interspecies and interstrain variability in sensitivity. This complicates dioxin risk assessment for humans. However, this variability also provides a means of characterizing mechanisms of dioxin toxicity. Long-Evans (Turku/AB) rats are orders of magnitude more susceptible to TCDD lethality than Han/Wistar (Kuopio) rats, and this difference constitutes a very useful model for identifying mechanisms of dioxin toxicity. We adopted a proteomic approach to identify the differential effects of TCDD exposure on liver protein expression in Han/Wistar rats as compared with Long-Evans rats. This allows determination of which, if any, protein markers are indicative of differences in dioxin susceptibility and/or responsible for conferring resistance. Differential protein expression in total liver protein was assessed using two-dimensional gel electrophoresis, computerized gel image analysis, in-gel digestion, and mass spectrometry. We observed significant changes in the abundance of several proteins, which fall into three general classes: (i) TCDD-independent and exclusively strain-specific (e.g. isoforms of the protein-disulfide isomerase A3, regucalcin, and agmatine ureohydrolase); (ii) strain-independent and only dependent on TCDD exposure (e.g. aldehyde dehydrogenase 3A1 and rat selenium-binding protein 2); (iii) dependent on both TCDD exposure and strain (e.g. oxidative stress-related proteins, apoptosis-inducing factor, and MAWD-binding protein). By integrating transcriptomic (microarray) data and genomic data (computational search of regulatory elements), we found that protein expression levels were mainly controlled at the level of transcription. These results reveal, for the first time, a subset of hepatic proteins that are differentially regulated in response to TCDD in a strain-specific manner. Some of these differential responses may play a role in establishing the major differences in TCDD response between these two strains of rats. As such, our work is expected to lead to new insights into the mechanism of TCDD toxicity and resistance.

Involvement of Retinoblastoma Protein and HBP1 in Histone H10 Gene Expression

PubMed Central

Lemercier, Claudie; Duncliffe, Kym; Boibessot, Isabelle; Zhang, Hui; Verdel, André; Angelov, Dimitar; Khochbin, Saadi

2000-01-01

The histone H10-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H10 promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H10 gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H10 H4 box were therefore expected to link differentiation-dependent expression of H10 to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H10 H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H10 gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H10, HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells. PMID:10958660

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro.

PubMed

Sassi, N; Laadhar, L; Allouche, M; Zandieh-Doulabi, B; Hamdoun, M; Klein-Nulend, J; Makni, S; Sellami, S

2014-01-01

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

Clinicopathological Significance of Vimentin and Cytokeratin Protein in the Genesis of Squamous Cell Carcinoma of Cervix.

PubMed

Husain, Nazik Elmalaika O S; Babiker, Ali Yousif; Albutti, Aqel S; Alsahli, Mohammed A; Aly, Salah M; Rahmani, Arshad H

2016-01-01

Cervical cancer is one of the commonest types of cancers worldwide especially in developing countries. Intermediate filaments protein family has shown a role in the diagnosis of various cancers, but a few studies are available about the vimentin and cytokeratin roles in the cervical cancer. This case control study aimed to interpret the expression of vimentin and cytokeratin proteins in the development and progression of cervical cancer and its correlation with clinicopathological features. The cytoplasmic expression of vimentin was observed in 40% of cases, but not in inflammatory lesions of cervix. It was noticed that vimentin expression was increasing significantly with high grade of the tumour. Cytokeratin expression was observed in 48.33% and it was noticed that the expression was 62.5% in well differentiated (G1), 45% in moderately differentiated (G2), and 41.66% in poorly differentiated carcinoma, yet statistically insignificant. The expression of vimentin and cytokeratin proteins was not significantly associated with age groups. The current findings concluded a possible role of vimentin in the development and progression of cervical cancer and vimentin marker will be useful in the diagnosis and grading of cervical cancer.

Clinicopathological Significance of Vimentin and Cytokeratin Protein in the Genesis of Squamous Cell Carcinoma of Cervix

PubMed Central

Husain, Nazik Elmalaika O. S.; Babiker, Ali Yousif; Albutti, Aqel S.; Alsahli, Mohammed A.; Aly, Salah M.; Rahmani, Arshad H.

2016-01-01

Cervical cancer is one of the commonest types of cancers worldwide especially in developing countries. Intermediate filaments protein family has shown a role in the diagnosis of various cancers, but a few studies are available about the vimentin and cytokeratin roles in the cervical cancer. This case control study aimed to interpret the expression of vimentin and cytokeratin proteins in the development and progression of cervical cancer and its correlation with clinicopathological features. The cytoplasmic expression of vimentin was observed in 40% of cases, but not in inflammatory lesions of cervix. It was noticed that vimentin expression was increasing significantly with high grade of the tumour. Cytokeratin expression was observed in 48.33% and it was noticed that the expression was 62.5% in well differentiated (G1), 45% in moderately differentiated (G2), and 41.66% in poorly differentiated carcinoma, yet statistically insignificant. The expression of vimentin and cytokeratin proteins was not significantly associated with age groups. The current findings concluded a possible role of vimentin in the development and progression of cervical cancer and vimentin marker will be useful in the diagnosis and grading of cervical cancer. PMID:27190522

The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

PubMed

Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A

2015-05-19

The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. © 2015 Authors.

Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours.

PubMed

Rudolph, Christiane; Melau, Cecilie; Nielsen, John E; Vile Jensen, Kristina; Liu, Dekang; Pena-Diaz, Javier; Rajpert-De Meyts, Ewa; Rasmussen, Lene Juel; Jørgensen, Anne

2017-08-01

Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 μM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined. MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p < 0.05) lower expression of the MMR and pluripotency factors, as well as a reduced MMR activity and cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p < 0.001) reduced cisplatin sensitivity. This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns.

PubMed

Azzam, Sausan; Broadwater, Laurie; Li, Shuo; Freeman, Ernest J; McDonough, Jennifer; Gregory, Roger B

2013-05-01

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns

PubMed Central

2013-01-01

Background Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Results Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. Conclusions SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights. PMID:23635033

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

PubMed Central

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-01-01

AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

Role of genomic architecture in the expression dynamics of long noncoding RNAs during differentiation of human neuroblastoma cells.

PubMed

Batagov, Arsen O; Yarmishyn, Aliaksandr A; Jenjaroenpun, Piroon; Tan, Jovina Z; Nishida, Yuichiro; Kurochkin, Igor V

2013-10-16

Mammalian genomes are extensively transcribed producing thousands of long non-protein-coding RNAs (lncRNAs). The biological significance and function of the vast majority of lncRNAs remain unclear. Recent studies have implicated several lncRNAs as playing important roles in embryonic development and cancer progression. LncRNAs are characterized with different genomic architectures in relationship with their associated protein-coding genes. Our study aimed at bridging lncRNA architecture with dynamical patterns of their expression using differentiating human neuroblastoma cells model. LncRNA expression was studied in a 120-hours timecourse of differentiation of human neuroblastoma SH-SY5Y cells into neurons upon treatment with retinoic acid (RA), the compound used for the treatment of neuroblastoma. A custom microarray chip was utilized to interrogate expression levels of 9,267 lncRNAs in the course of differentiation. We categorized lncRNAs into 19 architecture classes according to their position relatively to protein-coding genes. For each architecture class, dynamics of expression of lncRNAs was studied in association with their protein-coding partners. It allowed us to demonstrate positive correlation of lncRNAs with their associated protein-coding genes at bidirectional promoters and for sense-antisense transcript pairs. In contrast, lncRNAs located in the introns and downstream of the protein-coding genes were characterized with negative correlation modes. We further classified the lncRNAs by the temporal patterns of their expression dynamics. We found that intronic and bidirectional promoter architectures are associated with rapid RA-dependent induction or repression of the corresponding lncRNAs, followed by their constant expression. At the same time, lncRNAs expressed downstream of protein-coding genes are characterized by rapid induction, followed by transcriptional repression. Quantitative RT-PCR analysis confirmed the discovered functional modes for several selected lncRNAs associated with proteins involved in cancer and embryonic development. This is the first report detailing dynamical changes of multiple lncRNAs during RA-induced neuroblastoma differentiation. Integration of genomic and transcriptomic levels of information allowed us to demonstrate specific behavior of lncRNAs organized in different genomic architectures. This study also provides a list of lncRNAs with possible roles in neuroblastoma.

Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor.

PubMed

Tanoue, H; Morinaga, J; Yoshizawa, T; Yugami, M; Itoh, H; Nakamura, T; Uehara, Y; Masuda, T; Odagiri, H; Sugizaki, T; Kadomatsu, T; Miyata, K; Endo, M; Terada, K; Ochi, H; Takeda, S; Yamagata, K; Fukuda, T; Mizuta, H; Oike, Y

2018-01-01

Chondrocyte differentiation is crucial for long bone growth. Many cartilage extracellular matrix (ECM) proteins reportedly contribute to chondrocyte differentiation, indicating that mechanisms underlying chondrocyte differentiation are likely more complex than previously appreciated. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor normally abundantly produced in mesenchymal lineage cells such as adipocytes and fibroblasts, but its loss contributes to the pathogenesis of lifestyle- or aging-related diseases. However, the function of ANGPTL2 in chondrocytes, which are also differentiated from mesenchymal stem cells, remains unclear. Here, we investigate whether ANGPTL2 is expressed in or functions in chondrocytes. First, we evaluated Angptl2 expression during chondrocyte differentiation using chondrogenic ATDC5 cells and wild-type epiphyseal cartilage of newborn mice. We next assessed ANGPTL2 function in chondrogenic differentiation and associated signaling using Angptl2 knockdown ATDC5 cells and Angptl2 knockout mice. ANGPTL2 is expressed in chondrocytes, particularly those located in resting and proliferative zones, and accumulates in ECM surrounding chondrocytes. Interestingly, long bone growth was retarded in Angptl2 knockout mice from neonatal to adult stages via attenuation of chondrocyte differentiation. Both in vivo and in vitro experiments show that changes in ANGPTL2 expression can also alter p38 mitogen-activated protein kinase (MAPK) activity mediated by integrin α5β1. ANGPTL2 contributes to chondrocyte differentiation and subsequent endochondral ossification through α5β1 integrin and p38 MAPK signaling during bone growth. Our findings provide insight into molecular mechanisms governing communication between chondrocytes and surrounding ECM components in bone growth activities. Copyright © 2017. Published by Elsevier Ltd.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis.

PubMed

Chen, Chao; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

2018-01-01

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC.

Functional study of miR-27a in human hepatic stellate cells by proteomic analysis: comprehensive view and a role in myogenic tans-differentiation.

PubMed

Ji, Yuhua; Zhang, Jinsheng; Wang, Wenwen; Ji, Juling

2014-01-01

We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis

PubMed Central

Chen, Chao; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

2018-01-01

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC. PMID:29403558

Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells.

PubMed

Wang, Ruo-Chiau; Huang, Chien-Yu; Pan, Tai-Long; Chen, Wei-Yu; Ho, Chun-Te; Liu, Tsan-Zon; Chang, Yu-Jia

2015-01-01

To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells. Elevated levels of ANX-4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep-1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteomic Characterization of Yersinia pestis Virulence

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

Chromy, B; Murphy, G; Gonzales, A

2005-01-05

Yersinia pestis, the etiological agent of plague, functions via the Type III secretion mechanism whereby virulence factors are induced upon interactions with a mammalian host. Here, the Y. pestis proteome was studied by two-dimensional differential gel electrophoresis (2-D DIGE) under physiologically relevant growth conditions mimicking the calcium concentrations and temperatures that the pathogen would encounter in the flea vector and upon interaction with the mammalian host. Over 4100 individual protein spots were detected of which hundreds were differentially expressed in the entire comparative experiment. A total of 43 proteins that were differentially expressed between the vector and host growth conditionsmore » were identified by mass spectrometry. Expected differences in expression were observed for several known virulence factors including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as putative virulence factors. Chaperone proteins and signaling molecules hypothesized to be involved in virulence due to their role in Type III secretion were also identified. Other differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants. For example, several sugar metabolism proteins were differentially regulated in response to lower calcium and higher temperature, suggesting these proteins, while not directly connected to virulence, either represent a metabolic switch for survival in the host environment or may facilitate production of virulence factors. Results presented here contribute to a more thorough understanding of the virulence mechanism of Y. pestis through proteomic characterization of the pathogen under induced virulence.« less

Retained differentiation capacity of human skeletal muscle satellite cells from spinal cord-injured individuals.

PubMed

Savikj, Mladen; Ruby, Maxwell A; Kostovski, Emil; Iversen, Per O; Zierath, Juleen R; Krook, Anna; Widegren, Ulrika

2018-06-01

Despite the well-known role of satellite cells in skeletal muscle plasticity, the effect of spinal cord injury on their function in humans remains unknown. We determined whether spinal cord injury affects the intrinsic ability of satellite cells to differentiate and produce metabolically healthy myotubes. We obtained vastus lateralis biopsies from eight spinal cord-injured and six able-bodied individuals. Satellite cells were isolated, grown and differentiated in vitro. Gene expression was measured by quantitative PCR. Abundance of differentiation markers and regulatory proteins was determined by Western blotting. Protein synthesis and fatty acid oxidation were measured by radioactive tracer-based assays. Activated satellite cells (myoblasts) and differentiated myotubes derived from skeletal muscle of able-bodied and spinal cord-injured individuals expressed similar (P > 0.05) mRNA levels of myogenic regulatory factors. Myogenic differentiation factor 1 expression was higher in myoblasts from spinal cord-injured individuals. Desmin and myogenin protein content was increased upon differentiation in both groups, while myotubes from spinal cord-injured individuals contained more type I and II myosin heavy chain. Phosphorylated and total protein levels of Akt-mechanistic target of rapamycin and forkhead box protein O signalling axes and protein synthesis rate in myotubes were similar (P > 0.05) between groups. Additionally, fatty acid oxidation of myotubes from spinal cord-injured individuals was unchanged (P > 0.05) compared to able-bodied controls. Our results indicate that the intrinsic differentiation capacity of satellite cells and metabolic characteristics of myotubes are preserved following spinal cord injury. This may inform potential interventions targeting satellite cell activation to alleviate skeletal muscle atrophy. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Involvement of Rictor/mTORC2 in cardiomyocyte differentiation of mouse embryonic stem cells in vitro

PubMed Central

Zheng, Bei; Wang, Jiadan; Tang, Leilei; Tan, Chao; Zhao, Zhe; Xiao, Yi; Ge, Renshan; Zhu, Danyan

2017-01-01

Rictor is a key regulatory/structural subunit of the mammalian target of rapamycin complex 2 (mTORC2) and is required for phosphorylation of Akt at serine 473. It plays an important role in cell survival, actin cytoskeleton organization and other processes in embryogenesis. However, the role of Rictor/mTORC2 in the embryonic cardiac differentiation has been uncovered. In the present study, we examined a possible link between Rictor expression and cardiomyocyte differentiation of the mouse embryonic stem (mES) cells. Knockdown of Rictor by shRNA significantly reduced the phosphorylation of Akt at serine 473 followed by a decrease in cardiomyocyte differentiation detected by beating embryoid bodies. The protein levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein) and α-Actinin (cardiomyocyte biomarker) decreased in Rictor knockdown group during cardiogenesis. Furthermore, knockdown of Rictor specifically inhibited the ventricular-like cells differentiation of mES cells with reduced level of ventricular-specific protein, MLC-2v. Meanwhile, patch-clamp analysis revealed that shRNA-Rictor significantly increased the number of cardiomyocytes with abnormal electrophysiology. In addition, the expressions and distribution patterns of cell-cell junction proteins (Cx43/Desmoplakin/N-cadherin) were also affected in shRNA-Rictor cardiomyocytes. Taken together, the results demonstrated that Rictor/mTORC2 might play an important role in the cardiomyocyte differentiation of mES cells. Knockdown of Rictor resulted in inhibiting ventricular-like myocytes differentiation and induced arrhythmias symptom, which was accompanied by interfering the expression and distribution patterns of cell-cell junction proteins. Rictor/mTORC2 might become a new target for regulating cardiomyocyte differentiation and a useful reference for application of the induced pluripotent stem cells. PMID:28123351

Human periapical cyst-mesenchymal stem cells differentiate into neuronal cells.

PubMed

Marrelli, M; Paduano, F; Tatullo, M

2015-06-01

It was recently reported that human periapical cysts (hPCys), a commonly occurring odontogenic cystic lesion of inflammatory origin, contain mesenchymal stem cells (MSCs) with the capacity for self-renewal and multilineage differentiation. In this study, periapical inflammatory cysts were compared with dental pulp to determine whether this tissue may be an alternative accessible tissue source of MSCs that retain the potential for neurogenic differentiation. Flow cytometry and immunofluorescence analysis indicated that hPCy-MSCs and dental pulp stem cells spontaneously expressed the neuron-specific protein β-III tubulin and the neural stem-/astrocyte-specific protein glial fibrillary acidic protein (GFAP) in their basal state before differentiation occurs. Furthermore, undifferentiated hPCy-MSCs showed a higher expression of transcripts for neuronal markers (β-III tubulin, NF-M, MAP2) and neural-related transcription factors (MSX-1, Foxa2, En-1) as compared with dental pulp stem cells. After exposure to neurogenic differentiation conditions (neural media containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], and retinoic acid), the hPCy-MSCs showed enhanced expression of β-III tubulin and GFAP proteins, as well as increased expression of neurofilaments medium, neurofilaments heavy, and neuron-specific enolase at the transcript level. In addition, neurally differentiated hPCy-MSCs showed upregulated expression of the neural transcription factors Pitx3, Foxa2, Nurr1, and the dopamine-related genes tyrosine hydroxylase and dopamine transporter. The present study demonstrated for the first time that hPCy-MSCs have a predisposition toward the neural phenotype that is increased when exposed to neural differentiation cues, based on upregulation of a comprehensive set of proteins and genes that define neuronal cells. In conclusion, these results provide evidence that hPCy-MSCs might be another optimal source of neural/glial cells for cell-based therapies to treat neurologic diseases. © International & American Associations for Dental Research 2015.

Increased AAA-TOB3 correlates with lymph node metastasis and advanced stage of lung adenocarcinoma.

PubMed

Liu, Yanfeng; Bu, Lina; Li, Wei; Wu, Wei; Wang, Shengyu; Diao, Xin; Zhou, Jing; Chen, Guoan; Yang, Shuanying

2017-07-24

This study was to investigate the differential mitochondrial protein expressions in human lung adenocarcinoma and provide preliminary data for further exploration of the carcinogenic mechanism. Total proteins of A549 and 16HBE mitochondria were extracted through 2D polyacrylamide gel electrophoresis (2-DE). The differential mitochondria proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were further confirmed by Western blot, immunoelectron microscopy and immunohistochemistry (IHC) in A549 cells as well as lung adenocarcinoma tissues. A total of 41 differentially expressed protein spots were found in A549 mitochondria. Of them, 15 proteins were highly expressed and 26 proteins were lowly expressed in the mitochondria of A549 (by more than 1.5 times). Among the 15 more highly expressed proteins, AAA-TOB3 (by more than 3 times) was highly expressed in the mitochondria of A549 compared with the 16HBE, by LC-MS/MS identification. High electron density and clear circular colloidal gold-marked AAA-TOB3 particles were observed in the A549 cells via immunoelectron microscopy. Besides, AAA-TOB3 was confirmed to be elevated in lung adenocarcinoma by Western blot and IHC. Moreover, increased AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma (p<0.05). AAA-TOB3 was highly expressed in lung adenocarcinoma, and the up-regulation of AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma, which suggested that it could serve as a potential molecular marker for lung adenocarcinoma.

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

PubMed

Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

2001-03-15

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress.

PubMed

Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

2014-07-01

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential expression of genes in fetal brain as a consequence of maternal protein deficiency and nematode infection.

PubMed

Haque, Manjurul; Starr, Lisa M; Koski, Kristine G; Scott, Marilyn E

2018-01-01

Maternal dietary protein deficiency and gastrointestinal nematode infection during early pregnancy have negative impacts on both maternal placental gene expression and fetal growth in the mouse. Here we used next-generation RNA sequencing to test our hypothesis that maternal protein deficiency and/or nematode infection also alter the expression of genes in the developing fetal brain. Outbred pregnant CD1 mice were used in a 2×2 design with two levels of dietary protein (24% versus 6%) and two levels of infection (repeated sham versus Heligmosomoides bakeri beginning at gestation day 5). Pregnant dams were euthanized on gestation day 18 to harvest the whole fetal brain. Four fetal brains from each treatment group were analyzed using RNA Hi-Seq sequencing and the differential expression of genes was determined by the edgeR package using NetworkAnalyst. In response to maternal H. bakeri infection, 96 genes (88 up-regulated and eight down-regulated) were differentially expressed in the fetal brain. Differentially expressed genes were involved in metabolic processes, developmental processes and the immune system according to the PANTHER classification system. Among the important biological functions identified, several up-regulated genes have known neurological functions including neuro-development (Gdf15, Ing4), neural differentiation (miRNA let-7), synaptic plasticity (via suppression of NF-κβ), neuro-inflammation (S100A8, S100A9) and glucose metabolism (Tnnt1, Atf3). However, in response to maternal protein deficiency, brain-specific serine protease (Prss22) was the only up-regulated gene and only one gene (Dynlt1a) responded to the interaction of maternal nematode infection and protein deficiency. In conclusion, maternal exposure to GI nematode infection from day 5 to 18 of pregnancy may influence developmental programming of the fetal brain. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

RNA-seq Analysis of Host and Viral Gene Expression Highlights Interaction between Varicella Zoster Virus and Keratinocyte Differentiation

PubMed Central

Singh, Manuraj; Kanda, Ravinder K.; Yee, Michael B.; Kellam, Paul; Hollinshead, Michael; Kinchington, Paul R.; O'Toole, Edel A.; Breuer, Judith

2014-01-01

Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread. PMID:24497829

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation.

PubMed

Raschellà, G; Tanno, B; Bonetto, F; Negroni, A; Claudio, P P; Baldi, A; Amendola, R; Calabretta, B; Giordano, A; Paggi, M G

1998-05-01

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.

Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs

PubMed Central

2014-01-01

Background The genome is pervasively transcribed but most transcripts do not code for proteins, constituting non-protein-coding RNAs. Despite increasing numbers of functional reports of individual long non-coding RNAs (lncRNAs), assessing the extent of functionality among the non-coding transcriptional output of mammalian cells remains intricate. In the protein-coding world, transcripts differentially expressed in the context of processes essential for the survival of multicellular organisms have been instrumental in the discovery of functionally relevant proteins and their deregulation is frequently associated with diseases. We therefore systematically identified lncRNAs expressed differentially in response to oncologically relevant processes and cell-cycle, p53 and STAT3 pathways, using tiling arrays. Results We found that up to 80% of the pathway-triggered transcriptional responses are non-coding. Among these we identified very large macroRNAs with pathway-specific expression patterns and demonstrated that these are likely continuous transcripts. MacroRNAs contain elements conserved in mammals and sauropsids, which in part exhibit conserved RNA secondary structure. Comparing evolutionary rates of a macroRNA to adjacent protein-coding genes suggests a local action of the transcript. Finally, in different grades of astrocytoma, a tumor disease unrelated to the initially used cell lines, macroRNAs are differentially expressed. Conclusions It has been shown previously that the majority of expressed non-ribosomal transcripts are non-coding. We now conclude that differential expression triggered by signaling pathways gives rise to a similar abundance of non-coding content. It is thus unlikely that the prevalence of non-coding transcripts in the cell is a trivial consequence of leaky or random transcription events. PMID:24594072

Identification of the key molecules involved in chronic copper exposure-aggravated memory impairment in transgenic mice of Alzheimer's disease using proteomic analysis.

PubMed

Yu, Jun; Luo, Xiaobin; Xu, Hua; Ma, Quan; Yuan, Jianhui; Li, Xuling; Chang, Raymond Chuen-Chung; Qu, Zhongsen; Huang, Xinfeng; Zhuang, Zhixiong; Liu, Jianjun; Yang, Xifei

2015-01-01

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by a progressive impairment of cognitive functions including spatial learning and memory. Excess copper exposure accelerates the development of AD; however, the potential mechanisms by which copper exacerbates the symptoms of AD remain unknown. In this study, we explored the effects of chronic copper exposure on cognitive function by treating 6 month-old triple AD transgenic (3xTg-AD) mice with 250 ppm copper sulfate in drinking water for 6 months, and identified several potential key molecules involved in the effects of chronic copper exposure on memory by proteomic analysis. The behavioral test showed that chronic copper exposure aggravated memory impairment of 3xTg-AD mice. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry revealed a total of 44 differentially expressed proteins (18 upregulated and 26 down-regulated) in hippocampus between the wild-type (WT) mice and non-exposed 3xTg-AD mice. A total of 40 differentially expressed proteins were revealed (20 upregulated and 20 down-regulated) in hippocampus between copper exposed and non-exposed 3xTg-AD mice. Among these differentially expressed proteins, complexin-1 and complexin-2, two memory associated proteins, were significantly decreased in hippocampus of 3xTg-AD mice compared with the WT mice. Furthermore, the expression of these two proteins was further down-regulated in 3xTg-AD mice when exposed to copper. The abnormal expression of complexin-1 and complexin-2 identified by proteomic analysis was verified by western blot analysis. Taken together, our data showed that chronic copper exposure accelerated memory impairment and altered the expression of proteins in hippocampus in 3xTg-AD mice. The functional analysis on the differentially expressed proteins suggested that complexin-1 and complexin-2 may be the key molecules involved in chronic copper exposure-aggravated memory impairment in AD.

Up-regulation of hnRNP A1, Ezrin, tubulin β-2C and Annexin A1 in sentinel lymph nodes of colorectal cancer

PubMed Central

He, Zhen-Yu; Wen, Hao; Shi, Chuan-Bing; Wang, Jie

2010-01-01

AIM: To investigate the early metastasis-associated proteins in sentinel lymph node micrometastasis (SLNMM) of colorectal cancer (CRC) through comparative proteome. METHODS: Hydrophobic protein samples were extracted from individual-matched normal lymph nodes (NLN) and SLNMM of CRC. Differentially expressed protein spots were detected by two-dimensional electrophoresis and image analysis, and subsequently identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry-mass spectrometry and Western blotting, respectively. RESULTS: Forty proteins were differentially expressed in NLN and SLNMM, and 4 metastasis-concerned proteins highly expressed in SLNMM were identified to be hnRNP A1, Ezrin, tubulin β-2C and Annexin A1. Further immunohistochemistry staining of these four proteins showed their clinicopathological characteristics in lymph node metastasis of CRC. CONCLUSION: Variations of hydrophobic protein expression in NLN and SLNMM of CRC and increased expression of hnRNP A1, Ezrin, tubulin β-2C and Annexin A1 in SLNMM suggest a significantly elevated early CRC metastasis. PMID:20872967

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules.

PubMed

Kramer, Jan; Steinhoff, Jürgen; Klinger, Matthias; Fricke, Lutz; Rohwedel, Jürgen

2006-03-01

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.

Proteins related to the functions of fibroblast-like synoviocytes identified by proteomic analysis.

PubMed

Zhang, Hui; Fan, Lie Ying; Zong, Ming; Sun, Li Shan; Lu, Liu

2012-01-01

It is well known that the fibroblast-like synoviocytes (FLS) play a key role in pathogenesis of rheumatoid arthritis (RA). This study was performed to separate the differentially expressed proteins of FLS from the patients with RA or osteoarthritis (OA) by two-dimensional electrophoresis (2-DE), and found proteins associated with the functions of FLS by mass spectrometry (MS). Total proteins were extracted and quantified from the primary cultured FLS from patients of RA (n=8) or OA (n=6). Proteins were separated by high-resolution 2-DE, and identified the differentially expressed proteins by MS. Western blot analyses was used to validated the expression of candidate proteins. The mRNA of these proteins was detected by semi-quantitative fluorescent PCR. There are 1147 protein spots from RA and 1324 protein spots from OA showed on 2-DE graphs, respectively. We have selected 84 protein spots for MS analysis, and 27 protein spots were successfully identified. We have found that protein isoaspartyl methyltransferase (PIMT) and pirin (iron-binding nuclear protein, PIR) with lower expression in RA, and thioredoxin 1(Trx-1) only expressed in RA may be associated with functions of FLS. Western Blot confirmed the expression of PIMT and pirin lower in RA, and Trx-1 expressed only in RA. The results of semi-quantitative fluorescent PCR are also consistent with 2-DE graphs. PIMT, pirin and Trx-1 affect the functions of FLS in some style and can be the drug targets of RA.

The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts.

PubMed

Donker, Rogier B; Mouillet, Jean-François; Nelson, D Michael; Sadovsky, Yoel

2007-04-01

Endogenous microRNAs (miRNAs) post-transcriptionally regulate mRNA and protein expression during tissue development and function. Whereas adaptation to environmental insults are tightly regulated in human tissues, the role of miRNAs and miRNA biogenesis proteins in this context is inadequately explored. We sought to analyse the expression of the key RNAi enzyme Argonaute2 (Ago2) and other miRNA biogenesis proteins in human trophoblasts during differentiation and in hypoxic environment. Using an in vitro analysis of primary term human trophoblasts, we identified the expression of the core miRNA biogenesis proteins in human villous trophoblasts, with expression levels unaffected by cellular differentiation. We found that the miRNA biosynthetic pathway was functional and produced miRNAs, with miR-93 up-regulated and miR-424 down-regulated in hypoxic environment. In contrast, hypoxia did not alter the expression of key miRNA machinery proteins. The pivotal miRNA processing enzyme Ago2, along with its interacting protein DP103, were expressed in normal placentas as well as in placentas from pregnancies complicated by placental hypoperfusion that resulted in fetal growth restriction. Ago2 and DP103 co-immunoprecipitated, and did not limit trophoblast response to hypoxic stress. We concluded that the core miRNA machinery proteins are expressed and functional in human trophoblasts. The influence of hypoxia on the expression of a subset of placental miRNA species is unlikely to reflect altered expression of key miRNA biogenesis proteins.

Global differential gene expression in response to growth temperature alteration in group A Streptococcus.

PubMed

Smoot, L M; Smoot, J C; Graham, M R; Somerville, G A; Sturdevant, D E; Migliaccio, C A; Sylva, G L; Musser, J M

2001-08-28

Pathogens are exposed to different temperatures during an infection cycle and must regulate gene expression accordingly. However, the extent to which virulent bacteria alter gene expression in response to temperatures encountered in the host is unknown. Group A Streptococcus (GAS) is a human-specific pathogen that is responsible for illnesses ranging from superficial skin infections and pharyngitis to severe invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS survives and multiplies at different temperatures during human infection. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in a serotype M1 strain grown to exponential phase at 29 degrees C and 37 degrees C. Approximately 9% of genes were differentially expressed by at least 1.5-fold at 29 degrees C relative to 37 degrees C, including genes encoding transporter proteins, proteins involved in iron homeostasis, transcriptional regulators, phage-associated proteins, and proteins with no known homologue. Relatively few known virulence genes were differentially expressed at this threshold. However, transcription of 28 genes encoding proteins with predicted secretion signal sequences was altered, indicating that growth temperature substantially influences the extracellular proteome. TaqMan real-time reverse transcription-PCR assays confirmed the microarray data. We also discovered that transcription of genes encoding hemolysins, and proteins with inferred roles in iron regulation, transport, and homeostasis, was influenced by growth at 40 degrees C. Thus, GAS profoundly alters gene expression in response to temperature. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen lines of pathogenesis investigation.

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Jooho; Hengstschläger, Markus; Lubec, Gert

2006-05-15

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND. (c) 2006 Wiley-Liss, Inc.

[Expressions of neuropathic pain-related proteins in the spinal cord dorsal horn in rats with bilateral chronic constriction injury].

PubMed

Shen, Le; Li, Xu; Wang, Hai-tang; Yu, Xue-rong; Huang, Yu-guang

2013-12-01

To evaluate the pain-related behavioral changes in rats with bilateral chronic constriction injury(bCCI)and identify the expressions of neuropathic pain-related proteins. The bCCI models were established by ligating the sciatic nerves in female Sprague Dawley rats. Both mechanical hyperalgesia and cold hyperalgesia were evaluated through electronic von Frey and acetone method. Liquid chromatography-mass spectrometry/mass spectrometry was applied to characterize the differentially expressed proteins. Both mechanical withdrawal threshold and cold hyperalgesia threshold decreased significantly on the postoperative day 7 and 14, when compared with na ve or sham rats(P <0.05). Twenty five differentially expressed proteins associated with bilateral CCI were discovered, with eighteen of them were upregulated and seven of them downregulated. The bCCT rats have remarkably decreased mechanical and cold hyperalgesia thresholds. Twenty five neuropathic pain-related proteins are found in the spinal cord dorsal horn.

Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells.

PubMed

Yen, Andrew; Varvayanis, Susi; Smith, James L; Lamkin, Thomas J

2006-02-01

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.

Comparison of Pancreas Juice Proteins from Cancer Versus Pancreatitis Using Quantitative Proteomic Analysis

PubMed Central

Chen, Ru; Pan, Sheng; Cooke, Kelly; Moyes, Kara White; Bronner, Mary P.; Goodlett, David R.; Aebersold, Ruedi; Brentnall, Teresa A.

2008-01-01

Objectives Pancreatitis is an inflammatory condition of the pancreas. However, it often shares many molecular features with pancreatic cancer. Biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis. The efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false-positive involvement of pancreatitis. Methods In an attempt to develop protein biomarkers for pancreatic cancer, we previously use quantitative proteomics to identify and quantify the proteins from pancreatic cancer juice. Pancreatic juice is a rich source of proteins that are shed by the pancreatic ductal cells. In this study, we used a similar approach to identify and quantify proteins from pancreatitis juice. Results In total, 72 proteins were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice. Nineteen of the juice proteins were overexpressed, and 8 were underexpressed in pancreatitis juice by at least 2-fold compared with normal pancreatic juice. Of these 27 differentially expressed proteins in pancreatitis, 9 proteins were also differentially expressed in the pancreatic juice from pancreatic cancer patient. Conclusions Identification of these differentially expressed proteins from pancreatitis juice provides useful information for future study of specific pancreatitis-associated proteins and to eliminate potential false-positive biomarkers for pancreatic cancer. PMID:17198186

Comparison of pancreas juice proteins from cancer versus pancreatitis using quantitative proteomic analysis.

PubMed

Chen, Ru; Pan, Sheng; Cooke, Kelly; Moyes, Kara White; Bronner, Mary P; Goodlett, David R; Aebersold, Ruedi; Brentnall, Teresa A

2007-01-01

Pancreatitis is an inflammatory condition of the pancreas. However, it often shares many molecular features with pancreatic cancer. Biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis. The efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false-positive involvement of pancreatitis. In an attempt to develop protein biomarkers for pancreatic cancer, we previously use quantitative proteomics to identify and quantify the proteins from pancreatic cancer juice. Pancreatic juice is a rich source of proteins that are shed by the pancreatic ductal cells. In this study, we used a similar approach to identify and quantify proteins from pancreatitis juice. In total, 72 proteins were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice. Nineteen of the juice proteins were overexpressed, and 8 were underexpressed in pancreatitis juice by at least 2-fold compared with normal pancreatic juice. Of these 27 differentially expressed proteins in pancreatitis, 9 proteins were also differentially expressed in the pancreatic juice from pancreatic cancer patient. Identification of these differentially expressed proteins from pancreatitis juice provides useful information for future study of specific pancreatitis-associated proteins and to eliminate potential false-positive biomarkers for pancreatic cancer.

Presenilin expression during induced differentiation of the human neuroblastoma SH-SY5Y cell line.

PubMed

Flood, Fiona; Sundström, Erik; Samuelsson, Eva-Britt; Wiehager, Birgitta; Seiger, Ake; Johnston, Janet A; Cowburn, Richard F

2004-06-01

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.

Calcium: A novel and efficient inducer of differentiation of adipose-derived stem cells into neuron-like cells.

PubMed

Goudarzi, Farjam; Tayebinia, Heidar; Karimi, Jamshid; Habibitabar, Elahe; Khodadadi, Iraj

2018-06-05

This study comparatively investigated the effectiveness of calcium and other well-known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose-derived stem cells (ADSCs) into neuronal-like cells. ADSCs were immunophenotyped and differentiated into neuron-like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron-specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction (qRT-PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite-like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron-like cells and instantaneous increase in the expression of neuronal markers. © 2018 Wiley Periodicals, Inc.

Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells

PubMed Central

Cheng, Yu-Che; Huang, Chi-Jung; Lee, Yih-Jing; Tien, Lu-Tai; Ku, Wei-Chi; Chien, Raymond; Lee, Fa-Kung; Chien, Chih-Cheng

2016-01-01

This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons. PMID:27444754

Towards decrypting cryptobiosis--analyzing anhydrobiosis in the tardigrade Milnesium tardigradum using transcriptome sequencing.

PubMed

Wang, Chong; Grohme, Markus A; Mali, Brahim; Schill, Ralph O; Frohme, Marcus

2014-01-01

Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade Milnesium tardigradum Using Transcriptome Sequencing

PubMed Central

Wang, Chong; Grohme, Markus A.; Mali, Brahim; Schill, Ralph O.; Frohme, Marcus

2014-01-01

Background Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. Results A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Conclusions Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair. PMID:24651535

Global-scale profiling of differential expressed lysine acetylated proteins in colorectal cancer tumors and paired liver metastases.

PubMed

Shen, Zhanlong; Wang, Bo; Luo, Jianyuan; Jiang, Kewei; Zhang, Hui; Mustonen, Harri; Puolakkainen, Pauli; Zhu, Jun; Ye, Yingjiang; Wang, Shan

2016-06-16

Lysine acetylated modification was indicated to impact colorectal cancer (CRC)'s distant metastasis. However, the global acetylated proteins in CRC and the differential expressed acetylated proteins and acetylated sites between CRC primary and distant metastatic tumor remains unclear. Our aim was to construct a complete atlas of acetylome in CRC and paired liver metastases. Combining high affinity enrichment of acetylated peptides with high sensitive mass spectrometry, we identified 603 acetylation sites from 316 proteins, among which 462 acetylation sites corresponding to 243 proteins were quantified. We further classified them into groups according to cell component, molecular function and biological process and analyzed the metabolic pathways, domain structures and protein interaction networks. Finally, we evaluated the differentially expressed lysine acetylation sites and revealed that 31 acetylated sites of 22 proteins were downregulated in CRC liver metastases compared to that in primary CRC while 40 acetylated sites of 32 proteins were upregulated, of which HIST2H3AK19Ac and H2BLK121Ac were the acetylated histones most changed, while TPM2 K152Ac and ADH1B K331Ac were the acetylated non-histones most altered. These results provide an expanded understanding of acetylome in CRC and its distant metastasis, and might prove applicable in the molecular targeted therapy of metastatic CRC. This study described provides, for the first time, that full-scale profiling of lysine acetylated proteins were identified and quantified in colorectal cancer (CRC) and paired liver metastases. The novelty of the study is that we constructed a complete atlas of acetylome in CRC and paired liver metastases. Moreover, we analyzed these differentially expressed acetylated proteins in cell component, molecular function and biological process. In addition, metabolic pathways, domain structures and protein interaction networks of acetylated proteins were also investigated. Our approaches shows that of the differentially expressed proteins, HIST2H3AK19Ac and H2BLK121Ac were the acetylated histones most changed, while TPM2 K152Ac and ADH1B K331Ac were the acetylated non-histones most altered. Our findings provide an expanded understanding of acetylome in CRC and its distant metastasis, and might prove applicable in the molecular targeted therapy of metastatic CRC. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation

PubMed Central

van den Bogaard, Ellen; Podolsky, Michael; Smits, Jos; Cui, Xiao; John, Christian; Gowda, Krishne; Desai, Dhimant; Amin, Shantu; Schalkwijk, Joost; Perdew, Gary H.

2015-01-01

Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr-/- and Ahr+/+ murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr-/- keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr+/+ keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM), SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology. PMID:25602157

Determination of Protein Interactome of Transcription Factor Sox2 in Embryonic Stem Cells Engineered for Inducible Expression of Four Reprogramming Factors*

PubMed Central

Gao, Zhiguang; Cox, Jesse L.; Gilmore, Joshua M.; Ormsbee, Briana D.; Mallanna, Sunil K.; Washburn, Michael P.; Rizzino, Angie

2012-01-01

Unbiased proteomic screens provide a powerful tool for defining protein-protein interaction networks. Previous studies employed multidimensional protein identification technology to identify the Sox2-interactome in embryonic stem cells (ESC) undergoing differentiation in response to a small increase in the expression of epitope-tagged Sox2. Thus far the Sox2-interactome in ESC has not been determined. To identify the Sox2-interactome in ESC, we engineered ESC for inducible expression of different combinations of epitope-tagged Sox2 along with Oct4, Klf4, and c-Myc. Epitope-tagged Sox2 was used to circumvent the lack of suitable Sox2 antibodies needed to perform an unbiased proteomic screen of Sox2-associated proteins. Although i-OS-ESC differentiate when both Oct4 and Sox2 are elevated, i-OSKM-ESC do not differentiate even when the levels of the four transcription factors are coordinately elevated ∼2–3-fold. Our findings with i-OS-ESC and i-OSKM-ESC provide new insights into the reasons why ESC undergo differentiation when Sox2 and Oct4 are elevated in ESC. Importantly, the use of i-OSKM-ESC enabled us to identify the Sox2-interactome in undifferentiated ESC. Using multidimensional protein identification technology, we identified >70 proteins that associate with Sox2 in ESC. We extended these findings by testing the function of the Sox2-assoicated protein Smarcd1 and demonstrate that knockdown of Smarcd1 disrupts the self-renewal of ESC and induces their differentiation. Together, our work provides the first description of the Sox2-interactome in ESC and indicates that Sox2 along with other master regulators is part of a highly integrated protein-protein interaction landscape in ESC. PMID:22334693

[Expression and Significance of PI-PLCε1 in Colon Cancer].

PubMed

Li, Xiao-Ran; Yang, Kun; Huang, Xiao-Li

2017-11-01

To study the expression and clinical significance of phosphoinositide-specific phospholipase Cε1 (PI-PLCε1) in the pathogenesis of colon cancer. qRT-PCR and immunohistochemistry were used to detect the expression of PI-PLCε1 in the 42 cases of colon cancer tissues and their corresponding adjacent tissues. And the effects of tumor differentiation and tumor site on the expression PI-PLCε1 of colon cancer tissues were compared. The results of qRT-PCR showed that the expression of PI-PLCε1 in colon cancer tissue significantly lower than that in the adjacent tissue ( P <0.05). The expression of PI-PLCε1 gene of colon cancer tissue was not effected by tumor differentiation and tumor site ( P >0.05). The results of immuno-histochemistry showed that the positive expression rate of PI-PLCε1 protein in colon cancer tissue was significantly lower than that in the adjacent tissue ( P <0.05). The positive expression rate of PI-PLCε1 protein was not effected by tumor differentiation ( P >0.05),but the expression was different in tumor site ( P <0.05). Expression of PI-PLCε1 was reduced in colon tissue and barely to tumor differentiation.

Klf10 regulates odontoblast differentiation and mineralization via promoting expression of dentin matrix protein 1 and dentin sialophosphoprotein genes

PubMed Central

Chen, Zhuo; Li, Wentong; Wang, Han; Wan, Chunyan; Luo, Daoshu; Deng, Shuli

2016-01-01

Klf10, a member of the Krüppel-like family of transcription factors, is critical for osteoblast differentiation, bone formation and mineralization. However, whether Klf10 is involved in odontoblastic differentiation and tooth development has not been determined. In this study, we investigate the expression patterns of Klf10 during murine tooth development in vivo and its role in odontoblastic differentiation in vitro. Klf10 protein was expressed in the enamel organ and the underlying mesenchyme, ameloblasts and odontoblasts at early and later stages of murine molar formation. Furthermore, the expression of Klf10, Dmp1, Dspp and Runx2 was significantly elevated during the process of mouse dental papilla mesenchymal differentiation and mineralization. The overexpression of Klf10 induced dental papilla mesenchymal cell differentiation and mineralization as detected by alkaline phosphatase staining and alizarin red S assay. Klf10 additionally up-regulated the expression of odontoblastic differentiation marker genes Dmp1, Dspp and Runx2 in mouse dental papilla mesenchymal cells. The molecular mechanism of Klf10 in controlling Dmp1 and Dspp expression is thus to activate their regulatory regions in a dosage-dependent manner. Our results suggest that Klf10 is involved in tooth development and promotes odontoblastic differentiation via the up-regulation of Dmp1 and Dspp transcription. PMID:26310138

Transcriptome Analysis of Flower Sex Differentiation in Jatropha curcas L. Using RNA Sequencing.

