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PubMed2006

[The effect of Fuzheng Huayu Decoction on the hepatic proteome during the formation and resolution of rat liver fibrosis].

OBJECTIVE: To investigate the dynamic changes of the hepatic proteome during the formation and resolution of rat liver fibrosis induced by carbon tetrachloride and the effect of Fuzheng Huayu Decoction on it. METHODS: Liver fibrosis in Wistar rats was induced by subcutaneous injection of 40% CCl4 in olive oil while the rats in the treatment group received Fuzheng Huayu Decoction. Pathological examination, hydroxyproline content determination and protein extraction of the rats livers were performed at four time points, i.e. at the end of 4, 8, 12 and 16 weeks. After 2-dimensional electrophoresis (2-DE), the gels were analyzed using the PDQUEST 2-DE image analysis software and differentially expressed proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: (1) The 2-DE examination showed that the model group-specific proteins and differentially expressed proteins presented started at 4 weeks and reached their peaks at 12 weeks, and this was conformed by MALD-TOF-MS identification. (2) In addition, 18 proteins were present at all time points and another 19 proteins were absent in the model group among those identified by MALD-TOF-MS. (3) Fuzheng Huayu Decoction down-regulated proteins and their expressions increased at 4 and 8 weeks, whereas it up-regulated those which decreased at 8, 12 and 16 weeks. CONCLUSIONS: (1) The changes in protein expressions, which effect metabolism, neuroendocrine function and cell proliferation, are the basis of liver fibrosis. (2) The anti-fibrotic effect of Fuzheng Huayu Decoction is holistic, specifically because it can decrease the abnormal cell proliferation and can increase the synthesis of normal proteins.

Animals↗
PubMed2006

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

Animals↗
PubMed2006

Extreme skewing of annexin II and S100A6 expression identified by proteomic analysis of peritoneal B-1 cells.

B-1 cells differ phenotypically, biochemically and functionally from conventional B-2 cells. The origin of these differences remains uncertain. We hypothesized that unbiased analysis of differences in protein expression between B-1 and B-2 cells might provide information not otherwise available, and thus undertook 1-dimensional (1D) gel analysis combined with mass spectrometry. We identified annexin II and S100A6 in peritoneal B-1 cells (B-1P) but not in splenic B-2 cells (B-2S); these results were confirmed by western blot analysis and reflected in mRNA expression. Further analysis of mRNA indicated that elevated expression levels of annexin II and S100A6 were unique to B-1P among several naive lymphoid populations. However, expression of annexin II and S100A6 protein was up-regulated in mitogenically stimulated B-2S. In both naive B-1 cells and stimulated B-2 cells, annexin II and S100A6 formed Ca++-sensitive complexes. These results confirm that the emerging field of proteomics detects differentially expressed molecules independently of RNA screening methods. These results identify two proteins (annexin II and S100A6) that are unexpectedly differentially expressed in B-1 cells and, although members of larger families, may fulfill unique, subset-specific functions. These results also validate 1D GE/LC-MS/MS as a reliable screening tool in identifying final protein product expression differences between B-1P and B-2S.

Animals↗
PubMed2005

Characterization of a new NIH-registered variant human embryonic stem cell line, BG01V: a tool for human embryonic stem cell research.

Human embryonic stem cells (hESCs) offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent manipulations. For example, identifying genes expressed during culture, as well as their temporal expression order after passaging and conditions influencing the formation of all three germ layers may be helpful for the production of functional beta islet cells used in treating type I diabetes. Although several hESC lines have demonstrated karyotypic instability during extended time in culture, select variant lines exhibit characteristics similar to their normal parental lines. Such variant lines may be excellent tools and abundant sources of cells for pilot studies and in vitro differentiation research in which chromosome number is not a concern, similar to the role currently played by embryonal carcinoma cell lines. It is crucial that the cells be surveyed at a genetic and proteomic level during extensive propagation, expansion, and manipulation in vitro. Here we describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Our characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination.

Cell Culture Techniques↗
PubMed2007

Proteomic analysis of glutamate-induced toxicity in HT22 cells.