PubMed

Xu, Gang; Huang, Jian; Yang, Yong; Yao, Yin-an

2016-01-01

Jatropha curcas is thought to be a promising biofuel material, but its yield is restricted by a low ratio of instaminate/staminate flowers (1/10-1/30). Furthermore, valuable information about flower sex differentiation in this plant is scarce. To explore the mechanism of this process in J. curcas, transcriptome profiling of flower development was carried out, and certain genes related with sex differentiation were obtained through digital gene expression analysis of flower buds from different phases of floral development. After Illumina sequencing and clustering, 57,962 unigenes were identified. A total of 47,423 unigenes were annotated, with 85 being related to carpel and stamen differentiation, 126 involved in carpel and stamen development, and 592 functioning in the later development stage for the maturation of staminate or instaminate flowers. Annotation of these genes provided comprehensive information regarding the sex differentiation of flowers, including the signaling system, hormone biosynthesis and regulation, transcription regulation and ubiquitin-mediated proteolysis. A further expression pattern analysis of 15 sex-related genes using quantitative real-time PCR revealed that gibberellin-regulated protein 4-like protein and AMP-activated protein kinase are associated with stamen differentiation, whereas auxin response factor 6-like protein, AGAMOUS-like 20 protein, CLAVATA1, RING-H2 finger protein ATL3J, auxin-induced protein 22D, and r2r3-myb transcription factor contribute to embryo sac development in the instaminate flower. Cytokinin oxidase, Unigene28, auxin repressed-like protein ARP1, gibberellin receptor protein GID1 and auxin-induced protein X10A are involved in both stages mentioned above. In addition to its function in the differentiation and development of the stamens, the gibberellin signaling pathway also functions in embryo sac development for the instaminate flower. The auxin signaling pathway also participates in both stamen development and embryo sac development. Our transcriptome data provide a comprehensive gene expression profile for flower sex differentiation in Jatropha curcas, as well as new clues and information for further study in this field.

Transcriptome Analysis of Flower Sex Differentiation in Jatropha curcas L. Using RNA Sequencing

PubMed Central

Xu, Gang; Huang, Jian; Yang, Yong; Yao, Yin-an

2016-01-01

Background Jatropha curcas is thought to be a promising biofuel material, but its yield is restricted by a low ratio of instaminate / staminate flowers (1/10-1/30). Furthermore, valuable information about flower sex differentiation in this plant is scarce. To explore the mechanism of this process in J. curcas, transcriptome profiling of flower development was carried out, and certain genes related with sex differentiation were obtained through digital gene expression analysis of flower buds from different phases of floral development. Results After Illumina sequencing and clustering, 57,962 unigenes were identified. A total of 47,423 unigenes were annotated, with 85 being related to carpel and stamen differentiation, 126 involved in carpel and stamen development, and 592 functioning in the later development stage for the maturation of staminate or instaminate flowers. Annotation of these genes provided comprehensive information regarding the sex differentiation of flowers, including the signaling system, hormone biosynthesis and regulation, transcription regulation and ubiquitin-mediated proteolysis. A further expression pattern analysis of 15 sex-related genes using quantitative real-time PCR revealed that gibberellin-regulated protein 4-like protein and AMP-activated protein kinase are associated with stamen differentiation, whereas auxin response factor 6-like protein, AGAMOUS-like 20 protein, CLAVATA1, RING-H2 finger protein ATL3J, auxin-induced protein 22D, and r2r3-myb transcription factor contribute to embryo sac development in the instaminate flower. Cytokinin oxidase, Unigene28, auxin repressed-like protein ARP1, gibberellin receptor protein GID1 and auxin-induced protein X10A are involved in both stages mentioned above. In addition to its function in the differentiation and development of the stamens, the gibberellin signaling pathway also functions in embryo sac development for the instaminate flower. The auxin signaling pathway also participates in both stamen development and embryo sac development. Conclusions Our transcriptome data provide a comprehensive gene expression profile for flower sex differentiation in Jatropha curcas, as well as new clues and information for further study in this field. PMID:26848843

Antioxidants Maintain E-Cadherin Levels to Limit Drosophila Prohemocyte Differentiation

PubMed Central

Gao, Hongjuan; Wu, Xiaorong; Simon, LaTonya; Fossett, Nancy

2014-01-01

Mitochondrial reactive oxygen species (ROS) regulate a variety of biological processes by networking with signal transduction pathways to maintain homeostasis and support adaptation to stress. In this capacity, ROS have been shown to promote the differentiation of progenitor cells, including mammalian embryonic and hematopoietic stem cells and Drosophila hematopoietic progenitors (prohemocytes). However, many questions remain about how ROS alter the regulatory machinery to promote progenitor differentiation. Here, we provide evidence for the hypothesis that ROS reduce E-cadherin levels to promote Drosophila prohemocyte differentiation. Specifically, we show that knockdown of the antioxidants, Superoxide dismutatase 2 and Catalase reduce E-cadherin protein levels prior to the loss of Odd-skipped-expressing prohemocytes. Additionally, over-expression of E-cadherin limits prohemocyte differentiation resulting from paraquat-induced oxidative stress. Furthermore, two established targets of ROS, Enhancer of Polycomb and FOS, control the level of E-cadherin protein expression. Finally, we show that knockdown of either Superoxide dismutatase 2 or Catalase leads to an increase in the E-cadherin repressor, Serpent. As a result, antioxidants and targets of ROS can control E-cadherin protein levels, and over-expression of E-cadherin can ameliorate the prohemocyte response to oxidative stress. Collectively, these data strongly suggest that ROS promote differentiation by reducing E-cadherin levels. In mammalian systems, ROS promote embryonic stem cell differentiation, whereas E-cadherin blocks differentiation. However, it is not known if elevated ROS reduce E-cadherin to promote embryonic stem cell differentiation. Thus, our findings may have identified an important mechanism by which ROS promote stem/progenitor cell differentiation. PMID:25226030

Anterior Segment Alterations and Comparative Aqueous Humor Proteomics in the Buphthalmic Rabbit (An American Ophthalmological Society Thesis)

PubMed Central

Edward, Deepak P.; Bouhenni, Rachida

2011-01-01

Purpose To use an integrated proteohistologic approach to gain insight into the anterior segment alterations in the buphthalmic rabbit. Methods Eyes from 2- and 5-year-old buphthalmic and normal rabbits (n=20) were studied histologically. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) of aqueous humor (AH) was used to determine differential protein expression between animal groups. Western blot and immunohistochemistry were performed on selected differentially expressed proteins identified by LC-MS/MS. Results The buphthalmic rabbits manifested a mild clinical phenotype with typical angle anomalies that appeared progressive by histology. Significantly thickened Descemet’s membrane (DM) and anterior lens capsule in all buphthalmic rabbits showed increased fibronectin and collagen-IV immunolabeling. LC-MS/MS applying stringent filtering criteria revealed significant differential expression of several AH proteins in these rabbits. The protein of interest in the 2-year-old group was histidine-rich glycoprotein, and those in the 5-year-old group included alpha-2-HS-glycoprotein, clusterin, apolipoprotein E, interphotoreceptor retinoid-binding protein, transthyretin, cochlin, gelsolin, haptoglobin, hemopexin, and beta-2 microglobulin. The proteomic data for selected proteins was validated by Western blot and immunohistochemistry. A wide range of functional groups were affected by the altered AH proteins. These included extracellular matrix modulation, regulation of apoptosis, oxidative stress, and protein transport. Conclusions Multiple anterior segment alterations were histologically identified in the buphthalmic rabbits that showed progressive changes with age. The differentially expressed AH proteins in these rabbits suggest a multifunctional role for AH in modulating pathologic changes in DM, anterior lens capsule, and the angular meshwork in these animals. PMID:22253484

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

Zhang Bo; Huang Bo; School of Public Health, University of South China, Hengyang, Hunan 421001

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe,more » such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3{sigma} and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: > 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. > Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. > The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. > The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.« less

Differential expression profile of membrane proteins in L-02 cells exposed to trichloroethylene.

PubMed

Hong, Wen-Xu; Huang, Aibo; Lin, Sheng; Yang, Xifei; Yang, Linqing; Zhou, Li; Huang, Haiyan; Wu, Desheng; Huang, Xinfeng; Xu, Hua; Liu, Jianjun

2016-10-01

Trichloroethylene (TCE), a halogenated organic solvent widely used in industries, is known to cause severe hepatotoxicity. However, the mechanisms underlying TCE hepatotoxicity are still not well understood. It is predicted that membrane proteins are responsible for key biological functions, and recent studies have revealed that TCE exposure can induce abnormal levels of membrane proteins in body fluids and cultured cells. The aim of this study is to investigate the TCE-induced alterations of membrane proteins profiles in human hepatic L-02 liver cells. A comparative membrane proteomics analysis was performed in combination with two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 15 proteins were identified as differentially expressed (4 upregulated and 11 downregulated) between TCE-treated cells and normal controls. Among this, 14 of them are suggested as membrane-associated proteins by their transmembrane domain and/or subcellular location. Furthermore, the differential expression of β subunit of adenosine triphosphate synthase (ATP5B) and prolyl 4-hydroxylase, β polypeptide (P4HB) were verified by Western blot analysis in TCE-treated L-02 cells. Our work not only reveals the association between TCE exposure and altered expression of membrane proteins but also provides a novel strategy to discover membrane biomarkers and elucidate the potential mechanisms involving with membrane proteins response to chemical-induced toxic effect. © The Author(s) 2015.

Differential expression analysis of the broiler tracheal proteins responsible for the immune response and muscle contraction induced by high concentration of ammonia using iTRAQ-coupled 2D LC-MS/MS.

PubMed

Xiong, Yan; Tang, Xiangfang; Meng, Qingshi; Zhang, Hongfu

2016-11-01

Ammonia has been considered the contaminant primarily responsible for respiratory disease in poultry. Even though it can cause tracheal lesions, its adverse effects on the trachea have not been sufficiently studied. The present study investigated tracheal changes in Arbor Acres broilers (Gallus gallus) induced by high concentration of ammonia using isobaric tag for relative and absolute quantification (iTRAQ)-based proteome analysis. In total, 3,706 proteins within false discovery rate of 1% were identified, including 119 significantly differentially expressed proteins. Functional analysis revealed that proteins related to immune response and muscle contraction were significantly enriched. With respect to the immune response, up-regulated proteins (like FGA) were pro-inflammatory, while down-regulated proteins participated in antigen processing and antigen presenting (like MYO1G), immunoglobulin and cathelicidin production (like fowlicidin-2), and immunodeficiency (like PTPRC). Regarding muscle contraction, all differentially expressed proteins (like TPM1) were up-regulated. An over-expression of mucin, which is a common feature of airway disease, was also observed. Additionally, the transcriptional alterations of 6 selected proteins were analyzed by quantitative RT-PCR. Overall, proteomic changes suggested the onset of airway obstruction and diminished host defense in trachea after ammonia exposure. These results may serve as a valuable reference for future interventions against ammonia toxicity.

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation

PubMed Central

Veljkovic, D. Kika; Rivard, Georges E.; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M.

2009-01-01

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and α-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34+ progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD α-granules. Although QPD CD34+ progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIγ on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of α-granule proteins, which was normal. uPA was localized to QPD α-granules and it showed extensive colocalization with α-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, α-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with α-granule proteins prior to their proteolysis in QPD. PMID:19029443

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation.

PubMed

Veljkovic, D Kika; Rivard, Georges E; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M; Hayward, Catherine P M

2009-02-12

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and alpha-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34(+) progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD alpha-granules. Although QPD CD34(+) progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIgamma on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of alpha-granule proteins, which was normal. uPA was localized to QPD alpha-granules and it showed extensive colocalization with alpha-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, alpha-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with alpha-granule proteins prior to their proteolysis in QPD.

Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

USGS Publications Warehouse

Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

2011-01-01

The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

Differential expression of genes and proteins associated with wool follicle cycling.

PubMed

Liu, Nan; Li, Hegang; Liu, Kaidong; Yu, Juanjuan; Cheng, Ming; De, Wei; Liu, Jifeng; Shi, Shuyan; He, Yanghua; Zhao, Jinshan

2014-08-01

Sheep are valuable resources for the wool industry. Wool growth of Aohan fine wool sheep has cycled during different seasons in 1 year. Therefore, identifying genes that control wool growth cycling might lead to ways for improving the quality and yield of fine wool. In this study, we employed Agilent sheep gene expression microarray and proteomic technology to compare the gene expression patterns of the body side skins at August and December time points in Aohan fine wool sheep (a Chinese indigenous breed). Microarray study revealed that 2,223 transcripts were differentially expressed, including 1,162 up-regulated and 1,061 down-regulated transcripts, comparing body side skin at the August time point to the December one (A/D) in Aohan fine wool sheep. Then seven differentially expressed genes were selected to validated the reliability of the gene chip data. The majority of the genes possibly related to follicle development and wool growth could be assigned into the categories including regulation of receptor binding, extracellular region, protein binding and extracellular space. Proteomic study revealed that 84 protein spots showed significant differences in expression levels. Of the 84, 63 protein spots were upregulated and 21 were downregulated in A/D. Finally, 55 protein points were determined through MALDI-TOF/MS analyses. Furthermore, the regulation mechanism of hair follicle might resemble that of fetation.

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development.

PubMed

Santana, Flávia A; Nunes, Francis M F; Vieira, Carlos U; Machado, Maria Alice M S; Kerr, Warwick E; Silva, Wilson A; Bonetti, Ana Maria

2006-03-01

We have compared gene expression, using the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.

2D DIGE Does Not Reveal all: A Scotopic Report Suggests Differential Expression of a Single "Calponin Family Member" Protein for Tetany of Sphincters!

PubMed

Chaudhury, Arun

2015-01-01

Using 2D differential gel electrophoresis (DIGE) and mass spectrometry (MS), a recent report by Rattan and Ali (2015) compared proteome expression between tonically contracted sphincteric smooth muscles of the internal anal sphincter (IAS), in comparison to the adjacent rectum [rectal smooth muscles (RSM)] that contracts in a phasic fashion. The study showed the differential expression of a single 23 kDa protein SM22, which was 1.87 fold, overexpressed in RSM in comparison to IAS. Earlier studies have shown differences in expression of different proteins like Rho-associated protein kinase II, myosin light chain kinase, myosin phosphatase, and protein kinase C between IAS and RSM. The currently employed methods, despite its high-throughput potential, failed to identify these well-characterized differences between phasic and tonic muscles. This calls into question the fidelity and validatory potential of the otherwise powerful technology of 2D DIGE/MS. These discrepancies, when redressed in future studies, will evolve this recent report as an important baseline study of "sphincter proteome." Proteomics techniques are currently underutilized in examining pathophysiology of hypertensive/hypotensive disorders involving gastrointestinal sphincters, including achalasia, gastroesophageal reflux disease (GERD), spastic pylorus, seen during diabetes or chronic chemotherapy, intestinal pseudo-obstruction, and recto-anal incontinence. Global proteome mapping may provide instant snapshot of the complete repertoire of differential proteins, thus expediting to identify the molecular pathology of gastrointestinal motility disorders currently labeled "idiopathic" and facilitating practice of precision medicine.

Inactivation of EGFR/AKT signaling enhances TSA-induced ovarian cancer cell differentiation.

PubMed

Shao, Genbao; Lai, Wensheng; Wan, Xiaolei; Xue, Jing; Wei, Ye; Jin, Jie; Zhang, Liuping; Lin, Qiong; Shao, Qixiang; Zou, Shengqiang

2017-05-01

Ovarian tumor is one of the most lethal gynecologic cancers, but differentiation therapy for this cancer is poorly characterized. Here, we show that thrichostatin A (TSA), the well known inhibitor of histone deacetylases (HDACs), can induce cell differentiation in HO8910 ovarian cancer cells. TSA-induced cell differentiation is characterized by typical morphological change, increased expression of the differentiation marker FOXA2, decreased expression of the pluripotency markers SOX2 and OCT4, suppressing cell proliferation, and cell cycle arrest in the G1 phase. TSA also induces an elevated expression of cell cycle inhibitory protein p21Cip1 along with a decrease in cell cycle regulatory protein cyclin D1. Significantly, blockage of epidermal growth factor receptor (EGFR) signaling pathway with specific inhibitors of this signaling cascade promotes the TSA-induced differentiation of HO8910 cells. These results imply that the EGFR cascade inhibitors in combination with TSA may represent a promising differentiation therapy strategy for ovarian cancer.

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.

PubMed

Amjadi, Fatemehsadat; Mehdizadeh, Mehdi; Ashrafi, Mahnaz; Nasrabadi, Davood; Taleahmad, Sara; Mirzaei, Mehdi; Gupta, Vivek; Salekdeh, Ghasem Hosseini; Aflatoonian, Reza

2018-04-21

What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)? In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3-5 after ovulation, n = 6) from healthy women and PCOS patients (12-14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources. Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton. The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Comparative Proteomic Profiling Reveals Molecular Characteristics Associated with Oogenesis and Oocyte Maturation during Ovarian Development of Bactrocera dorsalis (Hendel)

PubMed Central

Li, Ran; Zhang, Meng-Yi; Liu, Yu-Wei; Zhang, Zheng; Smagghe, Guy; Wang, Jin-Jun

2017-01-01

Time-dependent expression of proteins in ovary is important to understand oogenesis in insects. Here, we profiled the proteomes of developing ovaries from Bactrocera dorsalis (Hendel) to obtain information about ovarian development with particular emphasis on differentially expressed proteins (DEPs) involved in oogenesis. A total of 4838 proteins were identified with an average peptide number of 8.15 and sequence coverage of 20.79%. Quantitative proteomic analysis showed that a total of 612 and 196 proteins were differentially expressed in developing and mature ovaries, respectively. Furthermore, 153, 196 and 59 potential target proteins were highly expressed in early, vitellogenic and mature ovaries and most tested DEPs had the similar trends consistent with the respective transcriptional profiles. These proteins were abundantly expressed in pre-vitellogenic and vitellogenic stages, including tropomyosin, vitellogenin, eukaryotic translation initiation factor, heat shock protein, importin protein, vitelline membrane protein, and chorion protein. Several hormone and signal pathway related proteins were also identified during ovarian development including piRNA, notch, insulin, juvenile, and ecdysone hormone signal pathways. This is the first report of a global ovary proteome of a tephritid fruit fly, and may contribute to understanding the complicate processes of ovarian development and exploring the potentially novel pest control targets. PMID:28665301

[Expression of Ki-67 and P53 protein in oral squamous cell carcinoma and its clinical significance].

PubMed

He, Wei; Xiao, Yan; Chen, Wei-min

2015-04-01

To investigate the clinical and pathological features and its relationship with the expression of Ki-67 and p53 protein in oral squamous cell carcinoma. Immunohistochemical SP staining method was used to quantify the protein expression levels of Ki-67 and p53 protein in 10 cases of normal oral mucosa, 16 cases of oral leukoplakia (OLK) tissue, and 48 cases of oral squamous cell carcinoma. The relationship of the expression of Ki-67 and p53 protein to clinical and pathological data was analyzed, and SPSS17.0 software package was used for statistical analysis. The positive expression rate of Ki-67 protein in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma was 30%, 56.3% and 79.2%, respectively; The positive expression rate of p53 was 0%, 43.8%, and 70.8%, respectively; Ki-67 and p53 expression had significant difference among normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma (P<0.05); The expression of Ki-67 protein was significantly elevated with tumor stage, differentiation and cervical lymph node metastasis (P<0.05); The expression of p53 protein was significantly related to the degree of tumor differentiation (P<0.05); The expression of Ki-67 and p53 was positively correlated in oral squamous cell carcinoma (P<0.05). The high expression of Ki-67 and p53 protein in oral squamous cell carcinoma tissues may play an important role in the development of oral squamous cell carcinoma.

A surface transporter family conveys the trypanosome differentiation signal.

PubMed

Dean, Samuel; Marchetti, Rosa; Kirk, Kiaran; Matthews, Keith R

2009-05-14

Microbial pathogens use environmental cues to trigger the developmental events needed to infect mammalian hosts or transmit to disease vectors. The parasites causing African sleeping sickness respond to citrate or cis-aconitate (CCA) to initiate life-cycle development when transmitted to their tsetse fly vector. This requires hypersensitization of the parasites to CCA by exposure to low temperature, conditions encountered after tsetse fly feeding at dusk or dawn. Here we identify a carboxylate-transporter family, PAD (proteins associated with differentiation), required for perception of this differentiation signal. Consistent with predictions for the response of trypanosomes to CCA, PAD proteins are expressed on the surface of the transmission-competent 'stumpy-form' parasites in the bloodstream, and at least one member is thermoregulated, showing elevated expression and surface access at low temperature. Moreover, RNA-interference-mediated ablation of PAD expression diminishes CCA-induced differentiation and eliminates CCA hypersensitivity under cold-shock conditions. As well as being molecular transducers of the differentiation signal in these parasites, PAD proteins provide the first example of a surface marker able to discriminate the transmission stage of trypanosomes in their mammalian host.

Comparative proteomic and bioinformatic analysis of Theileria luwenshuni and Theileria uilenbergi.

PubMed

Zhang, Xiao; Li, Youquan; Chen, Ze; Liu, Zhijie; Ren, Qiaoyun; Yang, Jifei; Zhu, Xinquan; Guan, Guiquan; Liu, Aihong; Luo, Jianxun; Yin, Hong

2016-07-01

Theileria is an obligatory intraerythrocytic protozoan parasite that causes economic losses to the cattle, sheep and goats industry. However, very little information is available on the genomes, transcriptomes, and proteomes of the ovine parasites, Theileria luwenshuni and Theileria uilenbergi. Differences in protein expression between these species were investigated to better understand their biology. Parasites were digested with trypsin, and the resulting peptides labeled with isobaric tags for relative and absolute quantification, followed by LC-MS/MS. More than 670 proteins, classified into categories primarily related to cellular process (29.78%), metabolic process (28.80%), localization (5.22%) and biological regulation (5.00%), were identified. Seventy-one proteins were differentially expressed; T. luwenshuni had 39 proteins more highly expressed than in T. uilenbergi, whereas T. uilenbergi had 32 that were more highly expressed. Several proteins related to parasite virulence and invasion (cysteine proteinase, histone deacetylase, pyruvate kinase, small nuclear ribonucleoprotein and orotate phosphoribosyltransferase) were differentially expressed. Real-time quantitative PCR validated protein expression changes at the transcript level. This is the first report on protein expression for the two most economically important Theileria species in China, and our findings may provide novel opportunities for ovine and caprine theileriosis control. Copyright © 2016 Elsevier Inc. All rights reserved.

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

PubMed

Aït Ghezali, Lamia; Arbabian, Atousa; Roudot, Hervé; Brouland, Jean-Philippe; Baran-Marszak, Fanny; Salvaris, Evelyn; Boyd, Andrew; Drexler, Hans G; Enyedi, Agnes; Letestu, Remi; Varin-Blank, Nadine; Papp, Bela

2017-06-26

Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Integrator complex plays an essential role in adipose differentiation

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

Otani, Yuichiro; Nakatsu, Yusuke; Sakoda, Hideyuki

2013-05-03

Highlights: •IntS6 and IntS11 are subunits of the Integrator complex. •Expression levels of IntS6 and IntS11 were very low in 3T3-L1 fibroblast. •IntS6 and IntS11 were upregulated during adipose differentiation. •Suppression of IntS6 or IntS11 expression inhibited adipose differentiation. -- Abstract: The dynamic process of adipose differentiation involves stepwise expressions of transcription factors and proteins specific to the mature fat cell phenotype. In this study, it was revealed that expression levels of IntS6 and IntS11, subunits of the Integrator complex, were increased in 3T3-L1 cells in the period when the cells reached confluence and differentiated into adipocytes, while being reducedmore » to basal levels after the completion of differentiation. Suppression of IntS6 or IntS11 expression using siRNAs in 3T3-L1 preadipocytes markedly inhibited differentiation into mature adipocytes, based on morphological findings as well as mRNA analysis of adipocyte-specific genes such as Glut4, perilipin and Fabp4. Although Pparγ2 protein expression was suppressed in IntS6 or IntS11-siRNA treated cells, adenoviral forced expression of Pparγ2 failed to restore the capacity for differentiation into mature adipocytes. Taken together, these findings demonstrate that increased expression of Integrator complex subunits is an indispensable event in adipose differentiation. Although further study is necessary to elucidate the underlying mechanism, the processing of U1, U2 small nuclear RNAs may be involved in cell differentiation steps.« less

Genome-wide Analysis of the H3K4 Histone Demethylase RBP2 Reveals a Transcriptional Program Controlling Differentiation

PubMed Central

Lopez-Bigas, Nuria; Kisiel, Tomasz A.; DeWaal, Dannielle C.; Holmes, Katie B.; Volkert, Tom L.; Gupta, Sumeet; Love, Jennifer; Murray, Heather L.; Young, Richard A.; Benevolenskaya, Elizaveta V.

2010-01-01

SUMMARY Retinoblastoma protein (pRB) mediates cell-cycle withdrawal and differentiation by interacting with a variety of proteins. RB-Binding Protein 2 (RBP2) has been shown to be a key effector. We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis and gene expression profiling experiments. We describe that RBP2 shows high correlation with the presence of H3K4me3 and its target genes are separated into two functionally distinct classes: differentiation-independent and differentiation-dependent genes. The former class is enriched by genes that encode mitochondrial proteins, while the latter is represented by cell-cycle genes. We demonstrate the role of RBP2 in mitochondrial biogenesis, which involves regulation of H3K4me3-modified nucleosomes. Analysis of expression changes upon RBP2 depletion depicted genes with a signature of differentiation control, analogous to the changes seen upon reintroduction of pRB. We conclude that, during differentiation, RBP2 exerts inhibitory effects on multiple genes through direct interaction with their promoters. PMID:18722178

Proteomics screening of adenosine triphosphate-interacting proteins in the liver of diazinon-treated rats.

PubMed

Pourtaji, A; Robati, R Yazdian; Lari, P; Hosseinzadeh, H; Ramezani, M; Abnous, K

2016-10-01

Diazinon (DZN) is one of the most important organophosphorus compounds used to control pests in agriculture in many countries. Several studies have shown that exposure to DZN may alter protein expression in the liver. In order to further investigate the mechanism of DZN toxicity, differentially expressed ATP-interacting proteins, following subacute exposure to toxin, were separated and identified in rat liver. Male rats were equally divided into four groups: control (corn oil) and DZN (15 mg/kg) by gavage once a day for 4 weeks. After homogenization of liver tissue, lysates were incubated ATP-sepharose beads. After several washes, ATP-interacting proteins were eluted and separated on 2-D polyacrylamide gels. Deferentially expressed proteins were cut and identified using matrix-assisted laser desorption/ionization/time-of-flight and Mascot database. Identified proteins were classified according to their biological process using protein analysis through evolutionary relationships (PANTHER) Web site. In this work, we showed that several key proteins involved in biological processes such as antioxidant system, oxidative stress, apoptosis, and metabolism were differentially expressed after subacute exposure to DZN. © The Author(s) 2015.

Comparative Proteomic Analysis of the Hepatic Response to Heat Stress in Muscovy and Pekin Ducks: Insight into Thermal Tolerance Related to Energy Metabolism

PubMed Central

Zeng, Tao; Jiang, Xueyuan; Li, Jinjun; Wang, Deqian; Li, Guoqin; Lu, Lizhi; Wang, Genlin

2013-01-01

The Pekin duck, bred from the mallard (Anas platyrhynchos) in china, is one of the most famous meat duck species in the world. However, it is more sensitive to heat stress than Muscovy duck, which is believed to have originated in South America. With temperature raising, mortality, laying performance, and meat quality of the Pekin duck are severely affected. This study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach. Duck was cultured under 39°C ± 0.5°C for 1 h, and then immediately returned to 20°C for a 3 h recovery period, the liver proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, 61 differentially expressed proteins were detected, 54 were clearly identified by MALDI TOF/TOF MS. Of the 54 differentially expressed protein spots identified, 7 were found in both species, whereas 47 were species specific (25 in Muscovy duck and 22 in Pekin duck). As is well known, chaperone proteins, such as heat shock protein (HSP) 70 and HSP10, were abundantly up-regulated in both species in response to heat stress. However, we also found that several proteins, such as α-enolase, and S-adenosylmethionine synthetase, showed different expression patterns in the 2 duck species. The enriched biological processes were grouped into 3 main categories according to gene ontology analysis: cell death and apoptosis (20.93%), amino acid metabolism (13.95%) and oxidation reduction (20.93%). The mRNA levels of several differentially expressed protein were investigated by real-time RT-PCR. To our knowledge, this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals. PMID:24116183

Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.

PubMed

Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S

2017-05-20

Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.

The TEL-AML1 fusion protein of acute lymphoblastic leukemia modulates IRF3 activity during early B-cell differentiation.

PubMed

de Laurentiis, A; Hiscott, J; Alcalay, M

2015-12-03

The t(12;21) translocation is the most common genetic rearrangement in childhood acute lymphoblastic leukemia (ALL) and gives rise to the TEL-AML1 fusion gene. Many studies on TEL-AML1 describe specific properties of the fusion protein, but a thorough understanding of its function is lacking. We exploited a pluripotent hematopoietic stem/progenitor cell line, EML1, and generated a cell line (EML-TA) stably expressing the TEL-AML1 fusion protein. EML1 cells differentiate to mature B-cells following treatment with IL7; whereas EML-TA display an impaired differentiation capacity and remain blocked at an early stage of maturation. Global gene expression profiling of EML1 cells at different stages of B-lymphoid differentiation, compared with EML-TA, identified the interferon (IFN)α/β pathway as a primary target of repression by TEL-AML1. In particular, expression and phosphorylation of interferon-regulatory factor 3 (IRF3) was decreased in EML-TA cells; strikingly, stable expression of IRF3 restored the capacity of EML-TA cells to differentiate into mature B-cells. Similarly, IRF3 silencing in EML1 cells by siRNA was sufficient to block B-lymphoid differentiation. The ability of TEL-AML1 to block B-cell differentiation and downregulate the IRF3-IFNα/β pathway was confirmed in mouse and human primary hematopoietic precursor cells (Lin- and CD34+ cells, respectively), and in a patient-derived cell line expressing TEL-AML1 (REH). Furthermore, treatment of TEL-AML1 expressing cells with IFNα/β was sufficient to overcome the maturation block. Our data provide new insight on TEL-AML1 function and may offer a new therapeutic opportunity for B-ALL.

Acute hypoxia stress induced abundant differential expression genes and alternative splicing events in heart of tilapia.

PubMed

Xia, Jun Hong; Li, Hong Lian; Li, Bi Jun; Gu, Xiao Hui; Lin, Hao Ran

2018-01-10

Hypoxia is one of the critical environmental stressors for fish in aquatic environments. Although accumulating evidences indicate that gene expression is regulated by hypoxia stress in fish, how genes undergoing differential gene expression and/or alternative splicing (AS) in response to hypoxia stress in heart are not well understood. Using RNA-seq, we surveyed and detected 289 differential expressed genes (DEG) and 103 genes that undergo differential usage of exons and splice junctions events (DUES) in heart of a hypoxia tolerant fish, Nile tilapia, Oreochromis niloticus following 12h hypoxic treatment. The spatio-temporal expression analysis validated the significant association of differential exon usages in two randomly selected DUES genes (fam162a and ndrg2) in 5 tissues (heart, liver, brain, gill and spleen) sampled at three time points (6h, 12h, and 24h) under acute hypoxia treatment. Functional analysis significantly associated the differential expressed genes with the categories related to energy conservation, protein synthesis and immune response. Different enrichment categories were found between the DEG and DUES dataset. The Isomerase activity, Oxidoreductase activity, Glycolysis and Oxidative stress process were significantly enriched for the DEG gene dataset, but the Structural constituent of ribosome and Structural molecule activity, Ribosomal protein and RNA binding protein were significantly enriched only for the DUES genes. Our comparative transcriptomic analysis reveals abundant stress responsive genes and their differential regulation function in the heart tissues of Nile tilapia under acute hypoxia stress. Our findings will facilitate future investigation on transcriptome complexity and AS regulation during hypoxia stress in fish. Copyright © 2017 Elsevier B.V. All rights reserved.

Correlation between Urothelial Differentiation and Sensory Proteins P2X3, P2X5, TRPV1, and TRPV4 in Normal Urothelium and Papillary Carcinoma of Human Bladder

PubMed Central

Sterle, Igor; Zupančič, Daša; Romih, Rok

2014-01-01

Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic receptors (P2X3, and P2X5) and transient receptor potential vanilloid channels (TRPV1, and TRPV4). Western blotting and immunofluorescence of uroplakins together with scanning electron microscopy of urothelial apical surface demonstrated terminal differentiation of normal urothelium, partial differentiation of low grade carcinoma, and poor differentiation of high grade carcinoma. P2X3 was expressed in normal urothelium as well as in low grade carcinoma and in both cases immunolabeling was stronger in the superficial cells. P2X3 expression decreased in high grade carcinoma. P2X5 expression was detected in normal urothelium and in high grade carcinoma, while in low grade carcinoma its expression was diminished. The expression of TRPV1 decreased in low grade and even more in high grade carcinoma when compared with normal urothelium, while TRPV4 expression was unchanged in all samples. Our results suggest that sensory proteins P2X3 and TRPV1 are in correlation with urothelial differentiation, while P2X5 and TRPV4 have unique expression patterns. PMID:24868547

Correlation between urothelial differentiation and sensory proteins P2X3, P2X5, TRPV1, and TRPV4 in normal urothelium and papillary carcinoma of human bladder.

PubMed

Sterle, Igor; Zupančič, Daša; Romih, Rok

2014-01-01

Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic receptors (P2X3, and P2X5) and transient receptor potential vanilloid channels (TRPV1, and TRPV4). Western blotting and immunofluorescence of uroplakins together with scanning electron microscopy of urothelial apical surface demonstrated terminal differentiation of normal urothelium, partial differentiation of low grade carcinoma, and poor differentiation of high grade carcinoma. P2X3 was expressed in normal urothelium as well as in low grade carcinoma and in both cases immunolabeling was stronger in the superficial cells. P2X3 expression decreased in high grade carcinoma. P2X5 expression was detected in normal urothelium and in high grade carcinoma, while in low grade carcinoma its expression was diminished. The expression of TRPV1 decreased in low grade and even more in high grade carcinoma when compared with normal urothelium, while TRPV4 expression was unchanged in all samples. Our results suggest that sensory proteins P2X3 and TRPV1 are in correlation with urothelial differentiation, while P2X5 and TRPV4 have unique expression patterns.

Comparative analysis of human protein-coding and noncoding RNAs between brain and 10 mixed cell lines by RNA-Seq.

PubMed

Chen, Geng; Yin, Kangping; Shi, Leming; Fang, Yuanzhang; Qi, Ya; Li, Peng; Luo, Jian; He, Bing; Liu, Mingyao; Shi, Tieliu

2011-01-01

In their expression process, different genes can generate diverse functional products, including various protein-coding or noncoding RNAs. Here, we investigated the protein-coding capacities and the expression levels of their isoforms for human known genes, the conservation and disease association of long noncoding RNAs (ncRNAs) with two transcriptome sequencing datasets from human brain tissues and 10 mixed cell lines. Comparative analysis revealed that about two-thirds of the genes expressed between brain and cell lines are the same, but less than one-third of their isoforms are identical. Besides those genes specially expressed in brain and cell lines, about 66% of genes expressed in common encoded different isoforms. Moreover, most genes dominantly expressed one isoform and some genes only generated protein-coding (or noncoding) RNAs in one sample but not in another. We found 282 human genes could encode both protein-coding and noncoding RNAs through alternative splicing in the two samples. We also identified more than 1,000 long ncRNAs, and most of those long ncRNAs contain conserved elements across either 46 vertebrates or 33 placental mammals or 10 primates. Further analysis showed that some long ncRNAs differentially expressed in human breast cancer or lung cancer, several of those differentially expressed long ncRNAs were validated by RT-PCR. In addition, those validated differentially expressed long ncRNAs were found significantly correlated with certain breast cancer or lung cancer related genes, indicating the important biological relevance between long ncRNAs and human cancers. Our findings reveal that the differences of gene expression profile between samples mainly result from the expressed gene isoforms, and highlight the importance of studying genes at the isoform level for completely illustrating the intricate transcriptome.

Prognostic Significance of Vascular Endothelial Growth Factor (VEGF) and Her-2 Protein in the Genesis of Cervical Carcinoma.

PubMed

Rahmani, Arshad H; Babiker, Ali Yousif; Alsahli, Mohammed A; Almatroodi, Saleh A; Husain, Nazik Elmalaika O S

2018-02-15

Angiogenesis plays a pivotal role in the progression of tumours through the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is a chief factor responsible for inducing and regulating angiogenesis. Additionally, the human epidermal growth factor receptor family of receptors also plays an important role in the pathogenesis of tumours. This study aimed to examine the association between VEGF and Her-2 protein expression and its correlation with clinic-pathological characteristics; in particular, prognosis. A total of 65 cases of cervical carcinoma and 10 samples of inflammatory lesions were evaluated for VEGF and Her-2 protein expression. Expression of VEGF and Her-2 was detected in 63.07% and 43.07% in cervical carcinoma cases respectively whereas control cases did not show any expression. The difference in the expression pattern of both markers comparing cancer and control cases was statistically significant (p < 0.05). However, no significant difference in the expression pattern of VEGF protein was observed among the different grades and stages of tumours (p > 0.05). Comparing different grades of a tumour, expression of Her-2 was detected in 31.8% of well-differentiated tumours, 36.0 % in moderately differentiated tumours and 66.66 % in poorly differentiated cancers. The expression of Her-2 was increased in high-grade tumours, and the difference of expression level between tumour grades was statistically significant (p < 0.05). The expression level of Her-2 protein was not correlated with the stage of a tumour (p > 0.05). The present study supports earlier findings that over-expression / up-regulation of VEGF and Her - 2 is linked with poor prognosis and may play a vital role in the development and progression of cervical cancer.

Prognostic Significance of Vascular Endothelial Growth Factor (VEGF) and Her-2 Protein in the Genesis of Cervical Carcinoma

PubMed Central

Rahmani, Arshad H.; Babiker, Ali Yousif; Alsahli, Mohammed A.; Almatroodi, Saleh A.; Husain, Nazik Elmalaika O. S.

2018-01-01

BACKGROUND: Angiogenesis plays a pivotal role in the progression of tumours through the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is a chief factor responsible for inducing and regulating angiogenesis. Additionally, the human epidermal growth factor receptor family of receptors also plays an important role in the pathogenesis of tumours. AIM: This study aimed to examine the association between VEGF and Her-2 protein expression and its correlation with clinic-pathological characteristics; in particular, prognosis. METHODS: A total of 65 cases of cervical carcinoma and 10 samples of inflammatory lesions were evaluated for VEGF and Her-2 protein expression. RESULTS: Expression of VEGF and Her-2 was detected in 63.07% and 43.07% in cervical carcinoma cases respectively whereas control cases did not show any expression. The difference in the expression pattern of both markers comparing cancer and control cases was statistically significant (p < 0.05). However, no significant difference in the expression pattern of VEGF protein was observed among the different grades and stages of tumours (p > 0.05). Comparing different grades of a tumour, expression of Her-2 was detected in 31.8% of well-differentiated tumours, 36.0 % in moderately differentiated tumours and 66.66 % in poorly differentiated cancers. The expression of Her-2 was increased in high-grade tumours, and the difference of expression level between tumour grades was statistically significant (p < 0.05). The expression level of Her-2 protein was not correlated with the stage of a tumour (p > 0.05). CONCLUSION: The present study supports earlier findings that over-expression / up-regulation of VEGF and Her - 2 is linked with poor prognosis and may play a vital role in the development and progression of cervical cancer. PMID:29531585

Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells

PubMed Central

Melidoni, Anna N.; Dyson, Michael R.; Wormald, Sam; McCafferty, John

2013-01-01

Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1β, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein “knock-down” and allows temporal control of stable signaling components at the protein level. PMID:24082130

MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis.

PubMed

Tagawa, Masashi; Ueyama, Tomomi; Ogata, Takehiro; Takehara, Naofumi; Nakajima, Norio; Isodono, Koji; Asada, Satoshi; Takahashi, Tomosaburo; Matsubara, Hiroaki; Oh, Hidemasa

2008-08-01

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.

Protein content and methyl donors in maternal diet interact to influence the proliferation rate and cell fate of neural stem cells in rat hippocampus.

PubMed

Amarger, Valérie; Lecouillard, Angèle; Ancellet, Laure; Grit, Isabelle; Castellano, Blandine; Hulin, Philippe; Parnet, Patricia

2014-10-14

Maternal diet during pregnancy and early postnatal life influences the setting up of normal physiological functions in the offspring. Epigenetic mechanisms regulate cell differentiation during embryonic development and may mediate gene/environment interactions. We showed here that high methyl donors associated with normal protein content in maternal diet increased the in vitro proliferation rate of neural stem/progenitor cells isolated from rat E19 fetuses. Gene expression on whole hippocampi at weaning confirmed this effect as evidenced by the higher expression of the Nestin and Igf2 genes, suggesting a higher amount of undifferentiated precursor cells. Additionally, protein restriction reduced the expression of the insulin receptor gene, which is essential to the action of IGFII. Inhibition of DNA methylation in neural stem/progenitor cells in vitro increased the expression of the astrocyte-specific Gfap gene and decreased the expression of the neuron-specific Dcx gene, suggesting an impact on cell differentiation. Our data suggest a complex interaction between methyl donors and protein content in maternal diet that influence the expression of major growth factors and their receptors and therefore impact the proliferation and differentiation capacities of neural stem cells, either through external hormone signals or internal genomic regulation.

Differentially expressed genes in the silk gland of silkworm (Bombyx mori) treated with TiO2 NPs.

PubMed

Xue, Bin; Li, Fanchi; Hu, Jingsheng; Tian, Jianghai; Li, Jinxin; Cheng, Xiaoyu; Hu, Jiahuan; Li, Bing

2017-05-05

Silk gland is a silkworm organ where silk proteins are synthesized and secreted. Dietary supplement of TiO 2 nanoparticles (NPs) promotes silk protein synthesis in silkworms. In this study, digital gene expression (DGE) tag was used to analyze the gene expression profile of the posterior silk gland of silkworms that were fed with TiO 2 NPs. In total, 5,702,823 and 6,150,719 clean tags, 55,096 and 74,715 distinct tags were detected in TiO 2 NPs treated and control groups, respectively. Compared with the control, TiO 2 NPs treated silkworms showed 306 differentially expressed genes, including 137 upregulated genes and 169 downregulated genes. Of these differentially expressed genes, 106 genes were related to silk protein synthesis, among which 97 genes were upregulated and 9 genes were downregulated. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 20 pathways were significantly enriched in TiO 2 NPs treated silkworms, and the metabolic pathway-related genes were the most significantly enriched. The DGE results were verified by qRT-PCR analysis of eight differentially expressed genes. The DGE and qRT-PCR results were consistent for all three upregulated genes and three of the five downregulated genes, but the expression trends of the remaining two genes were different between qRT-PCR and DGE analysis. This study enhances our understanding of the mechanism of TiO 2 NPs promoted silk protein synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

MMP‐2 and MMP‐14 Silencing Inhibits VEGFR2 Cleavage and Induces the Differentiation of Porcine Adipose‐Derived Mesenchymal Stem Cells to Endothelial Cells

PubMed Central

Almalki, Sami G.; Llamas Valle, Yovani

2017-01-01

Abstract The molecular mechanisms that control the ability of adipose‐derived mesenchymal stem cells (AMSCs) to remodel three‐dimensional extracellular matrix barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of AMSCs to endothelial cells (ECs) in vitro. MSCs were isolated from porcine abdominal adipose tissue, and characterized by immunopositivity to CD44, CD90, CD105, and immunonegativity to CD14 and CD45. Plasticity of AMSCs was confirmed by multilineage differentiation. The mRNA transcripts for MMPs and Tissue Inhibitor of Metalloproteinases (TIMPs), and protein expression of EC markers were analyzed. The enzyme activity and protein expression were analyzed by gelatin zymography, enzyme‐linked immunosorbent assay (ELISA), and Western blot. The differentiation of AMSCs to ECs was confirmed by mRNA and protein expressions of the endothelial markers. The mRNA transcripts for MMP‐2 and MMP‐14 were significantly increased during the differentiation of MSCs into ECs. Findings revealed an elevated MMP‐14 and MMP‐2 expression, and MMP2 enzyme activity. Silencing of MMP‐2 and MMP‐14 significantly increased the expression of EC markers, formation of capillary tubes, and acetylated‐low‐density lipoprotein uptake, and decreased the cleavage of vascular endothelial growth factor receptor type 2 (VEGFR2). Inhibition of VEGFR2 significantly decreased the expression of EC markers. These novel findings demonstrate that the upregulation of MMP2 and MMP14 has an inhibitory effect on the differentiation of AMSCs to ECs, and silencing these MMPs inhibit the cleavage of VEGFR2 and stimulate the differentiation of AMSCs to ECs. These findings provide a potential mechanism for the regulatory role of MMP‐2 and MMP‐14 in the re‐endothelialization of coronary arteries following intervention. Stem Cells Translational Medicine 2017;6:1385–1398 PMID:28213979

Differential proteomic analysis of Aspergillus fumigatus morphotypes reveals putative drug targets.

PubMed

Kubitschek-Barreira, Paula H; Curty, Nathalia; Neves, Gabriela W P; Gil, Concha; Lopes-Bezerra, Leila M

2013-01-14

Aspergillus fumigatus is the main etiological agent of invasive aspergillosis, an important opportunistic infection for neutropenic patients. The main risk groups are patients with acute leukemia and bone marrow transplantation recipients. The lack of an early diagnostic test together with the limited spectrum of antifungal drugs remains a setback to the successful treatment of this disease. During invasive infection the inhaled fungal conidia enter the morphogenic cycle leading to angioinvasive hyphae. This work aimed to study differentially expressed proteins of A. fumigatus during morphogenesis. To achieve this goal, a 2D-DIGE approach was applied to study surface proteins extractable by reducing agents of two A. fumigatus morphotypes: germlings and hyphae. Sixty-three differentially expressed proteins were identified by MALDI-ToF/MS. We observed that proteins associated with biosynthetic pathways and proteins with multiple functions (miscellaneous) were over-expressed in the early stages of germination, while in hyphae, the most abundant proteins detected were related to metabolic processes or have unknown functions. Among the most interesting proteins regulated during morphogenesis, two putative drug targets were identified, the translational factor, eEF3 and the CipC-like protein. Neither of these proteins are present in mammalian cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Persimmon tannin represses 3T3-L1 preadipocyte differentiation via up-regulating expression of miR-27 and down-regulating expression of peroxisome proliferator-activated receptor-γ in the early phase of adipogenesis.

PubMed

Zou, Bo; Ge, Zhenzhen; Zhu, Wei; Xu, Ze; Li, Chunmei

2015-12-01

Currently, obesity has become a worldwide health problem. Adipocyte differentiation is closely associated with the onset of obesity. Our previous studies suggested that persimmon tannin might be a potent anti-adipogenic dietary bioactive compound. However, the mechanism of persimmon tannin on adipocyte differentiation is still unknown. The purpose of this study was to investigate the effect of persimmon tannin on adipogenic differentiation in 3T3-L1 preadipocytes and the underlying mechanisms. Adipogenic differentiation was induced by cocktail in the presence or absence of persimmon tannin. Intracellular lipid accumulation was determined by Oil red O staining and enzymatic colorimetric methods. Gene expression and protein levels were measured by real time RT-PCR and Western blot. Persimmon tannin inhibited intracellular lipid accumulation markedly, and the inhibitory effect was largely limited to the early stage of adipocyte differentiation. Persimmon tannin suppressed the expression of C/EBPα and peroxisome proliferator-activated receptor-γ (PPARγ), significantly. Furthermore, genes related to lipogenesis, such as sterol regulatory element-binding protein 1, were down-regulated by persimmon tannin. In addition, adipocyte fatty acid binding protein (aP2), which is a target gene of PPARγ, was suppressed by persimmon tannin notably. Correspondingly, the expression of miR-27a and miR-27b were up-regulated by persimmon tannin from Day 2 to Day 8 significantly. Persimmon tannin inhibited adipocyte differentiation through regulation of PPARγ, C/EBPα and miR-27 in early stage of adipogenesis.

Myostatin inhibits porcine intramuscular preadipocyte differentiation in vitro.

PubMed

Sun, W X; Dodson, M V; Jiang, Z H; Yu, S G; Chu, W W; Chen, J

2016-04-01

This study assessed the effect of myostatin on adipogenesis by porcine intramuscular preadipocytes. Intramuscular preadipocytes were isolated from the longissimus dorsi muscle of newborn pigs. Myostatin inhibited intramuscular preadipocyte differentiation in a dose-dependent manner. Myostatin treatment during preadipocyte differentiation significantly (P < 0.05) inhibited the expression of the adipogenic marker genes CCAAT/enhancer-binding protein β, CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, fatty acid-binding protein, and adiponectin. Myostatin also significantly (P < 0.05) reduced the release of glycerol and decreased both adipose triglyceride lipase and hormone-sensitive lipase expression in intramuscular adipocytes. Our study suggests that myostatin acts as an extrinsic regulatory factor in regulating intramuscular adipogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential regulation of cyclo-oxygenase-2 and 5-lipoxygenase-activating protein (FLAP) expression by glucocorticoids in monocytic cells.

PubMed

Goppelt-Struebe, M; Schaefer, D; Habenicht, A J

1997-10-01

1. The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP). The human monocytic cell line THP-1 was used as a model system. mRNA and protein levels of COX-2 and FLAP were determined by Northern and Western blot analyses, respectively. 2. Low levels of COX-2 and FLAP mRNA were expressed in undifferentiated THP-1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days. 3. Coincubation of the undifferentiated cells with dexamethasone (10(-9) - 10(-6) M) and phorbol ester prevented induction of COX-2 mRNA, but did not affect the induction of FLAP mRNA. 4. Dexamethasone downregulated COX-2 mRNA and protein in differentiated, monocyte-like THP-1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP-1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5-lipoxygenase mRNA expression. 5. Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP-1 cells by ELISA. Calcium ionophore-dependent PGE2 synthesis was associated with COX-2 expression, whereas COX-1 and COX-2 seemed to participate in arachidonic acid-dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP-1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release. 6. These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid-mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5-lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis

PubMed Central

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K.

2012-01-01

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3′ untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior. PMID:22645327

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

PubMed

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

2012-06-12

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

Proteomic analysis of trans-hemispheric motor cortex reorganization following contralateral C7 nerve transfer

PubMed Central

Yuan, Yin; Xu, Xiu-yue; Lao, Jie; Zhao, Xin

2018-01-01

Nerve transfer is the most common treatment for total brachial plexus avulsion injury. After nerve transfer, the movement of the injured limb may be activated by certain movements of the healthy limb at the early stage of recovery, i.e., trans-hemispheric reorganization. Previous studies have focused on functional magnetic resonance imaging and changes in brain-derived neurotrophic factor and growth associated protein 43, but there have been no proteomics studies. In this study, we designed a rat model of total brachial plexus avulsion injury involving contralateral C7 nerve transfer. Isobaric tags for relative and absolute quantitation and western blot assay were then used to screen differentially expressed proteins in bilateral motor cortices. We found that most differentially expressed proteins in both cortices of upper limb were associated with nervous system development and function (including neuron differentiation and development, axonogenesis, and guidance), microtubule and cytoskeleton organization, synapse plasticity, and transmission of nerve impulses. Two key differentially expressed proteins, neurofilament light (NFL) and Thy-1, were identified. In contralateral cortex, the NFL level was upregulated 2 weeks after transfer and downregulated at 1 and 5 months. The Thy-1 level was upregulated from 1 to 5 months. In the affected cortex, the NFL level increased gradually from 1 to 5 months. Western blot results of key differentially expressed proteins were consistent with the proteomic findings. These results indicate that NFL and Thy-1 play an important role in trans-hemispheric organization following total brachial plexus root avulsion and contralateral C7 nerve transfer. PMID:29557385

Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells

PubMed Central

Sánchez-Mendoza, Eduardo H.; Bellver-Landete, Victor; Arce, Carmen; Doeppner, Thorsten R.; Hermann, Dirk M.

2017-01-01

The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs. PMID:28493916

Expression profiling of chickpea genes differentially regulated during a resistance response to Ascochyta rabiei.

PubMed

Coram, Tristan E; Pang, Edwin C K

2006-11-01

Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) expressed sequence tags (ESTs) and lentil (Lens culinaris Med.) resistance gene analogues, the ascochyta blight (Ascochyta rabiei (Pass.) L.) resistance response was studied in four chickpea genotypes, including resistant, moderately resistant, susceptible and wild relative (Cicer echinospermum L.) genotypes. The experimental system minimized environmental effects and was conducted in reference design, in which samples from mock-inoculated controls acted as reference against post-inoculation samples. Robust data quality was achieved through the use of three biological replicates (including a dye swap), the inclusion of negative controls and strict selection criteria for differentially expressed genes, including a fold change cut-off determined by self-self hybridizations, Student's t-test and multiple testing correction (P < 0.05). Microarray observations were also validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The time course expression patterns of 756 microarray features resulted in the differential expression of 97 genes in at least one genotype at one time point. k-means clustering grouped the genes into clusters of similar observations for each genotype, and comparisons between A. rabiei-resistant and A. rabiei-susceptible genotypes revealed potential gene 'signatures' predictive of effective A. rabiei resistance. These genes included several pathogenesis-related proteins, SNAKIN2 antimicrobial peptide, proline-rich protein, disease resistance response protein DRRG49-C, environmental stress-inducible protein, leucine-zipper protein, polymorphic antigen membrane protein, Ca-binding protein and several unknown proteins. The potential involvement of these genes and their pathways of induction are discussed. This study represents the first large-scale gene expression profiling in chickpea, and future work will focus on the functional validation of the genes of interest.

Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

PubMed

Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

2018-05-01

Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus

PubMed Central

Hu, Ping; Wang, Tao; Tao, Jing; Zong, Shixiang

2017-01-01

Seabuckthorn carpenter moth, Eogystia hippophaecolus (Lepidoptera: Cossidae), is an important pest of sea buckthorn (Hippophae rhamnoides), which is a shrub that has significant ecological and economic value in China. E. hippophaecolus is highly cold tolerant, but limited studies have been conducted to elucidate the molecular mechanisms underlying its cold resistance. Here we sequenced the E. hippophaecolus transcriptome using RNA-Seq technology and performed de novo assembly from the short paired-end reads. We investigated the larval response to cold stress by comparing gene expression profiles between treatments. We obtained 118,034 unigenes, of which 22,161 were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways. These resulted in 57 GO terms and 193 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparing transcriptome profiles for differential gene expression, we identified many differentially expressed proteins and genes, including heat shock proteins and cuticular proteins which have previously been reported to be involved in cold resistance of insects. This study provides a global transcriptome analysis and an assessment of differential gene expression in E. hippophaecolus under cold stress. We found seven differential expressed genes in common between developmental stages, which were verified with qPCR. Our findings facilitate future genomic studies aimed at improving our understanding of the molecular mechanisms underlying the response of insects to low temperatures. PMID:29131867

Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus.

PubMed

Cui, Mingming; Hu, Ping; Wang, Tao; Tao, Jing; Zong, Shixiang

2017-01-01

Seabuckthorn carpenter moth, Eogystia hippophaecolus (Lepidoptera: Cossidae), is an important pest of sea buckthorn (Hippophae rhamnoides), which is a shrub that has significant ecological and economic value in China. E. hippophaecolus is highly cold tolerant, but limited studies have been conducted to elucidate the molecular mechanisms underlying its cold resistance. Here we sequenced the E. hippophaecolus transcriptome using RNA-Seq technology and performed de novo assembly from the short paired-end reads. We investigated the larval response to cold stress by comparing gene expression profiles between treatments. We obtained 118,034 unigenes, of which 22,161 were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways. These resulted in 57 GO terms and 193 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparing transcriptome profiles for differential gene expression, we identified many differentially expressed proteins and genes, including heat shock proteins and cuticular proteins which have previously been reported to be involved in cold resistance of insects. This study provides a global transcriptome analysis and an assessment of differential gene expression in E. hippophaecolus under cold stress. We found seven differential expressed genes in common between developmental stages, which were verified with qPCR. Our findings facilitate future genomic studies aimed at improving our understanding of the molecular mechanisms underlying the response of insects to low temperatures.

Repression of Virus-Induced Interferon A Promoters by Homeodomain Transcription Factor Ptx1

PubMed Central

Lopez, Sébastien; Island, Marie-Laure; Drouin, Jacques; Bandu, Marie-Thérese; Christeff, Nicolas; Darracq, Nicole; Barbey, Régine; Doly, Janine; Thomas, Dominique; Navarro, Sébastien

2000-01-01

Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by substitutions in the proximal positive virus responsive element A (VRE-A) of their promoters and by the presence or absence of a distal negative regulatory element (DNRE). The functional feature of the DNRE is to specifically act by repression of VRE-A activity. With the use of the yeast one-hybrid system, we describe here the identification of a specific DNRE-binding protein, the pituitary homeobox 1 (Ptx1 or Pitx1). Ptx1 is detectable in different cell types that differentially express IFN-A genes, and the endogenous Ptx1 protein binds specifically to the DNRE. Upon virus induction, Ptx1 negatively regulates the transcription of DNRE-containing IFN-A promoters, and the C-terminal region, as well as the homeodomain of the Ptx1 protein, is required for this repression. After virus induction, the expression of the Ptx1 antisense RNA leads to a significant increase of endogenous IFN-A gene transcription and is able to modify the pattern of differential expression of individual IFN-A genes. These studies suggest that Ptx1 contributes to the differential transcriptional strength of the promoters of different IFN-A genes and that these genes may provide new targets for transcriptional regulation by a homeodomain transcription factor. PMID:11003649

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

NASA Technical Reports Server (NTRS)

Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

1996-01-01

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

Differences in MYB expression and gene abnormalities further confirm that salivary cribriform basal cell tumors and adenoid cystic carcinoma are two distinct tumor entities.

PubMed

Tian, Zhen; Li, Lei; Zhang, Chun-Ye; Gu, Ting; Li, Jiang

2016-10-01

In practices, some cases of salivary basal cell tumors that consist mainly of cribriform growth pattern are difficult to differentiate from adenoid cystic carcinoma (AdCC). Identification of reliable molecular biomarkers for the differential diagnosis between them is required. Twenty-two cases of cribriform salivary basal cell tumors (at least 10% cribriform pattern present in each tumor) comprising 18 cases of basal cell adenoma (BCA) and four cases of basal cell adenocarcinoma (BcAC) were collected between 1985 and 2008. Twenty cases of cribriform AdCC were retrieved from our archives. MYB protein expression and gene abnormalities were detected in all cases by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) analyses, respectively. Neither MYB protein nor split genes were detected in any of the cases of cribriform basal cell tumors, while 55% (11/20) of cases of cribriform AdCC had MYB protein expression. High MYB expression was detected in 81.8% (9/11) cases, while low expression was found in the remaining cases. FISH analysis indicated that nine AdCC tumors with high MYB protein expression were split gene-positive, while MYB gene splitting was not detected in the 11 cases with low or absent MYB protein expression. The molecular changes in AdCC differ from those associated with cribriform basal cell tumors, which further confirms that cribriform basal cell tumors and AdCC are two distinct tumor entities. Simultaneous detection of MYB protein expression and the associated molecular changes could be beneficial in differentiating salivary cribriform basal cell tumors from AdCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Comparative proteomic analysis in Aphis glycines Mutsumura under lambda-cyhalothrin insecticide stress.

PubMed

Bi, Rui; Pan, Yiou; Shang, Qingli; Peng, Tianfei; Yang, Shuang; Wang, Shang; Xin, Xuecheng; Liu, Yan; Xi, Jinghui

2016-09-01

Lambda-cyhalothrin is now widely used in China to control the soybean aphid Aphis glycines. To dissect the resistance mechanism, a laboratory-selected resistant soybean aphid strain (CRR) was established with a 43.42-fold resistance ratio to λ-cyhalothrin than the susceptible strain (CSS) in adult aphids. In this study, a comparative proteomic analysis between the CRR and CSS strains revealed important differences between the susceptible and resistant strains of soybean aphids for λ-cyhalothrin. Approximately 493 protein spots were detected in two-dimensional polyacrylamide gel electrophoresis (2-DE). Thirty-six protein spots displayed differential expression of >2-fold in the CRR strain compared to the CSS strain. Out of these 36 protein spots, 21 had elevated and 15 had decreased expression. Twenty-four differentially expressed proteins were identified by MALDI TOF MS/MS and categorized into the functional groups cytoskeleton-related protein, carbohydrate and energy metabolism, protein folding, antioxidant system, and nucleotide and amino acid metabolism. Function analysis showed that cytoskeleton-related proteins and energy metabolism proteins have been associated with the λ-cyhalothrin resistance of A. glycines. The differential expression of λ-cyhalothrin responsive proteins reflected the overall change in cellular structure and metabolism after insecticide treatment in aphids. In summary, our studies improve understanding of the molecular mechanism resistance of soybean aphid to lambda-cyhalothrin, which will facilitate the development of rational approaches to improve the management of this pest and to improve the yield of soybean. Copyright © 2016. Published by Elsevier Inc.

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms.

PubMed

Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Almeida, Igor C; Fernandes, Patricia M B; Zingali, Russolina B

2011-07-01

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors

PubMed Central

Pizzamiglio, Sara; De Bortoli, Maida; Taverna, Elena; Signore, Michele; Veneroni, Silvia; Chi-shing Cho, William; Orlandi, Rosaria; Verderio, Paolo; Bongarzone, Italia

2017-01-01

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer. PMID:28216608

Proteome analysis reveals that de novo regenerated mucosa over fibula flap-reconstructed mandibles resembles mature keratinized oral mucosa.

PubMed

Kumar, Vinay V; James, Bonney L; Ruß, Manuela; Mikkat, Stefan; Suresh, Amritha; Kämmerer, Peer W; Glocker, Michael O

2018-03-01

The aim of this study was to determine whether intra-oral de novo regenerated mucosa (D) that grew over free fibula flap reconstructed-mandibles resembled the donor tissue i.e. external skin (S) of the lateral leg, or the recipient site tissue, i.e. keratinized oral mucosa (K). Differential proteome analysis was performed with ten tissue samples from each of the three groups: de novo regenerated mucosa (D), external skin (S), and keratinized oral mucosa (K). Expression differences of cornulin and involucrin were validated by Western blot analysis and their spatial distributions in the respective tissues were ascertained by immunohistochemistry. From all three investigated tissue types a total of 1188 proteins were identified, 930 of which were reproducibly and robustly quantified by proteome analysis. The best differentiating proteins were assembled in an oral mucosa proteome signature that encompasses 56 differentially expressed proteins. Principal component analysis of both, the 930 quantifiable proteins and the 56 oral mucosa signature proteins revealed that the de novo regenerated mucosa resembles keratinized oral mucosa much closer than extra-oral skin. Differentially expressed cornification-related proteins comprise proteins from all subclasses of the cornified cell envelope. Prominently expressed in intra-oral mucosa tissues were (i) cornifin-A, cornifin-B, SPRR3, and involucrin from the cornified-cell-envelope precursor group, (ii) S100A9, S100A8 and S100A2 from the S100 group, and (iii) cornulin which belongs to the fused-gene-protein group. According to its proteome signature de novo regenerated mucosa over the free fibula flap not only presents a passive structural surface layer but has adopted active tissue function. Copyright © 2018 Elsevier Ltd. All rights reserved.

Liver proteomics in progressive alcoholic steatosis

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

Fernando, Harshica; Wiktorowicz, John E.; Soman, Kizhake V.

2013-02-01

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber–DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved inmore » alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (− 1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (− 1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified D-dopachrome tautomerase (− 1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum. -- Graphical abstract: The figure shows the Hierarchial cluster analysis of differentially expressed protein spots obtained after ethanol feeding for 1 (1–3) and 3 (4–6) months. C and E represent pair-fed control and ethanol-fed rats, respectively. Highlights: ► Proteins related to ethanol-induced steatosis and mild steatohepatitis are identified. ► ADH1C and ALDH2 involved in alcohol metabolism are differentially expressed at 1 and 3 months. ► Discovery proteomics identified a group of proteins to serve as potential biomarkers. ► Using nonparametric analysis DDT is identified as a possible marker for liver damage.« less

Optimization of Statistical Methods Impact on Quantitative Proteomics Data.

PubMed

Pursiheimo, Anna; Vehmas, Anni P; Afzal, Saira; Suomi, Tomi; Chand, Thaman; Strauss, Leena; Poutanen, Matti; Rokka, Anne; Corthals, Garry L; Elo, Laura L

2015-10-02

As tools for quantitative label-free mass spectrometry (MS) rapidly develop, a consensus about the best practices is not apparent. In the work described here we compared popular statistical methods for detecting differential protein expression from quantitative MS data using both controlled experiments with known quantitative differences for specific proteins used as standards as well as "real" experiments where differences in protein abundance are not known a priori. Our results suggest that data-driven reproducibility-optimization can consistently produce reliable differential expression rankings for label-free proteome tools and are straightforward in their application.

5-Hydroxyferulic acid methyl ester isolated from wasabi leaves inhibits 3T3-L1 adipocyte differentiation.

PubMed

Misawa, Naoki; Hosoya, Takahiro; Yoshida, Shuhei; Sugimoto, Osamu; Yamada-Kato, Tomoe; Kumazawa, Shigenori

2018-02-26

To investigate the compounds present in wasabi leaves (Wasabia japonica Matsumura) that inhibit the adipocyte differentiation, activity-guided fractionation was performed on these leaves. 5-Hydroxyferulic acid methyl ester (1: 5-HFA ester), one of the phenylpropanoids, was isolated from wasabi leaves as a compound that inhibits the adipocyte differentiation. Compound 1 suppressed the intracellular lipid accumulation of 3T3-L1 cells without significant cytotoxicity. Gene expression analysis revealed that 1 suppressed the mRNA expression of 2 master regulators of adipocyte differentiation, PPARγ and C/EBPα. Furthermore, 1 downregulated the expression of adipogenesis-related genes, GLUT4, LPL, SREBP-1c, ACC, and FAS. Protein expression analysis revealed that 1 suppressed PPARγ protein expression. Moreover, to investigate the relationship between the structure and activity of inhibiting the adipocyte differentiation, we synthesized 12 kinds of phenylpropanoid analog. Comparison of the activity among 1 and its analogs suggested that the compound containing the substructure that possess a common functional group at the ortho position such as a catechol group exhibits the activity of inhibiting the adipocyte differentiation. Taken together, our findings suggest that 1 from wasabi leaves inhibits adipocyte differentiation via the downregulation of PPARγ. Copyright © 2018 John Wiley & Sons, Ltd.

Differential protein expression during colonic adaptation in ultra-short bowel rats

PubMed Central

Jiang, Hai-Ping; Chen, Tao; Yan, Guang-Rong; Chen, Dan

2011-01-01

AIM: To investigate the proteins involved in colonic adaptation and molecular mechanisms of colonic adaptation in rats with ultra-short bowel syndrome (USBS). METHODS: Sprague Dawley rats were randomly assigned to three groups: USBS group (10 rats) undergoing an approximately 90%-95% small bowel resection; sham-operation group (10 rats) undergoing small bowel transaction and anastomosis; and control group (ten normal rats). Colon morphology and differential protein expression was analyzed after rats were given post-surgical enteral nutrition for 21 d. Protein expression in the colonic mucosa was analyzed by two-dimensional electrophoresis (2-DE) in all groups. Differential protein spots were detected by ImageMaster 2D Platinum software and were further analyzed with matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight-mass spectrometric (MALDI-TOF/TOF-MS) analysis. RESULTS: The colonic mucosal thickness significantly increased in the USBS group compared with the control group (302.1 ± 16.9 μm vs 273.7 ± 16.0 μm, P < 0.05). There was no statistically significant difference between the sham-operation group and control group (P > 0.05). The height of colon plica markedly improved in USBS group compared with the control group (998.4 ± 81.2 μm vs 883.4 ± 39.0 μm, P < 0.05). There was no statistically significant difference between the sham-operation and control groups (P > 0.05). A total of 141 differential protein spots were found in the USBS group. Forty-nine of these spots were down-regulated while 92 protein spots were up-regulated by over 2-folds. There were 133 differential protein spots in USBS group. Thirty of these spots were down-regulated and 103 were up-regulated. There were 47 common differential protein spots among the three groups, including 17 down-regulated protein spots and 30 up-regulated spots. Among 47 differential spots, eight up-regulated proteins were identified by MALDI-TOF/TOF-MS. These proteins were previously reported to be involved in sugar and fat metabolism, protein synthesis and oxidation reduction, which are associated with colonic adaption. CONCLUSION: Eight proteins found in this study play important roles in colonic compensation and are associated with sugar and fat metabolism, protein synthesis, and molecular chaperoning PMID:21633663

Differential protein expression during colonic adaptation in ultra-short bowel rats.

PubMed

Jiang, Hai-Ping; Chen, Tao; Yan, Guang-Rong; Chen, Dan

2011-05-28

To investigate the proteins involved in colonic adaptation and molecular mechanisms of colonic adaptation in rats with ultra-short bowel syndrome (USBS). Sprague Dawley rats were randomly assigned to three groups: USBS group (10 rats) undergoing an approximately 90%-95% small bowel resection; sham-operation group (10 rats) undergoing small bowel transaction and anastomosis; and control group (ten normal rats). Colon morphology and differential protein expression was analyzed after rats were given post-surgical enteral nutrition for 21 d. Protein expression in the colonic mucosa was analyzed by two-dimensional electrophoresis (2-DE) in all groups. Differential protein spots were detected by ImageMaster 2D Platinum software and were further analyzed with matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight-mass spectrometric (MALDI-TOF/TOF-MS) analysis. The colonic mucosal thickness significantly increased in the USBS group compared with the control group (302.1 ± 16.9 μm vs 273.7 ± 16.0 μm, P < 0.05). There was no statistically significant difference between the sham-operation group and control group (P > 0.05). The height of colon plica markedly improved in USBS group compared with the control group (998.4 ± 81.2 μm vs 883.4 ± 39.0 μm, P < 0.05). There was no statistically significant difference between the sham-operation and control groups (P > 0.05). A total of 141 differential protein spots were found in the USBS group. Forty-nine of these spots were down-regulated while 92 protein spots were up-regulated by over 2-folds. There were 133 differential protein spots in USBS group. Thirty of these spots were down-regulated and 103 were up-regulated. There were 47 common differential protein spots among the three groups, including 17 down-regulated protein spots and 30 up-regulated spots. Among 47 differential spots, eight up-regulated proteins were identified by MALDI-TOF/TOF-MS. These proteins were previously reported to be involved in sugar and fat metabolism, protein synthesis and oxidation reduction, which are associated with colonic adaption. Eight proteins found in this study play important roles in colonic compensation and are associated with sugar and fat metabolism, protein synthesis, and molecular chaperoning.

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

PubMed Central

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

1997-01-01

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

Proteomic study of the yeast Rhodotorula mucilaginosa RCL-11 under copper stress.

PubMed

Irazusta, Verónica; Estévez, Cristina; Amoroso, María Julia; de Figueroa, Lucía I C

2012-06-01

In order to understand the mechanism involved in Rhodotorula mucilaginosa RCL-11 resistance to copper a proteomic study was conducted. Atomic absorption spectroscopy showed that the copper concentration in the medium decreased from 0.5 to 0.19 mM 48 h after inoculation of the yeast. Analysis of one-dimensional gel electrophoresis of crude cell extracts revealed expression of differential bands between cells with and without copper. In order to study this difference, two-dimensional electrophoresis of R. mucilaginosa RCL-11 exposed to Cu for 16, 24, and 48 h was carried out. Identification of differentially expressed proteins was performed by MALDI-TOF/TOF. Ten of the 16 spots identified belonged to heat shock proteins. Superoxide dismutase, methionine synthase and beta-glucosidase were also found over-expressed at high copper concentrations. The results obtained in the present work show that when R. mucilaginosa RCL-11 is exposed to 0.5 mM copper, differential proteins, involved in cell resistance mechanisms, are expressed.

Control of developmentally primed erythroid genes by combinatorial co-repressor actions

PubMed Central

Stadhouders, Ralph; Cico, Alba; Stephen, Tharshana; Thongjuea, Supat; Kolovos, Petros; Baymaz, H. Irem; Yu, Xiao; Demmers, Jeroen; Bezstarosti, Karel; Maas, Alex; Barroca, Vilma; Kockx, Christel; Ozgur, Zeliha; van Ijcken, Wilfred; Arcangeli, Marie-Laure; Andrieu-Soler, Charlotte; Lenhard, Boris; Grosveld, Frank; Soler, Eric

2015-01-01

How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2–IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation. PMID:26593974

Protein profiling of preeclampsia placental tissues.

PubMed

Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; Jiang, Jing; Lee, Peng; Zhang, David Y; He, Jin; Ye, Fei

2014-01-01

Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia.

Protein Profiling of Preeclampsia Placental Tissues

PubMed Central

Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; Jiang, Jing; Lee, Peng; Zhang, David Y.

2014-01-01

Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia. PMID:25392996

Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/β-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

PubMed

Feng, Xiao-Yu; Wu, Xiao-Shan; Wang, Jin-Song; Zhang, Chun-Mei; Wang, Song-Lin

2018-02-01

Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway. © 2017 Eur J Oral Sci.

Changes in the regulation of heat shock gene expression in neuronal cell differentiation.

PubMed

Oza, Jay; Yang, Jingxian; Chen, Kuang Yu; Liu, Alice Y-C

2008-01-01

Neuronal differentiation of the NG108-15 neuroblastoma-glioma hybrid cells is accompanied by a marked attenuation in the heat shock induction of the Hsp70-firefly luciferase reporter gene activity. Analysis of the amount and activation of heat shock factor 1, induction of mRNA(hsp), and the synthesis and accumulation of heat shock proteins (HSPs) in the undifferentiated and differentiated cells suggest a transcriptional mechanism for this attenuation. Concomitant with a decreased induction of the 72-kDa Hsp70 protein in the differentiated cells, there is an increased abundance of the constitutive 73-kDa Hsc70, a protein known to function in vesicle trafficking. Assessment of sensitivity of the undifferentiated and differentiated cells against stress-induced cell death reveals a significantly greater vulnerability of the differentiated cells toward the cytotoxic effects of arsenite and glutamate/glycine. This study shows that changes in regulation of the HSP and HSC proteins are components of the neuronal cell differentiation program and that the attenuated induction of HSPs likely contributes to neuronal vulnerability whereas the increased expression of Hsc70 likely has a role in neural-specific functions.

Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers.

PubMed

Hawke, Thomas J; Atkinson, Daniel J; Kanatous, Shane B; Van der Ven, Peter F M; Goetsch, Sean C; Garry, Daniel J

2007-11-01

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.

Basic Fibroblast Growth Factor Regulates Gene and Protein Expression Related to Proliferation, Differentiation, and Matrix Production of Human Dental Pulp Cells.

PubMed

Chang, Ya-Ching; Chang, Mei-Chi; Chen, Yi-Jane; Liou, Ji-Uei; Chang, Hsiao-Hua; Huang, Wei-Ling; Liao, Wan-Chuen; Chan, Chiu-Po; Jeng, Po-Yuan; Jeng, Jiiang-Huei

2017-06-01

Basic fibroblast growth factor (bFGF) plays differential effects on the proliferation, differentiation, and extracellular matrix turnover in various tissues. However, limited information is known about the effect of bFGF on dental pulp cells. The purposes of this study were to investigate whether bFGF influences the cell differentiation and extracellular matrix turnover of human dental pulp cells (HDPCs) and the related gene and protein expression as well as the role of the mitogen-activated protein kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling pathway. The expression of fibroblast growth factor receptors (FGFRs) in HDPCs was also studied. The expression of FGFR1 and FGFR2 in HDPCs was investigated by reverse-transcription polymerase chain reaction. HDPCs were treated with different concentrations of bFGF. Cell proliferation was evaluated using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell differentiation was evaluated using alkaline phosphatase (ALP) staining. Changes in messenger expression of cyclin B1 and tissue inhibitor of metalloproteinase (TIMP) 1 were determined by reverse-transcription polymerase chain reaction. Changes in protein expression of cdc2, TIMP-1, TIMP-2, and collagen I were determined by Western blotting. U0126 was used to clarify the role of MEK/ERK signaling. HDPCs expressed both FGFR1 and FGFR2. Cell viability was stimulated by 50-250 ng/mL bFGF. The expression and enzyme activities of ALP were inhibited by 10-500 ng/mL bFGF. At similar concentrations, bFGF stimulates cdc2, cyclin B1, and TIMP-1 messenger RNA and protein expression. bFGF showed little effect on TIMP-2 and partly inhibited collagen I expression of pulp cells. U0126 (a MEK/ERK inhibitor) attenuated the bFGF-induced increase of cyclin B1, cdc2, and TIMP-1. bFGF may be involved in pulpal repair and regeneration by activation of FGFRs to regulate cell growth; stimulate cdc2, cyclin B1, and TIMP-1 expression; and inhibit ALP. These events are partly associated with MEK/ERK signaling. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Identification of Potential Biomarkers for Rhegmatogenous Retinal Detachment Associated with Choroidal Detachment by Vitreous iTRAQ-Based Proteomic Profiling

PubMed Central

Wu, Zhifeng; Ding, Nannan; Yu, Mengxi; Wang, Ke; Luo, Shasha; Zou, Wenjun; Zhou, Ying; Yan, Biao; Jiang, Qin

2016-01-01

Rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD) is a complicated and serious type of rhegmatogenous retinal detachment (RRD). In this study, we identified differentially expressed proteins in the vitreous humors of RRDCD and RRD using isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography-electrospray ion trap-mass spectrometry-mass spectrometry (nano-LC-ESI-MS/MS) and bioinformatic analysis. Our result shows that 103 differentially expressed proteins, including 54 up-regulated and 49 down-regulated proteins were identified in RRDCD. Gene ontology (GO) analysis suggested that most of the differentially expressed proteins were extracellular．The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis suggested that proteins related to complement and coagulation cascades were significantly enriched. iTRAQ-based proteomic profiling reveals that complement and coagulation cascades and inflammation may play important roles in the pathogenesis of RRDCD. This study may provide novel insights into the pathogenesis of RRDCD and offer potential opportunities for the diagnosis and treatment of RRDCD. PMID:27941623

Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation

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

Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.

2007-06-01

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, andmore » YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA regulation.« less

Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis.

PubMed

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J; Considine, Robert V; Sethi, Jaswinder K; Vidal-Puig, Antonio; O'Rahilly, Stephen

2004-03-19

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2-4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.

A microarray analysis of potential genes underlying the neurosensitivity of mice to propofol.

PubMed

Lowes, Damon A; Galley, Helen F; Lowe, Peter R; Rikke, Brad A; Johnson, Thomas E; Webster, Nigel R

2005-09-01

Establishing the mechanism of action of general anesthetics at the molecular level is difficult because of the multiple targets with which these drugs are associated. Inbred short sleep (ISS) and long sleep (ILS) mice are differentially sensitive in response to ethanol and other sedative hypnotics and contain a single quantitative trait locus (Lorp1) that accounts for the genetic variance of loss-of-righting reflex in response to propofol (LORP). In this study, we used high-density oligonucleotide microarrays to identify global gene expression and candidate genes differentially expressed within the Lorp1 region that may give insight into the molecular mechanism underlying LORP. Microarray analysis was performed using Affymetrix MG-U74Av2 Genechips and a selection of differentially expressed genes was confirmed by semiquantitative reverse transcription-polymerase chain reaction. Global expression in the brains of ILS and ISS mice revealed 3423 genes that were significantly expressed, of which 139 (4%) were differentially expressed. Analysis of genes located within the Lorp1 region showed that 26 genes were significantly expressed and that just 2 genes (7%) were differentially expressed. These genes encoded for the proteins AWP1 (associated with protein kinase 1) and "BTB (POZ) domain containing 1," whose functions are largely uncharacterized. Genes differentially expressed outside Lorp1 included seven genes with previously characterized neuronal functions and thus stand out as additional candidate genes that may be involved in mediating the neurosensitivity differences between ISS and ILS.

Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo.

PubMed

Novoa-Herran, Susana; Umaña-Perez, Adriana; Canals, Francesc; Sanchez-Gomez, Myriam

2016-01-01

How nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line. The viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test ( n  = 3, significance level 0.10, D > 0.642) and/or ANOVA ( n  = 3, p  < 0.05). The results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells. This comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.

Prmt7 is dispensable in tissue culture models for adipogenic differentiation.

PubMed

Hu, Yu-Jie; Sif, Saïd; Imbalzano, Anthony N

2013-01-01

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models.

Prmt7 is dispensable in tissue culture models for adipogenic differentiation

PubMed Central

Imbalzano, Anthony N.

2013-01-01

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models. PMID:24715966

Expressed proteins of Herbaspirillum seropedicae in maize (DKB240) roots-bacteria interaction revealed using proteomics.

PubMed

Ferrari, Cibele Santos; Amaral, Fernanda Plucani; Bueno, Jessica Cavalheiro Ferreira; Scariot, Mirella Christine; Valentim-Neto, Pedro Alexandre; Arisi, Ana Carolina Maisonnave

2014-11-01

Several molecular tools have been used to clarify the basis of plant-bacteria interaction; however, the mechanism behind the association is still unclear. In this study, we used a proteomic approach to investigate the root proteome of Zea mays (cv. DKB240) inoculated with Herbaspirillum seropedicae strain SmR1 grown in vitro and harvested 7 days after inoculation. Eighteen differentially accumulated proteins were observed in root samples, ten of which were identified by MALDI-TOF mass spectrometry peptide mass fingerprint. Among the identified proteins, we observed three proteins present exclusively in inoculated root samples and six upregulated proteins and one downregulated protein relative to control. Differentially expressed maize proteins were identified as hypothetical protein ZEAMMB73_483204, hypothetical protein ZEAMMB73_269466, and tubulin beta-7 chain. The following were identified as H. seropedicae proteins: peroxiredoxin protein, EF-Tu elongation factor protein, cation transport ATPase, NADPH:quinone oxidoreductase, dinitrogenase reductase, and type III secretion ATP synthase. Our results presented the first evidence of type III secretion ATP synthase expression during H. seropedicae-maize root interaction.

A subset of replication-dependent histone mRNAs are expressed as polyadenylated RNAs in terminally differentiated tissues.

PubMed

Lyons, Shawn M; Cunningham, Clark H; Welch, Joshua D; Groh, Beezly; Guo, Andrew Y; Wei, Bruce; Whitfield, Michael L; Xiong, Yue; Marzluff, William F

2016-11-02

Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of avocado (Persea americana) root proteins induced by infection with the oomycete Phytophthora cinnamomi using a proteomic approach.

PubMed

Acosta-Muñiz, Carlos H; Escobar-Tovar, Lina; Valdes-Rodríguez, Silvia; Fernández-Pavia, Silvia; Arias-Saucedo, Luis J; de la Cruz Espindola Barquera, Maria; Gómez Lim, Miguel Á

2012-01-01

Avocado root rot, caused by Phytophthora cinnamomi, is the most important disease that limits avocado production. A proteomic approach was employed to identify proteins that are upregulated by infection with P. cinnamomi. Different proteins were shown to be differentially expressed after challenge with the pathogen by two-dimensional (2-D) gel electrophoresis. A densitometric evaluation of protein expression indicated differential regulation during the time-course analyzed. Some proteins induced in response to the infection were identified by standard peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of the 400 protein spots detected on 2-D gels, 21 seemed to change in abundance by 3 hours after infection. Sixteen proteins were upregulated, 5 of these were only detected in infected roots and 11 showed an increased abundance. Among the differentially expressed proteins identified are homologs to isoflavone reductase, glutathione S-transferase, several abscisic acid stress-ripening proteins, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, cysteine synthase and quinone reductase. A 17.3-kDa small heat-shock protein and a glycine-rich RNA-binding protein were identified as downregulated. Our group is the first to report on gene induction in response to oomycete infection in roots from avocado, using proteomic techniques. Copyright © Physiologia Plantarum 2011.

Proteomic identification of altered cerebral proteins in the complex regional pain syndrome animal model.

PubMed

Nahm, Francis Sahngun; Park, Zee-Yong; Nahm, Sang-Soep; Kim, Yong Chul; Lee, Pyung Bok

2014-01-01

Complex regional pain syndrome (CRPS) is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP) model, a novel experimental model of CRPS. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS.

[Relationship of GSTP1 lower expression and multidrug resistance reversing of curcumin on human colon carcinoma cells].

PubMed

Li, He; Li, Lei

2015-08-11

To explore the proteomic differences among with and without curcumin treatment of vincristine-resistance HCT-8/VCR cells of human colon carcinoma by using mass spectrometry and two-dimensional gel electrophore-sis (2-DE). The total proteins of the both groups were extracted from serum were run in immobilized pH gradient isoelectic focusing (IPG-IEF) at the first dimension.The proteins pots in gels were visualized by silver staining protocol, scanned by using a molecular imager GS-800 calibrated densitometer. The differentially expressed proteins were identified and analyzed by PDQuest 8.0 software. The diferentially displayed protein spots were searched and identifiyed by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS), the interested proteins were further validated by RT-PCR and Western blot. The 2-DE HCT-8/VCR cells patterns were acquired with clear background, well-resolution and reproduction. And 1 070±96 protein spots were detected in control HCT-8/VCR cells and 1 030±69 in curcumin-treated HCT-8/VCR cells. Twenty-nine differential protein spots were found to be differentially expressed. Glutathione S-transferase pi1 gene (GSTP1), a diferentiaI expression protein was identified which one of these proteins. RT-PCR and Western blotting results showed that the expressions of GSTP1 mRNA (0.49±0.09) and protein (0.29±0.07) in curcumin-treated group were significantly lower than in control group (GSTP1 mRNA 1.19±0.21 and protein 0.70±0.13, both P<0.05), indicating that curcumin down regulated these expressions. The suppression of GSTP1 by curcumin could enhance the vincristine chemosensitivity in HCT-8/VCR. GSTP1 overexpression may be involved in the vincristine -resistance of human colon carcinoma cells.

Proteomic analysis of cerebrospinal fluid in canine cervical spondylomyelopathy.

PubMed

Martin-Vaquero, Paula; da Costa, Ronaldo C; Allen, Matthew J; Moore, Sarah A; Keirsey, Jeremy K; Green, Kari B

2015-05-01

Prospective study. To identify proteins with differential expression in the cerebrospinal fluid (CSF) from 15 clinically normal (control) dogs and 15 dogs with cervical spondylomyelopathy (CSM). Canine CSM is a spontaneous, chronic, compressive cervical myelopathy similar to human cervical spondylotic myelopathy. There is a limited knowledge of the molecular mechanisms underlying these conditions. Differentially expressed CSF proteins may contribute with novel information about the disease pathogenesis in both dogs and humans. Protein separation was performed with 2-dimensional electrophoresis. A Student t test was used to detect significant differences between groups (P < 0.05). Three comparisons were made: (1) control versus CSM-affected dogs, (2) control versus non-corticosteroid-treated CSM-affected dogs, and (3) non-corticosteroid-treated CSM-affected versus corticosteroid-treated CSM-affected dogs. Protein spots exhibiting at least a statistically significant 1.25-fold change between groups were selected for subsequent identification with capillary-liquid chromatography tandem mass spectrometry. A total of 96 spots had a significant average change of at least 1.25-fold in 1 of the 3 comparisons. Compared with the CSF of control dogs, CSM-affected dogs demonstrated increased CSF expression of 8 proteins including vitamin D-binding protein, gelsolin, creatine kinase B-type, angiotensinogen, α-2-HS-glycoprotein, SPARC (secreted protein, acidic, rich in cysteine), calsyntenin-1, and complement C3, and decreased expression of pigment epithelium-derived factor, prostaglandin-H2 D-isomerase, apolipoprotein E, and clusterin. In the CSF of CSM-affected dogs, corticosteroid treatment increased the expression of haptoglobin, transthyretin isoform 2, cystatin C-like, apolipoprotein E, and clusterin, and decreased the expression of angiotensinogen, α-2-HS-glycoprotein, and gelsolin. Many of the differentially expressed proteins are associated with damaged neural tissue, bone turnover, and/or compromised blood-spinal cord barrier. The knowledge of the protein changes that occur in CSM and upon corticosteroid treatment of CSM-affected patients will aid in further understanding the pathomechanisms underlying this disease. N/A.