In the present study, we have investigated the proteome changes associated with glutamate-induced HT22 cell death, a model system to study oxidative stress-mediated toxicity. Among a number of HT22 proteins exhibiting altered expression, several molecular chaperones demonstrated substantial changes. For example, the levels of Hsp90 and Hsp70 decreased as cell death progressed whereas that of Hsp60 increased dramatically. Interestingly, cytosolic Hsp60 increased more prominently than mitochondrial Hsp60. Concomitantly, the accumulation of poly-ubiquitylated proteins and differential regulation of the peptidase activities and the subunits of 26S proteasomes were observed in glutamate-treated HT22 cells. Our findings that the molecular chaperones and the ubiquitin-proteasome system undergo changes during glutamate-induced HT22 cell death may suggest the importance of a protein quality control system in oxidative damage-mediated toxicity.

Animals↗
PubMed2006

Proteomics of xenografted human breast cancer indicates novel targets related to tamoxifen resistance.

Tamoxifen is the most frequently used drug for hormone therapy of breast cancer patients, even though a high percentage of women are (or become) refractory to this treatment. The proteins involved in tamoxifen resistance of breast tumor cells as well as the mechanisms by which they interact, are still unknown. Some years ago, we established the xenograft breast tumor 3366, sensitive to tamoxifen and the 3366/TAM, resistant to tamoxifen, derived after two years of in vivo passages of the parental 3366 under tamoxifen treatment. Here, we compare the protein expression levels of both xenografts. 2-DE of proteins from total cell extracts showed very high reproducibility among tumors from each group (tamoxifen sensitive and tamoxifen resistant). The heuristic clustering analysis of these gels pooled them correctly in both groups. Twelve proteins were found up-regulated in the tamoxifen-resistant line, while nine were down-regulated. The proteins differentially expressed were identified by MS and sequence database analysis. Biological functions of these proteins are related to cell-cell adhesion and interaction, signal transduction, DNA and protein synthesis machinery, mitochondrial respiratory chain, oxidative stress processes and apoptosis. Three of the identified proteins (ALG-2 interacting protein and two GDP-dissociation inhibitors) could be directly involved in the resistance phenomenon.

Amino Acid Sequence↗
PubMed2000

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines.

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.

Blotting, Western↗
PubMed2005

Two-dimensional polyacrylamide gel electrophoresis analysis of indomethacin-treated human colon cancer cells.

AIM: To establish the two-dimensional gel electrophoresis (2-DE) profiles of indomethacin (IN)-treated human colon cancer cell line HCT116, and to provide a new way to study its anti-tumor molecular mechanism through analyzing a variety of protein maps. METHODS: Two-DE profiles of HCT116 were established in IN-treated and untreated groups. Total proteins were separated by immobilized pH gradient-based 2-DE. The gels were stained by silver, scanned by ImageScanner, and analyzed with Image Master software. RESULTS: Clear background, well-resolved and reproducible 2-DE patterns of HCT116 cells were acquired in IN-treated and untreated group. The average deviation of spot position was 0.896+/-0.177 mm in IEF direction and 1.106+/-0.289 mm in SDS-PAGE direction respectively. In IN-treated group, 1169+/-36 spots were detected and 1061+/-32 spots were matched, the average matching rate was 90.6% in three gels. In untreated group, 1256+/-50 spots were detected and 1168+/-46 spots were matched, the average matching rate was 93.0% in three gels. Forty-five differential protein spots were displayed between IN-treated and untreated groups. Of which, 34 protein spots decreased and 9 showed higher expression in IN-treated group, and only two protein spots showed an expression in untreated cells. CONCLUSION: Two-DE profiles of IN-treated and untreated HCT116 cells were established. Apparent 45 different protein spots were detected in IN-treated and untreated HCT116 cells. The analysis on differential protein spots may serve as a new way to study the molecule mechanism of IN-treated colon cancer.

Antineoplastic Agents↗
PubMed2006

A phylogenomic analysis of the Ascomycota.