Dexamethasone Regulates Cochlear Expression of Deafness-associated Proteins Myelin Protein Zero and Heat Shock Protein 70, as Revealed by iTRAQ Proteomics.

PubMed

Maeda, Yukihide; Fukushima, Kunihiro; Kariya, Shin; Orita, Yorihisa; Nishizaki, Kazunori

2015-08-01

Using proteomics, we aimed to identify the proteins differentially regulated by dexamethasone in the mouse cochlea based on mass-spectrometry data. Glucocorticoid therapy is widely used for many forms of sensorineural hearing loss; however, the molecular mechanism of its action in the cochlea remains poorly understood. Dexamethasone or control saline was intratympanically applied to the cochleae of mice. Twelve hours after application, proteins differentially regulated by dexamethasone in the cochlea were analyzed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-mass spectrometry. Next, dexamethasone-dependent regulation of these proteins was verified in the cochleae of mice with noise-induced hearing loss (NIHL) and systemic administration of dexamethasone by western blotting. Immunolocalizations of these proteins were examined in cochleae with NIHL. A total of 247 proteins with a greater than 95% confidence interval of protein identification were found, and 11 differentially expressed proteins by dexamethasone were identified by the iTRAQ-mass spectrometry. One protein, myelin protein zero (Mpz), was upregulated (1.870 ± 0.201-fold change, p < 0.01) at 6 hours post-systemic dexamethasone and noise exposure in a mouse model of NIHL. Heat shock protein 70 (Hsp70) was downregulated (0.511 ± 0.274-fold change, p < 0.05) at 12 hours post-systemic dexamethasone. Immunohistochemistry confirmed Mpz localization to the efferent and afferent processes of the spiral neurons, whereas Hsp70 showed a more ubiquitous expression pattern in the cochlea. Both Mpz and Hsp70 have been reported to be closely associated with sensorineural hearing loss in humans. Dexamethasone significantly modulated the expression levels of these proteins in the cochleae of mice.

Simultaneous detection of changes in protein expression and oxidative modification as function of age and APOE genotype in human APOE-targeted replacement mice

EPA Science Inventory

Background The purpose of this study was to improve the current method for detecting differentially-oxidized (carbonyl-modified) proteins by 2D-DIGE, while at the same time determining changes in total protein expression. Protein oxidation is a widely accepted model of aging and...

Protein Expression Profile using Two-Dimensional Gel Analysis in Squamous Cervical Cancer Patients

PubMed Central

Bae, Su-Mi; Min, Hyun-Jin; Ding, Guo Hua; Kwak, Sun-Young; Cho, Young-Lae; Nam, Kye-Hyun; Park, Choong Hak; Kim, Yong-Wan; Kim, Chong-Kook; Han, Byoung-Don; Lee, Young-Joo; Kim, Do Kang

2006-01-01

Purpose Screening in cervical cancer is now progressing to discover candidate genes and proteins that may serve as biological markers and that play a role in tumor progression. We examined the protein expression patterns of the squamous cell carcinoma (SCC) tissues from Korean women with using two- dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight (MALDI- TOF) mass spectrometer. Materials and Methods Normal cervix and SCC tissues were solubilized and 2-DE was performed using pH 3~10 linear IPG strips of 17 cm length. The protein expression was evaluated using PDQuest 2-D software™. The differentially expressed protein spots were identified with a MALDI-TOF mass spectrometer, and the peptide mass spectra identifications were performed using the Mascot program and by searching the Swiss-prot or NCBInr databases. Results A total of 35 proteins were detected in SCC. 17 proteins were up-regulated and 18 proteins weredown-regulated. Among the proteins that were identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, α-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase and apolipoprotein a1) were protein previously known to be involved in tumor, and 21 proteins were newly identified in this study. Conclusion 2-DE offers the total protein expression profiles of SCC tissues; further characterization of these differentially expressed proteins will give a chance to identify the badly needed tumor-specific diagnostic markers for SCC. PMID:19771267

p16 expression is not associated with human papillomavirus in urinary bladder squamous cell carcinoma.

PubMed

Alexander, Riley E; Hu, Yingchuan; Kum, Jennifer B; Montironi, Rodolfo; Lopez-Beltran, Antonio; Maclennan, Gregory T; Idrees, Muhammad T; Emerson, Robert E; Ulbright, Thomas M; Grignon, David G; Eble, John N; Cheng, Liang

2012-11-01

Squamous cell carcinoma of the urinary bladder is unusual and of unknown etiology. There is a well-established association between human papillomavirus (HPV) infection and the development of cervical and head/neck squamous cell carcinomas. However, the role of HPV in the pathogenesis of squamous cell carcinoma of the urinary bladder is uncertain. The purposes of this study were to investigate the possible role of HPV in the development of squamous cell carcinoma of the urinary bladder and to determine if p16 expression could serve as a surrogate marker for HPV in this malignancy. In all, 42 cases of squamous cell carcinoma of the urinary bladder and 27 cases of urothelial carcinoma with squamous differentiation were investigated. HPV infection was analyzed by both in situ hybridization at the DNA level and immunohistochemistry at the protein level. p16 protein expression was analyzed by immunohistochemistry. HPV DNA and protein were not detected in 42 cases of squamous cell carcinoma (0%, 0/42) or 27 cases of urothelial carcinoma with squamous differentiation (0%, 0/15). p16 expression was detected in 13 cases (31%, 13/42) of squamous cell carcinoma and 9 cases (33%, 9/27) of urothelial carcinoma with squamous differentiation. There was no correlation between p16 expression and the presence of HPV infection in squamous cell carcinoma of the bladder or urothelial carcinoma with squamous differentiation. Our data suggest that HPV does not play a role in the development of squamous cell carcinoma of the urinary bladder or urothelial carcinoma with squamous differentiation. p16 expression should not be used as a surrogate marker for evidence of HVP infection in either squamous cell carcinoma of the urinary bladder or urothelial carcinoma with squamous differentiation as neither HVP DNA nor protein is detectable in these neoplasms.

Characterization and comparison of proteomes of albino sea cucumber Apostichopus japonicus (Selenka) by iTRAQ analysis.

PubMed

Xia, Chang-Ge; Zhang, Dijun; Ma, Chengnv; Zhou, Jun; He, Shan; Su, Xiu-Rong

2016-04-01

Sea cucumber is a commercially important marine organism in China. Of the different colored varieties sold in China, albino sea cucumber has the greatest appeal among consumers. Identification of factors contributing to albinism in sea cucumber is therefore likely to provide a scientific basis for improving the cultivability of these strains. In this study, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification labeling was used for the first time to quantitatively define the proteome of sea cucumbers and reveal proteomic characteristics unique to albino sea cucumbers. A total of 549 proteins were identified and quantified in albino sea cucumber and the functional annotations of 485 proteins have been exhibited based on COG database. Compared with green sea cucumber, 12 proteins were identified as differentially expressed in the intestine and 16 proteins in the body wall of albino sea cucumber. Among them, 5 proteins were up-regulated in the intestine and 8 proteins were down-regulated in body wall. Gene ontology annotations of these differentially expressed proteins consisted mostly of 'biological process'. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying albinism in sea cucumber. Copyright © 2015 Elsevier Ltd. All rights reserved.

miR-21 promotes the differentiation of hair follicle-derived neural crest stem cells into Schwann cells

PubMed Central

Ni, Yuxin; Zhang, Kaizhi; Liu, Xuejuan; Yang, Tingting; Wang, Baixiang; Fu, Li; A, Lan; Zhou, Yanmin

2014-01-01

Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair follicles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA (miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist (agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist (antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regulating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA. PMID:25206896

Identification and Validation of Selected Universal Stress Protein Domain Containing Drought-Responsive Genes in Pigeonpea (Cajanus cajan L.)

PubMed Central

Sinha, Pallavi; Pazhamala, Lekha T.; Singh, Vikas K.; Saxena, Rachit K.; Krishnamurthy, L.; Azam, Sarwar; Khan, Aamir W.; Varshney, Rajeev K.

2016-01-01

Pigeonpea is a resilient crop, which is relatively more drought tolerant than many other legume crops. To understand the molecular mechanisms of this unique feature of pigeonpea, 51 genes were selected using the Hidden Markov Models (HMM) those codes for proteins having close similarity to universal stress protein domain. Validation of these genes was conducted on three pigeonpea genotypes (ICPL 151, ICPL 8755, and ICPL 227) having different levels of drought tolerance. Gene expression analysis using qRT-PCR revealed 6, 8, and 18 genes to be ≥2-fold differentially expressed in ICPL 151, ICPL 8755, and ICPL 227, respectively. A total of 10 differentially expressed genes showed ≥2-fold up-regulation in the more drought tolerant genotype, which encoded four different classes of proteins. These include plant U-box protein (four genes), universal stress protein A-like protein (four genes), cation/H(+) antiporter protein (one gene) and an uncharacterized protein (one gene). Genes C.cajan_29830 and C.cajan_33874 belonging to uspA, were found significantly expressed in all the three genotypes with ≥2-fold expression variations. Expression profiling of these two genes on the four other legume crops revealed their specific role in pigeonpea. Therefore, these genes seem to be promising candidates for conferring drought tolerance specifically to pigeonpea. PMID:26779199

Induction of motor neuron differentiation by transduction of Olig2 protein.

PubMed

Mie, Masayasu; Kaneko, Mami; Henmi, Fumiaki; Kobatake, Eiry

2012-10-26

Olig2 protein, a member of the basic helix-loop-helix transcription factor family, was introduced into the mouse embryonic carcinoma cell line P19 for induction of motor neuron differentiation. We show that Olig2 protein has the ability to permeate the cell membrane without the addition of a protein transduction domain (PTD), similar to other basic helix-loop-helix transcription factors such as MyoD and NeuroD2. Motor neuron differentiation was evaluated for the elongation of neurites and the expression of choline acetyltransferase (ChAT) mRNA, a differentiation marker of motor neurons. By addition of Olig2 protein, motor neuron differentiation was induced in P19 cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Transcriptome analysis reveals differential gene expression in intramuscular adipose tissues of Jinhua and Landrace pigs.

PubMed

Miao, Zhiguo; Wei, Panpeng; Khan, Muhammad Akram; Zhang, Jinzhou; Guo, Liping; Liu, Dongyang; Zhang, Xiaojian; Bai, Yueyu; Wang, Shan

2018-05-01

Meat is a rich source of protein, fatty acids and carbohydrates for human needs. In addition to necessary nutrients, high fat contents in pork increase the tenderness and juiciness of the meat, featuring diverse application in various dishes. This study investigated the transcriptomic profiles of intramuscular adipose tissues in Jinhua and Landrace pigs by employing advanced RNA sequencing. Results showed significant interesting to note that there were significant differences in the expression of genes. 1,632 genes showed significant differential expression, 837 genes were up-regulated and 195 genes were down-regulated. Variations in genes responsible for cell aggregation, extracellular matrix formation, cellular lipid catabolic process, and fatty acid binding strongly supported that both pig breeds feature variable fat and muscle metabolism. Certain differentially expressed genes are included in the pathway of mitogen-activated protein kinase signaling pathway, Ras signaling pathway and insulin pathway. Results from real-time quantitative polymerase chain reaction also validated the differential expression of 17 mRNAs between meats of the two pig breeds. Overall, these findings reveal significant differences in fat and protein metabolism of intramuscular adipose tissues of two pig breeds at the transcriptomic level and suggest diversification at the genetic level between breeds of the same species.

Proteomics-based approach identified differentially expressed proteins with potential roles in endometrial carcinoma.

PubMed

Li, Zhengyu; Min, Wenjiao; Huang, Canhua; Bai, Shujun; Tang, Minghai; Zhao, Xia

2010-01-01

We used proteomic approaches to identify altered expressed proteins in endometrial carcinoma, with the aim of discovering potential biomarkers or therapeutic targets for endometrial carcinoma. The global proteins extracted from endometrial carcinoma and normal endometrial tissues were separated by 2-dimensional electrophoresis and analyzed with PDQuest (Bio-Rad, Hercules, Calif) software. The differentially expressed spots were identified by mass spectrometry and searched against NCBInr protein database. Those proteins with potential roles were confirmed by Western blotting and immunohistochemical assays. Ninety-nine proteins were identified by mass spectrometry, and a cluster diagram analysis indicated that these proteins were involved in metabolism, cell transformation, protein folding, translation and modification, proliferation and apoptosis, signal transduction, cytoskeleton, and so on. In confirmatory immunoblotting and immunohistochemical analyses, overexpressions of epidermal fatty acid-binding protein, calcyphosine, and cyclophilin A were also observed in endometrial carcinoma tissues, which were consistent with the proteomic results. Our results suggested that these identified proteins, including epidermal fatty acid-binding protein, calcyphosine, and cyclophilin A, might be of potential values in the studies of endometrial carcinogenesis or investigations of diagnostic biomarkers or treatment targets for endometrial carcinoma.

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

PubMed

Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

2016-09-01

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Tetramethylpyrazine induces SH-SY5Y cell differentiation toward the neuronal phenotype through activation of the PI3K/Akt/Sp1/TopoIIβ pathway.

PubMed

Yan, Yong-Xin; Zhao, Jun-Xia; Han, Shuo; Zhou, Na-Jing; Jia, Zhi-Qiang; Yao, Sheng-Jie; Cao, Cui-Li; Wang, Yan-Ling; Xu, Yan-Nan; Zhao, Juan; Yan, Yun-Li; Cui, Hui-Xian

2015-12-01

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Previously, we have shown that TMP induces human SH-SY5Y neuroblastoma cell differentiation toward the neuronal phenotype by targeting topoisomeraseIIβ (TopoIIβ), a protein implicated in neural development. In the present study, we aimed to elucidate whether the transcriptional factors specificity protein 1 (Sp1) and nuclear factor Y (NF-Y), in addition to the upstream signaling pathways ERK1/2 and PI3K/Akt, are involved in modulating TopoIIβ expression in the neuronal differentiation process. We demonstrated that SH-SY5Y cells treated with TMP (80μM) terminally differentiated into neurons, characterized by increased neuronal markers, tubulin βIII and microtubule associated protein 2 (MAP2), and increased neurite outgrowth, with no negative effect on cell survival. TMP also increased the expression of TopoIIβ, which was accompanied by increased expression of Sp1 in the differentiated neuron-like cells, whereas NF-Y protein levels remained unchanged following the differentiation progression. We also found that the phosphorylation level of Akt, but not ERK1/2, was significantly increased as a result of TMP stimulation. Furthermore, as established by chromatin immunoprecipitation (ChIP) assay, activation of the PI3K/Akt pathway increased Sp1 binding to the promoter of the TopoIIβ gene. Blockage of PI3K/Akt was shown to lead to subsequent inhibition of TopoIIβ expression and neuronal differentiation. Collectively, the results indicate that the PI3K/Akt/Sp1/TopoIIβ signaling pathway is necessary for TMP-induced neuronal differentiation. Our findings offer mechanistic insights into understanding the upstream regulation of TopoIIβ in neuronal differentiation, and suggest potential applications of TMP both in neuroscience research and clinical practice to treat relevant diseases of the nervous system. Copyright © 2015 Elsevier GmbH. All rights reserved.

Development of a Targeted Urine Proteome Assay for kidney diseases.

PubMed

Cantley, Lloyd G; Colangelo, Christopher M; Stone, Kathryn L; Chung, Lisa; Belcher, Justin; Abbott, Thomas; Cantley, Jennifer L; Williams, Kenneth R; Parikh, Chirag R

2016-01-01

Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases. Potential biomarkers were identified by using a multiproteomics workflow to compare urine proteomes of kidney transplant patients with immediate and delayed graft function. Differentially expressed proteins were identified, and corresponding stable isotope labeled internal peptide standards were synthesized for scheduled MRM. The Targeted Urine Proteome Assay (TUPA) was then developed by identifying those peptides for which there were at least two transitions for which interference in a urine matrix across 156 MRM runs was <30%. This resulted in an assay that monitors 224 peptides from 167 quantifiable proteins. TUPA opens the way for using a robust mass spectrometric technology, MRM, for quantifying and validating biomarkers from among 167 urinary proteins. This approach, while developed using differentially expressed urinary proteins from patients with delayed versus immediate graft function after kidney transplant, can be expanded to include differentially expressed urinary proteins in multiple kidney diseases. Thus, TUPA could provide a single assay to help diagnose, prognose, and manage many kidney diseases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RNA-Seq reveals 10 novel promising candidate genes affecting milk protein concentration in the Chinese Holstein population.

PubMed

Li, Cong; Cai, Wentao; Zhou, Chenghao; Yin, Hongwei; Zhang, Ziqi; Loor, Juan J; Sun, Dongxiao; Zhang, Qin; Liu, Jianfeng; Zhang, Shengli

2016-06-02

Paired-end RNA sequencing (RNA-Seq) was used to explore the bovine transcriptome from the mammary tissue of 12 Chinese Holstein cows with 6 extremely high and 6 low phenotypic values for milk protein percentage. We defined the differentially expressed transcripts between the two comparison groups, extremely high and low milk protein percentage during the peak lactation (HP vs LP) and during the non-lactating period (HD vs LD), respectively. Within the differentially expressed genes (DEGs), we detected 157 at peak lactation and 497 in the non-lactating period with a highly significant correlation with milk protein concentration. Integrated interpretation of differential gene expression indicated that SERPINA1, CLU, CNTFR, ERBB2, NEDD4L, ANG, GALE, HSPA8, LPAR6 and CD14 are the most promising candidate genes affecting milk protein concentration. Similarly, LTF, FCGR3A, MEGF10, RRM2 and UBE2C are the most promising candidates that in the non-lactating period could help the mammary tissue prevent issues with inflammation and udder disorders. Putative genes will be valuable resources for designing better breeding strategies to optimize the content of milk protein and also to provide new insights into regulation of lactogenesis.

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

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

Barboro, Paola; D'Arrigo, Cristina; Repaci, Erica

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentiallymore » expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.« less

Necdin interacts with the Msx2 homeodomain protein via MAGE-D1 to promote myogenic differentiation of C2C12 cells.

PubMed

Kuwajima, Takaaki; Taniura, Hideo; Nishimura, Isao; Yoshikawa, Kazuaki

2004-09-24

Necdin is a potent growth suppressor that is expressed predominantly in postmitotic cells such as neurons and skeletal muscle cells. Necdin shows a significant homology to MAGE (melanoma antigen) family proteins, all of which contain a large homology domain. MAGE-D1 (NRAGE, Dlxin-1) interacts with the Dlx/Msx family homeodomain proteins via an interspersed hexapeptide repeat domain distinct from the homology domain. Here we report that necdin associates with the Msx homeodomain proteins via MAGE-D1 to modulate their function. In vitro binding and co-immunoprecipitation analyses revealed that MAGE-D1 directly interacted with necdin via the homology domain and Msx1 (or Msx2) via the repeat domain. A ternary complex of necdin, MAGE-D1, and Msx2 was formed in vitro, and an endogenous complex containing these three proteins was detected in differentiating embryonal carcinoma cells. Co-expression of necdin and MAGE-D1 released Msx-dependent transcriptional repression. C2C12 myoblast cells that were stably transfected with Msx2 cDNA showed a marked reduction in myogenic differentiation, and co-expression of necdin and MAGE-D1 canceled the Msx2-dependent repression. These results suggest that necdin and MAGE-D1 cooperate to modulate the function of Dlx/Msx homeodomain proteins in cellular differentiation. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

Proteomic analysis of high yield rice variety mutated from spaceflight

NASA Astrophysics Data System (ADS)

Ma, Y.; Cheng, Z.; Wang, W.; Sun, Y.

Seeds of pure rice varieties were flown on Chinese recoverable satellite, JB-1, for a 15-day flight in 1996. Many mutant rice varieties with various phenotypes were generated after continuous selection and breeding. Among the mutants, a variety 971-5 showed a significant increase in grain yield compared to its control (971ck). In this study, proteomic analysis of both mutant variety 971-5 and control variety 971ck were carried out to investigate the changes of protein expression level in their leaves at three different growth stages (early and middle stage of tillering, and booting stage). Results showed that (1) almost all differentially expressed proteins were down-regulated in 971-5 with only one exception, (2) the percentages of differentially expressed proteins were 3.1%, 2.1% and 3.1% at the three stages, respectively, and (3) one protein showed a significant alteration in its molecular weight (MW). These data demonstrated that the space environment can alter the expression level of rice proteins both quantitatively and qualitatively.

Novel leukocyte protein, Trojan, differentially expressed during thymocyte development.

PubMed

Petrov, Petar; Motobu, Maki; Salmi, Jussi; Uchida, Tatsuya; Vainio, Olli

2010-04-01

"Trojan" is a novel cell surface protein, discovered from chicken embryonic thymocytes on the purpose to identify molecules involved in T cell differentiation. The molecule is predicted as a type I transmembrane protein having a Sushi and two fibronectin type III domains and a pair of intracellular phosphorylation sites. Its transcript expression is specific for lymphoid tissues and the presence of the protein on the surface of recirculating lymphocytes and macrophages was confirmed by immunofluorescence analysis. In thymus, about half of the double negative (CD4(-) CD8(-)) and CD8 single positive and the majority of CD4 single positive cells express Trojan with a relatively high intensity. However, only a minority of the double positive (CD4(+) CD8(+)) cells are positive for Trojan. This expression pattern, similar to that of some proteins with anti-apoptotic and function, like IL-7Ralpha, makes Trojan an attractive candidate of having an anti-apoptotic role. Copyright 2010 Elsevier Ltd. All rights reserved.

Exploring of the molecular mechanism of rhinitis via bioinformatics methods

PubMed Central

Song, Yufen; Yan, Zhaohui

2018-01-01

The aim of this study was to analyze gene expression profiles for exploring the function and regulatory network of differentially expressed genes (DEGs) in pathogenesis of rhinitis by a bioinformatics method. The gene expression profile of GSE43523 was downloaded from the Gene Expression Omnibus database. The dataset contained 7 seasonal allergic rhinitis samples and 5 non-allergic normal samples. DEGs between rhinitis samples and normal samples were identified via the limma package of R. The webGestal database was used to identify enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the DEGs. The differentially co-expressed pairs of the DEGs were identified via the DCGL package in R, and the differential co-expression network was constructed based on these pairs. A protein-protein interaction (PPI) network of the DEGs was constructed based on the Search Tool for the Retrieval of Interacting Genes database. A total of 263 DEGs were identified in rhinitis samples compared with normal samples, including 125 downregulated ones and 138 upregulated ones. The DEGs were enriched in 7 KEGG pathways. 308 differential co-expression gene pairs were obtained. A differential co-expression network was constructed, containing 212 nodes. In total, 148 PPI pairs of the DEGs were identified, and a PPI network was constructed based on these pairs. Bioinformatics methods could help us identify significant genes and pathways related to the pathogenesis of rhinitis. Steroid biosynthesis pathway and metabolic pathways might play important roles in the development of allergic rhinitis (AR). Genes such as CDC42 effector protein 5, solute carrier family 39 member A11 and PR/SET domain 10 might be also associated with the pathogenesis of AR, which provided references for the molecular mechanisms of AR. PMID:29257233

Identification of differentially expressed proteins of Arthrospira (Spirulina) plantensis-YZ under salt-stress conditions by proteomics and qRT-PCR analysis

PubMed Central

2013-01-01

Arthrospira (Spirulina) platensis as a representative species of cyanobacteria has been recognized and used worldwide as a source of protein in the food, which possesses some unusual and valuable physiological characteristics, such as alkali and salt tolerance. Based on complete genome sequencing of Arthrospira (Spirulina) plantensis-YZ, we compared the protein expression profiles of this organism under different salt-stress conditions (i.e. 0.02 M, 0.5 M and 1.0 M NaCl, respectively), using 2-D electrophoresis and peptide mass fingerprinting, and retrieved 141 proteins showing significantly differential expression in response to salt-stress. Of the 141 proteins, 114 Arthrospira (Spirulina) plantensis-YZ proteins were found with significant homology to those found in Arthrospira (76 proteins in Arthrospira platensis str. Paraca and 38 in Arthrospira maxima CS-328). The remaining 27 proteins belong to other bacteria. Subsequently, we determined the transcriptional level of 29 genes in vivo in response to NaCl treatments and verified them by qRT-PCR. We found that 12 genes keep consistency at both transcription and protein levels, and transcription of all of them but one were up-regulated. We classified the 141 differentially expressed proteins into 18 types of function categories using COG database, and linked them to their respective KEGG metabolism pathways. These proteins are involved in 31 metabolism pathways, such as photosynthesis, glucose metabolism, cysteine and methionine metabolism, lysine synthesis, fatty acid metabolism, glutathione metabolism. Additionally, the SRPs, heat shock protein and ABC transporter proteins were identified, which probably render Arthrospira (Spirulina) plantensis’s resistance against high salt stress. PMID:23363438

Cassava (Manihot esculenta Krantz) genome harbors KNOX genes differentially expressed during storage root development.

PubMed

Guo, D; Li, H L; Tang, X; Peng, S Q

2014-12-18

In plants, homeodomain proteins play a critical role in regulating various aspects of plant growth and development. KNOX proteins are members of the homeodomain protein family. The KNOX transcription factors have been reported from Arabidopsis, rice, and other higher plants. The recent publication of the draft genome sequence of cassava (Manihot esculenta Krantz) has allowed a genome-wide search for M. esculenta KNOX (MeKNOX) transcription factors and the comparison of these positively identified proteins with their homologs in model plants. In the present study, we identified 12 MeKNOX genes in the cassava genome and grouped them into two distinct subfamilies based on their domain composition and phylogenetic analysis. Furthermore, semi-quantitative reverse transcription polymerase chain reaction analysis was performed to elucidate the expression profiles of these genes in different tissues and during various stages of root development. The analysis of MeKNOX expression profiles of indicated that 12 MeKNOX genes display differential expressions either in their transcript abundance or expression patterns.

[Cloning and characterization of a novel rat gene RSD-7 differentially expressed in testis].

PubMed

Zhang, Xiao-dong; Gou, Da-wei; Miao, Shi-ying; Zhang, Jian-chao; Zong, Shu-dong; Wang, Lin-fang

2003-06-01

To isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis. Screening of the cDNA library, Northern blot, expression and purification in E. coli with GST expression system, immunocytochemical staining of testis sections were used. (1) A cDNA fragment designated as RSD-7 was isolated from rat testis cDNA library. It was 1,238 bp in length, coding a protein of 232 amino acids with the GenBank accession number AF315467. The encoding protein of RSD-7 cDNA had a Ubiquitin-like domain. (2) Northern blot indicated that RSD-7 was uniquely expressed in rat testis, and in the testis RSD-7 emerged on the 30th postnatal day and expressed until 120th postnatal day. (3) Expression and purification of RSD-7 protein in E. coli with GST expression system and were used to obtain anti-RSD-7 antibody. (4) Immunolocalization of RSD-7 in rat testis revealed that it is expressed only in Sertoli cells. Transcription pattern of RSD-7 and localization of RSD-7 protein in testis have been made, which established the base for the functional study of RSD-7.

Proteomic analysis of plasma proteins in diabetic retinopathy patients by two dimensional electrophoresis and MALDI-Tof-MS.

PubMed

Gopalakrishnan, Vidhya; Purushothaman, Parthiban; Bhaskar, Anusha

2015-01-01

Diabetic retinopathy is a highly specific vascular complication of diabetes mellitus and progresses from mild non-proliferative abnormalities characterized by increased vascular permeability to moderate and severe proliferative diabetic retinopathy characterized by the growth of blood vessels on the retina. The aim of the study was to identify the differentially expressed proteins in diabetic retinopathy using two-dimensional electrophoresis. Blood sample was drawn from subjects with diabetes mellitus (without retinopathy) who served as controls and patients with diabetic retinopathy in tubes containing EDTA as anticoagulant. Albumin and immunoglobulin IgG collectively removed to enrich proteins of lower abundance. 2de was carried out to see if there are any differentially expressed proteins. Approximately 48 and 61 spots were identified in control and diabetic retinopathy respectively, of which three protein spots RBP1 (retinol-binding protein 1), NUD10 (Diphosphoinositol polyphosphohydrolase 3 alpha), NGB (neuroglobin) were down regulated and HBG2 (hemoglobin) and BY55 (CD 160 antigen) were upregulated in diabetic retinopathy. These five protein spots were excised and were subjected to in-gel tryptic digestion, and their identities were determined by ultraflex MALDI-TOF-MS. We report a comprehensive patient-based plasma proteomic approach to the identification of potential biomarkers for diabetic retinopathy screening and detection. We identified 5 different proteins that were differentially expressed in the plasma of control diabetic patients (without retinopathy). Among these five proteins the expression of neuroglobin (NGB) protein varied significantly and may be a potential biomarker in diabetic retinopathy. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteomic changes in female rat hippocampus following exposure to a terrified sound stress.

PubMed

Yang, Juan; Hu, Lili; Song, Tusheng; Liu, Yong; Wu, Qiuhua; Zhao, Lingyu; Liu, Liying; Zhao, Xiaoge; Zhang, Dianzeng; Huang, Chen

2014-06-01

Stress plays a profound role in the onset of affective disorders, including an elevation in risk factors for depression and anxiety. Women are twice as vulnerable to stress as men because of greater sensitivity to a substance produced during times of anxiety. To better define the abnormal proteins implicated in cognitive deficits and other stress-induced dysfunction, female rats were exposed to terrified sound stress, and two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) were utilized to determine the differential protein expression in the hippocampus in sound-stressed female rats compared with controls. Quantitative differences were found in 44 protein spots which were differentially expressed between the stressed and control groups (fold change of >2; p < 0.01). Eighteen protein spots were downregulated, and 26 protein spots were upregulated in the stressed group. The seven most differentially expressed proteins were identified and validated as follows: dihydropyrimidinase-related protein 2 (DRP-2), creatine kinase B type, dynamin-1 protein, alpha-internexin, glial fibrillary acidic protein beta, gamma-enolase, and peptidyl-prolyl cis-trans isomerase A. Changes in protein levels were detected in the hippocampus of female rats subjected to terrified sound stress. The findings herein may open new opportunities for further investigations on the modulation induced in the hippocampus by stress at the molecular level, especially with respect to females stress.

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

PubMed

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

2016-01-01

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

Ucma--A novel secreted factor represents a highly specific marker for distal chondrocytes.

PubMed

Tagariello, Andreas; Luther, Julia; Streiter, Melanie; Didt-Koziel, Lydia; Wuelling, Manuela; Surmann-Schmitt, Cordula; Stock, Michael; Adam, Nadia; Vortkamp, Andrea; Winterpacht, Andreas

2008-01-01

Growth and development of most parts of the vertebrate skeleton takes place by endochondral ossification, a process during which chondrocytes undergo distinct stages of differentiation resulting in a successive replacement of the cartilage anlagen by bone. In the context of an EST project we isolated a novel transcript from a human fetal growth plate cartilage cDNA library. The transcript which we called Ucma (unique cartilage matrix-associated protein) encodes a short protein of 138 amino acids. The protein sequence is evolutionary conserved throughout vertebrates and comprises a signal peptide, a coiled-coil domain, and a putative dibasic cleavage site for proprotein convertases. Using RNA in situ hybridization and immunohistochemistry with a polyclonal anti-Ucma antibody we found high expression of Ucma uniquely in distal (resting) chondrocytes in developing long bones of wildtype mice. This restricted expression could also be observed in Ihh(-/-), Ihh(-/-); Gli3(-/-), Gli3(-/-) mice, and in mice that overexpress Ihh under the control of the Col2a1 promoter indicating that expression of Ucma is regulated independent of hedgehog signaling. During insulin-induced differentiation of ATDC5 cells we found gradual increase of Ucma expression at day 21 with a maximum at day 24 and a decrease correlating with a simultaneous increase in the expression of cartilage link protein (Crtl1), a protein with maximum expression in column-forming proliferating chondrocytes. The present data strongly suggest an important function of Ucma in the early phase of chondrocyte differentiation.

Megakaryocyte polyploidization is associated with decreased expression of polo-like kinase (PLK).

PubMed

Yagi, M; Roth, G J

2006-09-01

During differentiation, megakaryocytes (MK), the bone marrow precursors of circulating blood platelets, undergo polyploidization, repeated rounds of DNA replication without cell division. Mature normal MK may contain a DNA content of up to 128N, in contrast to normal diploid (2N) cells. The extent of polyploidy may influence the number of platelets produced by the MK. Therefore, understanding the molecular mechanisms regulating polyploidization could identify events involved in controlling both cell division and thrombopoiesis. We investigated the expression of several proteins involved in mitosis in cultured mouse MK, and tested the effect of expression on polyploidization. Western blot and immunofluorescent analyses were used to assess expression of cell cycle proteins in cultured MK. Populations of polyploidizing MK were separated on the basis of DNA content by flow cytometry. The gene encoding mouse polo-like kinase 1 (PLK-1) was introduced into MK by retroviral transduction, and its effects measured by flow cytometry. Polyploid mouse MK expressed lower levels of two proteins, p55CDC and PLK-1, whose activity is necessary for cell cycle progression and completion of mitosis. Comparison of sorted 2N/4N and polyploid MK indicated that PLK-1 expression was absent in polyploid MK, while expression of other cell cycle proteins was similar in both populations. Forced expression of PLK-1 during MK differentiation was associated with decreased polyploidization. These experiments suggest that PLK-1 is an important regulator of polyploidization in differentiating MK.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

PubMed

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration

PubMed Central

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds. PMID:21994173

Novel isoforms of Dlg are fundamental for neuronal development in Drosophila.

PubMed

Mendoza, Carolina; Olguín, Patricio; Lafferte, Gabriela; Thomas, Ulrich; Ebitsch, Susanne; Gundelfinger, Eckart D; Kukuljan, Manuel; Sierralta, Jimena

2003-03-15

Drosophila discs-large (dlg) mutants exhibit multiple developmental abnormalities, including severe defects in neuronal differentiation and synaptic structure and function. These defects have been ascribed to the loss of a single gene product, Dlg-A, a scaffold protein thought to be expressed in many cell types. Here, we describe that additional isoforms arise as a consequence of different transcription start points and alternative splicing of dlg. At least five different dlg gene products are predicted. We identified a subset of dlg-derived cDNAs that include novel exons encoding a peptide homologous to the N terminus of the mammalian protein SAP97/hDLG (S97N). Dlg isoforms containing the S97N domain are expressed at larval neuromuscular junctions and within the CNS of both embryos and larvae but are not detectable in epithelial tissues. Strong hypomorphic dlg alleles exhibit decreased expression of S97N, which may account for neural-specific aspects of the pleiomorphic dlg mutant phenotype. Selective inhibition of the expression of S97N-containing proteins in embryos by double-strand RNA leads to severe defects in neuronal differentiation and axon guidance, without overt perturbations in epithelia. These results indicate that the differential expression of dlg products correlates with distinct functions in non-neural and neural cells. During embryonic development, proteins that include the S97N domain are essential for proper neuronal differentiation and organization, acting through mechanisms that may include the adequate localization of cell fate determinants.

TULA-2, a novel histidine phosphatase regulates bone remodeling by modulating osteoclast function

PubMed Central

Back, Steven H.; Adapala, Naga Suresh; Barbe, Mary F.; Carpino, Nick C.; Tsygankov, Alexander Y.; Sanjay, Archana

2013-01-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The novel protein T-cell Ubiquitin Ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO Mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of Immune-receptor-Tyrosine-based-Activation-Motif (ITAM)–mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function. PMID:23149425

TULA-2, a novel histidine phosphatase, regulates bone remodeling by modulating osteoclast function.

PubMed

Back, Steven H; Adapala, Naga Suresh; Barbe, Mary F; Carpino, Nick C; Tsygankov, Alexander Y; Sanjay, Archana

2013-04-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The protein T cell ubiquitin ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members, only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation, suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of immune-receptor-tyrosine-based-activation-motif (ITAM)-mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function.

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase.

PubMed

Spencer, Michael L; Shao, Haipeng; Andres, Douglas A

2002-06-07

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.

[Adrenal protein expressions after Pinggan Qianyang Formula treatment in hypertensive rats with liver-yang hyperactivity: a comparative proteomic analysis].

PubMed

Zhang, Ying; Chen, Ze-qi; Zhong, Guang-wei

2008-07-01

To explore the pathogenic mechanism of liver-yang hyperactivity type of hypertension and to observe the effects of Pinggan Qianyang Formula (PGQYF), a compound of traditional Chinese herbals for calming the liver and suppressing yang, so as to provide experimental evidence for new marker proteins of drug therapy. A rat model of liver-yang hyperactivity was prepared with spontaneous hypertensive rats (SHRs) by administration of Aconiti Praeparatae Decoction. Adrenal proteins were separated by 2D gel electrophoresis (2-DE). The differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database analysis. The rat model of liver-yang hyperactivity was successfully reproduced, and the PGQYF could decrease the grades of irritability, conjunctival congestion and systolic blood pressure of the rats (P<0.05, P<0.01). After analysis, twelve obviously differentially expressed proteins were found, eight of which were identified. The expression levels of isocitrate dehydrogenase and steroidogenic acute regulatory protein in the untreated group were up-regulated as compared with those in the normal control group, and down-regulated in the treatment group. The expression levels of ferritin light chain, elongation factor Tu, Rho GDP disassociation inhibitor 1, flavin reductase and basic transcription factor 3 in the untreated group were down-regulated as compared with those in the normal control group, and up-regulated in the treatment group. Differentially expressed adrenal proteins in SHRs with live-yang hyperactivity are successfully identified. This approach may lay a foundation for the further investigation of pathogenic mechanisms in hypertension with liver-yang hyperactivity and the mechanisms of PGQYF treatment.

Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis

PubMed Central

Au, Catherine E.; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Simon, Paul H. G.; Kearney, Robert E.; Cameron, Pamela H.; Smith, Charles E.; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Ma, Kewei; Nilsson, Tommy; Bergeron, John J. M.

2015-01-01

The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell–specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation. PMID:25808494

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype.

PubMed

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

2017-03-01

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4 + T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Exploring the Therapeutic Mechanism of Desmodium styracifolium on Oxalate Crystal-Induced Kidney Injuries Using Comprehensive Approaches Based on Proteomics and Network Pharmacology.

PubMed

Hou, Jiebin; Chen, Wei; Lu, Hongtao; Zhao, Hongxia; Gao, Songyan; Liu, Wenrui; Dong, Xin; Guo, Zhiyong

2018-01-01

Purpose: As a Chinese medicinal herb, Desmodium styracifolium (Osb.) Merr (DS) has been applied clinically to alleviate crystal-induced kidney injuries, but its effective components and their specific mechanisms still need further exploration. This research first combined the methods of network pharmacology and proteomics to explore the therapeutic protein targets of DS on oxalate crystal-induced kidney injuries to provide a reference for relevant clinical use. Methods: Oxalate-induced kidney injury mouse, rat, and HK-2 cell models were established. Proteins differentially expressed between the oxalate and control groups were respectively screened using iTRAQ combined with MALDI-TOF-MS. The common differential proteins of the three models were further analyzed by molecular docking with DS compounds to acquire differential targets. The inverse docking targets of DS were predicted through the platform of PharmMapper. The protein-protein interaction (PPI) relationship between the inverse docking targets and the differential proteins was established by STRING. Potential targets were further validated by western blot based on a mouse model with DS treatment. The effects of constituent compounds, including luteolin, apigenin, and genistein, were investigated based on an oxalate-stimulated HK-2 cell model. Results: Thirty-six common differentially expressed proteins were identified by proteomic analysis. According to previous research, the 3D structures of 15 major constituents of DS were acquired. Nineteen differential targets, including cathepsin D (CTSD), were found using molecular docking, and the component-differential target network was established. Inverse-docking targets including p38 MAPK and CDK-2 were found, and the network of component-reverse docking target was established. Through PPI analysis, 17 inverse-docking targets were linked to differential proteins. The combined network of component-inverse docking target-differential proteins was then constructed. The expressions of CTSD, p-p38 MAPK, and p-CDK-2 were shown to be increased in the oxalate group and decreased in kidney tissue by the DS treatment. Luteolin, apigenin, and genistein could protect oxalate-stimulated tubular cells as active components of DS. Conclusion: The potential targets including the CTSD, p38 MAPK, and CDK2 of DS in oxalate-induced kidney injuries and the active components (luteolin, apigenin, and genistein) of DS were successfully identified in this study by combining proteomics analysis, network pharmacology prediction, and experimental validation.

Systematic Analysis of Cell-Type Differences in the Epithelial Secretome Reveals Insights into the Pathogenesis of RSV-Induced Lower Respiratory Tract Infections

PubMed Central

Zhao, Yingxin; Jamaluddin, Mohammad; Zhang, Yueqing; Sun, Hong; Ivanciuc, Teodora; Garofalo, Roberto P.; Brasier, Allan R.

2017-01-01

Lower respiratory tract infections (LRTIs) from Respiratory Syncytial Virus (RSV) are due, in part, to secreted signals from lower airway cells that modify immune response and trigger airway remodeling. To understand this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea (hBECs) vs small airway bronchiolar cells (hSAECs). A workflow was established using telomerase- immortalized human epithelial cells that revealed highly reproducible cell type-specific differences in both secreted proteins and nanoparticles (exosomes). Approximately one-third of secretome proteins are exosomal, with the remainder from lysosomal and vacuolar compartments. We applied this workflow to three independently derived primary human cultures from trachea (phBECs) vs bronchioles (phSAECs). 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate (FDR). Fifteen proteins unique to RSV-infected phBECs were regulated by epithelial-specific ets homology factor (EHF). 106 proteins unique to RSV-infected hSAECs were regulated by the transcription factor NFκB. In this latter group, we validated the differential expression of Chemokine (C-C Motif) Ligand 20 (CCL20)/macrophage-inducible protein (MIP)3α, thymic stromal lymphopoietin (TSLP) and chemokine (CC) ligand 3-like 1(CCL3-L1) because of their roles in Th2 polarization. CCL20/MIP3α was the most active mucin-inducing factor in the RSV-infected hSAEC secretome, and was differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insights into the complexity of innate responses, and regional differences in epithelial secretome participating in RSV LRTI-induced airway remodeling. PMID:28258195

In vitro-microenvironment directs preconditioning of human chorion derived MSC promoting differentiation of OPC-like cells.

PubMed

Periasamy, Ramesh; Surbek, Daniel V; Schoeberlein, Andreina

2018-06-01

The loss of oligodendrocyte progenitor cells (OPC) is a hallmark of perinatal brain injury. Our aim was to develop an in vitro culture condition for human chorion-derived mesenchymal stem cells (MSC) that enhances their stem cell properties and their capability to differentiate towards OPC-like cells. MSC were grown either in serum replacement medium (SRM) or serum-containing medium (SM) and tested for their morphology, proliferation, secretome, migration, protein expression and differentiation into OPC-like cells. MSC cultured in SRM condition have distinct morphology/protein expression profile, increased cell proliferation/migration and capacity to differentiate into OPC-like cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

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

Cambier, Linda; Pomies, Pascal, E-mail: pascal.pomies@crbm.cnrs.fr

2011-06-17

Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletalmore » muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.« less

Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome.

PubMed

Judelson, Howard S; Ah-Fong, Audrey M V; Aux, George; Avrova, Anna O; Bruce, Catherine; Cakir, Cahid; da Cunha, Luis; Grenville-Briggs, Laura; Latijnhouwers, Maita; Ligterink, Wilco; Meijer, Harold J G; Roberts, Samuel; Thurber, Carrie S; Whisson, Stephen C; Birch, Paul R J; Govers, Francine; Kamoun, Sophien; van West, Pieter; Windass, John

2008-04-01

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately (1)/(10) being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

PubMed Central

Gaines, Peter; Lamoureux, James; Marisetty, Anantha; Chi, Jeffrey; Berliner, Nancy

2008-01-01

Objective The function of neutrophils as primary mediators of innate immunity depends on the activity of granule proteins and critical components of the NADPH oxidase complex. Expression of their cognate genes is regulated during neutrophil differentiation by a complex network of intracellular signaling pathways. In this study we have investigated the role of two members of the calcium/calmodulin-dependent protein kinase (CaMK) signaling cascade, CaMKI-like kinase (CKLiK) and CaMKKα, in regulating neutrophil differentiation and functional activation. Materials and Methods Mouse myeloid cell lines were used to examine the expression of a CaMK cascade in developing neutrophils and to examine the effects of constitutive activation versus inhibition of CaMKs on neutrophil maturation. Results Expression of CaMKKα was shown to increase during neutrophil differentiation in multiple cell lines, whereas expression of CKLiK increased as multipotent progenitors committed to promyelocytes but then decreased as cells differentiated into mature neutrophils. Expression of constitutively active CKLiKs did not affect morphologic maturation, but caused dramatic decreases in both respiratory burst responses and chemotaxis. This loss of neutrophil function was accompanied by reduced secondary granule and gp91phox gene expression. The CaMK inhibitor KN93 attenuated cytokine-stimulated proliferative responses in promyelocytic cell lines, and inhibited the respiratory burst. Similar data were observed with the CaMKKα inhibitor, STO-609. Conclusions Overactivation of a cascade of CaMKs inhibits neutrophil maturation, suggesting that these kinases play an antagonistic role during neutrophil differentiation, but at least one CaMK is required for myeloid cell expansion and functional activation. PMID:18400360

A Comprehensive Analysis of Chromoplast Differentiation Reveals Complex Protein Changes Associated with Plastoglobule Biogenesis and Remodeling of Protein Systems in Sweet Orange Flesh1[OPEN

PubMed Central

Wang, Lun; Deng, Xiuxin

2015-01-01

Globular and crystalloid chromoplasts were observed to be region specifically formed in sweet orange (Citrus sinensis) flesh and converted from amyloplasts during fruit maturation, which was associated with the composition of specific carotenoids and the expression of carotenogenic genes. Subsequent isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analyses of purified plastids from the flesh during chromoplast differentiation and senescence identified 1,386 putative plastid-localized proteins, 1,016 of which were quantified by spectral counting. The iTRAQ values reflecting the expression abundance of three identified proteins were validated by immunoblotting. Based on iTRAQ data, chromoplastogenesis appeared to be associated with three major protein expression patterns: (1) marked decrease in abundance of the proteins participating in the translation machinery through ribosome assembly; (2) increase in abundance of the proteins involved in terpenoid biosynthesis (including carotenoids), stress responses (redox, ascorbate, and glutathione), and development; and (3) maintenance of the proteins for signaling and DNA and RNA. Interestingly, a strong increase in abundance of several plastoglobule-localized proteins coincided with the formation of plastoglobules in the chromoplast. The proteomic data also showed that stable functioning of protein import, suppression of ribosome assembly, and accumulation of chromoplast proteases are correlated with the amyloplast-to-chromoplast transition; thus, these processes may play a collective role in chromoplast biogenesis and differentiation. By contrast, the chromoplast senescence process was inferred to be associated with significant increases in stress response and energy supply. In conclusion, this comprehensive proteomic study identified many potentially new plastid-localized proteins and provides insights into the potential developmental and molecular mechanisms underlying chromoplast biogenesis, differentiation, and senescence in sweet orange flesh. PMID:26056088

Changes in inflorescence protein during advanced stages of floret development in Buchloe dactyloides (Poaceae).

PubMed

Zhou, Y-J; Xue, J-G; Wang, X-G; Zhang, X-Q

2012-11-12

Buffalograss, Buchloe dactyloides, is a dioecious species native to the Great Plains of North America. The florets at the early stages of development possess both gynoecium and androecium organ primordia but later become unisexual. Very little is known about the proteomic changes that occur when the florets change from hermaphroditism to unisexuality. We compared the protein composition of florets at the hermaphroditic stage with that at the unisexual stage. The development stage of the floret was determined by stereomicroscopic observation. Two-dimensional gel electrophoresis was used to separate the proteins extracted from female and male inflorescences. Stage- specific protein maps, with an average of about 400 spots per map, were analyzed with the protein analysis software. Eighteen spots were found to be differentially expressed between the hermaphrodite and unisexual stages. Of these, 12 were present at both stages but with a different expression value. Four specific spots appeared at the hermaphrodite stage and disappeared at the unisexual stage. Two specific protein spots were associated with female and male floret differentiation. One appears to be associated with contabescence in the female floret and the final protein appears to lead to the abortion of gynoecium in the male floret. The MALDI TOF/TOF technique was used for peptide mass fingerprinting of the differentially expressed proteins and the MASCOT software was used to search the protein database. However, only two protein spots were identified from the database. These were aldolase1 and Os05g0574400 (similar to malate dehydrogenase). This type of proteomic study can help to identify novel protein products and determine the mechanisms involved in the floral sex differentiation process in buffalo grass.

Differential expression of homeobox-containing genes Msx-1 and Msx-2 and homeoprotein Msx-2 expression during chick craniofacial development.

PubMed

Nishikawa, K; Nakanishi, T; Aoki, C; Hattori, T; Takahashi, K; Taniguchi, S

1994-03-01

The expression pattern of chick Msx-1 and Msx-2 homeobox genes in craniofacial primordia was examined by in situ hybridization using cRNA probes. Both genes were expressed in the distal region of the facial primordia, where the distribution of Msx-2 expression was restricted distally within the Msx-1 expression domain. On the contrary, Msx-2 expression in the lateral choroid plexus and cranial skull was broader and more intensive than Msx-1 expression. Our findings suggest that these two genes cooperate to play differential roles in craniofacial development. Msx-2 protein was detected immunohistochemically, and its localization essentially corresponded to the mRNA expression pattern, substantiating the involvement of Msx-2 protein as a transcriptional regulator in developing limb and face.

Low-dose/dose-rate γ radiation depresses neural differentiation and alters protein expression profiles in neuroblastoma SH-SY5Y cells and C17.2 neural stem cells.

PubMed

Bajinskis, Ainars; Lindegren, Heléne; Johansson, Lotta; Harms-Ringdahl, Mats; Forsby, Anna

2011-02-01

The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose γ-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs γ rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate γ rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism.

A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization.

PubMed

Américo-Da-Silva, Luan; Diaz, Jheimmy; Bustamante, Mario; Mancilla, Georthan; Oyarzún, Ingrid; Verdejo, Hugo E; Quiroga, Clara

2018-03-23

Bone integrity depends on a finely tuned balance between bone synthesis by osteoblasts and resorption by osteoclasts. The secretion capacity of mature osteoblasts requires strict control of proteostasis. Endoplasmic reticulum-associated degradation (ERAD) prevents the accumulation of unfolded ER proteins via dislocation to the cytosol and degradation by the proteasome. The ER membrane protein, homocysteine-inducible endoplasmic reticulum protein with ubiquitin-like domain 1 (HERPUD1), is a key component of the ERAD multiprotein complex which helps to stabilize the complex and facilitate the efficient degradation of unfolded proteins. HERPUD1 expression is strongly up-regulated by the unfolded protein response and cellular stress. The aim of the current study was to establish whether HERPUD1 and ERAD play roles in osteoblast differentiation and maturation. We evaluated preosteoblastic MC3T3-E1 cell and primary rat osteoblast differentiation by measuring calcium deposit levels, alkaline phosphatase activity, and runt-related transcription factor 2 and osterix expression. We found that ERAD and proteasomal degradation were activated and that HERPUD1 expression was increased as osteoblast differentiation progressed. The absence of HERPUD1 blocked osteoblast mineralization in vitro and significantly reduced alkaline phosphatase activity. In contrast, HERPUD1 overexpression activated the osteoblast differentiation program. Our results demonstrate that HERPUD1 and ERAD are important for the activation of the osteoblast maturation program and may be useful new targets for elucidating bone physiology.-Américo-Da-Silva, L., Diaz, J., Bustamante, M., Mancilla, G., Oyarzún, I., Verdejo, H. E., Quiroga, C. A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization.

Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

PubMed

Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

2013-01-14

The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

New insights into the mechanisms of acetic acid resistance in Acetobacter pasteurianus using iTRAQ-dependent quantitative proteomic analysis.

PubMed

Xia, Kai; Zang, Ning; Zhang, Junmei; Zhang, Hong; Li, Yudong; Liu, Ye; Feng, Wei; Liang, Xinle

2016-12-05

Acetobacter pasteurianus is the main starter in rice vinegar manufacturing due to its remarkable abilities to resist and produce acetic acid. Although several mechanisms of acetic acid resistance have been proposed and only a few effector proteins have been identified, a comprehensive depiction of the biological processes involved in acetic acid resistance is needed. In this study, iTRAQ-based quantitative proteomic analysis was adopted to investigate the whole proteome of different acidic titers (3.6, 7.1 and 9.3%, w/v) of Acetobacter pasteurianus Ab3 during the vinegar fermentation process. Consequently, 1386 proteins, including 318 differentially expressed proteins (p<0.05), were identified. Compared to that in the low titer circumstance, cells conducted distinct biological processes under high acetic acid stress, where >150 proteins were differentially expressed. Specifically, proteins involved in amino acid metabolic processes and fatty acid biosynthesis were differentially expressed, which may contribute to the acetic acid resistance of Acetobacter. Transcription factors, two component systems and toxin-antitoxin systems were implicated in the modulatory network at multiple levels. In addition, the identification of proteins involved in redox homeostasis, protein metabolism, and the cell envelope suggested that the whole cellular system is mobilized in response to acid stress. These findings provide a differential proteomic profile of acetic acid resistance in Acetobacter pasteurianus and have potential application to highly acidic rice vinegar manufacturing. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of Gene Expression Signatures in the Chicken Intestinal Intraepithelial Lymphocytes in Response to Herb Additive Supplementations

PubMed Central

Won, Kyeong-Hye; Song, Ki-Duk; Park, Jong-Eun; Kim, Duk-Kyung; Na, Chong-Sam

2016-01-01

Anethole and garlic have an immune modulatory effects on avian coccidiosis, and these effects are correlated with gene expression changes in intestinal epithelial lymphocytes (IELs). In this study, we integrated gene expression datasets from two independent experiments and investigated gene expression profile changes by anethole and garlic respectively, and identified gene expression signatures, which are common targets of these herbs as they might be used for the evaluation of the effect of plant herbs on immunity toward avian coccidiosis. We identified 4,382 and 371 genes, which were differentially expressed in IELs of chickens supplemented with garlic and anethole respectively. The gene ontology (GO) term of differentially expressed genes (DEGs) from garlic treatment resulted in the biological processes (BPs) related to proteolysis, e.g., “modification-dependent protein catabolic process”, “proteolysis involved in cellular protein catabolic process”, “cellular protein catabolic process”, “protein catabolic process”, and “ubiquitin-dependent protein catabolic process”. In GO analysis, one BP term, “Proteolysis”, was obtained. Among DEGs, 300 genes were differentially regulated in response to both garlic and anethole, and 234 and 59 genes were either up- or down-regulated in supplementation with both herbs. Pathway analysis resulted in enrichment of the pathways related to digestion such as “Starch and sucrose metabolism” and “Insulin signaling pathway”. Taken together, the results obtained in the present study could contribute to the effective development of evaluation system of plant herbs based on molecular signatures related with their immunological functions in chicken IELs. PMID:26954117

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

PubMed

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

1994-09-01

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

Differentiation of embryonic stem cells into hepatocytes that coexpress coagulation factors VIII and IX.

PubMed

Cao, Jun; Shang, Chang-zhen; Lü, Li-hong; Qiu, De-chuan; Ren, Meng; Chen, Ya-jin; Min, Jun

2010-11-01

To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX. Mouse E14 ES cells were cultured in differentiation medium containing sodium butyrate (SB), basic fibroblast growth factor (bFGF), and/or bone morphogenetic protein 4 (BMP4) to induce the differentiation of endoderm cells and hepatic progenitor cells. Hepatocyte growth factor, oncostatin M, and dexamethasone were then used to induce the maturation of ES cell-derived hepatocytes. The mRNA expression levels of endoderm-specific genes and hepatocyte-specific genes, including the levels of F-VIII and F-IX, were detected by RT-PCR and real-time PCR during various stages of differentiation. Protein expression was examined by immunofluorescence and Western blot. At the final stage of differentiation, flow cytometry was performed to determine the percentage of cells coexpressing F-VIII and F-IX, and ELISA was used to detect the levels of F-VIII and F-IX protein secreted into the culture medium. The expression of endoderm-specific and hepatocyte-specific markers was upregulated to highest level in response to the combination of SB, bFGF, and BMP4. Treatment with the three inducers during hepatic progenitor differentiation significantly enhanced the mRNA and protein levels of F-VIII and F-IX in ES cell-derived hepatocytes. More importantly, F-VIII and F-IX were coexpressed with high efficiency at the final stage of differentiation, and they were also secreted into the culture medium. We have established a novel in vitro differentiation protocol for ES-derived hepatocytes that coexpress F-VIII and F-IX that may provide a foundation for stem cell replacement therapy for hemophilia.

Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation.

PubMed

Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

2016-07-07

Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes.

Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

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

Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi

2014-03-15

Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction ofmore » A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.« less

Proteomics of inflammatory and oxidative stress response in cows with subclinical and clinical mastitis.

PubMed

Turk, Romana; Piras, Cristian; Kovačić, Mislav; Samardžija, Marko; Ahmed, Hany; De Canio, Michele; Urbani, Andrea; Meštrić, Zlata Flegar; Soggiu, Alessio; Bonizzi, Luigi; Roncada, Paola

2012-07-19

Cow serum proteome was evaluated by three different complementary approaches in the control group, subclinical and clinical mastitis in order to possibly find differential protein expression useful for a better understanding of the pathophysiology of mastitis as well as for an early diagnosis of the disease. The systemic inflammatory and oxidative stress response in cows with subclinical and clinical mastitis were observed. The collected evidence shows a differential protein expression of serpin A3-1, vitronectin-like protein and complement factor H in subclinical mastitis in comparison with the control. It was also found a differential protein expression of inter-alpha-trypsin inhibitor heavy chain H4, serpin A3-1, C4b-binding protein alpha chain, haptoglobin and apolipoprotein A-I in clinical mastitis compared to the control. Among the inflammatory proteins up-regulated in clinical mastitis, vitronectin is over-expressed in both subclinical and clinical mastitis indicating a strong bacterial infection. This suggests vitronectin as an important mediator in the pathogenesis of the onset of mastitis as well as a valuable marker for diagnosis of the subclinical form of the disease. Obtained data could be useful for the detection of mastitis during the subclinical phase and for a better comprehension of the pathophysiological mechanisms involved in the onset of the disease. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparative proteomic analysis of differentially expressed proteins between peripheral sensory and motor nerves.

PubMed

He, Qianru; Man, Lili; Ji, Yuhua; Zhang, Shuqiang; Jiang, Maorong; Ding, Fei; Gu, Xiaosong

2012-06-01

Peripheral sensory and motor nerves have different functions and different approaches to regeneration, especially their distinct ability to accurately reinervate terminal nerve pathways. To understand the molecular aspects underlying these differences, the proteomics technique by coupling isobaric tags for relative and absolute quantitation (iTRAQ) with online two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) was used to investigate the protein profile of sensory and motor nerve samples from rats. A total of 1472 proteins were identified in either sensory or motor nerve. Of them, 100 proteins showed differential expressions between both nerves, and some of them were validated by quantitative real time RT-PCR, Western blot analysis, and immunohistochemistry. In the light of functional categorization, the differentially expressed proteins in sensory and motor nerves, belonging to a broad range of classes, were related to a diverse array of biological functions, which included cell adhesion, cytoskeleton, neuronal plasticity, neurotrophic activity, calcium-binding, signal transduction, transport, enzyme catalysis, lipid metabolism, DNA-binding, synaptosome function, actin-binding, ATP-binding, extracellular matrix, and commitment to other lineages. The relatively higher expressed proteins in either sensory or motor nerve were tentatively discussed in combination with their specific molecular characteristics. It is anticipated that the database generated in this study will provide a solid foundation for further comprehensive investigation of functional differences between sensory and motor nerves, including the specificity of their regeneration.

The HMX/NKX homeodomain protein MLS-2 specifies the identity of the AWC sensory neuron type via regulation of the ceh-36 Otx gene in C. elegans

PubMed Central

Kim, Kyuhyung; Kim, Rinho; Sengupta, Piali

2010-01-01

The differentiated features of postmitotic neurons are dictated by the expression of specific transcription factors. The mechanisms by which the precise spatiotemporal expression patterns of these factors are regulated are poorly understood. In C. elegans, the ceh-36 Otx homeobox gene is expressed in the AWC sensory neurons throughout postembryonic development, and regulates terminal differentiation of this neuronal subtype. Here, we show that the HMX/NKX homeodomain protein MLS-2 regulates ceh-36 expression specifically in the AWC neurons. Consequently, the AWC neurons fail to express neuron type-specific characteristics in mls-2 mutants. mls-2 is expressed transiently in postmitotic AWC neurons, and directly initiates ceh-36 expression. CEH-36 subsequently interacts with a distinct site in its cis-regulatory sequences to maintain its own expression, and also directly regulates the expression of AWC-specific terminal differentiation genes. We also show that MLS-2 acts in additional neuron types to regulate their development and differentiation. Our analysis describes a transcription factor cascade that defines the unique postmitotic characteristics of a sensory neuron subtype, and provides insights into the spatiotemporal regulatory mechanisms that generate functional diversity in the sensory nervous system. PMID:20150279

Microarray Meta-Analysis of RNA-Binding Protein Functions in Alternative Polyadenylation

PubMed Central

Hu, Wenchao; Liu, Yuting; Yan, Jun

2014-01-01

Alternative polyadenylation (APA) is a post-transcriptional mechanism to generate diverse mRNA transcripts with different 3′UTRs from the same gene. In this study, we systematically searched for the APA events with differential expression in public mouse microarray data. Hundreds of genes with over-represented differential APA events and the corresponding experiments were identified. We further revealed that global APA differential expression occurred prevalently in tissues such as brain comparing to peripheral tissues, and biological processes such as development, differentiation and immune responses. Interestingly, we also observed widespread differential APA events in RNA-binding protein (RBP) genes such as Rbm3, Eif4e2 and Elavl1. Given the fact that RBPs are considered as the main regulators of differential APA expression, we constructed a co-expression network between APAs and RBPs using the microarray data. Further incorporation of CLIP-seq data of selected RBPs showed that Nova2 represses and Mbnl1 promotes the polyadenylation of closest poly(A) sites respectively. Altogether, our study is the first microarray meta-analysis in a mammal on the regulation of APA by RBPs that integrated massive mRNA expression data under a wide-range of biological conditions. Finally, we present our results as a comprehensive resource in an online website for the research community. PMID:24622240

Developmental Considerations of Sperm Protein 17 Gene Expression in Rheumatoid Arthritis Synoviocytes

PubMed Central

Takeoka, Yuichi; Kenny, Thomas P.; Yago, Hisashi; Naiki, Mitsuru; Gershwin, M. Eric; Robbins, Dick L.

2002-01-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by proliferative synovial tissue. We used mRNA differential display and library subtraction to compare mRNA expression in RA and osteoarthritis (OA) synoviocytes. We initially compared the mRNA expression patterns in 1 female RA and 1 OA synovia and found a differentially expressed 350 bp transcript in the RA synoviocytes which was, by sequence analysis, 100% homologous to sperm protein 17 (Sp17). Moreover, the Sp17 transcript was found differentially expressed in a RA synovial library that was subtracted with an OA synovial library. Using specific primers for full length Sp17, a 1.1 kb transcript was amplified from the synoviocytes of 7 additional female RA patients, sequenced and found to 100% homologous to Sp17. Thus, we found the unexpected expression of Sp17, a thought to be gamete-specific protein, in the synoviocytes of 8/8 female RA patients in contrast to control OA synoviocytes. Interestingly, Sp17's structural relationship with cell-binding and recognition proteins, suggests that Sp17 may function in cell-cell recognition and signaling in the RA synoviocyte. Further, Sp17 could have a significant regulatory role in RA synoviocyte gene transcription and/or signal transduction. Thus, Sp17 could have an important role in RA synoviocyte proliferation or defective apoptosis. Finally, the presence of Sp17 in synoviocytes has interesting developmental considerations. PMID:12739786

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

PubMed

Han, Qianqian; Yang, Pishan; Wu, Yuwei; Meng, Shu; Sui, Lei; Zhang, Lan; Yu, Liming; Tang, Yin; Jiang, Hua; Xuan, Dongying; Kaplan, David L; Kim, Sung Hoon; Tu, Qisheng; Chen, Jake

2015-08-01

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds.

Identification of Proteins Modulated in the Date Palm Stem Infested with Red Palm Weevil (Rhynchophorus ferrugineus Oliv.) Using Two Dimensional Differential Gel Electrophoresis and Mass Spectrometry

PubMed Central

Rasool, Khawaja Ghulam; Khan, Muhammad Altaf; Aldawood, Abdulrahman Saad; Tufail, Muhammad; Mukhtar, Muhammad; Takeda, Makio

2015-01-01

A state of the art proteomic methodology using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI TOF) has been employed to characterize peptides modulated in the date palm stem subsequent to infestation with red palm weevil (RPW). Our analyses revealed 32 differentially expressed peptides associated with RPW infestation in date palm stem. To identify RPW infestation associated peptides (I), artificially wounded plants (W) were used as additional control beside uninfested plants, a conventional control (C). A constant unique pattern of differential expression in infested (I), wounded (W) stem samples compared to control (C) was observed. The upregulated proteins showed relative fold intensity in order of I > W and downregulated spots trend as W > I, a quite interesting pattern. This study also reveals that artificially wounding of date palm stem affects almost the same proteins as infestation; however, relative intensity is quite lower than in infested samples both in up and downregulated spots. All 32 differentially expressed spots were subjected to MALDI-TOF analysis for their identification and we were able to match 21 proteins in the already existing databases. Relatively significant modulated expression pattern of a number of peptides in infested plants predicts the possibility of developing a quick and reliable molecular methodology for detecting plants infested with date palm. PMID:26287180

Identification of Proteins Modulated in the Date Palm Stem Infested with Red Palm Weevil (Rhynchophorus ferrugineus Oliv.) Using Two Dimensional Differential Gel Electrophoresis and Mass Spectrometry.

PubMed

Rasool, Khawaja Ghulam; Khan, Muhammad Altaf; Aldawood, Abdulrahman Saad; Tufail, Muhammad; Mukhtar, Muhammad; Takeda, Makio

2015-08-17

A state of the art proteomic methodology using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI TOF) has been employed to characterize peptides modulated in the date palm stem subsequent to infestation with red palm weevil (RPW). Our analyses revealed 32 differentially expressed peptides associated with RPW infestation in date palm stem. To identify RPW infestation associated peptides (I), artificially wounded plants (W) were used as additional control beside uninfested plants, a conventional control (C). A constant unique pattern of differential expression in infested (I), wounded (W) stem samples compared to control (C) was observed. The upregulated proteins showed relative fold intensity in order of I > W and downregulated spots trend as W > I, a quite interesting pattern. This study also reveals that artificially wounding of date palm stem affects almost the same proteins as infestation; however, relative intensity is quite lower than in infested samples both in up and downregulated spots. All 32 differentially expressed spots were subjected to MALDI-TOF analysis for their identification and we were able to match 21 proteins in the already existing databases. Relatively significant modulated expression pattern of a number of peptides in infested plants predicts the possibility of developing a quick and reliable molecular methodology for detecting plants infested with date palm.

Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus.

PubMed

Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

2016-06-09

Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH.

Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus

PubMed Central

Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

2016-01-01

Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH. PMID:27277140

Neuroblastoma differentiation involves the expression of two isoforms of the alpha-subunit of Go.

PubMed

Brabet, P; Pantaloni, C; Rodriguez, M; Martinez, J; Bockaert, J; Homburger, V

1990-04-01

The regulation of GTP-binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E-115 cells. N1E-115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to alpha-subunits (G alpha) of Go (a G protein of unknown function) and "Gi (a G protein inhibitory to adenylate cyclase)-like" proteins and one substrate of Vibrio cholerae toxin corresponding to an alpha-subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Go alpha was found, having a pI of 5.8 Go alpha content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Go alpha form having a pI of 5.55. Both Go alpha isoforms have similar sizes on sodium dodecyl sulfate-polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Go alpha, are ADP-ribosylated by PTX, and are covalently myristylated in whole N1E-115 cells. In addition, immunofluorescent staining of N1E-115 cells with Go alpha antibodies revealed that association of Go alpha with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Gi alpha and Gs alpha did not significantly change, whereas that of the common beta-subunit increased by approximately 30% over the same period. These results demonstrate specific regulation of the expression of Go alpha during neuronal differentiation.

Differential Expression of Ccn4 and Other Genes Between Metastatic and Non-metastatic EL4 Mouse Lymphoma Cells

PubMed Central

S. CHAHAL, MANPREET; TERESA KU, H.; ZHANG, ZHIHONG; M. LEGASPI, CHRISTIAN; LUO, ANGELA; M. HOPKINS, MANDI; E. MEIER, KATHRYN

2016-01-01

Background: Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. Materials and Methods: Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. Results: Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). Conclusion: The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells. PMID:27807066

Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

PubMed Central

Pérez-Pérez, Rafael; López, Juan A.; García-Santos, Eva; Camafeita, Emilio; Gómez-Serrano, María; Ortega-Delgado, Francisco J.; Ricart, Wifredo; Fernández-Real, José M.; Peral, Belén

2012-01-01

Background Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. Methodology and Findings To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. Conclusions ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study. PMID:22272336

Differential expressed protein in developing stages of Nepenthes gracilis Korth. pitcher.

PubMed

Pinthong, Krit; Chaveerach, Arunrat; Tanee, Tawatchai; Sudmoon, Runglawan; Mokkamul, Piya

2009-03-15

Nepenthes gracilis Korth. is a member of carnivorous plants in family Nepenthaceae. The plants have beautiful and economically important pitchers. It is interesting to study the protein(s) correlated with the pitcher. Crude proteins were extracted from leaf, leaf with developing pitcher and developed pitcher of the same plant and analyzed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Two protein bands with molecular weights of 42.7 and 38 kDa were obtained from young leaf and leaf with developing pitcher, respectively. The 42.7 kDa protein was identified as phosphoglycerate kinase (PGK) by Liquid Chromatography Mass Spectrometry (LC-MS/MS), but the 38 kDa band is an unknown protein. Both proteins were differentially expressed in each developing stage of the pitcher, thus may be powerful candidates play role in development pathway of leaf and pitcher.

iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis.

PubMed

Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

2016-01-01

Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Expression analyses of metabolism-related protein groups belonging to different functional categories and subcategories indicated that significantly upregulated proteins were related to flavonoid and starch synthesis. On the other hand, the downregulated proteins were determined to be related to nitrogen metabolism, as well as other functional categories and subcategories, including photosynthesis, redox homeostasis, tocopherol biosynthetic, and signal transduction. The results provide valuable new insights into the characterization and understanding of ACN pigment production in black rice.

Comparative transcriptome analysis to investigate the potential role of miRNAs in milk protein/fat quality.

PubMed

Wang, Xuehui; Zhang, Li; Jin, Jing; Xia, Anting; Wang, Chunmei; Cui, Yingjun; Qu, Bo; Li, Qingzhang; Sheng, Chunyan

2018-04-19

miRNAs play an important role in the processes of cell differentiation, biological development, and physiology. Here we investigated the molecular mechanisms regulating milk secretion and quality in dairy cows via transcriptome analyses of mammary gland tissues from dairy cows during the high-protein/high-fat, low-protein/low-fat or dry periods. To characterize the important roles of miRNAs and mRNAs in milk quality and to elucidate their regulatory networks in relation to milk secretion and quality, an integrated analysis was performed. A total of 25 core miRNAs were found to be differentially expressed (DE) during lactation compared to non-lactation, and these miRNAs were involved in epithelial cell terminal differentiation and mammary gland development. In addition, comprehensive analysis of mRNA and miRNA expression between high-protein/high-fat group and low-protein/low-fat groups indicated that, 38 miRNAs and 944 mRNAs were differentially expressed between them. Furthermore, 38 DE miRNAs putatively negatively regulated 253 DE mRNAs. The putative genes (253 DE mRNAs) were enriched in lipid biosynthetic process and amino acid transmembrane transporter activity. Moreover, putative DE genes were significantly enriched in fatty acid (FA) metabolism, biosynthesis of amino acids, synthesis and degradation of ketone bodies and biosynthesis of unsaturated FAs. Our results suggest that DE miRNAs might play roles as regulators of milk quality and milk secretion during mammary gland differentiation.

Comparative and quantitative proteomic analysis of normal and degenerated human annulus fibrosus cells.

PubMed

Ye, Dongping; Liang, Weiguo; Dai, Libing; Zhou, Longqiang; Yao, Yicun; Zhong, Xin; Chen, Honghui; Xu, Jiake

2015-05-01

Degeneration of the intervertebral disc (IVD) is a major chronic medical condition associated with back pain. To better understand the pathogenesis of IVD degeneration, we performed comparative and quantitative proteomic analyses of normal and degenerated human annulus fibrosus (AF) cells and identified proteins that are differentially expressed between them. Annulus fibrosus cells were isolated and cultured from patients with lumbar disc herniation (the experimental group, degenerated AF cells) and scoliosis patients who underwent orthopaedic surgery (the control group, normal AF cells). Comparative proteomic analyses of normal and degenerated cultured AF cells were carried out using 2-D electrophoresis, mass spectrometric analyses, and database searching. Quantitative analyses of silver-stained 2-D electrophoresis gels of normal and degenerated cultured AF cells identified 10 protein spots that showed the most altered differential expression levels between the two groups. Among these, three proteins were decreased, including heat shock cognate 71-kDa protein, glucose-6-phosphate 1-dehydrogenase, and protocadherin-23, whereas seven proteins were increased, including guanine nucleotide-binding protein G(i) subunit α-2, superoxide dismutase, transmembrane protein 51, adenosine receptor A3, 26S protease regulatory subunit 8, lipid phosphate phosphatase-related protein, and fatty acyl-crotonic acid reductase 1. These differentially expressed proteins might be involved in the pathophysiological process of IVD degeneration and have potential values as biomarkers of the degeneration of IVD. © 2015 Wiley Publishing Asia Pty Ltd.

Proteomic Analysis of Serum from Patients with Major Depressive Disorder to Compare Their Depressive and Remission Statuses

PubMed Central

Lee, Jiyeong; Joo, Eun-Jeong; Lim, Hee-Joung; Park, Jong-Moon; Lee, Kyu Young; Park, Arum; Seok, AeEun

2015-01-01

Objective Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. Methods Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. Results The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. Conclusion This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation. PMID:25866527

CDK5 Regulatory Subunit-Associated Protein 1-like 1 Negatively Regulates Adipocyte Differentiation through Activation of Wnt Signaling Pathway.

PubMed

Take, Kazumi; Waki, Hironori; Sun, Wei; Wada, Takahito; Yu, Jing; Nakamura, Masahiro; Aoyama, Tomohisa; Yamauchi, Toshimasa; Kadowaki, Takashi

2017-08-04

CDK5 Regulatory Subunit-Associated Protein 1-like 1 (CDKAL1) was identified as a susceptibility gene for type 2 diabetes and body mass index in genome-wide association studies. Although it was reported that CDKAL1 is a methylthiotransferase essential for tRNA Lys (UUU) and faithful translation of proinsulin generated in pancreatic β cells, the role of CDKAL1 in adipocytes has not been understood well. In this study, we found that CDKAL1 is expressed in adipose tissue and its expression is increased during differentiation. Stable overexpression of CDKAL1, however, inhibited adipocyte differentiation of 3T3-L1 cells, whereas knockdown of CDKAL1 promoted differentiation. CDKAL1 increased protein levels of β-catenin and its active unphosphorylated form in the nucleus, thereby promoting Wnt target gene expression, suggesting that CDKAL1 activated the Wnt/β-catenin pathway-a well-characterized inhibitory regulator of adipocyte differentiation. Mutant experiments show that conserved cysteine residues of Fe-S clusters of CDKAL1 are essential for its anti-adipogenic action. Our results identify CDKAL1 as novel negative regulator of adipocyte differentiation and provide insights into the link between CDKAL1 and metabolic diseases such as type 2 diabetes and obesity.

In vitro approaches to evaluate placental drug transport by using differentiating JEG-3 human choriocarcinoma cells.

PubMed

Ikeda, Kenji; Utoguchi, Naoki; Tsutsui, Hidenobu; Yamaue, Satoko; Homemoto, Manami; Nakao, Erina; Hukunaga, Yumi; Yamasaki, Kyohei; Myotoku, Michiaki; Hirotani, Yoshihiko

2011-02-01

Human choriocarcinoma cells have been used as models for studying transcellular drug transport through placental trophoblasts. However, these models allow the transport of low-molecular-weight drugs through intercellular gap junctions. This study aimed at investigating the differentiation patterns of JEG-3 choriocarcinoma cells under different culture conditions and establishing the appropriate model of in vitro syncytiotrophoblast drug transport. Paracellular permeability was estimated by measuring the transepithelial electrical resistance (TEER) across JEG-3 cell layers. The mRNA expression levels of non-expressed in choriocarcinoma clone 1 (NECC1) and breast cancer resistance protein (BCRP), and those of E-cadherin (ECAD) and cadherin-11 (CDH11), which are adherens junction-associated proteins related to fusogenic ability of syncytiotrophoblasts differentiated from cytotrophoblasts, protein expression levels were considered as the differentiation signals. The highest TEER values were obtained in the JEG-3 cells cultured in the Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (1:1) mixed medium (CS-C(®) ; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan). By comparing the TEER values and the differentiation signals, the authors identified at least five JEG-3 cell-differentiation patterns. The differentiation pattern of JEG-3 cultured in CS-C resembled the syncytiotrophoblast-like differentiation signal characterizations in vivo. In conclusion, the syncytiotrophoblast-like models of differentiating JEG-3 cells cultured in CS-C might be appropriate for evaluating drug transport across the placental trophoblast. © 2010 The Authors. Basic & Clinical Pharmacology & Toxicology © 2010 Nordic Pharmacological Society.

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

PubMed Central

Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Sun, Guochang

2012-01-01

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium. PMID:23166741

Differential expression of utrophin-A and -B promoters in the central nervous system (CNS) of normal and dystrophic mdx mice.

PubMed

Baby, Santhosh M; Bogdanovich, Sasha; Willmann, Gabriel; Basu, Utpal; Lozynska, Olga; Khurana, Tejvir S

2010-03-01

Utrophin (Utrn) is the autosomal homolog of dystrophin, the Duchene Muscular Dystrophy (DMD) locus product and of therapeutic interest, as its overexpression can compensate dystrophin's absence. Utrn is transcribed by Utrn-A and -B promoters with mRNAs differing at their 5' ends. However, previous central nervous system (CNS) studies used C-terminal antibodies recognizing both isoforms. As this distinction may impact upregulation strategies, we generated Utrn-A and -B promoter-specific antibodies, Taqman Polymerase chain reaction (PCR)-based absolute copy number assays, and luciferase-reporter constructs to study CNS of normal and dystrophic mdx mice. Differential expression of Utrn-A and -B was noted in microdissected and capillary-enriched fractions. At the protein level, Utrn-B was predominantly expressed in vasculature and ependymal lining, whereas Utrn-A was expressed in neurons, astrocytes, choroid plexus and pia mater. mRNA quantification demonstrated matching patterns of differential expression; however, transcription-translation mismatch was noted for Utrn-B in caudal brain regions. Utrn-A and Utrn-B proteins were significantly upregulated in olfactory bulb and cerebellum of mdx brain. Differential promoter activity, mRNA and protein expressions were studied in cultured C2C12, bEnd3, neurons and astrocytes. Promoter activity ranking for Utrn-A and -B was neurons > astrocytes > C2C12 > bEnd3 and bEnd3 > astrocytes > neurons > C2C12, respectively. Our results identify promoter usage patterns for therapeutic targeting and define promoter-specific differential distribution of Utrn isoforms in normal and dystrophic CNS.

Mechanical stimuli differentially control stem cell behavior: morphology, proliferation, and differentiation

PubMed Central

Maul, Timothy M.; Chew, Douglas W.; Nieponice, Alejandro

2011-01-01

Mesenchymal stem cell (MSC) therapy has demonstrated applications in vascular regenerative medicine. Although blood vessels exist in a mechanically dynamic environment, there has been no rigorous, systematic analysis of mechanical stimulation on stem cell differentiation. We hypothesize that mechanical stimuli, relevant to the vasculature, can differentiate MSCs toward smooth muscle (SMCs) and endothelial cells (ECs). This was tested using a unique experimental platform to differentially apply various mechanical stimuli in parallel. Three forces, cyclic stretch, cyclic pressure, and laminar shear stress, were applied independently to mimic several vascular physiologic conditions. Experiments were conducted using subconfluent MSCs for 5 days and demonstrated significant effects on morphology and proliferation depending upon the type, magnitude, frequency, and duration of applied stimulation. We have defined thresholds of cyclic stretch that potentiate SMC protein expression, but did not find EC protein expression under any condition tested. However, a second set of experiments performed at confluence and aimed to elicit the temporal gene expression response of a select magnitude of each stimulus revealed that EC gene expression can be increased with cyclic pressure and shear stress in a cell-contact-dependent manner. Further, these MSCs also appear to express genes from multiple lineages simultaneously which may warrant further investigation into post-transcriptional mechanisms for controlling protein expression. To our knowledge, this is the first systematic examination of the effects of mechanical stimulation on MSCs and has implications for the understanding of stem cell biology, as well as potential bioreactor designs for tissue engineering and cell therapy applications. PMID:21253809

Threonine Affects Intestinal Function, Protein Synthesis and Gene Expression of TOR in Jian Carp (Cyprinus carpio var. Jian)

PubMed Central

Feng, Lin; Peng, Yan; Wu, Pei; Hu, Kai; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Li, Shu-Hong; Zhou, Xiao-Qiu

2013-01-01

This study aimed to investigate the effects of threonine (Thr) on the digestive and absorptive ability, proliferation and differentiation of enterocytes, and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian). First, seven isonitrogenous diets containing graded levels of Thr (7.4–25.2 g/kg diet) were fed to the fishes for 60 days. Second, enterocyte proliferation and differentiation were assayed by culturing enterocytes with graded levels of Thr (0–275 mg/l) in vitro. Finally, enterocytes were cultured with 0 and 205 mg/l Thr to determine protein synthesis. The percent weight gain (PWG), specific growth rate, feed intake, feed efficiency, protein retention value, activities of trypsin, lipase and amylase, weights and protein contents of hepatopancreas and intestine, folds heights, activities of alkaline phosphatase (AKP), γ- glutamyl transpeptidase and Na+/K+-ATPase in all intestinal segments, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities in hepatopancreas, and 4E-BP2 gene expression in muscle, hepatopancreas and intestinal segments were significantly enhanced by Thr (p<0.05). However, the plasma ammonia concentration and TOR gene expression decreased (p<0.05). In vitro, Thr supplement significantly increased cell numbers, protein content, the activities of GOT, GPT, AKP and Na+/K+-ATPase, and protein synthesis rate of enterocytes, and decreased LDH activity and ammonia content in cell medium (p<0.05). In conclusion, Thr improved growth, digestive and absorptive capacity, enterocyte proliferation and differentiation, and protein synthesis and regulated TOR and 4E-BP2 gene expression in juvenile Jian carp. The dietary Thr requirement of juvenile Jian carp was 16.25 g/kg diet (51.3 g/kg protein) based on quadratic regression analysis of PWG. PMID:23922879

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses.

PubMed

Alves-Costa, Fernanda A; Wasko, A P

2010-03-01

Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues. Therefore, we attempted to identify sexually dimorphic gene expression in the brains of adult males and females of Leporinus macrocephalus, a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase-PCR and real-time PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragments presented no significant identity with previously reported gene sequences. The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2) and to the Rab 37 gene, a member of the Ras oncogene family. The overexpression of these genes has been associated to a greater production of the beta-amyloid protein which, in turns, is the major factor that leads to Alzheimer's disease, and to the development of brain-tumors, respectively. Quantitative RT-PCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus may represent an interesting animal model to the understanding of the function of several vertebrate genes, including those involved in neurodegenerative and cancer diseases.

Tim2 is expressed in mouse fetal hepatocytes and regulates their differentiation.

PubMed

Watanabe, Natsumi; Tanaka, Minoru; Suzuki, Kaori; Kumanogoh, Atsushi; Kikutani, Hitoshi; Miyajima, Atsushi

2007-05-01

Liver development is regulated by various extracellular molecules such as cytokines and cell surface proteins. Although several such regulators have been identified, additional molecules are likely to be involved in liver development. To identify such molecules, we employed the signal sequence trap (SST) method to screen cDNAs encoding a secreted or membrane protein from fetal liver and obtained a number of clones. Among them, we found that T cell immunoglobulin and mucin domain 2 (Tim2) was expressed specifically on immature hepatocytes in the fetal liver. Tim2 has been shown to regulate immune responses, but its role in liver development had not been studied. We have examined the possible role of Tim2 in hepatocyte differentiation. At first, we prepared a soluble Tim2 fusion protein consisting of its extracellular domain and the Fc domain of human IgG (Tim2-hFc) and found that it bound to fetal and adult hepatocytes, suggesting that there are Tim2-binding molecules on hepatocytes. Second, Tim2-hFc inhibited the differentiation of hepatocytes in fetal liver primary culture, i.e., the expression of mature hepatic enzymes and accumulation of glycogen were severely reduced. Third, Tim2-hFc also inhibited proliferation of fetal hepatocytes. Fourth, down-regulation of Tim2 expression by small interfering RNA (siRNA) enhanced the expression of liver differentiation marker genes. It is strongly suggested that Tim2 is involved in the differentiation of fetal hepatocytes.

EMMPRIN expression in oral squamous cell carcinomas: correlation with tumor proliferation and patient survival.

PubMed

Monteiro, Luís Silva; Delgado, Maria Leonor; Ricardo, Sara; Garcez, Fernanda; do Amaral, Barbas; Pacheco, José Júlio; Lopes, Carlos; Bousbaa, Hassan

2014-01-01

The aim of our study was to explore the clinicopathological and prognostic significance of extracellular matrix metalloproteinase inducer (EMMPRIN) expression in oral squamous cell carcinomas (OSCC), and its relation with the proliferative tumor status of OSCC. We examined EMMPRIN and Ki-67 proteins expression by immunohistochemistry in 74 cases with OSCC. Statistical analysis was conducted to examine their clinicopathological and prognostic significance in OSCC. EMMPRIN membrane expression was observed in all cases, with both membrane and cytoplasmic tumor expression in 61 cases (82.4%). EMMPRIN overexpression was observed in 56 cases (75.7%). Moderately or poorly differentiated tumors showed EMMPRIN overexpression more frequently than well-differentiated tumors (P = 0.002). Overexpression of EMMPRIN was correlated with high Ki-67 expression (P = 0.004). In the multivariate analysis, EMMPRIN overexpression reveals an adverse independent prognostic value for cancer-specific survival (CSS) (P = 0.034). Our results reveal that EMMPRIN protein is overexpressed in more than two-thirds of OSCC cases, especially in high proliferative and less differentiated tumors. The independent value of EMMPRIN overexpression in CSS suggests that this protein could be used as an important biological prognostic marker for patients with OSCC. Moreover, the high expression of EMMPRIN makes it a possible therapeutic target in OSCC patients.

Two domains in vertebral development: antagonistic regulation by SHH and BMP4 proteins.

PubMed

Watanabe, Y; Duprez, D; Monsoro-Burq, A H; Vincent, C; Le Douarin, N M

1998-07-01

It has previously been shown that the notochord grafted laterally to the neural tube enhances the differentiation of the vertebral cartilage at the expense of the derivatives of the dermomyotome. In contrast, the dorsomedial graft of a notochord inhibits cartilage differentiation of the dorsal part of the vertebra carrying the spinous process. Cartilage differentiation is preceded by the expression of transcription factors of the Pax family (Pax1/Pax9) in the ventrolateral domain and of the Msx family in the dorsal domain. The proliferation and differentiation of Msx-expressing cells in the dorsal precartilaginous domain of the vertebra are stimulated by BMP4, which acts upstream of Msx genes. It has previously been shown that the SHH protein arising from the notochord (and floor plate) is necessary for the survival and further development of Pax1/Pax9-expressing sclerotomal cells. We show here that SHH acts antagonistically to BMP4. SHH-producing cells grafted dorsally to the neural tube at E2 inhibit expression of Bmp4 and Msx genes and also inhibits the differentiation of the spinous process. We present a model that accounts for cartilage differentiation in the vertebra.