An automated procedure was developed to extract orthologous sequences from fungal genomes and incorporate them into phylogenomic analyses in a timely and efficient manner. This approach involves parsing an all versus all BLASTP search of 17 proteomes and creating a similarity matrix from e-values, which is then used to cluster proteins into related groups by means of a Markov Clustering algorithm. After performing this analysis at different stringency levels, 854 single copy protein clusters, which were ubiquitously distributed in all 17 proteomes, were identified. These clusters were culled to include only those clusters where all proteins had best hits to and received hits from a protein within the same cluster. The final data set included gapless alignments for 781 clusters of orthologous sequences that were concatenated into one super alignment containing 195,664 amino acid characters. Neighbor-joining distance and maximum likelihood analyses resulted in identical topologies and all except one node received 100% bootstrap support. The node supporting Stagonospora nodorum's position received 83% support or higher; it was also the only taxon differentially resolved in the maximum parsimony analyses. All analyses resolved the two derived subphyla Pezizomycotina and Saccharomycotina, and Schizosaccharomyces pombe as an early diverging lineage of the Ascomycota. Importantly, these analyses resolved the Leotiomycetes as the sister group to the Sordariomycetes, a region of the Ascomycota phylogeny that has remained problematic in molecular phylogenetic studies of more limited character sampling. Additional phylogenetic analyses which included orthologous sequences from an unannotated ascomycotan genome (e.g., Coccidioides immitis) and subsets of orthologs with different characteristics supported this topology. Phylogenetic analyses of the 595 orthologs which included C. immitis resulted in an identical topology to the previous 781 ortholog analysis and correctly placed C. immitis in the Eurotiomycetes. This demonstrated the correct identification of orthologs and the ability to incorporate unannotated genomic data into a common phylogenetic analysis.

Ascomycota↗
PubMed2004

An integrated approach to the detection of colorectal cancer utilizing proteomics and bioinformatics.

AIM: To find new potential biomarkers and to establish patterns for early detection of colorectal cancer. METHODS: One hundred and eighty-two serum samples including 55 from colorectal cancer (CRC) patients, 35 from colorectal adenoma (CRA) patients and 92 from healthy persons (HP) were detected by surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS). The data of spectra were analyzed by bioinformatics tools like artificial neural network (ANN) and support vector machine (SVM). RESULTS: The diagnostic pattern combined with 7 potential biomarkers could differentiate CRC patients from CRA patients with a specificity of 83%, sensitivity of 89% and positive predictive value of 89%. The diagnostic pattern combined with 4 potential biomarkers could differentiate CRC patients from HP with a specificity of 92%, sensitivity of 89% and positive predictive value of 86%. CONCLUSION: The combination of SELDI with bioinformatics tools could help find new biomarkers and establish patterns with high sensitivity and specificity for the detection of CRC.

Biomarkers, Tumor↗
PubMed2004

Human agmatinase is diminished in the clear cell type of renal cell carcinoma.

The proteome of RCC was analyzed by 2D PAGE to search for tumor-associated proteins. Agmatinase, which hydrolyzes agmatine to putrescine and urea, was identified by mass spectrometry and database searches and shown to be downregulated in tumor cells. Additionally, RT-PCR and Northern blot analyses demonstrated a clearly decreased amount of agmatinase mRNA in tumor cells. The differential expression of agmatinase mRNA was confirmed at the protein level. Western blot analysis showed almost no detectable agmatinase protein in tumor cells compared to corresponding normal renal tissue. Agmatinase mRNA is most abundant in human liver and kidney but expressed to a lesser extent in several other tissues, including skeletal muscle and small intestine. The human agmatinase gene encodes a 352-residue protein with a putative mitochondrial targeting sequence at the N-terminus. Using transfection and immunohistochemical studies, we show that agmatinase is localized in the mitochondria. Immunohistochemical studies revealed that agmatinase in the normal kidney is restricted to tubulus epithelial cells, while in tumors staining was low and heterogeneous. Thus, expression of human agmatinase is altered in RCC. We discuss the consequences of these findings in terms of polyamine, NO metabolism and macrophage function.

Adenocarcinoma, Clear Cell↗
PubMed2006

Identification and analysis of the mouse basic/Helix-Loop-Helix transcription factor family.

The basic/Helix-Loop-Helix (bHLH) proteins are a family of transcription factors that regulates a variety of biological processes. Based on a previously defined consensus motif, we identified the complete set of bHLH protein family from the mouse proteome databases and carried out a series of bioinformatics analysis. As results, 124 mouse bHLH proteins were identified in this study, and 28 of them were additional bHLH proteins beyond the previous report. These 124 mouse bHLH proteins were classified into groups from A to F by the nomenclature and phylogenetic analysis. Statistic analysis of the Gene Ontology annotation of these proteins showed that the bHLH proteins tend to perform functions related to cell differentiation and development. Gene function enrichment analysis among six groups illuminated that the proteins in certain group tend to have special biology functions, so that the molecular function of the uncharacterized proteins in groups could be inferred.

Amino Acid Sequence↗
PubMed2026

Protective effects of seminal exosomes on cryopreserved sperm via inhibiting oxidative damage.

This study aimed to explore the protective effect of seminal plasma exosomes (SPEs) on human sperm structure and function during cryopreservation and its potential mechanism. The samples were divided into two groups: the control group was treated solely with sperm cryoprotectant before freezing, while the exosome group was supplemented with SPEs. After cryopreservation and thawing, sperm progressive motility, normal morphological rate, and survival rate were evaluated. Furthermore, PKH67 labeling experiments were performed, and oxidative stress markers as well as energy metabolism indicators in sperm were detected. Subsequent mechanism exploration was conducted via proteomic analysis and protein validation assays. This work reveals that adding SPEs at a concentration of 1 or 2 mg/ml effectively improves sperm progressive motility after cryopreservation. After supplementing with SPEs, sperm glucose levels are reduced and mitochondrial membrane potential is enhanced. Simultaneously, SPEs alleviate oxidative stress by decreasing reactive oxygen species (ROS) and DNA fragment index (DFI) while increasing superoxide dismutase (SOD) activity. Functional annotation of proteomics reveals that 14 of the differentially expressed proteins (DEPs) are associated with sperm motility. Enriched metabolic pathways related to sperm motility and sperm protein validation experiments indicate that the expression of MAPK, p-MAPK, and p-JNK proteins in sperm is higher in the Exosome group than in the Control group. This study provides important theoretical support for the application of SPEs in mitigating cryopreservation damage to sperm by enhancing antioxidant capacity. The specific mechanism may be mediated by the MAPK/p-JNK pathway.

Male↗
PubMed2005

Lotus japonicus metabolic profiling. Development of gas chromatography-mass spectrometry resources for the study of plant-microbe interactions.

Symbiotic nitrogen fixation (SNF) in legume root nodules requires differentiation and integration of both plant and bacterial metabolism. Classical approaches of biochemistry, molecular biology, and genetics have revealed many aspects of primary metabolism in legume nodules that underpin SNF. Functional genomics approaches, especially transcriptomics and proteomics, are beginning to provide a more holistic picture of the metabolic potential of nodules in model legumes like Medicago truncatula and Lotus japonicus. To extend these approaches, we have established protocols for nonbiased measurement and analysis of hundreds of metabolites from L. japonicus, using gas chromatography coupled with mass spectrometry. Following creation of mass spectral tag libraries, which represent both known and unknown metabolites, we measured and compared relative metabolite levels in nodules, roots, leaves, and flowers of symbiotic plants. Principal component analysis of the data revealed distinct metabolic phenotypes for the different organs and led to the identification of marker metabolites for each. Metabolites that were enriched in nodules included: octadecanoic acid, asparagine, glutamate, homoserine, cysteine, putrescine, mannitol, threonic acid, gluconic acid, glyceric acid-3-P, and glycerol-3-P. Hierarchical cluster analysis enabled discrimination of 10 groups of metabolites, based on distribution patterns in diverse Lotus organs. The resources and tools described here, together with ongoing efforts in the areas of genome sequencing, and transcriptome and proteome analysis of L. japonicus and Mesorhizobium loti, should lead to a better understanding of nodule metabolism that underpins SNF.

Gas Chromatography-Mass Spectrometry↗
PubMed2006

Quantitative proteomics identifies a change in glial glutamate metabolism at the time of female puberty.