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

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

Williams, K.; Chubb, C.; Huberman, E.

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

Secretagogin is a novel marker for neuroendocrine differentiation.

PubMed

Birkenkamp-Demtröder, Karin; Wagner, Ludwig; Brandt Sørensen, Flemming; Bording Astrup, Lone; Gartner, Wolfgang; Scherübl, Hans; Heine, Bernhard; Christiansen, Peer; Ørntoft, Torben Falck

2005-01-01

Our previous microarray-based studies identified secretagogin to be highly expressed in normal colon mucosa compared to basal expression in colon adenocarcinomas. The aim of this study was to analyze the differential expression of secretagogin in normal mucosa, adenocarcinomas, and neuroendocrine tumors. Western blotting, immunohistochemistry, immunofluorescence microscopy and ELISA were applied. Western blot analysis detected a 32-kDa secretagogin band in samples from normal mucosa. Immunohistochemical analyses on tissue specimens showed that secretagogin is exclusively expressed in neuroendocrine cells and nerve cells in normal mucosa of the digestive tract. Tissues adjacent to benign hyperplasic polyps and adenomas showed a decreased number of secretagogin-expressing neuroendocrine cells. Secretagogin co-localized with neuroendocrine markers (chromogranin A, neuron-specific enolase, synaptophysin) in neuroendocrine cells in crypts of normal mucosa, and in tumor cells of carcinoids. Secretagogin was strongly expressed in the cytosol and the nucleus of 19 well-differentiated neuroendocrine carcinoids and carcinoid metastases, as well as in neuroendocrine tumors from the lung, pancreas and adrenal gland. Secretagogin was detected in plasma from carcinoid patients with distant metastasis. Combined immunohistochemical analysis of secretagogin and FK506-binding protein 65, a protein de novo synthesized in adenocarcinomas, distinguished well-differentiated carcinoids, adenocarcinoids and undifferentiated carcinomas. We conclude that secretagogin is a novel marker for neuroendocrine differentiation.

Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

NASA Technical Reports Server (NTRS)

Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

2000-01-01

Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in CaR protein expression, occurring at a translational level during their differentiation into cells with a monocyte/macrophage phenotype in response to treatment with PMA or 1, 25(OH)(2)D(3), which is functionally linked to activation of a nonselective cation channel.

Expression of the transforming growth factor alpha protooncogene in differentiating human promyelocytic leukemia (HL-60) cells.

PubMed

Walz, T M; Malm, C; Wasteson, A

1993-01-01

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

Protein interaction networks at the host-microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen.

PubMed

Ramsey, J S; Chavez, J D; Johnson, R; Hosseinzadeh, S; Mahoney, J E; Mohr, J P; Robison, F; Zhong, X; Hall, D G; MacCoss, M; Bruce, J; Cilia, M

2017-02-01

The Asian citrus psyllid ( Diaphorina citri) is the insect vector responsible for the worldwide spread of ' Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host-microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host-microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening.

Protein interaction networks at the host–microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen

PubMed Central

Chavez, J. D.; Johnson, R.; Hosseinzadeh, S.; Mahoney, J. E.; Mohr, J. P.; Robison, F.; Zhong, X.; Hall, D. G.; MacCoss, M.; Bruce, J.; Cilia, M.

2017-01-01

The Asian citrus psyllid (Diaphorina citri) is the insect vector responsible for the worldwide spread of ‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host–microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host–microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening. PMID:28386418

Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog.

PubMed

Pérez, J; Bautista, M J; Rollón, E; de Lara, F C; Carrasco, L; Martin de las Mulas, J

1996-07-01

The immunohistochemical expression of muscle actin has been studied in 45 canine hemangiopericytomas (CHP) using a monoclonal antibody (HHF35) and formalin-fixed, paraffin-embedded specimens. The distribution of vimentin, desmin, cytokeratins, lysozyme, factor VIII-related antigen, S-100 protein, and glial fibrillary acidic protein was studied both in CHP and in some canine soft-tissue neoplasms (seven fibrosarcomas, seven benign schwannomas, seven benign fibrous histiocytomas, and six leiomyosarcomas) used as controls for differential diagnosis. All CHP and control tumors expressed vimentin. Twenty-three CHP expressed muscle actin, whereas all control tumors analyzed were muscle actin-negative, with the exception of leiomyosarcomas. Among muscle actin- and vimentin-positive CHP, one case could be reclassified as leiomyosarcoma because it was desmin-positive, two cases expressed lysozyme, and nine cases expressed S-100 protein. Among muscle actin-negative and vimentin-positive CHP, seven expressed S-100 protein. In addition, S-100 protein was detected in five schwannomas. All CHP and control tumors analyzed were negative for cytokeratins, factor VIII-related antigen, and glial fibrillary acidic protein. Our results support the hypothesis of a pericytic origin of CHP, and suggest that muscle actin, desmin, vimentin, and lysozyme could be useful for the differential diagnosis of canine spindle cell tumors, but not all these neoplasms can be identified with these tumor tissue markers.

Altered Protein Expression in the Ileum of Mice Associated with the Development of Chronic Infections with Echinostoma caproni (Trematoda)

PubMed Central

Cortés, Alba; Sotillo, Javier; Muñoz-Antoli, Carla; Fried, Bernard; Esteban, J. Guillermo; Toledo, Rafael

2015-01-01

Background Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been extensively used as experimental model to investigate the factors determining the expulsion of intestinal helminths or, in contrast, the development of chronic infections. Herein, we analyze the changes in protein expression induced by E. caproni infection in ICR mice, a host of high compatibility in which the parasites develop chronic infections. Methodology/Principal Findings To determine the changes in protein expression, a two-dimensional DIGE approach using protein extracts from the intestine of naïve and infected mice was employed; and spots showing significant differential expression were analyzed by mass spectrometry. A total of 37 spots were identified differentially expressed in infected mice (10 were found to be over-expressed and 27 down-regulated). These proteins were related to the restoration of the intestinal epithelium and the control of homeostatic dysregulation, concomitantly with mitochondrial and cytoskeletal proteins among others. Conclusion/Significance Our results suggests that changes in these processes in the ileal epithelium of ICR mice may facilitate the establishment of the parasite and the development of chronic infections. These results may serve to explain the factors determining the development of chronicity in intestinal helminth infection. PMID:26390031

Autocrine class 3 semaphorin system regulates slit diaphragm proteins and podocyte survival.

PubMed

Guan, F; Villegas, G; Teichman, J; Mundel, P; Tufro, A

2006-05-01

Class 3 semaphorins are guidance proteins that play crucial roles during development. Semaphorins 3A (sema 3A) and 3F are expressed by podocytes in vivo throughout ontogeny and their function is unknown. Here we examined the expression of class 3 semaphorins (3A, 3B, 3C, 3D, 3E, and 3F) and their receptors (neuropilins 1 and 2, plexins A1, A2, A3, B2, and D1) in undifferentiated and differentiated mouse podocytes using reverse transcriptase-polymerase chain reaction (RT-PCR). All class 3 semaphorins, neuropilins 1 and 2 are expressed by undifferentiated and differentiated podocytes at similar levels. Differentiated podocytes expressed 2-4-fold higher plexin A1, A2, and A3 mRNA levels than undifferentiated podocytes. To examine semaphorin regulation, we exposed podocytes to recombinant sema 3A. Sema 3A decreased semaphorin 3B, plexin A1, A3, and D1 >/=50% and reduced plexin A2 mRNA to undetectable levels. To identify sema 3A function in podocytes, we examined whether sema 3A regulates slit diaphragm proteins and podocyte survival. Sema 3A induced a dose-response podocin downregulation and decreased its interaction with CD2-associated protein and nephrin, as determined by Western analysis and co-immunoprecipitation. To evaluate sema 3A role in podocyte survival, we quantified podocyte apoptosis using a caspase 3 activity marker. Sema 3A induced a 10-fold increase in podocyte apoptosis and significantly decreased the activity of the Akt survival pathway. Our data indicate that (1) immortalized podocytes in culture have a functional autocrine semaphorin system that is regulated by differentiation and ligand availability; (2) sema 3A signaling regulates the expression and interactions of slit-diaphragm proteins and decreases podocyte survival.

Growth and differentiation of human lens epithelial cells in vitro on matrix

NASA Technical Reports Server (NTRS)

Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.; Aragon, G.; Lin, S. P.; Lui, G.; Polansky, J. R.

2000-01-01

PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis.

PubMed

Ramalho-Ortigão, J M; Temporal, P; de Oliveira , S M; Barbosa, A F; Vilela, M L; Rangel, E F; Brazil, R P; Traub-Cseko, Y M

2001-01-01

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Effects of fibroblast growth factor-2 on the expression and regulation of chemokines in human dental pulp cells.

PubMed

Kim, Young-Suk; Min, Kyung-San; Jeong, Dong-Ho; Jang, Jun-Hyeog; Kim, Hae-Won; Kim, Eun-Cheol

2010-11-01

Fibroblast growth factor-2 (FGF-2) participates in both hematopoiesis and osteogenesis; however, the effects of FGF-2 on chemokines during odontoblastic differentiation have not been reported. This study investigated whether human dental pulp cells (HDPCs) treated with FGF-2 could express chemokines during differentiation into odontoblastic cells and sought to identify its underlying mechanism of action. To analyze differentiation, we measured alkaline phosphatase (ALP) activity, calcified nodule formation by alizarin red staining, and marker RNA (mRNA) expression by reverse-transcriptase polymerase chain reaction (RT-PCR). Expression of chemokines, such as interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and MIP-3α, were evaluated by RT-PCR. ALP activity, the mineralization, and mRNA expression for odontoblastic markers were enhanced by FGF-2 in HDPCs. FGF-2 also up-regulated the expression of IL-6, IL-8, MCP-1, MIP-1α, and MIP-3α mRNAs, which were attenuated by inhibitors of p38, ERK1/2 and p38 MAP kinases, protein kinase C, phosphoinositide-3 kinase, and NF-κB. Taken together, these data suggest that FGF-2 plays a role not only as a differentiation inducing factor in the injury repair processes of pulpal tissue but also as a positive regulator of chemokine expression, which may help in tissue engineering and pulp regeneration using HDPCs. However, the fate of odontoblastic or osteoblastic differentiation, effective local delivery for FGF-2, interaction of chemotatic and odontogenic factors, and other limitations will need to be overcome before a major modality for the treatment of pulp disease. Copyright © 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath

We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment proteinmore » (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.« less

Differential proteome analysis of serum proteins associated with the development of type 2 diabetes mellitus in the KK-A(y) mouse model using the iTRAQ technique.

PubMed

Takahashi, Eri; Okumura, Akinori; Unoki-Kubota, Hiroyuki; Hirano, Hisashi; Kasuga, Masato; Kaburagi, Yasushi

2013-06-12

To identify candidate serum molecules associated with the progression of type 2 diabetes mellitus (T2DM), we carried out differential proteomic analysis using the KK-A(y) mouse, an animal model of T2DM with obesity. We employed an iTRAQ-based quantitative proteomic approach to analyze the proteomic changes in the sera collected from a pair of 4-week-old KK-A(y) versus C57BL/6 mice. Among the 227 proteins identified, a total of 45 proteins were differentially expressed in KK-A(y) versus C57BL/6 mice. We comparatively analyzed a series of the sera collected at 4 and 12weeks of age from KK-A(y) and C57BL/6 mice for the target protein using multiple reaction monitoring analysis, and identified 8 differentially expressed proteins between the sera of these mice at both time points. Among them, serine (or cysteine) peptidase inhibitor, clade A, member 3K (SERPINA3K) levels were elevated significantly in the sera of KK-A(y) mice compared to C57BL/6 mice. An in vitro assay revealed that the human homologue SERPINA3 increased the transendothelial permeability of retinal microvascular endothelial cells, which may be involved in the pathogenesis of diabetes and/or diabetic retinopathy. With the identified proteins, our proteomics study could provide valuable clues for a better understanding of the underlying mechanisms associated with T2DM. In this paper, we investigated the serum proteome of KK-A(y) mice in a pre-diabetic state compared to that of wild type controls in an attempt to uncover early diagnostic markers of diabetes that are maintained through a diabetic phenotype. We used iTRAQ-based two-dimensional LC-MS/MS serum profiling, and identified several differentially expressed proteins at the pre-diabetic stage. The differential expression was confirmed by multiple reaction monitoring assay, which is fast gaining ground as a sensitive, specific, and cost-effective methodology for relative quantification of the candidate proteins. Using these techniques, we have identified eight candidate proteins of interest including SERPINA3K, which may be important in the pathology of T2DM and/or diabetic retinopathy. Copyright © 2013 Elsevier B.V. All rights reserved.

Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells.

PubMed

Gu, Hanna; Song, Mina; Boonanantanasarn, Kanitsak; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

2018-01-09

Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O -linked-β- N -acetylglucosamine glycosylation ( O -GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O -GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N -acetylglucosamine concentrations or by O -GlcNAc transferase (OGT) overexpression. The effect of O -GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N -acetylglucosamine increased O -GlcNAcylation of Runx2 and the total levels of O -GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O -GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β- N -acetylglucosaminidase. Our findings suggest that excessive protein O -GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

Proteomic response of mouse pituitary gland under heat stress revealed active regulation of stress responsive proteins.

PubMed

Memon, Shahar Bano; Lian, Li; Gadahi, Javaid Ali; Genlin, Wang

2016-10-01

The mapping of tissue proteomes can identify the molecular regulators and effectors of their physiological activity. However, proteomic response of a mammalian tissue against heat stress (HS) particularly of the pituitary gland has not yet been resolved. The proteomic response of the mouse pituitary gland against HS at 40 o C was evaluated by iTRAQ. We found that, HS actively regulates stress-related proteins. Among 375 differentially expressed proteins, 26 up and 46 downregulated proteins were found as stress responsive proteins. Two proteins belonging to the HSP70 and one to HSP90 family were found upregulated. Meanwhile, the expression of HSP90α (Cytosolic), HSP60, and HSP84b were observed to be downregulated. A neuroprotective enzyme Nmnat3 was observed to be significantly upregulated. Three proteins related to the intermediate filament (IF) proteins (lamins, vimentin and keratins) were also found to be upregulated. We reported, an association between the IF proteins and HSPs as a biological marker of HS. The expression of Apo A-IV was upregulated and might be one explanation for low food intake during HS. Our findings indicated that, differentially expressed proteins might be played important roles in combating HS. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative Proteomic Analysis of Yak Follicular Fluid during Estrus

PubMed Central

Guo, Xian; Pei, Jie; Ding, Xuezhi; Chu, Min; Bao, Pengjia; Wu, Xiaoyun; Liang, Chunnian; Yan, Ping

2016-01-01

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development. PMID:26954118

Role of the POZ Zinc Finger Transcription Factor FBI-1 in Human and Murine Adipogenesis

PubMed Central

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J.; Considine, Robert V.; Sethi, Jaswinder K.; Vidal-Puig, Antonio; O’Rahilly, Stephen

2015-01-01

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2–4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation. PMID:14701838

Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line.

PubMed

Schelman, William R; Andres, Robert D; Sipe, Kimberly J; Kang, Evan; Weyhenmeyer, James A

2004-09-28

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of control. Taken together, these findings provide additional evidence for the reciprocal regulation of Bcl-2 and Bax expression by glutamate and suggest that neuronal excitotoxicity may, in part, result from the inverse regulation of these proteins.

Describing the Diapause-Preparatory Proteome of the Beetle Colaphellus bowringi and Identifying Candidates Affecting Lipid Accumulation Using Isobaric Tags for Mass Spectrometry-Based Proteome Quantification (iTRAQ)

PubMed Central

Tan, Qian-Qian; Liu, Wen; Zhu, Fen; Lei, Chao-Liang; Hahn, Daniel A.; Wang, Xiao-Ping

2017-01-01

Prior to entering diapause, insects must prepare themselves physiologically to withstand the stresses of arresting their development for a lengthy period. While studies describing the biochemical and cellular milieu of the maintenance phase of diapause are accumulating, few studies have taken an “omics” approach to describing molecular events during the diapause preparatory phase. We used isobaric tags and mass spectrometry (iTRAQ) to quantitatively compare the expression profiles of proteins identified during the onset of diapause preparation phase in the heads of adult female cabbage beetles, Colaphellus bowringi. A total of 3,175 proteins were identified, 297 of which were differentially expressed between diapause-destined and non-diapause-destined female adults and could therefore be involved in diapause preparation in this species. Comparison of identified proteins with protein function databases shows that many of these differentially expressed proteins enhanced in diapause destined beetles are involved in energy production and conversion, carbohydrate metabolism and transport, and lipid metabolism. Further hand annotation of differentially abundant peptides nominates several associated with stress hardiness, including HSPs and antioxidants, as well as neural development. In contrast, non-diapause destined beetles show substantial increases in cuticle proteins, suggesting additional post-emergence growth. Using RNA interference to silence a fatty acid-binding protein (FABP) that was highly abundant in the head of diapause-destined females prevented the accumulation of lipids in the fat body, a common product of diapause preparation in this species and others. Surprisingly, RNAi against the FABP also affected the transcript abundance of several heat shock proteins. These results suggest that the identified differentially expressed proteins that play vital roles in lipid metabolism may also contribute somehow to enhanced hardiness to environmental stress that is characteristic of diapause. PMID:28491041

Induced pluripotent stem cells with NOTCH1 gene mutation show impaired differentiation into smooth muscle and endothelial cells: Implications for bicuspid aortic valve-related aortopathy.

PubMed

Jiao, Jiao; Tian, Weihua; Qiu, Ping; Norton, Elizabeth L; Wang, Michael M; Chen, Y Eugene; Yang, Bo

2018-03-12

The NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. The iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1 -/- (NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1 -/- and wild type cells. The expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1 -/- NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1 -/- NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1 -/- CVPCs, the expression of ISL1, NKX2.5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1 -/- ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. NOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Role of H1 Linker Histones in Mammalian Development and Stem Cell Differentiation

PubMed Central

Pan, Chenyi; Fan, Yuhong

2016-01-01

H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. PMID:26689747

Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

PubMed

Radhakrishnan, Karthika; Bhagya, Kongattu P; Kumar, Anil Tr; Devi, Anandavalli N; Sengottaiyan, Jeeva; Kumar, Pradeep G

2016-08-01

Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein localization, and protein degradation, thus setting the foundation in understanding the functional role of AIRE in germ cell biology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

PubMed

Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

2016-01-01

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a 'function follows form' model of differentiation.

PubMed

Kankeu, Cynthia; Clarke, Kylie; Van Haver, Delphi; Gevaert, Kris; Impens, Francis; Dittrich, Anna; Roderick, H Llewelyn; Passante, Egle; Huber, Heinrich J

2018-05-17

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.

iTRAQ reveals proteomic changes during intestine regeneration in the sea cucumber Apostichopus japonicus.

PubMed

Sun, Lina; Xu, Dongxue; Xu, Qinzeng; Sun, Jingchun; Xing, Lili; Zhang, Libin; Yang, Hongsheng

2017-06-01

Sea cucumbers have a striking capacity to regenerate most of their viscera after evisceration, which has drawn the interest of many researchers. In this study, the isobaric tag for relative and absolute quantitation (iTRAQ) was utilized to investigate protein abundance changes during intestine regeneration in sea cucumbers. A total of 4073 proteins were identified, and 2321 proteins exhibited significantly differential expressions, with 1100 upregulated and 1221 downregulated proteins. Our results suggest that intestine regeneration constitutes a complex life activity regulated by the cooperation of various biological processes, including cytoskeletal changes, extracellular matrix (ECM) remodeling and ECM-receptor interactions, protein synthesis, signal recognition and transduction, energy production and conversion, and substance transport and metabolism. Additionally, real-time PCR showed mRNA expression of differentially expressed genes correlated positively with their protein levels. Our results provided a basis for studying the regulatory mechanisms associated with sea cucumber regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of black soybean koji extract on glucose utilization and adipocyte differentiation in 3T3-L1 cells.

PubMed

Huang, Chi-Chang; Huang, Wen-Ching; Hou, Chien-Wen; Chi, Yu-Wei; Huang, Hui-Yu

2014-05-09

Adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and progression of diseases such as insulin resistance and obesity. Black soybean koji (BSK) is produced by the fermentation of black soybean with Aspergilllus awamori. Previous study indicated that BSK extract has antioxidative and multifunctional bioactivities, however, the role of BSK in the regulation of energy metabolism is still unclear. We aimed to investigate the effect of glucose utilization on insulin-resistant 3T3-L1 preadipocytes and adipogenesis-related protein expression in differentiated adipocytes with BSK treatment. Cytoxicity assay revealed that BSK did not adversely affect cell viability at levels up to 200 µg/mL. The potential for glucose utilization was increased by increased glucose transporter 1 (GLUT1), GLUT4 and protein kinase B (AKT) protein expression in insulin-resistant 3T3-L1 cells in response to BSK treatment. Simultaneously, BSK inhibited lipid droplet accumulation in differentiated 3T3-L1 cells. The inhibitory effect of adipogenesis was associated with downregulated peroxisome proliferator-activated receptor g (PPARγ) level and upregulated Acrp30 protein expression. Our results suggest that BSK extract could improve glucose uptake by modulating GLUT1 and GLUT4 expression in a 3T3-L1 insulin-resistance cell model. In addition, BSK suppressed differentiation and lipid accumulation in mature 3T3-L1 adipocytes, which may suggest its potential for food supplementation to prevent obesity and related metabolic abnormalities.

Cdx1 and cdx2 expression during intestinal development.

PubMed

Silberg, D G; Swain, G P; Suh, E R; Traber, P G

2000-10-01

The intestine-specific transcription factors Cdx1 and Cdx2 are candidate genes for directing intestinal development, differentiation, and maintenance of the intestinal phenotype. This study focused on the complex patterns of expression of Cdx1 and Cdx2 during mouse gastrointestinal development. Embryonic and postnatal mouse tissues were analyzed by immunohistochemistry to determine protein expression of Cdx1 and Cdx2 in the developing intestinal tract. Cdx2 protein expression was observed at 9. 5 postcoitum (pc), whereas weak expression of Cdx1 protein was first seen at 12.5 pc in the distal developing intestine (hindgut). Expression of Cdx1 increased from 13.5 to 14.5 pc during the endoderm/epithelial transition with predominately distal expression. In contrast to Cdx1, there was intense expression of Cdx2 in all but the distal portions of the developing intestine. Cdx2 expression remained low in the distal colon throughout postnatal development. A gradient of expression formed in the crypt-villus axis, with Cdx1 primarily in the crypt and Cdx2 primarily in the villus. Direct comparison of the patterns of Cdx1 and Cdx2 protein expression during development as performed in this study provides new insights into their potential functional roles. The relative expression of Cdx1 to Cdx2 protein may be important in the anterior to posterior patterning of the intestinal epithelium and in defining patterns of proliferation and differentiation along the crypt-villus axis.

[The in vitro differentiation and the variant expression of protein of bone marrow stromal stem cells when treating the spinal cord injury].

PubMed

Shao, Ming; Bi, Zheng-Gang; Sun, Gang

2008-12-01

To explore the differentiation and the variant expression of protein of the bone marrow stromal stem cells (BMSCs) when the BMSCs differentiated into the neuronal cells in the analogous micro-environment of spinal cord injury. BMSCs were isolated from bone marrow of Wistar rats and labeled with PKH26 (control group), and then were cocultured with neural cells, which were isolated from the spinal cord of the fetal rats, in the same plate well (co-culture group) or in the two-layer Petri well (two-layer group). Eight days later, the BMSCs were identified by immunofluorescence staining of NSE and GFAP respectively. The apparently changing proteins were analyzed by SELDI-TOF-MS while the BMSCs differentiated into neurons. Eight days after co-culturing with neural cells in the same plate well or in the two-layer Petri well, BMSCs appeared more similar with neural cells. The immunofluorescence identification showed that, NSE and GFAP of which the BMSCs of the two-layer group expressed were obviously higher than control group (P < 0.05); and these two proteins of co-culture group were also obviously higher than the other two groups (P < 0.05). Five proteins in the co-culture group changed obviously as followed: TIP39_RAT and CALC_RAT were 5.360 and 2.807 times of that in the control group; INSL6_RAT, PNOC_RAT and PCSK1_RAT were 38.0, 49.9 and 43.8 percent of those in the control group. BMSCs could differentiate into neural cells in vitro, and the differentiation ratio of BMSCs in the co-culture group is higher than that of the two-layer group. Five proteins, including TIP39_RAT, CALC_RAT, INSL6_RAT, PNOC_RAT and PCSK1_RAT, are correlated closely to the mechanisms of which the BMSCs differentiated into neurons.

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

PubMed

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

2016-06-21

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

A fucose containing polymer-rich fraction from the brown alga Ascophyllum nodosum mediates lifespan increase and thermal-tolerance in Caenorhabditis elegans, by differential effects on gene and protein expression.

PubMed

Kandasamy, Saveetha; Khan, Wajahatullah; Evans, Franklin D; Critchley, Alan T; Zhang, Junzeng; Fitton, J H; Stringer, Damien N; Gardiner, Vicki-Anne; Prithiviraj, Balakrishnan

2014-02-01

The extracts of the brown alga, Ascophyllum nodosum, which contains several bioactive compounds, have been shown to impart biotic and abiotic stress tolerance properties when consumed by animals. However, the physiological, biochemical and molecular mechanism underlying such effects remain elusive. We investigated the effect of A. nodosum fucose-containing polymer (FCP) on tolerance to thermally induced stress using the invertebrate animal model, Caenorhabditis elegans. FCP at a concentration of 150 μg mL(-1) significantly improved the life span and tolerance against thermally induced stress in C. elegans. The treatment increased the C. elegans survival by approximately 24%, when the animals were under severe thermally induced stress (i.e. 35 °C) and 27% under mild stress (i.e. 30 °C) conditions. The FCP induced differential expression of genes and proteins is associated with stress response pathways. Under thermal stress, FCP treatment significantly altered the expression of 65 proteins (54 up-regulated & 11 down-regulated). Putative functional analysis of FCP-induced differential proteins signified an association of altered proteins in stress-related molecular and biochemical pathways of the model worm.

Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus.

PubMed

Rachinsky, Anna; Guerrero, Felix D; Scoles, Glen A

2007-12-01

We used gel electrophoresis and mass spectrometry to investigate differences in protein expression in ovarian tissues from Babesia bovis-infected and uninfected southern cattle tick, Rhipicephalus (Boophilus) microplus. Soluble and membrane proteins were extracted from ovaries of adult female ticks, and analyzed by isoelectric focusing (IEF) and one-dimensional or two-dimensional (2-D) gel electrophoresis. Protein patterns were analyzed for differences in expression between infected and uninfected ticks. 2-D separation of proteins revealed a number of proteins that appeared to be up- or down-regulated in response to infection with Babesia, in particular membrane/membrane-associated proteins and proteins in a low molecular mass range between 6 and 36kDa. A selection of differentially expressed proteins was subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the ovarian proteins that were up-regulated in infected ticks were calreticulin, two myosin subunits, an endoplasmic reticulum protein, a peptidyl-prolyl cis-trans isomerase (PPIase), a cytochrome c oxidase subunit, a glutamine synthetase, and a family of Kunitz-type serine protease inhibitors. Among the down-regulated ovarian proteins were another PPIase, a hemoglobin subunit, and a lysozyme. This study is part of an ongoing effort to establish a proteome database that can be utilized to investigate specific proteins involved in successful pathogen transmission.

Low-intensity pulsed ultrasound stimulation facilitates in vitro osteogenic differentiation of human adipose-derived stem cells via up-regulation of heat shock protein (HSP)70, HSP90, and bone morphogenetic protein (BMP) signaling pathway.

PubMed

Zhang, Zhonglei; Ma, Yalin; Guo, Shaowen; He, Yi; Bai, Gang; Zhang, Wenjun

2018-05-29

Low-intensity pulsed ultrasound (LIPUS) has positive effects on osteogenic differentiation. However, the effect of LIPUS on osteogenic differentiation of human adipose-derived stem cells (hASCs) is unclear. In the present study, we investigated whether LIPUS could promote the proliferation and osteogenic differentiation of hASCs. hASCs were isolated and osteogenically induced with LIPUS stimulation at 20 and 30 mW cm -2 for 30 min day -1 Cell proliferation and osteogenic differentiation potential of hASCs were respectively analyzed by cell counting kit-8 assay, Alizarin Red S staining, real-time polymerase chain reaction, and Western blotting. The results indicated that LIPUS stimulation did not significantly affect the proliferation of hASCs, but significantly increased their alkaline phosphatase activity on day 6 of culture and markedly promoted the formation of mineralized nodules on day 21 of culture. The mRNA expression levels of runt-related transcription factor, osteopontin, and osteocalcin were significantly up-regulated by LIPUS stimulation. LIPUS stimulation did not affect the expression of heat shock protein (HSP) 27, HSP40, bone morphogenetic protein (BMP)-6 and BMP-9, but significantly up-regulated the protein levels of HSP70, HSP90, BMP-2, and BMP-7 in the hASCs. Further studies found that LIPUS increased the mRNA levels of Smad 1 and Smad 5, elevated the phosphorylation of Smad 1/5, and suppressed the expression of BMP antagonist Noggin. These findings indicated that LIPUS stimulation enhanced osteogenic differentiation of hASCs possibly through the up-regulation of HSP70 and HSP90 expression and activation of BMP signaling pathway. Therefore, LIPUS might have the potential to promote the repair of bone defect. © 2018 The Author(s).

HSP60, a protein downregulated by IGFBP7 in colorectal carcinoma

PubMed Central

2010-01-01

Background In our previous study, it was well defined that IGFBP7 was an important tumor suppressor gene in colorectal cancer (CRC). We aimed to uncover the downstream molecules responsible for IGFBP7's behaviour in this study. Methods Differentially expressed protein profiles between PcDNA3.1(IGFBP7)-transfected RKO cells and the empty vector transfected controls were generated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) identification. The selected differentially expressed protein induced by IGFBP7 was confirmed by western blot and ELISA. The biological behaviour of the protein was explored by cell growth assay and colony formation assay. Results Six unique proteins were found differentially expressed in PcDNA3.1(IGFBP7)-transfected RKO cells, including albumin (ALB), 60 kDa heat shock protein(HSP60), Actin cytoplasmic 1 or 2, pyruvate kinase muscle 2(PKM2), beta subunit of phenylalanyl-tRNA synthetase(FARSB) and hypothetical protein. The downregulation of HSP60 by IGFBP7 was confirmed by western blot and ELISA. Recombinant human HSP60 protein could increase the proliferation rate and the colony formation ability of PcDNA3.1(IGFBP7)-RKO cells. Conclusion HSP60 was an important downstream molecule of IGFBP7. The downregulation of HSP60 induced by IGFBP7 may be, at least in part, responsible for IGFBP7's tumor suppressive biological behaviour in CRC. PMID:20433702

Biochemical analysis of 'kerosene tree' Hymenaea courbaril L. under heat stress.

PubMed

Gupta, Dinesh; Eldakak, Moustafa; Rohila, Jai S; Basu, Chhandak

2014-01-01

Hymenaea courbaril or jatoba is a tropical tree known for its medically important secondary metabolites production. Considering climate change, the goal of this study was to investigate differential expression of proteins and lipids produced by this tree under heat stress conditions. Total lipid was extracted from heat stressed plant leaves and various sesquiterpenes produced by the tree under heat stress were identified. Gas chromatographic and mass spectrometric analysis were used to study lipid and volatile compounds produced by the plant. Several volatiles, isoprene, 2-methyl butanenitrile, β ocimene and a numbers of sesquiterpenes differentially produced by the plant under heat stress were identified. We propose these compounds were produced by the tree to cope up with heat stress. A protein gel electrophoresis (2-D DIGE) was performed to study differential expression of proteins in heat stressed plants. Several proteins were found to be expressed many folds different in heat stressed plants compared to the control. These proteins included heat shock proteins, histone proteins, oxygen evolving complex, and photosynthetic proteins, which, we believe, played key roles in imparting thermotolerance in Hymenaea tree. To the best of our knowledge, this is the first report of extensive molecular physiological study of Hymenaea trees under heat stress. This work will open avenues of further research on effects of heat stress in Hymenaea and the findings can be applied to understand how global warming can affect physiology of other plants.

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21

PubMed Central

Park, Mi Hee; Lee, Hwa-Jeong; Lee, Hye Lim; Son, Dong Ju; Ju, Jung Hoon; Hyun, Byung Kook; Jung, Sung Hee; Song, Ju-Kyoung; Lee, Dong Hun; Hwang, Chul Ju; Han, Sang Bae; Kim, Sanghyeon; Hong, Jin Tae

2017-01-01

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21. PMID:28656059

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21.

PubMed

Park, Mi Hee; Lee, Hwa-Jeong; Lee, Hye Lim; Son, Dong Ju; Ju, Jung Hoon; Hyun, Byung Kook; Jung, Sung Hee; Song, Ju-Kyoung; Lee, Dong Hun; Hwang, Chul Ju; Han, Sang Bae; Kim, Sanghyeon; Hong, Jin Tae

2017-01-01

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

Shotgun Proteomic Analysis of Plasma from Dairy Cattle Suffering from Footrot: Characterization of Potential Disease-Associated Factors

PubMed Central

Sun, Dongbo; Zhang, Hong; Guo, Donghua; Sun, Anguo; Wang, Hongbin

2013-01-01

The plasma proteome of healthy dairy cattle and those with footrot was investigated using a shotgun LC-MS/MS approach. In total, 648 proteins were identified in healthy plasma samples, of which 234 were non-redundant proteins and 123 were high-confidence proteins; 712 proteins were identified from footrot plasma samples, of which 272 were non-redundant proteins and 138 were high-confidence proteins. The high-confidence proteins showed significant differences between healthy and footrot plasma samples in molecular weight, isoelectric points and the Gene Ontology categories. 22 proteins were found that may differentiate between the two sets of plasma proteins, of which 16 potential differential expression (PDE) proteins from footrot plasma involved in immunoglobulins, innate immune recognition molecules, acute phase proteins, regulatory proteins, and cell adhesion and cytoskeletal proteins; 6 PDE proteins from healthy plasma involved in regulatory proteins, cytoskeletal proteins and coagulation factors. Of these PDE proteins, haptoglobin, SERPINA10 protein, afamin precursor, haptoglobin precursor, apolipoprotein D, predicted peptidoglycan recognition protein L (PGRP-L) and keratan sulfate proteoglycan (KS-PG) were suggested to be potential footrot-associated factors. The PDE proteins PGRP-L and KS-PG were highlighted as potential biomarkers of footrot in cattle. The resulting protein lists and potential differentially expressed proteins may provide valuable information to increase understanding of plasma protein profiles in cattle and to assist studies of footrot-associated factors. PMID:23418487

Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis.

PubMed

Oldereid, N B; Angelis, P D; Wiger, R; Clausen, O P

2001-05-01

We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.

PGPR strain Paenibacillus polymyxa SQR-21 potentially benefits watermelon growth by re-shaping root protein expression.

PubMed

E, Yaoyao; Yuan, Jun; Yang, Fang; Wang, Lei; Ma, Jinghua; Li, Jing; Pu, Xiaowei; Raza, Waseem; Huang, Qiwei; Shen, Qirong

2017-12-01

Paenibacillus polymyxa (SQR-21) is not only a plant growth-promoting rhizobacteria, but also an effective biocontrol agent against Fusarium wilt disease of watermelon. For the better understanding and clarifying the potential mechanisms of SQR-21 to improve watermelon growth and disease resistance, a split-root methodology in hydroponic and LC-MS technology with the label free method was used to analyze the key root proteins involved in watermelon metabolism and disease resistance after the inoculation of SQR-21. Out of 623 identified proteins, 119 proteins were differentially expressed when treatment (SQR-21 inoculation) and control (no bacterial inoculation) were compared. Among those, 57 and 62 proteins were up-regulated and down-regulated, respectively. These differentially expressed proteins were identified to be involved in signal transduction (ADP-ribosylation factor, phospholipase D), transport (aspartate amino-transferase), carbohydratemetabolic (glucose-6-phosphate dehydrogenase, UDP-glucose pyrophosphorylase), defense and response to stress (glutathione S-transferase, Ubiquitin-activating enzyme E1), and oxidation-reduction process (thioredoxin peroxidase, ascorbate peroxidase). The results of this study indicated that SQR-21 inoculation on the watermelon roots benefits plant by inducing the expression of several proteins involved in growth, photosynthesis, and other metabolic and physiological activities.

Involvement of CRF2 signaling in enterocyte differentiation

PubMed Central

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-01-01

AIM To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. RESULTS CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. CONCLUSION Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases. PMID:28811708

Involvement of CRF2 signaling in enterocyte differentiation.

PubMed

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-07-28

To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology.

PubMed

Armenta, Jenny M; Hoeschele, Ina; Lazar, Iulia M

2009-07-01

An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17beta-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of <4%. The reproducibility of protein identifications was approximately 60%-67% between duplicate, and approximately 50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P < 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate approximately 2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, approximately 16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated.

Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

PubMed Central

Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

2013-01-01

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

PubMed

Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

2015-11-01

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3 as a novel lung cancer-related gene

PubMed Central

2013-01-01

Background To understand the carcinogenesis caused by accumulated genetic and epigenetic alterations and seek novel biomarkers for various cancers, studying differentially expressed genes between cancerous and normal tissues is crucial. In the study, two cDNA libraries of lung cancer were constructed and screened for identification of differentially expressed genes. Methods Two cDNA libraries of differentially expressed genes were constructed using lung adenocarcinoma tissue and adjacent nonmalignant lung tissue by suppression subtractive hybridization. The data of the cDNA libraries were then analyzed and compared using bioinformatics analysis. Levels of mRNA and protein were measured by quantitative real-time polymerase chain reaction (q-RT-PCR) and western blot respectively, as well as expression and localization of proteins were determined by immunostaining. Gene functions were investigated using proliferation and migration assays after gene silencing and gene over-expression. Results Two libraries of differentially expressed genes were obtained. The forward-subtracted library (FSL) and the reverse-subtracted library (RSL) contained 177 and 59 genes, respectively. Bioinformatic analysis demonstrated that these genes were involved in a wide range of cellular functions. The vast majority of these genes were newly identified to be abnormally expressed in lung cancer. In the first stage of the screening for 16 genes, we compared lung cancer tissues with their adjacent non-malignant tissues at the mRNA level, and found six genes (ERGIC3, DDR1, HSP90B1, SDC1, RPSA, and LPCAT1) from the FSL were significantly up-regulated while two genes (GPX3 and TIMP3) from the RSL were significantly down-regulated (P < 0.05). The ERGIC3 protein was also over-expressed in lung cancer tissues and cultured cells, and expression of ERGIC3 was correlated with the differentiated degree and histological type of lung cancer. The up-regulation of ERGIC3 could promote cellular migration and proliferation in vitro. Conclusions The two libraries of differentially expressed genes may provide the basis for new insights or clues for finding novel lung cancer-related genes; several genes were newly found in lung cancer with ERGIC3 seeming a novel lung cancer-related gene. ERGIC3 may play an active role in the development and progression of lung cancer. PMID:23374247

Terminal differentiation of murine resident peritoneal macrophages is characterized by expression of the STK protein tyrosine kinase, a receptor for macrophage-stimulating protein.

PubMed

Iwama, A; Wang, M H; Yamaguchi, N; Ohno, N; Okano, K; Sudo, T; Takeya, M; Gervais, F; Morissette, C; Leonard, E J; Suda, T

1995-11-01

STK, a new member of the hepatocyte growth factor receptor family, is the receptor for macrophage-stimulating protein (MSP), which acts on murine resident peritoneal macrophages. We established polyclonal and monoclonal antibodies against STK and characterized the structure of STK protein and STK expression on cells of the mononuclear phagocyte system. Western blotting showed that the STK transcript is translated into a single-chain precursor and then cleaved into a 165-kD disulfide-linked heterodimer composed of a 35-kD alpha-chain and a 144-kD beta-chain. Western blotting detected STK protein on resident peritoneal macrophages, a target of MSP, and showed that it was autophosphorylated in cells stimulated by MSP. By flow cytometric analysis using a monoclonal anti-STK antibody, we showed that STK protein is expressed on restricted macrophage populations such as resident peritoneal macrophages, but not on exudate peritoneal macrophages or mononuclear phagocytes of the bone marrow, peripheral blood, spleen, or alveoli. Resident peritoneal macrophages were classified into two fractions according to their reactivity with an anti-STK antibody and a marker antibody for macrophages: STKhigh-F4/80high cells and STKnegative-F4/80low cells. Acute exudative macrophages were all STKnegative-F4/80low, but they gradually became predominantly STKhigh-F4/80high several days after entrance into the peritoneal cavity. These results showed that after monocytes migrate into the peritoneal cavity, they undergo terminal differentiation in the peritoneal microenvironment. This is the first evidence of tissue-specific terminal differentiation of peritoneal macrophages, and this terminal differentiation can be characterized by the expression of STK receptor tyrosine kinase.

Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

PubMed

Eaton, Samantha L; Roche, Sarah L; Llavero Hurtado, Maica; Oldknow, Karla J; Farquharson, Colin; Gillingwater, Thomas H; Wishart, Thomas M

2013-01-01

Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.

Transcriptome interrogation of human myometrium identifies differentially expressed sense-antisense pairs of protein-coding and long non-coding RNA genes in spontaneous labor at term

PubMed Central

Romero, Roberto; Tarca, Adi; Chaemsaithong, Piya; Miranda, Jezid; Chaiworapongsa, Tinnakorn; Jia, Hui; Hassan, Sonia S.; Kalita, Cynthia A.; Cai, Juan; Yeo, Lami; Lipovich, Leonard

2014-01-01

Objective The mechanisms responsible for normal and abnormal parturition are poorly understood. Myometrial activation leading to regular uterine contractions is a key component of labor. Dysfunctional labor (arrest of dilatation and/or descent) is a leading indication for cesarean delivery. Compelling evidence suggests that most of these disorders are functional in nature, and not the result of cephalopelvic disproportion. The methodology and the datasets afforded by the post-genomic era provide novel opportunities to understand and target gene functions in these disorders. In 2012, the ENCODE Consortium elucidated the extraordinary abundance and functional complexity of long non-coding RNA genes in the human genome. The purpose of the study was to identify differentially expressed long non-coding RNA genes in human myometrium in women in spontaneous labor at term. Materials and Methods Myometrium was obtained from women undergoing cesarean deliveries who were not in labor (n=19) and women in spontaneous labor at term (n=20). RNA was extracted and profiled using an Illumina® microarray platform. The analysis of the protein coding genes from this study has been previously reported. Here, we have used computational approaches to bound the extent of long non-coding RNA representation on this platform, and to identify co-differentially expressed and correlated pairs of long non-coding RNA genes and protein-coding genes sharing the same genomic loci. Results Upon considering more than 18,498 distinct lncRNA genes compiled nonredundantly from public experimental data sources, and interrogating 2,634 that matched Illumina microarray probes, we identified co-differential expression and correlation at two genomic loci that contain coding-lncRNA gene pairs: SOCS2-AK054607 and LMCD1-NR_024065 in women in spontaneous labor at term. This co-differential expression and correlation was validated by qRT-PCR, an independent experimental method. Intriguingly, one of the two lncRNA genes differentially expressed in term labor had a key genomic structure element, a splice site that lacked evolutionary conservation beyond primates. Conclusions We provide for the first time evidence for coordinated differential expression and correlation of cis-encoded antisense lncRNAs and protein-coding genes with known, as well as novel roles in pregnancy in the myometrium of women in spontaneous labor at term. PMID:24168098

Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

PubMed

Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

2015-08-01

Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

Protein Kinase Inhibitor γ reciprocally regulates osteoblast and adipocyte differentiation by downregulating Leukemia Inhibitory Factor

PubMed Central

Chen, Xin; Hausman, Bryan S.; Luo, Guangbin; Zhou, Guang; Murakami, Shunichi; Rubin, Janet; Greenfield, Edward M.

2013-01-01

The Protein Kinase Inhibitor (Pki) gene family inactivates nuclear PKA and terminates PKA-induced gene expression. We previously showed that Pkig is the primary family member expressed in osteoblasts and that Pkig knockdown increases the effects of parathyroid hormone and isoproterenol on PKA activation, gene expression, and inhibition of apoptosis. Here, we determined whether endogenous levels of Pkig regulate osteoblast differentiation. Pkig is the primary family member in MEFs, murine marrow-derived mesenchymal stem cells, and human mesenchymal stem cells. Pkig deletion increased forskolin-dependent nuclear PKA activation and gene expression and Pkig deletion or knockdown increased osteoblast differentiation. PKA signaling is known to stimulate adipogenesis; however, adipogenesis and osteogenesis are often reciprocally regulated. We found that the reciprocal regulation predominates over the direct effects of PKA since adipogenesis was decreased by Pkig deletion or knockdown. Pkig deletion or knockdown simultaneously increased osteogenesis and decreased adipogenesis in mixed osteogenic/adipogenic medium. Pkig deletion increased PKA-induced expression of Leukemia Inhibitory Factor (Lif) mRNA and LIF protein. LIF neutralizing antibodies inhibited the effects on osteogenesis and adipogenesis of either Pkig deletion in MEFs or PKIγ knockdown in both murine and human mesenchymal stem cells. Collectively, our results show that endogenous levels of Pkig reciprocally regulate osteoblast and adipocyte differentiation and that this reciprocal regulation is mediated in part by LIF. PMID:23963683

Signal transducer and activator of transcription 5B (STAT5B) modulates adipocyte differentiation via MOF.

PubMed

Gao, Peng; Zhang, Yuchao; Liu, Yuantao; Chen, Jicui; Zong, Chen; Yu, Cong; Cui, Shang; Gao, Weina; Qin, Dandan; Sun, Wenchuan; Li, Xia; Wang, Xiangdong

2015-12-01

The role and mechanism of signal transducer and activator of transcription 5B (STAT5B) in adipogenesis remain unclear. In this study, our data showed that Males absent on the first (MOF) protein expression was increased during 3 T3-L1 preadipocytes differentiation accompanied with STAT5B expression increasing. Over-expression STAT5B enhanced MOF promoter trans-activation in HeLa cells. Mutagenesis assay and ChIP analysis exhibited that STAT5B was able to bind MOF promoter. Knocking-down STAT5B in 3 T3-L1 preadipocytes led to decreased expression of MOF, but resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid-binding protein 4 (Fabp4), which were important factors or enzymes for adipogenesis. We also found that knocking-down MOF in 3 T3-L1 preadipocytes resulted in increased expression of PPARγ, C/EBPα and Fabp4, which was in the same trend as STAT5B knocking-down. Over-expression MOF resulted in reduced promoter trans-activation activity of C/EBPα. These results suggest that STAT5B and MOF work as negative regulators in adipogenesis, and STAT5B modulates preadipocytes differentiation partially by regulating MOF expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Integrated Analyses of Gene Expression Profiles Digs out Common Markers for Rheumatic Diseases

PubMed Central

Wang, Lan; Wu, Long-Fei; Lu, Xin; Mo, Xing-Bo; Tang, Zai-Xiang; Lei, Shu-Feng; Deng, Fei-Yan

2015-01-01

Objective Rheumatic diseases have some common symptoms. Extensive gene expression studies, accumulated thus far, have successfully identified signature molecules for each rheumatic disease, individually. However, whether there exist shared factors across rheumatic diseases has yet to be tested. Methods We collected and utilized 6 public microarray datasets covering 4 types of representative rheumatic diseases including rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and osteoarthritis. Then we detected overlaps of differentially expressed genes across datasets and performed a meta-analysis aiming at identifying common differentially expressed genes that discriminate between pathological cases and normal controls. To further gain insights into the functions of the identified common differentially expressed genes, we conducted gene ontology enrichment analysis and protein-protein interaction analysis. Results We identified a total of eight differentially expressed genes (TNFSF10, CX3CR1, LY96, TLR5, TXN, TIA1, PRKCH, PRF1), each associated with at least 3 of the 4 studied rheumatic diseases. Meta-analysis warranted the significance of the eight genes and highlighted the general significance of four genes (CX3CR1, LY96, TLR5, and PRF1). Protein-protein interaction and gene ontology enrichment analyses indicated that the eight genes interact with each other to exert functions related to immune response and immune regulation. Conclusion The findings support that there exist common factors underlying rheumatic diseases. For rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and osteoarthritis diseases, those common factors include TNFSF10, CX3CR1, LY96, TLR5, TXN, TIA1, PRKCH, and PRF1. In-depth studies on these common factors may provide keys to understanding the pathogenesis and developing intervention strategies for rheumatic diseases. PMID:26352601

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

PubMed

Chen, B; Han, B H; Sun, X H; Lim, R W

1997-01-15

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

PubMed Central

Chen, B; Han, B H; Sun, X H; Lim, R W

1997-01-01

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected. PMID:9016574

Proteomic analysis reveals the important roles of alpha-5-collagen and ATP5β during skin ulceration syndrome progression of sea cucumber Apostichopus japonicus.

PubMed

Zhao, Zelong; Jiang, Jingwei; Pan, Yongjia; Sun, Hongjuan; Guan, Xiaoyan; Gao, Shan; Chen, Zhong; Dong, Ying; Zhou, Zunchun

2018-03-20

Apostichopus japonicus is one of the most important aquaculture species in China. Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease affected the development of A. japonicus culture industry. To better understand the response mechanisms of A. japonicus during SUS progression, the protein variations in the body wall of A. japonicus at different stages of SUS were investigated by a comparative proteomic approach based on isobaric tags for relative and absolute quantification. A total of 1449 proteins were identified from the samples at different SUS stages. Among these proteins, 145 proteins were differentially expressed in the SUS-related samples compared to those of healthy A. japonicus. These differentially expressed proteins involved a wide range of functions. Among these differentially expressed proteins, only two proteins, alpha-5-collagen and an unknown function protein, were differentially expressed during the whole progression of SUS compared with healthy A. japonicus. In addition, ATP synthase subunit beta (ATP5β) interacted with a variety of proteins with different functions during the SUS progression. These results implied that alpha-5-collagen and ATP5β could play important roles during the SUS progression of A. japonicus. Our study provided a new sight to understand the molecular responses of sea cucumber during the SUS progression and accumulated data for the prevention of SUS in sea cucumber aquaculture. The current study aimed to reveal how the body wall of Apostichopus japonicus response to skin ulceration syndrome (SUS). To the best of our knowledge, this is the first proteomic study analyzing the differences in protein profile of sea cucumber during the whole SUS progression. By analyzing the expression differences of the proteome via isobaric labeling-based quantitative proteomic, we identified some proteins which may play important roles during the SUS progression. According to the enrichment analyses of these proteins based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, a draft view of how the sea cucumber affected by SUS has been drawn. The common and unique differentially expressed proteins by Venn analysis showed that alpha-5-collagen was down-regulated at all stages of SUS, which had the potential as a target component for the host-directed SUS therapy. In addition, ATP5β, a subunit of mitochondrial ATP synthase, interacting with a variety of proteins with different functions during the SUS progression. This result illustrated that energy production and metabolism could play an important role in the formation of skin ulceration and resistance to pathogens in sea cucumber. The results of this study will be helpful for researchers to gain insights into the complex molecular mechanism of SUS in sea cucumber. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell

PubMed Central

Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A.; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

2016-01-01

The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. PMID:26601937

Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

PubMed Central

Wagenknecht, Nadine; Reuter, Nina; Scherer, Myriam; Reichel, Anna; Müller, Regina; Stamminger, Thomas

2015-01-01

Promyelocytic leukemia nuclear bodies, also termed nuclear domain 10 (ND10), have emerged as nuclear protein accumulations mediating an intrinsic cellular defense against viral infections via chromatin-based mechanisms, however, their contribution to the control of herpesviral latency is still controversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence and Western blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency. PMID:26057166

Changes in Global Transcriptional Profiling of Women Following Obesity Surgery Bypass.

PubMed

Pinhel, Marcela Augusta de Souza; Noronha, Natalia Yumi; Nicoletti, Carolina Ferreira; de Oliveira, Bruno Affonso Parente; Cortes-Oliveira, Cristiana; Pinhanelli, Vitor Caressato; Salgado Junior, Wilson; Machry, Ana Julia; da Silva Junior, Wilson Araújo; Souza, Dorotéia Rossi Silva; Marchini, Júlio Sérgio; Nonino, Carla Barbosa

2018-01-01

Differential gene expression in peripheral blood mononuclear cells (PBMCs) after Roux-en-Y gastric bypass (RYGB) is poorly characterized. Markers of these processes may provide a deeper understanding of the mechanisms that underlie these events. The main goal of this study was to identify changes in PBMC gene expression in women with obesity before and 6 months after RYGB-induced weight loss. The ribonucleic acid (RNA) of PBMCs from 13 obese women was analyzed before and 6 months after RYGB; the RNA of PBMCs from nine healthy women served as control. The gene expression levels were determined by microarray analysis. Significant differences in gene expression were validated by real-time quantitative polymerase chain reaction (RT-qPCR). Microarray analysis for comparison of the pre- and postoperative periods showed that 1366 genes were differentially expressed genes (DEGs). The main pathways were related to gene transcription; lipid, energy, and glycide metabolism; inflammatory and immunological response; cell differentiation; oxidative stress regulation; response to endogenous and exogenous stimuli; substrate oxidation; mTOR signaling pathway; interferon signaling; mitogen-activated protein kinases (MAPK), cAMP response element binding protein (CREB1), heat shock factor 1 (HSF1), and sterol regulatory element binding protein 1c (SREBP-1c) gene expression; adipocyte differentiation; and methylation. Six months after bariatric surgery and significant weight loss, many molecular pathways involved in obesity and metabolic diseases change. These findings are an important tool to identify potential targets for therapeutic intervention and clinical practice of nutritional genomics in obesity.

Distribution of syndecan-1 protein in developing mouse teeth

PubMed Central

Filatova, Anna; Pagella, Pierfrancesco; Mitsiadis, Thimios A.

2014-01-01

Syndecan-1 is a cell surface proteoglycan involved in the regulation of various biological processes such as proliferation, migration, condensation and differentiation of cells, intercellular communication, and morphogenesis. The extracellular domain of syndecan-1 can bind to extracellular matrix components and signaling molecules, while its intracellular domain interacts with cytoskeletal proteins, thus allowing the transfer of information about extracellular environment changes into the cell that consequently affect cellular behavior. Although previous studies have shown syndecan-1 expression during precise stages of tooth development, there is no equivalent study regrouping the expression patterns of syndecan-1 during all stages of odontogenesis. Here we examined the distribution of syndecan-1 protein in embryonic and post-natal developing mouse molars and incisors. Syndecan-1 distribution in mesenchymal tissues such as dental papilla and dental follicle was correlated with proliferating events and its expression was often linked to stem cell niche territories. Syndecan-1 was also expressed in mesenchymal cells that will differentiate into the dentin producing odontoblasts, but not in differentiated functional odontoblasts. In the epithelium, syndecan-1 was detected in all cell layers, by the exception of differentiated ameloblasts that form the enamel. Furthermore, syndecan-1 was expressed in osteoblast precursors and osteoclasts of the alveolar bone that surrounds the developing tooth germs. Taken together these results show the dynamic nature of syndecan-1 expression during odontogenesis and suggest its implication in various processes of tooth development and homeostasis. PMID:25642191

RNA-Seq reveals 10 novel promising candidate genes affecting milk protein concentration in the Chinese Holstein population

PubMed Central

Li, Cong; Cai, Wentao; Zhou, Chenghao; Yin, Hongwei; Zhang, Ziqi; Loor, Juan J.; Sun, Dongxiao; Zhang, Qin; Liu, Jianfeng; Zhang, Shengli

2016-01-01

Paired-end RNA sequencing (RNA-Seq) was used to explore the bovine transcriptome from the mammary tissue of 12 Chinese Holstein cows with 6 extremely high and 6 low phenotypic values for milk protein percentage. We defined the differentially expressed transcripts between the two comparison groups, extremely high and low milk protein percentage during the peak lactation (HP vs LP) and during the non-lactating period (HD vs LD), respectively. Within the differentially expressed genes (DEGs), we detected 157 at peak lactation and 497 in the non-lactating period with a highly significant correlation with milk protein concentration. Integrated interpretation of differential gene expression indicated that SERPINA1, CLU, CNTFR, ERBB2, NEDD4L, ANG, GALE, HSPA8, LPAR6 and CD14 are the most promising candidate genes affecting milk protein concentration. Similarly, LTF, FCGR3A, MEGF10, RRM2 and UBE2C are the most promising candidates that in the non-lactating period could help the mammary tissue prevent issues with inflammation and udder disorders. Putative genes will be valuable resources for designing better breeding strategies to optimize the content of milk protein and also to provide new insights into regulation of lactogenesis. PMID:27254118

1,25-DIHYDROXYVITAMIN D3 INDUCES MONOCYTIC DIFFERENTIATION OF HUMAN MYELOID LEUKEMIA CELLS BY REGULATING C/EBPβ EXPRESSION THROUGH MEF2C

PubMed Central

Zheng, Ruifang; Wang, Xuening; Studzinski, George P.

2015-01-01

Myogenic enhancer factor2 (Mef2) consists of a family of transcription factors involved in morphogenesis of skeletal, cardiac and smooth muscle cells. Among the four isoforms (Mef2A, 2B, 2C, and 2D), Mef2C was also found to play important roles in hematopoiesis. At myeloid progenitor level, Mef2C expression favors monocytic differentiation. Previous studies from our laboratory demonstrated that ERK5 was activated in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation in AML cells and ERK5 activation was accompanied by increased Mef2C phosphorylation. We therefore examined the role of Mef2C in 1,25D-induced monocytic differentiation in AML cell lines (HL60, U937 and THP1) and found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreases the expression of the monocytic marker, CD14, without affecting the expression of the general myeloid marker, CD11b. CCAAT/Enhancer-binding protein (C/EBP) β, which can bind to CD14 promoter and increase its transcription, has been shown to be the downstream effector of 1,25D-induced monocytic differentiation in AML cells. When Mef2C was knocked down, expression of C/EBPβ was reduced at both mRNA and protein levels. The protein expression levels of cell cycle regulators, p27Kip1 and cyclin D1, were not affected by Mef2C knockdown, nor the monopoiesis related transcription factor, ATF2 (Activating Transcription Factor 2). Thus, we conclude that 1,25D-induced monocytic differentiation, and CD14 expression in particular, is mediated through activation of ERK5-Mef2C-C/EBPβ signaling pathway, and that Mef2C does not seem to modulate cell cycle progression. PMID:25448741

A new invertebrate member of the p53 gene family is developmentally expressed and responds to polychlorinated biphenyls.

PubMed Central

Jessen-Eller, Kathryn; Kreiling, Jill A; Begley, Gail S; Steele, Marjorie E; Walker, Charles W; Stephens, Raymond E; Reinisch, Carol L

2002-01-01

The cell-cycle checkpoint protein p53 both directs terminal differentiation and protects embryos from DNA damage. To study invertebrate p53 during early development, we identified three differentially expressed p53 family members (p53, p97, p120) in the surf clam, Spisula solidissima. In these mollusks, p53 and p97 occur in both embryonic and adult tissue, whereas p120 is exclusively embryonic. We sequenced, cloned, and characterized p120 cDNA. The predicted protein, p120, resembles p53 across all evolutionarily conserved regions and contains a C-terminal extension with a sterile alpha motif (SAM) as in p63 and p73. These vertebrate forms of p53 are required for normal inflammatory, epithelial, and neuronal development. Unlike clam p53 and p97, p120 mRNA and protein levels are temporally expressed in embryos, with mRNA levels decreasing with increasing p120 protein (R(2) = 0.97). Highest surf clam p120 mRNA levels coincide with the onset of neuronal growth. In earlier work we have shown that neuronal development is altered by exposure to polychlorinated biphenyls (PCBs), a neurotoxic environmental contaminant. In this study we show that PCBs differentially affect expression of the three surf clam p53 family members. p120 mRNA and protein are reduced the most and earliest in development, p97 protein shows a smaller and later reduction, and p53 protein levels do not change. For the first time we report that unlike p53 and p97, p120 is specifically embryonic and expressed in a time-dependent manner. Furthermore, p120 responds to PCBs by 48 hr when PCB-induced suppression of the serotonergic nervous system occurs. PMID:11940455

Differential expression and activation of Rab27A in human eosinophils: relationship to blood eosinophilia.

PubMed

Coughlin, Jason J; Odemuyiwa, Solomon O; Davidson, Courtney E; Moqbel, Redwan

2008-08-29

Eosinophil degranulation is thought to play a pathophysiological role in asthma. Rab27A is a GTP-binding protein that is known to be essential for the degranulation of several leukocyte subsets and thus may be essential for eosinophil granule exocytosis. Here, we show that Rab27A mRNA and protein are expressed in human eosinophils. We have developed a novel assay to assess Rab27A activation and have found a similar activation pattern of this protein upon stimulation of eosinophils, neutrophils and NK cells suggesting a similar function in these cell types. Interestingly, Rab27A expression was elevated in eosinophils from asthmatic donors. Furthermore, eosinophils from eosinophilic donors displayed more rapid Rab27A activation kinetics than those from donors with lower eosinophil counts. Given that elevated blood eosinophil numbers correlate with increased priming of eosinophils, this pattern of Rab27A activation suggests differential protein expression in activated cells may allow eosinophils to degranulate more rapidly upon stimulation.

Host response to intravenous injection of epsilon toxin in mouse model: a proteomic view.

PubMed

Kumar, Bhoj; Alam, Syed Imteyaz; Kumar, Om

2013-01-01

Epsilon toxin (ETX) is an extremely potent pore-forming toxin and a category B biological agent. ETX is a major virulence determinant of Clostridium perfringens toxinotypes B and D, and is implicated in pathogenesis of rapidly fatal economically important pulpy kidney disease in lambs caused by toxinotype D. Despite being a toxin, ETX can be utilized as a tool to target glutamatergic neurons and for drug delivery into the CNS. 2DE-MS approach was employed to elucidate the host response to ETX following intravenous injection in mouse model. In total, 136 proteins were identified either differentially expressed in brain (18) and kidney (33); showing specific interaction with ETX from lysates of brain (4), kidney (21), or from plasma (42); and urine markers (18) of intoxication. Differentially expressed proteins in kidney included those involved in calcium homeostasis and cytoskeletal organization. Proteins involved in ER and oxidative stress and energy metabolism also showed differential levels in the target tissue after ETX treatment. The known functions of the proteins differentially expressed and those interacting with ETX indicate involvement of interlinked pathways. This study provides first proteomic account of host response to ETX exposure providing clues to mechanism of toxicity and potential therapeutic targets. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Effect of Yiguan Decoction on differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells: an experimental research].

PubMed

Ping, Jian; Chen, Hong-Yun; Yang, Zhou; Yang, Cheng; Xu, Lie-Ming

2014-03-01

To observe the effect of Yiguan Decoction (YGD) on differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro. Rat BMSCs were isolated using whole bone marrow adherent method. The properties of BMSCs were identified by analyzing the expression of surface cytokines by flow cytometry. The third passage cells were differentiated into fat cells to identify their features. BMSCs were incubated with hepatocyte growth factor (HGF) plus fibroblast growth factor 4 (FGF4) or YGD containing serum YGD for 21 days. The mRNA expression of alpha-fetoprotein (alphaAFP), albumin (Alb), and hepatocyte nuclear factor 4alpha (HNF4alpha) were detected by real time PCR. Expression of AFP and cytokeratin 18 (CK18) protein was detected by cell immunofluorescence. Glycogen synthesis was observed using periodic acid-Schiff stain (PAS). CK18, Wnt 3alpha, and alphacatenin protein expressions were detected by Western blot. High expression of CD90, CD29, and CD44, and low expression of CD34 and CD11b were observed in BMSCs isolated by whole bone mar- row adherent method, and numerous lipid droplets were observed in BMSCs using oil red O staining. Both YGD containing serum and growth factor stimulated the expression levels of Alb, AFP, HNF4alpha mRNA and CK18 protein. The down-regulated expression of Wnt 3alpha and beta-catenin could be detected at 21 days after induction. The synthesized glycogen granule could be seen. Down-regulated Wnt 3alpha and beta-catenin expression could also be observed. YGD could induce the differentiation of rat BMSCs into hepatocyte-like cells, which was related to down-regulating Wnt/beta-catenin signal pathway.

Distinct profiles of expressed sequence tags during intestinal regeneration in the sea cucumber Holothuria glaberrima

PubMed Central

Rojas-Cartagena, Carmencita; Ortíz-Pineda, Pablo; Ramírez-Gómez, Francisco; Suárez-Castillo, Edna C.; Matos-Cruz, Vanessa; Rodríguez, Carlos; Ortíz-Zuazaga, Humberto; García-Arrarás, José E.

2010-01-01

Repair and regeneration are key processes for tissue maintenance, and their disruption may lead to disease states. Little is known about the molecular mechanisms that underline the repair and regeneration of the digestive tract. The sea cucumber Holothuria glaberrima represents an excellent model to dissect and characterize the molecular events during intestinal regeneration. To study the gene expression profile, cDNA libraries were constructed from normal, 3-day, and 7-day regenerating intestines of H. glaberrima. Clones were randomly sequenced and queried against the nonredundant protein database at the National Center for Biotechnology Information. RT-PCR analyses were made of several genes to determine their expression profile during intestinal regeneration. A total of 5,173 sequences from three cDNA libraries were obtained. About 46.2, 35.6, and 26.2% of the sequences for the normal, 3-days, and 7-days cDNA libraries, respectively, shared significant similarity with known sequences in the protein database of GenBank but only present 10% of similarity among them. Analysis of the libraries in terms of functional processes, protein domains, and most common sequences suggests that a differential expression profile is taking place during the regeneration process. Further examination of the expressed sequence tag dataset revealed that 12 putative genes are differentially expressed at significant level (R > 6). Experimental validation by RT-PCR analysis reveals that at least three genes (unknown C-4677-1, melanotransferrin, and centaurin) present a differential expression during regeneration. These findings strongly suggest that the gene expression profile varies among regeneration stages and provide evidence for the existence of differential gene expression. PMID:17579180

Melatonin ameliorates anxiety and depression-like behaviors and modulates proteomic changes in triple transgenic mice of Alzheimer's disease.

PubMed

Nie, Lulin; Wei, Gang; Peng, Shengming; Qu, Zhongsen; Yang, Ying; Yang, Qian; Huang, Xinfeng; Liu, Jianjun; Zhuang, Zhixiong; Yang, Xifei

2017-07-08

Alzheimer's disease (AD) is a devastating neurodegenerative disease accompanied by neuropsychiatric symptoms, such as anxiety and depression. The levels of melatonin decrease in brains of AD patients. The potential effect of melatonin on anxiety and depression behaviors in AD and the underlying mechanisms remain unclear. In this study, we treated 10-month-old triple transgenic mice of AD (3xTg-AD) with melatonin (10 mg/kg body weight/day) for 1 month and explored the effects of melatonin on anxiety and depression-like behaviors in 3xTg-AD mice and the protein expression of hippocampal tissues. The behavioral test showed that melatonin ameliorated anxiety and depression-like behaviors of 3xTg-AD mice as measured by open field test, elevated plus maze test, forced swimming test, and tail suspension test. By carrying out two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, we revealed a total of 46 differentially expressed proteins in hippocampus between the wild-type (WT) mice and non-treated 3xTg-AD mice. A total of 21 differentially expressed proteins were revealed in hippocampus between melatonin-treated and non-treated 3xTg-AD mice. Among these differentially expressed proteins, glutathione S-transferase P 1 (GSTP1) (an anxiety-associated protein) and complexin-1 (CPLX1) (a depression-associated protein) were significantly down-regulated in hippocampus of 3xTg-AD mice compared with the WT mice. The expression of these two proteins was modulated by melatonin treatment. Our study suggested that melatonin could be used as a potential candidate drug to improve the neuropsychiatric behaviors in AD via modulating the expression of the proteins (i.e. GSTP1 and CPLX1) involved in anxiety and depression behaviors. © 2017 BioFactors, 43(4):593-611, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Differences in osmotolerance in freshwater and brackish water populations of Theodoxus fluviatilis (Gastropoda: Neritidae) are associated with differential protein expression.

PubMed

Symanowski, Frauke; Hildebrandt, Jan-Peter

2010-03-01

The euryhaline gastropod Theodoxus fluviatilis is found in northern Germany in freshwater or in brackish water habitats in the Baltic Sea. Previous studies have revealed that individuals from both habitats are not distinguishable by morphological characters or by sequence comparison of DNA encoding 16S RNA or cytochrome C. As reported in this study, animals collected in the two habitats differ substantially in their physiological ability to adapt to different salinities. Comparison of accumulation rates of ninhydrin-positive substances (NPS) in foot muscle upon transfer of animals to higher medium salinities revealed that brackish water animals were perfectly able to mobilize NPS, while freshwater animals had only limited ability to do so. In an attempt to explore whether this difference in physiology may be caused by genetic differentiation, we compared protein expression patterns of soluble foot muscle proteins using 2D gel electrophoresis and silver staining. Of the 40 consistently detected protein spots, 27 showed similar levels in protein expression in animals collected from freshwater or brackish water habitats, respectively. In 12 spots, however, protein concentration was higher in brackish water than in freshwater animals. In four of these spots, expression levels followed increases or decreases in medium salinities. In a different set of 4 of these 12 spots, protein levels were always higher in brackish water as compared to freshwater animals, regardless of their physiological situation (14 days in artificial pond water or in medium with a salinity of 16 per thousand). The remaining 4 of the 12 spots had complex expression patterns. Protein levels of the remaining single spot were generally higher in freshwater animals than in brackish water animals. These expression patterns may indicate that freshwater and brackish water animals of T. fluviatilis belong to different locally adapted populations with subtle genetic differentiation.

In Vivo Genome-Wide Expression Study on Human Circulating B Cells Suggests a Novel ESR1 and MAPK3 Network for Postmenopausal Osteoporosis

PubMed Central

Xiao, Peng; Chen, Yuan; Jiang, Hui; Liu, Yao-Zhong; Pan, Feng; Yang, Tie-Lin; Tang, Zi-Hui; Larsen, Jennifer A; Lappe, Joan M; Recker, Robert R; Deng, Hong-Wen

2008-01-01

Introduction Osteoporosis is characterized by low BMD. Studies have shown that B cells may participate in osteoclastogenesis through expression of osteoclast-related factors, such as RANKL, transforming growth factor β (TGFB), and osteoprotegerin (OPG). However, the in vivo significance of B cells in human bone metabolism and osteoporosis is still largely unknown, particularly at the systematic gene expression level. Materials and Methods In this study, Affymetrix HG-U133A GeneChip arrays were used to identify genes differentially expressed in B cells between 10 low and 10 high BMD postmenopausal women. Significance of differential expression was tested by t-test and adjusted for multiple testing with the Benjamini and Hochberg (BH) procedure (adjusted p ≤ 0.05). Results Twenty-nine genes were downregulated in the low versus high BMD group. These genes were further analyzed using Ingenuity Pathways Analysis (Ingenuity Systems). A network involving estrogen receptor 1 (ESR1) and mitogen activated protein kinase 3 (MAPK3) was identified. Real-time RT-PCR confirmed differential expression of eight genes, including ESR1, MAPK3, methyl CpG binding protein 2 (MECP2), proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1), Scr-like-adaptor (SLA), serine/threonine kinase 11 (STK11), WNK lysine-deficient protein kinase 1 (WNK1), and zinc finger protein 446 (ZNF446). Conclusions This is the first in vivo genome-wide expression study on human B cells in relation to osteoporosis. Our results highlight the significance of B cells in the etiology of osteoporosis and suggest a novel mechanism for postmenopausal osteoporosis (i.e., that downregulation of ESR1 and MAPK3 in B cells regulates secretion of factors, leading to increased osteoclastogenesis or decreased osteoblastogenesis). PMID:18433299

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

PubMed

Ma, Yu-Hua; Ye, Gui-Sheng

2018-06-11

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Gene expression profiling reveals different molecular patterns in G-protein coupled receptor signaling pathways between early- and late-onset preeclampsia.

PubMed

Liang, Mengmeng; Niu, Jianmin; Zhang, Liang; Deng, Hua; Ma, Jian; Zhou, Weiping; Duan, Dongmei; Zhou, Yuheng; Xu, Huikun; Chen, Longding

2016-04-01

Early-onset preeclampsia and late-onset preeclampsia have been regarded as two different phenotypes with heterogeneous manifestations; To gain insights into the pathogenesis of the two traits, we analyzed the gene expression profiles in preeclamptic placentas. A whole genome-wide microarray was used to determine the gene expression profiles in placental tissues from patients with early-onset (n = 7; <34 weeks), and late-onset (n = 8; >36 weeks) preeclampsia and their controls who delivered preterm (n = 5; <34 weeks) or at term (n = 5; >36 weeks). Genes were termed differentially expressed if they showed a fold-change ≥ 2 and q-value < 0.05. Quantitative real-time reverse transcriptase PCR was used to verify the results. Western blotting was performed to verify the expressions of secreted genes at the protein level. Six hundred twenty-seven genes were differentially expressed in early-compared with late-onset preeclampsia (177 genes were up-regulated and 450 were down-regulated). Gene ontology analysis identified significant alterations in several biological processes; the top two were immune response and cell surface receptor linked signal transduction. Among the cell surface receptor linked signal transduction-related, differentially expressed genes, those involved in the G-protein coupled receptor protein signaling pathway were significantly enriched. G-protein coupled receptor signaling pathway related genes, such as GPR124 and MRGPRF, were both found to be down-regulated in early-onset preeclampsia. The results were consistent with those of western blotting that the abundance of GPR124 was lower in early-onset compared with late-onset preeclampsia. The different gene expression profiles reflect the different levels of transcription regulation between the two conditions and supported the hypothesis that they are separate disease entities. Moreover, the G-protein coupled receptor signaling pathway related genes may contribute to the mechanism underlying early- and late-onset preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modulation of Gene Expression in Contextual Fear Conditioning in the Rat

PubMed Central

Macchi, Monica; Ciampini, Cristina; Bernardi, Rodolfo; Baldi, Elisabetta; Bucherelli, Corrado; Brunelli, Marcello; Scuri, Rossana

2013-01-01

In contextual fear conditioning (CFC) a single training leads to long-term memory of context-aversive electrical foot-shocks association. Mid-temporal regions of the brain of trained and naive rats were obtained 2 days after conditioning and screened by two-directional suppression subtractive hybridization. A pool of differentially expressed genes was identified and some of them were randomly selected and confirmed with qRT-PCR assay. These transcripts showed high homology for rat gene sequences coding for proteins involved in different cellular processes. The expression of the selected transcripts was also tested in rats which had freely explored the experimental apparatus (exploration) and in rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make the association between painful stimuli and the apparatus difficult (shock-only). Some genes resulted differentially expressed only in the rats subjected to CFC, others only in exploration or shock-only rats, whereas the gene coding for translocase of outer mitochondrial membrane 20 protein and nardilysin were differentially expressed in both CFC and exploration rats. For example, the expression of stathmin 1 whose transcripts resulted up regulated was also tested to evaluate the transduction and protein localization after conditioning. PMID:24278235

Differential display cloning of a novel rat cDNA (RNB6) that shows high expression in the neonatal brain revealed a member of Ena/VASP family.

PubMed

Ohta, S; Mineta, T; Kimoto, M; Tabuchi, K

1997-08-18

We have used the differential display method to identify genes that control the neural cell development in CNS. Screening of the differential display bands that showed higher expression at neonate than at adult age enabled us to identify a novel rat cDNA (RNB6) coding for a protein of 393 amino acid residues. Database search revealed this gene as a rat homologue of the murine EVL, a member of Ena/VASP protein family that is implicated to be involved in the control of cell motility through actin filament assembly by their GP5 motifs. Although the precise characterization of EVL was not reported, our Northern blot and immunoblot analyses demonstrated that RNB6 expression in the brain gradually increases during embryonic development, reaches maximum at postnatal day 1 and decreases thereafter. Studies of tissue distribution revealed the expression of RNB6 not only in the brain but also in the spleen, thymus and testis. Histochemical analyses showed that RNB6 protein is mainly expressed in neurons and may be expressed in neural fibers. Our analyses suggest that RNB6 is critically involved in the development of CNS probably through the control of neural cell motility and/or including neuronal fiber extension.

UV laser radiation alters the embryonic protein profile of adrenal-kidney-gonadal complex and gonadal differentiation in the lizard, Calotes Versicolor.

PubMed

Khodnapur, Bharati S; Inamdar, Laxmi S; Nindi, Robertraj S; Math, Shivkumar A; Mulimani, B G; Inamdar, Sanjeev R

2015-02-01

To examine the impact of ultraviolet (UV) laser radiation on the embryos of Calotes versicolor in terms of its effects on the protein profile of the adrenal-kidney-gonadal complex (AKG), sex determination and differentiation, embryonic development and hatching synchrony. The eggs of C. versicolor, during thermo-sensitive period (TSP), were exposed to third harmonic laser pulses at 355 nm from a Q-switched Nd:YAG laser for 180 sec. Subsequent to the exposure they were incubated at the male-producing temperature (MPT) of 25.5 ± 0.5°C. The AKG of hatchlings was subjected to protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and to histology. The UV laser radiation altered the expression of the protein banding pattern in the AKG complex of hatchlings and it also affected the gonadal sex differentiation. SDS-PAGE of AKG of one-day-old hatchlings revealed a total of nine protein bands in the control group whereas UV laser irradiated hatchlings expressed a total of seven protein bands only one of which had the same Rf as a control band. The UV laser treated hatchlings have an ovotestes kind of gonad exhibiting a tendency towards femaleness instead of the typical testes. It is inferred that 355 nm UV laser radiation during TSP induces changes in the expression of proteins as well as their secretions. UV laser radiation had an impact on the gonadal differentiation pathway but no morphological anomalies were noticed.

Proteomic profiling for plasma biomarkers of tuberculosis progression.

PubMed

Liu, Qiuyue; Pan, Liping; Han, Fen; Luo, Baojian; Jia, Hongyan; Xing, Aiying; Li, Qi; Zhang, Zongde

2018-06-05

Severe pulmonary tuberculosis (STB) is a life‑threatening condition with high economic and social burden. The present study aimed to screen for distinct proteins in different stages of TB and identify biomarkers for a better understanding of TB progression and pathogenesis. Blood samples were obtained from 81 patients with STB, 80 with mild TB (MTB) and 50 healthy controls. Differentially expressed proteins were identified using liquid chromatography‑tandem mass spectrometry‑based label‑free quantitative proteomic analysis. Functional and pathway enrichment analyses were performed for the identified proteins. The expression of potential biomarkers was further validated by western blot analysis and enzyme‑linked immunosorbent assays. The accuracy, sensitivity and specificity for selected protein biomarkers in diagnosing STB were also evaluated. A total of 1,011 proteins were identified in all three groups, and 153 differentially expressed proteins were identified in patients with STB. These proteins were involved in 'cellular process', 'response to stimulus', 'apoptotic process', 'immune system process' and 'select metabolic process'. Significant differences in protein expression were detected in α‑1‑acid glycoprotein 2 (ORM2), interleukin‑36α (IL‑36α), S100 calcium binding protein A9 (S100‑A9), superoxide dismutase (SOD)1 in the STB group, compared with the MTB and control groups. The combination of plasma ORM2, IL‑36α, S100A9 and SOD1 levels achieved 90.00% sensitivity and 92.16% specificity to discriminate between patients with STB and MTB, and 89.66% sensitivity and 98.9% specificity to discriminate between patients with STB and healthy controls. ORM2, S100A9, IL‑36α and SOD1 were associated with the development of TB, and have the potential to distinguish between different stages of TB. Differential protein expression during disease progression may improve the current understanding of STB pathogenesis.

Coatomer subunit beta 2 (COPB2), identified by label-free quantitative proteomics, regulates cell proliferation and apoptosis in human prostate carcinoma cells.

PubMed

Mi, Yuanyuan; Sun, Chuanyu; Wei, Bingbing; Sun, Feiyu; Guo, Yijun; Hu, Qingfeng; Ding, Weihong; Zhu, Lijie; Xia, Guowei

2018-01-01

Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G 1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA. Copyright © 2017 Elsevier Inc. All rights reserved.

Skeletal muscle tissue transcriptome differences in lean and obese female beagle dogs.

PubMed

Grant, R W; Vester Boler, B M; Ridge, T K; Graves, T K; Swanson, K S

2013-08-01

Skeletal muscle is a large and insulin-sensitive tissue that is an important contributor to metabolic homeostasis and energy expenditure. Many metabolic processes are altered with obesity, but the contribution of muscle tissue in this regard is unclear. A limited number of studies have compared skeletal muscle gene expression of lean and obese dogs. Using microarray technology, our objective was to identify genes and functional classes differentially expressed in skeletal muscle of obese (14.6 kg; 8.2 body condition score; 44.5% body fat) vs. lean (8.6 kg; 4.1 body condition score; 22.9% body fat) female beagle adult dogs. Alterations in 77 transcripts was observed in genes pertaining to the functional classes of signaling, transport, protein catabolism and proteolysis, protein modification, development, transcription and apoptosis, cell cycle and differentiation. Genes differentially expressed in obese vs. lean dog skeletal muscle indicate oxidative stress and altered skeletal muscle cell differentiation. Many genes traditionally associated with lipid, protein and carbohydrate metabolism were not altered in obese vs. lean dogs, but genes pertaining to endocannabinoid metabolism, insulin signaling, type II diabetes mellitus and carnitine transport were differentially expressed. The relatively small response of skeletal muscle could indicate that changes are occurring at a post-transcriptional level, that other tissues (e.g., adipose tissue) were buffering skeletal muscle from metabolic dysfunction or that obesity-induced changes in skeletal muscle require a longer period of time and that the length of our study was not sufficient to detect them. Although only a limited number of differentially expressed genes were detected, these results highlight genes and functional classes that may be important in determining the etiology of obesity-induced derangement of skeletal muscle function. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

PTEN loss represses glioblastoma tumor initiating cell differentiation via inactivation of Lgl1.

PubMed

Gont, Alexander; Hanson, Jennifer E L; Lavictoire, Sylvie J; Parolin, Doris A; Daneshmand, Manijeh; Restall, Ian J; Soucie, Mathieu; Nicholas, Garth; Woulfe, John; Kassam, Amin; Da Silva, Vasco F; Lorimer, Ian A J

2013-08-01

Glioblastoma multiforme is an aggressive and incurable type of brain tumor. A subset of undifferentiated glioblastoma cells, known as glioblastoma tumor initiating cells (GTICs), has an essential role in the malignancy of this disease and also appears to mediate resistance to radiation therapy and chemotherapy. GTICs retain the ability to differentiate into cells with reduced malignant potential, but the signaling pathways controlling differentiation are not fully understood at this time. PTEN loss is a very common in glioblastoma multiforme and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Increased signalling through this pathway leads to activation of multiple protein kinases, including atypical protein kinase C. In Drosophila, active atypical protein kinase C has been shown to promote the self-renewal of neuroblasts, inhibiting their differentiation along a neuronal lineage. This effect is mediated by atypical protein kinase c-mediated phosphorylation and inactivation of Lgl, a protein that was first characterized as a tumour suppressor in Drosophila. The effects of the atypical protein kinase C/Lgl pathway on the differentiation status of GTICs, and its potential link to PTEN loss, have not been assessed previously. Here we show that PTEN loss leads to the phosphorylation and inactivation of Lgl by atypical protein kinase C in glioblastoma cells. Re-expression of PTEN in GTICs promoted their differentiation along a neuronal lineage. This effect was also seen when atypical protein kinase C was knocked down using RNA interference, and when a non-phosphorylatable, constitutively active form of Lgl was expressed in GTICs. Thus PTEN loss, acting via atypical protein kinase C activation and Lgl inactivation, helps to maintain GTICs in an undifferentiated state.

Annotated Differentially Expressed Salivary Proteins of Susceptible and Insecticide-Resistant Mosquitoes of Anopheles stephensi

PubMed Central

Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Raghavendra, Kamaraju; Sharma, Arun

2015-01-01

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito—parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins may play a role in regulating mosquito biting behavior patterns and may have implications in the development of malaria parasites in resistant mosquitoes during parasite transmission. PMID:25742511

Annotated differentially expressed salivary proteins of susceptible and insecticide-resistant mosquitoes of Anopheles stephensi.

PubMed

Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Raghavendra, Kamaraju; Sharma, Arun

2015-01-01

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito-parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins may play a role in regulating mosquito biting behavior patterns and may have implications in the development of malaria parasites in resistant mosquitoes during parasite transmission.

The C. elegans SoxC protein SEM-2 opposes differentiation factors to promote a proliferative blast cell fate in the postembryonic mesoderm

PubMed Central

Tian, Chenxi; Shi, Herong; Colledge, Clark; Stern, Michael; Waterston, Robert; Liu, Jun

2011-01-01

The proper development of multicellular organisms requires precise regulation and coordination of cell fate specification, cell proliferation and differentiation. Abnormal regulation and coordination of these processes could lead to disease, including cancer. We have examined the function of the sole C. elegans SoxC protein, SEM-2, in the M lineage, which produces the postembryonic mesoderm. We found that SEM-2/SoxC is both necessary and sufficient to promote a proliferating blast cell fate, the sex myoblast fate, over a differentiated striated bodywall muscle fate. A number of factors control the specific expression of sem-2 in the sex myoblast precursors and their descendants. This includes direct control of sem-2 expression by a Hox-PBC complex. The crucial nature of the HOX/PBC factors in directly enhancing expression of this proliferative factor in the C. elegans M lineage suggests a possible more general link between Hox-PBC factors and SoxC proteins in regulating cell proliferation. PMID:21307099

PMA Induces SnoN Proteolysis and CD61 Expression through an Autocrine Mechanism

PubMed Central

Li, Chonghua; Peart, Natoya; Xuan, Zhenyu; Lewis, Dorothy E; Xia, Yang; Jin, Jianping

2014-01-01

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-β family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APCCdh1 ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation. PMID:24637302

The caspase-1 inhibitor CARD18 is specifically expressed during late differentiation of keratinocytes and its expression is lost in lichen planus.

PubMed

Qin, Haihong; Jin, Jiang; Fischer, Heinz; Mildner, Michael; Gschwandtner, Maria; Mlitz, Veronika; Eckhart, Leopold; Tschachler, Erwin

2017-08-01

CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.

PubMed

Roberts, Thomas C; Johansson, Henrik J; McClorey, Graham; Godfrey, Caroline; Blomberg, K Emelie M; Coursindel, Thibault; Gait, Michael J; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A

2015-12-01

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle. © The Author 2015. Published by Oxford University Press.

Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

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

Kodavanti, Prasada Rao S., E-mail: kodavanti.prasada@epa.gov; Osorio, Cristina; Program on Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, North Carolina

2011-11-15

The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca{sup 2+}-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studiesmore » showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0 mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit {beta} (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity. -- Highlights: Black-Right-Pointing-Pointer We performed brain proteomic analysis of rats exposed to the neurotoxicant, Aroclor 1254. Black-Right-Pointing-Pointer Cerebellum and hippocampus were analyzed by 2D DIGE and Mass spectrometry. Black-Right-Pointing-Pointer Proteins affected participate in Energy metabolism, calcium signaling and nervous system growth.« less

Gene expression profiling in the hippocampus of learned helpless and nonhelpless rats.

PubMed

Kohen, R; Kirov, S; Navaja, G P; Happe, H Kevin; Hamblin, M W; Snoddy, J R; Neumaier, J F; Petty, F

2005-01-01

In the learned helplessness (LH) animal model of depression, failure to attempt escape from avoidable environmental stress, LH, indicates behavioral despair, whereas nonhelpless (NH) behavior reflects behavioral resilience to the effects of environmental stress. Comparing hippocampal gene expression with large-scale oligonucleotide microarrays, we found that stress-resilient (NH) rats, although behaviorally indistinguishable from controls, showed a distinct gene expression profile compared to LH, sham stressed, and naïve control animals. Genes that were confirmed as differentially expressed in the NH group by quantitative PCR strongly correlated in their levels of expression across all four animal groups. Differential expression could not be confirmed at the protein level. We identified several shared degenerate sequence motifs in the 3' untranslated region (3'UTR) of differentially expressed genes that could be a factor in this tight correlation of expression levels among differentially expressed genes.