Mammalian puberty requires activation of luteinizing hormone-releasing hormone (LHRH) neurons. In turn, these neurons are controlled by transsynaptic and glia-to-neuron communication pathways, which employ diverse cellular proteins for proper function. We have now used a high throughput relative quantitative proteomics technique to identify such proteins. We selected the method of two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) and cleavable isotope-coded affinity tags (cICAT), to both identify and quantify individual proteins within a complex protein mixture. The proteins used derived from the hypothalamus of juvenile (25-day-old) and peripubertal (first proestrus, LP) female rats, and their identity was established by analyzing their mass spectra via database searching. Five proteins involved in glutamate metabolism were detected and two of them appeared to be differentially expressed. They were selected for further analysis, because of their importance in controlling glutamate synthesis and degradation, and their preferential expression in astroglial cells. One, glutamate dehydrogenase (GDH) catalyzes glutamate synthesis; its hypothalamic content detected by 2DLC-MS/MS increases at first proestrus. The other, glutamine synthetase (GS), catalyzes the metabolism of glutamate to glutamine; its content decreases in proestrus. Western blot analysis verified these results. Because these changes suggested an increased glutamate production at puberty, we measured glutamate release from hypothalamic fragments from juvenile 29-day old rats, and from rats treated with PMSG to induce a premature proestrus surge of luteinizing hormone (LH). To determine the net output of glutamate in the absence of re-uptake we used the excitatory amino acid transporter (EAAT) inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). PDC elicited significantly more glutamate- and LHRH-release from the proestrus hypothalamus. Thus, an increase excitatory drive to the LHRH neuronal network provided by glutamatergic inputs of glial origin, is an event contributing to the pubertal activation of LHRH secretion.

Age Factors↗
PubMed2004

[Effects of siwu tang on protein expression of bone marrow of blood deficiency mice induced by irradiation].

OBJECTIVE: To observe the effects of siwu tang on protein expression of bone marrow of blood deficiency mice and provide the theoretical and experimental basis for understanding the molecular mechanism of blood enriching function of siwu tang. METHOD: Blood deficiency mice were established by using 3.5 Gy 60Co gamma-ray. With proteomic technologies including two-dimensional electrophoresis, image analysis, in-gel digestion, peptide mass fingerprinting and bioinformatics the proteins of bone marrow of blood deficiency mice were isolated, analyzed, and identified. RESULT: Siwu tang could reverse 10 up-regulated and 4 down-regulated proteins of blood deficiency mice bone marrow. Seven of the proteins were likely to be lymphocyte specific protein 1, proteasome 26S ATPase subunit 4, hematopoietic cell protein-tyrosine phosphatase, glyceraldehyde-3-phosphate dehydrogenase, growth factor receptor binding protein 14 and lgals12 respectively. CONCLUSION: Siwu tang can regulate the protein expression of bone marrow of blood deficiency mice and thus promote the growth and differentiation of hematopoietic cells and then exert its effects on blood enriching function.

Adaptor Proteins, Signal Transducing↗
PubMed2024

Gradient-Elution Nanoflow Liquid Chromatography Without a Binary Pump: Smoothed Step Gradients Enable Reproducible, Sensitive, and Low-Cost Separations for Single-Cell Proteomics.

Mass spectrometry-based proteome profiling of trace analytes including single cells benefits from liquid chromatography separations operated at low flow rates (e.g., <50&#xa0;nl/min). However, high-pressure binary pumps needed to achieve such flow rates are not commercially available, and instead require splitting of the gradient flow to achieve low-nanoliter-per-minute flow rates. Gradient flow splitting can waste solvent and lead to flow inconsistencies. To address this, we have developed a method for creating gradients by combining segments of mobile phase having increasing solvent strength together in an open capillary, and then relying on Taylor dispersion to form the desired smooth gradient profile. Our method dramatically reduces costs, as only a single isocratic high-pressure pump is required. Following development of gradient profiles for both 10- and 20-min active gradients, we measured 200 pg injections of HeLa digest using a timsTOF mass spectrometer. Finally, we investigated differences in protein expression between single cells originating from two different colonies of ATG-KO HeLa cells. Thousands of proteins were quantified, and a potential mechanism explaining differential immune responses of these two colonies upon exposure to viral DNA treatment was determined.

Humans↗
PubMed2006

Robust method for detecting differential gene expression in twin studies.

MOTIVATION: A steadily increasing number of experiments with microarrays stimulate the further development of the statistical methods of the analysis of gene expression data. One of the central problems in this area is detecting differential gene expression under two or more conditions. Unfortunately, up to now it has not been studied how the correlations between related individuals, such as twins influence the estimates of differential gene expression. RESULTS: In this paper, we discuss this problem and propose a new method that is robust with respect to correlations of gene expression data for twins.

Algorithms↗