Proteomic Profiling of Liver and Plasma in Chronic Ethanol Feeding Model of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-10-01

Chronic alcohol abuse, a major risk factor for such diseases as hepatitis and cirrhosis, impairs hepatic alcohol dehydrogenase (ADH; key ethanol [EtOH]-metabolizing enzyme). Therefore, differentially altered hepatic and plasma proteomes were identified in chronic EtOH feeding model of hepatic ADH-deficient (ADH - ) deer mice to understand the metabolic basis of alcoholic liver disease (ALD). ADH - deer mice were fed 3.5 g% EtOH via Lieber-DeCarli liquid diet daily for 3 months and histology of the liver assessed. Liver and plasma proteins were separated by 2-dimensional gel electrophoresis. The proteins differentially expressed were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Histology of the liver showed panlobular steatosis and infiltration of T lymphocytes. Using the criteria of ≥1.5 for fold change (p-value ≤0.05) with expectation value (E ≤10 -3 ) and protein score (≥64), 18 proteins in the livers and 5 in the plasma of EtOH-fed mice were differentially expressed and identified. Prolyl 4-hydroxylase, cytochrome b-5, endo A cytokeratin, ATP synthase, heat-shock 70 kD proteins, enoyl CoA hydratase, stress-70 protein, peroxiredoxin 1, and ornithine carbamoyl transferase were up-regulated in the livers. However, carbonic anhydrase 3, mitochondrial ATP synthase, aldolase 2, actin γ, laminin receptor, and carbamoyl phosphate synthase were down-regulated. Contrary to the increased expression of creatine kinase M-type, a decreased expression of serine protease inhibitor A3A precursor, sulfated glycoprotein-2 (clusterin), and apolipoprotein E isoforms were found in the plasma of EtOH group. Chronic EtOH feeding in ADH - deer mice causes steatosis and infiltration of T lymphocytes in the livers along with increased expression of proteins involved in endoplasmic reticulum (ER) stress, fibrosis, fatty acid β oxidation and biogenesis, and decreased expression of proteins involved in ATP synthesis, carbohydrate metabolism, in cell regulation and architecture. Reduced expression of various carrier proteins as found in the plasma of EtOH group has a biomarker potential. Copyright © 2017 by the Research Society on Alcoholism.

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

PubMed

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

2014-05-01

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

Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice

NASA Technical Reports Server (NTRS)

Dodig, M.; Kronenberg, M. S.; Bedalov, A.; Kream, B. E.; Gronowicz, G.; Clark, S. H.; Mack, K.; Liu, Y. H.; Maxon, R.; Pan, Z. Z.;

Quantitative proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in Down syndrome

PubMed Central

2013-01-01

Background Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Results Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression. Conclusions The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21. PMID:23394617

Proteomic analysis of PSD-93 knockout mice following the induction of ischemic cerebral injury.

PubMed

Rong, Rong; Yang, Hui; Rong, Liangqun; Wei, Xiue; Li, Qingjie; Liu, Xiaomei; Gao, Hong; Xu, Yun; Zhang, Qingxiu

2016-03-01

Postsynaptic density protein-93 (PSD-93) is enriched in the postsynaptic density and is involved in N-methyl-d-aspartate receptor (NMDAR) triggered neurotoxicity through PSD-93/NMDAR/nNOS signaling pathway. In the present study, we found that PSD-93 deficiency reduced infarcted volume and neurological deficits induced by transient middle cerebral artery occlusion (tMCAO) in the mice. To identify novel targets of PSD-93 related neurotoxicity, we applied isobaric tags for relative and absolute quantitative (iTRAQ) labeling and combined this labeling with on-line two-dimensional LC/MS/MS technology to elucidate the changes in protein expression in PSD-93 knockout mice following tMCAO. The proteomic data set consisted of 1892 proteins. Compared to control group, differences in expression levels in ischemic group >1.5-fold and <0.66-fold were considered as differential expression. A total of 104 unique proteins with differential abundance levels were identified, among which 17 proteins were selected for further validation. Gene ontology analysis using UniProt database revealed that these differentially expressed proteins are involved in diverse function such as synaptic transmission, neuronal neurotransmitter and ion transport, modification of organelle membrane components. Moreover, network analysis revealed that the interacting proteins were involved in the transport of synaptic vesicles, the integrity of synaptic membranes and the activation of the ionotropic glutamate receptors NMDAR1 and NMDAR2B. Finally, RT-PCR and Western blot analysis showed that SynGAP, syntaxin-1A, protein kinase C β, and voltage-dependent L-type calcium channels were inhibited by ischemia-reperfusion. Identification of these proteins provides valuable clues to elucidate the mechanisms underlying the actions of PSD-93 in ischemia-reperfusion induced neurotoxicity. Copyright © 2015 Elsevier Inc. All rights reserved.

6-Gingerol Suppresses Adipocyte-Derived Mediators of Inflammation In Vitro and in High-Fat Diet-Induced Obese Zebra Fish.

PubMed

Choi, Jia; Kim, Kui-Jin; Kim, Byung-Hak; Koh, Eun-Jeong; Seo, Min-Jung; Lee, Boo-Yong

2017-02-01

The present study was performed to investigate the molecular mechanism of 6-gingerol on adipocyte-mediated systemic inflammation in vitro and in high-fat diet-induced obese zebra fish. 6-Gingerol decreased adipogenesis due to the suppression of adipocyte differentiation markers, including peroxisome proliferator-activated receptor gamma, CCAATT enhancer binding protein α , and adipocyte protein 2, and triglyceride synthesis enzymes, including sterol regulatory element-binding protein-1, fatty acid synthase, lysophosphatidic acid acyltransferase, and acyl-coA : diacylglycerol acyltransferase 1, in 3T3-L1. A coculture insert system using 3T3-L1 with RAW 264.7 (coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages) revealed that 6-gingerol increased anti-inflammatory cytokine interleukin-10. The expression of TNF α , monocyte chemotactic protein-1, interleukin-1 β , and interleukin-6 were decreased in the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages treated with 6-gingerol. Moreover, the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages treated with 6-gingerol inhibited the protein expression of TNF α and monocyte chemotactic protein-1 in RAW 264.7. 6-Gingerol decreased c-JUN N-terminal kinase and I kappa B kinase beta and its downstream target AP-1 expression in the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages. Furthermore, 6-gingerol decreased the expression of inducible nitric oxide synthase stimulated by the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages in RAW 264.7 and attenuated nitric oxide production in diet-induced obese zebra fish. Our results suggest that 6-gingerol suppresses inflammation through the regulation of the c-JUN N-terminal kinase-I kappa B kinase beta and its downstream targets. Georg Thieme Verlag KG Stuttgart · New York.

Differential regulation of oligodendrocyte markers by glucocorticoids: Post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase

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

Kumar, S.; Cole, R.; Chiappelli, F.

During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expression of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase. The actual locus at which hydrocortisone exerts its developmental influence on glial physiology is, however, not well understood. Gycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2- to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunostaining demonstrates that this increase inmore » mRNAs is followed by a 2- to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogeneis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving post-transcriptional regulation.« less

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

PubMed

Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L. interrogans to adapt to conditions encountered in the host and to cause disease. Our results suggest down-regulation of protein expression in response to temperature, and decreased expression of outer membrane proteins may facilitate minimal interaction with host immune mechanisms.

Nandrolone, an anabolic steroid, stabilizes Numb protein through inhibition of mdm2 in C2C12 myoblasts.

PubMed

Liu, Xin-Hua; Yao, Shen; Levine, Alice C; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Collier, Lauren; Bauman, William A; Cardozo, Christopher P

2012-01-01

Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.

Pleiotrophin Expression during Odontogenesis

PubMed Central

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

2012-01-01

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

Supplementation with tocotrienol-rich fraction alters the plasma levels of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor from young and old individuals.

PubMed

Heng, Eng Chee; Karsani, Saiful Anuar; Abdul Rahman, Mariati; Abdul Hamid, Noor Aini; Hamid, Zalina; Wan Ngah, Wan Zurinah

2013-10-01

Tocotrienol possess beneficial effects not exhibited by tocopherol. In vitro studies using animal models have suggested that these effects are caused via modulation of gene and protein expression. However, human supplementation studies using tocotrienol-rich isomers are limited. This study aims to identify plasma proteins that changed in expression following tocotrienol-rich fraction (TRF) supplementation within two different age groups. Subjects were divided into two age groups-32 ± 2 (young) and 52 ± 2 (old) years old. Four subjects from each group were assigned with TRF (78% tocotrienol and 22% tocopherol, 150 mg/day) or placebo capsules for 6 months. Fasting plasma were obtained at 0, 3, and 6 months. Plasma tocopherol and tocotrienol levels were determined. Plasma proteome was resolved by 2DE, and differentially expressed proteins identified by MS. The expressions of three proteins were validated by Western blotting. Six months of TRF supplementation significantly increased plasma levels of tocopherols and tocotrienols. Proteins identified as being differentially expressed were related to cholesterol homeostasis, acute-phase response, protease inhibitor, and immune response. The expressions of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor were validated. The old groups showed more proteins changing in expression. TRF appears to not only affect plasma levels of tocopherols and tocotrienols, but also the levels of plasma proteins. The identity of these proteins may provide insights into how TRF exerts its beneficial effects. They may also be potentially developed into biomarkers for the study of the effects and effectiveness of TRF supplementation.

All-Trans-Retinoic Acid Stimulates Overexpression of Tumor Protein D52 (TPD52, Isoform 3) and Neuronal Differentiation of IMR-32 Cells.

PubMed

Kotapalli, Sudha Sravanti; Dasari, Chandrashekhar; Duscharla, Divya; Kami Reddy, Karthik Reddy; Kasula, Manjula; Ummanni, Ramesh

2017-12-01

Tumor protein D52 (TPD52), a proto-oncogene is overexpressed in a variety of epithelial carcinomas and plays an important role in cell proliferation, migration, and cell death. In the present study we found that the treatment of IMR-32 neuroblastoma (NB) cells with retinoic acid (RA) stimulates an increase in expression of TPD52. TPD52 expression is detectable after 72 h, can be maintained till differentiation of NB cells suggesting that TPD52 is involved in differentiation. Here, we demonstrate that TPD52 is essential for RA to promote differentiation of NB cells. Our results show that exogenous expression of EGFP-TPD52 in IMR-32 cells resulted cell differentiation even without RA. RA by itself and with overexpression of TPD52 can increase the ability of NB cells differentiation. Interestingly, transfection of IMR-32 cells with a specific small hairpin RNA for efficient knockdown of TPD52 attenuated RA induced NB cells differentiation. Transcriptional and translational level expression of neurotropic (BDNF, NGF, Nestin) and differentiation (β III tubulin, NSE, TH) factors in NB cells with altered TPD52 expression and/or RA treatment confirmed essential function of TPD52 in cellular differentiation. Furthermore, we show that TPD52 protects cells from apoptosis and arrest cell proliferation by varying expression of p27Kip1, activation of Akt and ERK1/2 thus promoting cell differentiation. Additionally, inhibition of STAT3 activation by its specific inhibitor arrested NB cells differentiation by EGFP-TPD52 overexpression with or without RA. Taken together, our data reveal that TPD52 act through activation of JAK/STAT signaling pathway to undertake NB cells differentiation induced by RA. J. Cell. Biochem. 118: 4358-4369, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Paradoxical Role of DNA Methylation in Activation of FoxA2 Gene Expression during Endoderm Development*

PubMed Central

Bahar Halpern, Keren; Vana, Tal; Walker, Michael D.

2014-01-01

The transcription factor FoxA2 is a master regulator of endoderm development and pancreatic beta cell gene expression. To elucidate the mechanisms underlying the activation of the FoxA2 gene during differentiation, we have compared the epigenetic status of undifferentiated human embryonic stem cells (hESCs), hESC-derived early endoderm stage cells (CXCR4+ cells), and pancreatic islet cells. Unexpectedly, a CpG island in the promoter region of the FoxA2 gene displayed paradoxically high levels of DNA methylation in expressing tissues (CXCR4+, islets) and low levels in nonexpressing tissues. This CpG island region was found to repress reporter gene expression and bind the Polycomb group protein SUZ12 and the DNA methyltransferase (DNMT)3b preferentially in undifferentiated hESCs as compared with CXCR4+ or islets cells. Consistent with this, activation of FoxA2 gene expression, but not CXCR4 or SOX17, was strongly inhibited by 5-aza-2′-deoxycytidine and by knockdown of DNMT3b. We hypothesize that in nonexpressing tissues, the lack of DNA methylation allows the binding of DNA methyltransferases and repressing proteins, such as Polycomb group proteins; upon differentiation, DNMT activation leads to CpG island methylation, causing loss of repressor protein binding. These results suggest a novel and unexpected role for DNA methylation in the activation of FoxA2 gene expression during differentiation. PMID:25016019

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing

PubMed Central

Han, Qianqian; Yang, Pishan; Wu, Yuwei; Meng, Shu; Sui, Lei; Zhang, Lan; Yu, Liming; Tang, Yin; Jiang, Hua; Xuan, Dongying; Kaplan, David L.; Kim, Sung Hoon

2015-01-01

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds. PMID:25923143

Expression of receptor protein tyrosine kinase tif is regulated during leukemia cell differentiation.

PubMed

Dai, W; Pan, H Q; Ouyang, B; Greenberg, J M; Means, R T; Li, B; Cardie, J

1996-06-01

tif is a recently cloned and characterized cDNA predicting a transmembrane protein with a putative tyrosine kinase structure in its cytoplasmic domain. By analysis of the purified tif cytoplasmic domain expressed in Escherichia coli, we have demonstrated that tif is an active protein tyrosine kinase capable of autophosphorylation on tyrosine residues and this phosphorylation is inhibited by a tyrosine-specific inhibitor genistein. Northern blot analyses of various leukemia cell lines have revealed that tif mRNA expression is primarily confined to those bearing erythroid and megakaryocytic phenotypes. Megakaryocytic differentiation of K562 and HEL cells induced by phorbol 12-myristate 13-acetate is accompanied by down-regulation of tif mRNA expression. In addition, treatment of K562 and HEL with hexamethylene bis-acetamide, but not with hemin, decreases the steady-state level of tif mRNA. These combined results suggest that the receptor tyrosine kinase tif is involved in hematopoietic development.

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

PubMed Central

Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

2015-01-01

Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

Identification of differentially expressed genes of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) in response to Tetracapsuloides bryosalmonae (Myxozoa).

PubMed

Kumar, Gokhlesh; Abd-Elfattah, Ahmed; El-Matbouli, Mansour

2015-03-01

Tetracapsuloides bryosalmonae Canning et al., 1999 (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids in Europe and North America. We have shown previously that the development and distribution of the European strain of T. bryosalmonae differs in the kidney of brown trout (Salmo trutta) Linnaeus, 1758 and rainbow trout (Oncorhynchus mykiss) Walbaum, 1792, and that intra-luminal sporogonic stages were found in brown trout but not in rainbow trout. We have now compared transcriptomes from kidneys of brown trout and rainbow trout infected with T. bryosalmonae using suppressive subtractive hybridization (SSH). The differentially expressed transcripts produced by SSH were cloned, transformed, and tested by colony PCR. Differential expression screening of PCR products was validated using dot blot, and positive clones having different signal intensities were sequenced. Differential screening and a subsequent NCBI-BLAST analysis of expressed sequence tags revealed nine clones expressed differently between both fish species. These differentially expressed genes were validated by quantitative real-time PCR of kidney samples from both fish species at different time points of infection. Expression of anti-inflammatory (TSC22 domain family protein 3) and cell proliferation (Prothymin alpha) genes were upregulated significantly in brown trout but downregulated in rainbow trout. The expression of humoral immune response (immunoglobulin mu) and endocytic pathway (Ras-related protein Rab-11b) genes were significantly upregulated in rainbow trout but downregulated in brown trout. This study suggests that differential expression of host anti-inflammatory, humoral immune and endocytic pathway responses, cell proliferation, and cell growth processes do not inhibit the development of intra-luminal sporogonic stages of the European strain of T. bryosalmonae in brown trout but may suppress it in rainbow trout.

Vitamin K2 promotes mesenchymal stem cell differentiation by inhibiting miR‑133a expression.

PubMed

Zhang, Yuelei; Weng, Shiyang; Yin, Junhui; Ding, Hao; Zhang, Changqing; Gao, Youshui

2017-05-01

Vitamin K2 has been demonstrated to promote the osteogenic differentiation of mesenchymal stem cells; however, the mechanisms underlying this effect remain unclear. As microRNA (miR)‑133a has been identified as a negative regulator of osteogenic differentiation, the present study hypothesized that vitamin K2 promoted osteogenesis by inhibiting miR‑133a. Using human bone marrow stromal cells (hBMSCs) overexpressing miR‑133a, or a control, the expression levels of osteogenesis‑associated proteins, including runt‑related transcription factor 2, alkaline phosphatase and osteocalcin, were analyzed. miR‑133a significantly suppressed the osteogenic differentiation of hBMSCs. To determine the effect of vitamin K2 on miR‑133a expression and osteogenesis, hBMSCs were treated with vitamin K2. Vitamin K2 inhibited miR‑133a expression, which was accompanied by enhanced osteogenic differentiation. Furthermore, the expression levels of vitamin K epoxide reductase complex subunit 1, the key protein in γ‑carboxylation, were downregulated by miR‑133a overexpression and upregulated by vitamin K2 treatment, indicating a positive feedback on γ‑carboxylation. The results of the present study suggested that vitamin K2 targets miR‑133a to regulate osteogenesis.

Expression of calcium binding protein S100 A7 (psoriasin) in laryngeal carcinoma.

PubMed

Tiveron, Rogério Costa; de Freitas, Luiz Carlos Conti; Figueiredo, David L; Serafini, Luciano N; Mamede, Rui Celso Martins; Zago, Marco A

2012-01-01

Many studies have reported increased expression of S100 A7 (psoriasin) in neoplastic lesions. Among them are studies on breast carcinoma, bladder squamous cell carcinoma, skin tumors and oral cavity squamous cell carcinoma. The expression of S100 A7 has not been described for laryngeal cancer. This study aims to identify the expression of the calcium-binding protein S100 A7 and its correlation with squamous cell carcinomas of the larynx. Specimens from 63 patients were submitted to immunohistochemistry testing with antibody S100 A7. Results were classified and compared. The group with highly differentiated tumors had the highest treatment failure scores. Moderately differentiated tumors had higher treatment failure scores than poorly differentiated tumors. Higher scores were predominantly seen on stages I and II in moderately differentiated tumors, whereas score distribution was more homogeneous in advanced stage disease (III and IV). Regarding failure in treatment, the group scoring zero (3/4 complications: 75%) differed significantly from the remaining groups (13/59: 22%). S100 A7 marker was expressed in 93.7% of laryngeal cancer cases, with higher positive correlation rates in more differentiated tumors and significantly lower rates of treatment failure. Scores had no impact on survival rates.

Differentiation of neuroblastoma cell line N1E-115 involves several signaling cascades.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Joo-ho; Pollak, Arnold; Freilinger, Angelika; Hengstschläger, Markus; Lubec, Gert

2005-03-01

No systematic searches for differential expression of signaling proteins (SP) in undifferentiated vs. differentiated cell lineages were published and herein we used protein profiling for this purpose. The NIE-115 cell line was cultivated and an aliquot was differentiated with dimethylsulfoxide (DMSO), that is known to lead to a neuronal phenotype. Cell lysates were prepared, run on two-dimensional gel electrophoresis followed by MALDI-TOF-TOF identification of proteins and maps of identified SPs were generated. Seven SPs were comparable, 27 SPs: GTP-binding/Ras-related proteins, kinases, growth factors, calcium binding proteins, phosphatase-related proteins were observed in differentiated NIE-115 cells and eight SPs of the groups mentioned above were observed in undifferentiated cells only. Switching-on/off of several individual SPs from different signaling cascades during the differentiation process is a key to understand mechanisms involved. The findings reported herein are challenging in vitro and in vivo studies to confirm a functional role for deranged SPs.

Knock-in strategy at 3'-end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation.

PubMed

Homma, Kohei; Usui, Sumiko; Kaneda, Makoto

2017-03-01

Fluorescent reporter gene knock-in induced pluripotent stem cell (iPSC) lines have been used to evaluate the efficiency of differentiation into specific cell lineages. Here, we report a knock-in strategy for the generation of human iPSC reporter lines in which a 2A peptide sequence and a red fluorescent protein (E2-Crimson) gene were inserted at the termination codon of the cone-rod homeobox (Crx) gene, a photoreceptor-specific transcriptional factor gene. The knock-in iPSC lines were differentiated into fluorescence-expressing cells in 3D retinal differentiation culture, and the fluorescent cells also expressed Crx specifically in the nucleus. We found that the fluorescence intensity was positively correlated with the expression levels of Crx mRNA and that fluorescent cells expressed rod photoreceptor-specific genes in the later stage of differentiation. Finally, we treated the fluorescent cells with DAPT, a Notch inhibitor, and found that DAPT-enhanced retinal differentiation was associated with up-regulation of Crx, Otx2 and NeuroD1, and down-regulation of Hes5 and Ngn2. These suggest that this knock-in strategy at the 3'-end of the target gene, combined with the 2A peptide linked to fluorescent proteins, offers a useful tool for labeling specific cell lineages or monitoring expression of any marker genes without affecting the function of the target gene. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

EB1 protein alteration characterizes sporadic but not ulcerative colitis associated colorectal cancer.

PubMed

Gemoll, Timo; Kollbeck, Sophie L; Karstens, Karl F; Hò, Gia G; Hartwig, Sonja; Strohkamp, Sarah; Schillo, Katharina; Thorns, Christoph; Oberländer, Martina; Kalies, Kathrin; Lehr, Stefan; Habermann, Jens K

2017-08-15

While carcinogenesis in Sporadic Colorectal Cancer (SCC) has been thoroughly studied, less is known about Ulcerative Colitis associated Colorectal Cancer (UCC). This study aimed to identify and validate differentially expressed proteins between clinical samples of SCC and UCC to elucidate new insights of UCC/SCC carcinogenesis and progression. Multiplex-fluorescence two-dimensional gel electrophoresis (2-D DIGE) and mass spectrometry identified 67 proteoforms representing 43 distinct proteins. After analysis by Ingenuity Pathway Analysis ® (IPA), subsequent Western blot validation proofed the differential expression of Heat shock 27 kDA protein 1 (HSPB1) and Microtubule-associated protein R/EB family, member 1 (EB1) while the latter one showed also expression differences by immunohistochemistry. Fresh frozen tissue of UCC ( n = 10) matched with SCC ( n = 10) was investigated. Proteins of cancerous intestinal mucosal cells were obtained by Laser Capture Microdissection (LCM) and compared by 2-D DIGE. Significant spots were identified by mass spectrometry. After IPA, three proteins [EB1, HSPB1, and Annexin 5 (ANXA5)] were chosen for further validation by Western blotting and tissue microarray-based immunohistochemistry. This study identified significant differences in protein expression of colorectal carcinoma cells from UCC patients compared to patients with SCC. Particularly, EB1 was validated in an independent clinical cohort.

Multiway modeling and analysis in stem cell systems biology

PubMed Central

2008-01-01

Background Systems biology refers to multidisciplinary approaches designed to uncover emergent properties of biological systems. Stem cells are an attractive target for this analysis, due to their broad therapeutic potential. A central theme of systems biology is the use of computational modeling to reconstruct complex systems from a wealth of reductionist, molecular data (e.g., gene/protein expression, signal transduction activity, metabolic activity, etc.). A number of deterministic, probabilistic, and statistical learning models are used to understand sophisticated cellular behaviors such as protein expression during cellular differentiation and the activity of signaling networks. However, many of these models are bimodal i.e., they only consider row-column relationships. In contrast, multiway modeling techniques (also known as tensor models) can analyze multimodal data, which capture much more information about complex behaviors such as cell differentiation. In particular, tensors can be very powerful tools for modeling the dynamic activity of biological networks over time. Here, we review the application of systems biology to stem cells and illustrate application of tensor analysis to model collagen-induced osteogenic differentiation of human mesenchymal stem cells. Results We applied Tucker1, Tucker3, and Parallel Factor Analysis (PARAFAC) models to identify protein/gene expression patterns during extracellular matrix-induced osteogenic differentiation of human mesenchymal stem cells. In one case, we organized our data into a tensor of type protein/gene locus link × gene ontology category × osteogenic stimulant, and found that our cells expressed two distinct, stimulus-dependent sets of functionally related genes as they underwent osteogenic differentiation. In a second case, we organized DNA microarray data in a three-way tensor of gene IDs × osteogenic stimulus × replicates, and found that application of tensile strain to a collagen I substrate accelerated the osteogenic differentiation induced by a static collagen I substrate. Conclusion Our results suggest gene- and protein-level models whereby stem cells undergo transdifferentiation to osteoblasts, and lay the foundation for mechanistic, hypothesis-driven studies. Our analysis methods are applicable to a wide range of stem cell differentiation models. PMID:18625054

Differential Expression of Ccn4 and Other Genes Between Metastatic and Non-metastatic EL4 Mouse Lymphoma Cells.

PubMed

Chahal, Manpreet S; Ku, H Teresa; Zhang, Zhihong; Legaspi, Christian M; Luo, Angela; Hopkins, Mandi M; Meier, Kathryn E

Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Decreased RB1 mRNA, Protein, and Activity Reflect Obesity-Induced Altered Adipogenic Capacity in Human Adipose Tissue

PubMed Central

Moreno-Navarrete, José María; Petrov, Petar; Serrano, Marta; Ortega, Francisco; García-Ruiz, Estefanía; Oliver, Paula; Ribot, Joan; Ricart, Wifredo; Palou, Andreu; Bonet, Mª Luisa; Fernández-Real, José Manuel

2013-01-01

Retinoblastoma (Rb1) has been described as an essential player in white adipocyte differentiation in mice. No studies have been reported thus far in human adipose tissue or human adipocytes. We aimed to investigate the possible role and regulation of RB1 in adipose tissue in obesity using human samples and animal and cell models. Adipose RB1 (mRNA, protein, and activity) was negatively associated with BMI and insulin resistance (HOMA-IR) while positively associated with the expression of adipogenic genes (PPARγ and IRS1) in both visceral and subcutaneous human adipose tissue. BMI increase was the main contributor to adipose RB1 downregulation. In rats, adipose Rb1 gene expression and activity decreased in parallel to dietary-induced weight gain and returned to baseline with weight loss. RB1 gene and protein expression and activity increased significantly during human adipocyte differentiation. In fully differentiated adipocytes, transient knockdown of Rb1 led to loss of the adipogenic phenotype. In conclusion, Rb1 seems to play a permissive role for human adipose tissue function, being downregulated in obesity and increased during differentiation of human adipocytes. Rb1 knockdown findings further implicate Rb1 as necessary for maintenance of adipogenic characteristics in fully differentiated adipocytes. PMID:23315497

Chronic ethanol exposure changes dopamine D2 receptor splicing during retinoic acid-induced differentiation of human SH-SY5Y cells.

PubMed

Wernicke, Catrin; Hellmann, Julian; Finckh, Ulrich; Rommelspacher, Hans

2010-01-01

There is evidence for ethanol-induced impairment of the dopaminergic system in the brain during development. The dopamine D2 receptor (DRD2) and the dopamine transporter (DAT) are decisively involved in dopaminergic signaling. Two splice variants of DRD2 are known, with the short one (DRD2s) representing the autoreceptor and the long one (DRD2l) the postsynaptic receptor. We searched for a model to investigate the impact of chronic ethanol exposure and withdrawal on the expression of these proteins during neuronal differentiation. RA-induced differentiation of human neuroblastoma SH-SY5Y cells seems to represent such a model. Our real-time RT-PCR, Western blot, and immunocytochemistry analyses of undifferentiated and RA-differentiated cells have demonstrated the enhanced expression of both splice variants of DRD2, with the short one being stronger enhanced than the long one under RA-treatment, and the DRD2 distribution on cell bodies and neurites under both conditions. In contrast, DAT was down-regulated by RA. The DAT is functional both in undifferentiated and RA-differentiated cells as demonstrated by [(3)H]dopamine uptake. Chronic ethanol exposure during differentiation for up to 4 weeks resulted in a delayed up-regulation of DRD2s. Ethanol withdrawal caused an increased expression of DRD2l and a normalization of DRD2s. Thus the DRD2s/DRD2l ratio was still disturbed. The dopamine level was increased by RA-differentiation compared to controls and was diminished under RA/ethanol treatment and ethanol withdrawal compared to RA-only treated cells. In conclusion, chronic ethanol exposure impairs differentiation-dependent adaptation of dopaminergic proteins, specifically of DRD2s. RA-differentiating SH-SY5Y cells are suited to study the impact of chronic ethanol exposure and withdrawal on expression of dopaminergic proteins during neuronal differentiation.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2*

PubMed Central

Dudakovic, Amel; Camilleri, Emily T.; Xu, Fuhua; Riester, Scott M.; McGee-Lawrence, Meghan E.; Bradley, Elizabeth W.; Paradise, Christopher R.; Lewallen, Eric A.; Thaler, Roman; Deyle, David R.; Larson, A. Noelle; Lewallen, David G.; Dietz, Allan B.; Stein, Gary S.; Montecino, Martin A.; Westendorf, Jennifer J.; van Wijnen, Andre J.

2015-01-01

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production. PMID:26424790

Comparative proteomic analysis of somatic embryo maturation in Carica papaya L.

PubMed Central

2014-01-01

Background Somatic embryogenesis is a complex process regulated by numerous factors. The identification of proteins that are differentially expressed during plant development could result in the development of molecular markers of plant metabolism and provide information contributing to the monitoring and understanding of different biological responses. In addition, the identification of molecular markers could lead to the optimization of protocols allowing the use of biotechnology for papaya propagation and reproduction. This work aimed to investigate the effects of polyethylene glycol (PEG) on somatic embryo development and the protein expression profile during somatic embryo maturation in papaya (Carica papaya L.). Results The maturation treatment supplemented with 6% PEG (PEG6) resulted in the greatest number of somatic embryos and induced differential protein expression compared with cultures grown under the control treatment. Among 135 spots selected for MS/MS analysis, 76 spots were successfully identified, 38 of which were common to both treatments, while 14 spots were unique to the control treatment, and 24 spots were unique to the PEG6 treatment. The identified proteins were assigned to seven categories or were unclassified. The most representative class of proteins observed in the control treatment was associated with the stress response (25.8%), while those under PEG6 treatment were carbohydrate and energy metabolism (18.4%) and the stress response (18.4%). Conclusions The differential expression of three proteins (enolase, esterase and ADH3) induced by PEG6 treatment could play an important role in maturation, and these proteins could be characterized as candidate biomarkers of somatic embryogenesis in papaya. PMID:25076862

Proteomics Analysis of Alfalfa Response to Heat Stress

PubMed Central

Li, Weimin; Wei, Zhenwu; Qiao, Zhihong; Wu, Zinian; Cheng, Lixiang; Wang, Yuyang

2013-01-01

The proteome responses to heat stress have not been well understood. In this study, alfalfa (Medicago sativa L. cv. Huaiyin) seedlings were exposed to 25°C (control) and 40°C (heat stress) in growth chambers, and leaves were collected at 24, 48 and 72 h after treatment, respectively. The morphological, physiological and proteomic processes were negatively affected under heat stress. Proteins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), and differentially expressed protein spots were identified by mass spectrometry (MS). Totally, 81 differentially expressed proteins were identified successfully by MALDI-TOF/TOF. These proteins were categorized into nine classes: including metabolism, energy, protein synthesis, protein destination/storage, transporters, intracellular traffic, cell structure, signal transduction and disease/defence. Five proteins were further analyzed for mRNA levels. The results of the proteomics analyses provide a better understanding of the molecular basis of heat-stress responses in alfalfa. PMID:24324825

Differential global gene expression in red and white skeletal muscle

NASA Technical Reports Server (NTRS)

Campbell, W. G.; Gordon, S. E.; Carlson, C. J.; Pattison, J. S.; Hamilton, M. T.; Booth, F. W.

2001-01-01

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

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

Tsukasaki, Masayuki; Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp; Suzuki, Dai

2011-07-15

Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- andmore » dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.« less

Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

PubMed

Merchant, Mark; Kinney, Clint; Sanders, Paige

2009-12-01

Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation.

PubMed

Balint, Eva; Lapointe, David; Drissi, Hicham; van der Meijden, Caroline; Young, Daniel W; van Wijnen, Andre J; Stein, Janet L; Stein, Gary S; Lian, Jane B

2003-05-15

Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP-2, a member of the TGFbeta superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP-mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP-2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP-2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP-2 and expression was modulated from 3- to 14-fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up- or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP-2 immediately responsive genes (1-4 h), which exhibited either transient or sustained expression, reflect activation and repression of non-osseous BMP-2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2-8 h). Commitment to the osteogenic phenotype is recognized by 8 h, reflected by downregulation of most myogenic-related genes and induction of a spectrum of signaling proteins and enzymes facilitating synthesis and assembly of an extracellular skeletal environment. These genes included collagens Type I and VI and the small leucine rich repeat family of proteoglycans (e.g., decorin, biglycan, osteomodulin, fibromodulin, and osteoadherin/osteoglycin) that reached peak expression at 24 h. With extracellular matrix development, the bone phenotype was further established from 16 to 24 h by induction of genes for cell adhesion and communication and enzymes that organize the bone ECM. Our microarray analysis resulted in the discovery of a class of genes, initially described in relation to differentiation of astrocytes and oligodendrocytes that are functionally coupled to signals for cellular extensions. They include nexin, neuropilin, latexin, neuroglian, neuron specific gene 1, and Ulip; suggesting novel roles for these genes in the bone microenvironment. This global analysis identified a multistage molecular and cellular cascade that supports BMP-2-mediated osteoblast differentiation. Copyright 2003 Wiley-Liss, Inc.

Nitric oxide enhances Oct-4 expression in bone marrow stem cells and promotes endothelial differentiation.

PubMed

Chu, Ling; Jiang, Yuehua; Hao, Hong; Xia, Yong; Xu, Jian; Liu, Zehao; Verfaillie, Catherine M; Zweier, Jay L; Liu, Zhenguo

2008-09-04

This study was designed to investigate the role of nitric oxide (NO) in bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells. Oct-4 expression (both mRNA and protein) was significantly increased by up to 68.0% in MAPCs when incubated with NO donors DETA-NONOate or sodium nitroprusside (SNP) in a concentration-dependant manner (n=3, P<0.05). However, the cell proliferation was dramatically decreased by over 3-folds when treated with DETA-NONOate or SNP for 48 h (n=3, P<0.05). When MAPCs were exposed to DETA-NONOate (100 microM) for the first 48 h during differentiation, the expression (both mRNA and protein) of vWF was significantly increased at day 14 in the differentiating cells. The effects of DETA-NONOate or SNP on cell proliferation, Oct-4 expression and endothelial differentiation of MAPCs were not affected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or cGMP analog 8-Br-cGMP. These data indicate that NO may regulate both the pluripotency and differentiation of MAPCs via a cGMP-independent mechanism.

Fine regulation of RhoA and Rock is required for skeletal muscle differentiation.

PubMed

Castellani, Loriana; Salvati, Erica; Alemà, Stefano; Falcone, Germana

2006-06-02

The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.

A gene expression signature that correlates with CD8+T cell expansion in acute Epstein Barr virus infection1

PubMed Central

Greenough, Thomas C.; Straubhaar, Juerg R.; Kamga, Larisa; Weiss, Eric R.; Brody, Robin M.; McManus, Margaret M.; Lambrecht, Linda K.; Somasundaran, Mohan; Luzuriaga, Katherine F.

2015-01-01

Virus specific CD8+ T cells expand dramatically during acute Epstein Barr virus (EBV) infection, and their persistence is important for lifelong control of EBV-related disease. To better define the generation and maintenance of these effective CD8+ T cell responses, we used microarrays to characterize gene expression in total and EBV-specific CD8+ T cells isolated from the peripheral blood of ten individuals followed from acute infectious mononucleosis (AIM) into convalescence (CONV). In total CD8+ T cells, differential expression of genes in AIM and CONV was most pronounced among those encoding proteins important in T cell activation/differentiation, cell division/metabolism, chemokines/cytokines and receptors, signaling and transcription factors (TF), immune effector functions, and negative regulators. Within these categories, we identified 28 genes that correlated with CD8+ T cell expansion in response to an acute EBV infection. In EBV-specific CD8+ T cells, we identified 33 genes that were differentially expressed in AIM and CONV. Two important TF, T-bet and Eomesodermin (Eomes), were upregulated and maintained at similar levels in both AIM and CONV; by contrast, protein expression declined from AIM to CONV. Expression of these TF varied among cells with different epitope specificities. Altogether, gene and protein expression patterns suggest that a large proportion, if not a majority of CD8+ T cells in AIM are virus-specific, activated, dividing, and primed to exert effector activities. High expression of T-bet and Eomes may help to maintain effector mechanisms in activated cells, and to enable proliferation and transition to earlier differentiation states in CONV. PMID:26416268

Brca1 regulates in vitro differentiation of mammary epithelial cells.

PubMed

Kubista, Marion; Rosner, Margit; Kubista, Ernst; Bernaschek, Gerhard; Hengstschläger, Markus

2002-07-18

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.

Identification of Phosphoproteins as Possible Differentiation Markers in All-Trans-Retinoic Acid-Treated Neuroblastoma Cells

PubMed Central

Mandili, Giorgia; Marini, Cristina; Carta, Franco; Zanini, Cristina; Prato, Mauro; Khadjavi, Amina

2011-01-01

Background Neuroblastic tumors account for 9–10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation. Methodology/Principal Findings In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied. Conclusions/Significance Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins. PMID:21573212

Gene expression studies of developing bovine longissimus muscle from two different beef cattle breeds

PubMed Central

Lehnert, Sigrid A; Reverter, Antonio; Byrne, Keren A; Wang, Yonghong; Nattrass, Greg S; Hudson, Nicholas J; Greenwood, Paul L

2007-01-01

Background The muscle fiber number and fiber composition of muscle is largely determined during prenatal development. In order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cDNA microarray. Fetal longissimus muscle was sampled at 4 stages of myogenesis and muscle maturation: primary myogenesis (d 60), secondary myogenesis (d 135), as well as beginning (d 195) and final stages (birth) of functional differentiation of muscle fibers. All fetuses and newborns (total n = 24) were from Hereford dams and crossed with either Wagyu (high intramuscular fat) or Piedmontese (GDF8 mutant) sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life. Results We obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. Quantitative reverse transcription-PCR analysis of RNA from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. We detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. FSTL1 and IGFBP5 were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. An abundantly expressed gene with no functional annotation was found to be developmentally regulated in the same manner as muscle structural proteins. We also observed differences in gene expression profiles between the two different sire breeds. Wagyu-sired calves showed higher expression of fatty acid binding protein 5 (FABP5) RNA at birth. The developing longissimus muscle of fetuses carrying the Piedmontese mutation shows an emphasis on glycolytic muscle biochemistry and a large-scale up-regulation of the translational machinery at birth. We also document evidence for timing differences in differentiation events between the two breeds. Conclusion Taken together, these findings provide a detailed description of molecular events accompanying skeletal muscle differentiation in the bovine, as well as gene expression differences that may underpin the phenotype differences between the two breeds. In addition, this study has highlighted a non-coding RNA, which is abundantly expressed and developmentally regulated in bovine fetal muscle. PMID:17697390

Proteomics Identification of Potential Candidates Involved in Cell Proliferation for Early Stage of Brain Regeneration in the Adult Zebrafish.

PubMed

Lim, Fei Tieng; Ogawa, Satoshi; Smith, A Ian; Parhar, Ishwar S

2017-02-01

The central nervous system (CNS) of the non-mammalian vertebrates has better neuroregenerative capability as compared with the mammalian CNS. Regeneration of habenula was observed 40 days after damage in zebrafish. During the early stage of regeneration, we found a significant increase of apoptotic cells on day-1 post-damage and of proliferative cells on day-3 post-damage. To identify the molecular factor(s) involved in the early stages of neuroregeneration, differentially expressed proteins during sham, 20- and 40-h post-habenula damage were investigated by proteomic approach by using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-ToF) and tandem mass spectrometry. Protein profiles revealed 17 differentially (>1.5-fold) expressed proteins: 10 upregulated, 4 downregulated, 2 proteins were found to be downregulated at the early stage but upregulated at a later stage, and 1 protein was found to be upregulated at 2 different time points. All proteins identified can be summarized under few molecular processes involved in the early stages of neuroregeneration in zebrafish CNS: apoptosis regulation (Wnt inhibitory factor 1 [WIF1]), neuroprotection (metallothionein), cell proliferation (Spred2, ependymin, Lhx1, and Wnts), differentiation (Spred2, Lhx9, and Wnts), and morphogenesis (cytoplasmic actins and draculin). These protein profiling results suggest that drastic molecular changes occur in the neuroregenerative process during this period, which includes cell proliferation, differentiation, and protection.

Proteins involved in neuronal differentiation of neuroblastoma cell line N1E-115.

PubMed

Oh, Ji-Eun; Freilinger, Angelika; Gelpi, Ellen; Pollak, Arnold; Hengstschläger, Markus; Lubec, Gert

2007-06-01

Neuronal differentiation (ND) represents a well-defined phenomenon in biological terms but proteins involved have not been studied systematically. We therefore aimed to study ND by retinoic acid (RA) in a widely used neuroblastoma cell line by comparative proteomics. The ND was induced in the N1E-115 cell line by serum deprivation and RA treatment. Undifferentiated cells and cells undergoing serum deprivation served as controls. Protein extracts were run on 2-DE followed by MALDI-TOF or MALDI-TOF-TOF analysis. Quantification was carried out using specific software and stringent statistical analysis was performed. Tubulin beta 5, cat eye syndrome critical region protein 5 homolog, putative GTP-binding protein PTD004 homolog, and the metabolic proteins glyceraldehyde-3-phosphate dehydrogenase and transketolase were differentially regulated. Differential protein levels of cytoskeleton proteins including tubulins and metabolic proteins have been reported to be regulated by ND. Herein, specific signaling differences as reflected by putative GTP-binding protein PTD004 changes in differentiated cells are shown and a possible role for the Cat eye syndrome critical region protein 5 homolog is proposed. The protein disulfide isomerase associated 3 protein fits the already proposed findings of chaperon regulation by ND. The study forms the molecular basis for further evaluation of the functional roles of the differentially expressed proteins in ND.

Differential gene expression in Staphylococcus aureus exposed to Orange II and Sudan III azo dyes

PubMed Central

Pan, Hongmiao; Xu, Joshua; Kweon, Oh-Gew; Zou, Wen; Feng, Jinhui; He, Gui-Xin; Cerniglia, Carl E.

2018-01-01

We previously demonstrated the effects of azo dyes and their reduction metabolites on bacterial cell growth and cell viability. In this report, the effects of Orange II and Sudan III on gene expression profiling in Staphylococcus aureus ATCC BAA 1556 were analyzed using microarray and quantitative RT-PCR technology. Upon exposure to 6 μg/ml Orange II for 18 h, 21 genes were found to be differently expressed. Among them, 8 and 13 genes were up- and down-regulated, respectively. Most proteins encoded by these differentially expressed genes involve stress response caused by drug metabolism, oxidation, and alkaline shock indicating that S. aureus could adapt to Orange II exposure through a balance between up and down regulated gene expression. Whereas, after exposure to 6 μg/ml Sudan III for 18 h, 57 genes were differentially expressed. In which, 51 genes were up-regulated and 6 were down-regulated. Most proteins encoded by these differentially expressed genes involve in cell wall/membrane biogenesis and biosynthesis, nutrient uptake, transport and metabolite, and stress response, suggesting that Sudan III damages the bacterial cell wall or/and membrane due to binding of the dye. Further analysis indicated that all differentially expressed genes encoded membrane proteins were up-regulated and most of them serve as transporters. The result suggested that these genes might contribute to survival, persistence and growth in the presence of Sudan III. Only one gene msrA, which plays an important role in oxidative stress resistance, was found to be down-regulated after exposure to both Orange II and Sudan III. The present results suggested that both these two azo dyes can cause stress in S. aureus and the response of the bacterium to the stress is mainly related to characteristics of the azo dyes. PMID:25720844