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Results for “differential proteomics analysis”

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At least 757 records · Page 42Linked to original sources
PubMed2003

Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1.

Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation.

Animals↗
PubMed2005

Proteomic profiling identifies an UV-induced activation of cofilin-1 and destrin in human epidermis.

The human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin.

Actin Depolymerizing Factors↗
PubMed2006

Quantitative proteomic and genomic profiling reveals metastasis-related protein expression patterns in gastric cancer cells.

Gastric cancer is a leading cause of death worldwide, and patients have an overall 5-year survival rate of less than 10%. Using quantitative proteomic techniques together with microarray chips, we have established comprehensive proteome and transcriptome profiles of the metastatic gastric cancer TMC-1 cells and the noninvasive gastric cancer SC-M1 cell. Our qualitative protein profiling strategy offers the first comprehensive analysis of the gastric cancer cell proteome, identifying 926 and 909 proteins from SC-M1 and TMC-1 cells, respectively. Cleavable isotope-coded affinity tagging analysis allows quantitation of a total of 559 proteins (with a protein false-positive rate of <0.005), and 240 proteins were differentially expressed (>1.3-fold) between the SC-M1 and TMC-1 cells. We identified numerous proteins not previously associated with gastric cancer. Notably, a large subset of differentially expressed proteins was associated with tumor metastasis, including proteins functioning in cell-cell and cell-extracellular matrix (cell-ECM) adhesion, cell motility, proliferation, and tumor immunity. Gene expression profiling by DNA microarray revealed differential expression (of >2-fold) of about 1000 genes. The weak correlation observed between protein and mRNA profiles highlights the important complementarities of DNA microarray and proteomics approaches. These comparative data enabled us to map the disease-perturbed cell-cell and cell-ECM adhesion and Rho GTPase-mediated cytoskeletal pathways. Further validation of a subset of genes suggests the potential use of vimentin and galectin 1 as markers for metastasis. We demonstrate that combining proteomic and genomic approaches not only provides a rapid, robust, and sensitive platform to elucidate the molecular mechanisms underlying gastric cancer metastasis but also may identify candidate diagnostic markers and therapeutic targets.

Biomarkers, Tumor↗
PubMedDate not supplied

Proteomic analysis of pancreatic ductal carcinoma cells after combined treatment with gemcitabine and trichostatin A.

The human pancreatic adenocarcinoma cell line T3M4 has been treated with two agents, gemcitabine (2',2'-difluorodeoxycytidine, a drug interfering with DNA synthesis) and trichostatin A (a drug interfering with histone acetylation), both separately and in association. The association of the two drugs showed a marked cooperative effect in inhibiting proliferation and inducing apoptosis of the cells. In an effort to identify differentially expressed proteins in the different drug treatments, the proteomic expression has been studied by two-dimensional gel electrophoresis comparing untreated cells with cells treated with trichostatin A and/or gemcitabine. A total of 81 differentially expressed polypeptide chains have been visualized by setting a 2.5-fold threshold value. Of these, 56 were identified via MALDI-TOF and Q-TOF MS analyses. Most of the regulated proteins are involved in two major biological processes, namely apoptotic cell death and proliferation. Our results demonstrate that the level of activation/repression of the proteins involved in these processes correlates with the growth inhibition and the apoptotic response of the cells subjected to single or combined drug treatment.

Antibiotics, Antineoplastic↗
PubMed2006

Global analysis of heat shock response in Desulfovibrio vulgaris Hildenborough.

Desulfovibrio vulgaris Hildenborough belongs to a class of sulfate-reducing bacteria (SRB) and is found ubiquitously in nature. Given the importance of SRB-mediated reduction for bioremediation of metal ion contaminants, ongoing research on D. vulgaris has been in the direction of elucidating regulatory mechanisms for this organism under a variety of stress conditions. This work presents a global view of this organism's response to elevated growth temperature using whole-cell transcriptomics and proteomics tools. Transcriptional response (1.7-fold change or greater; Z >/= 1.5) ranged from 1,135 genes at 15 min to 1,463 genes at 120 min for a temperature up-shift of 13 degrees C from a growth temperature of 37 degrees C for this organism and suggested both direct and indirect modes of heat sensing. Clusters of orthologous group categories that were significantly affected included posttranslational modifications; protein turnover and chaperones (up-regulated); energy production and conversion (down-regulated), nucleotide transport, metabolism (down-regulated), and translation; ribosomal structure; and biogenesis (down-regulated). Analysis of the genome sequence revealed the presence of features of both negative and positive regulation which included the CIRCE element and promoter sequences corresponding to the alternate sigma factors sigma(32) and sigma(54). While mechanisms of heat shock control for some genes appeared to coincide with those established for Escherichia coli and Bacillus subtilis, the presence of unique control schemes for several other genes was also evident. Analysis of protein expression levels using differential in-gel electrophoresis suggested good agreement with transcriptional profiles of several heat shock proteins, including DnaK (DVU0811), HtpG (DVU2643), HtrA (DVU1468), and AhpC (DVU2247). The proteomics study also suggested the possibility of posttranslational modifications in the chaperones DnaK, AhpC, GroES (DVU1977), and GroEL (DVU1976) and also several periplasmic ABC transporters.

Bacterial Proteins↗
PubMed2025

Identification of Differential Proteins in Thrombi of Cardioembolic and Atherothrombotic Etiology in Patients with Ischemic Stroke.

Knowing the precise etiology in ischemic stroke is necessary to ensure accurate diagnosis and decide on appropriate preventive treatments, especially in those of undetermined cause. Analysis of the thrombus protein composition could be useful to identify diagnostic biomarkers to help determine the stroke origin. Thrombi from 54 ischemic stroke patients with large vessel occlusion (LVO), of cardioembolic and atherothrombotic etiology, were analyzed using a proteomics approach. The proteome profile was compared between them to detect differential proteins of each etiology. Peptides of those differential proteins were quantified and related to the neurological function and clinical status of the patients. Of the 516 proteins identified, three showed significant differences between atherothrombotic and cardioembolic thrombi. These were fibronectin (FINC), 2,3-bisphosphoglycerate mutase (PMGE), and tropomyosin-1 (TPM1). Combining these proteins in a biomarker panel provided good sensitivity and high specificity for differentiating cardioembolic and atherothrombotic strokes. In addition, several of the quantified peptide levels correlated with clinical parameters related to stroke severity and prognosis. Three proteins differentially detected in ischemic stroke thrombi could be useful tools for accurately diagnosing ischemic stroke etiology, particularly in cases of undetermined cause. These biomarkers should be further analyzed in prospective multicenter studies to demonstrate their usefulness.

Humans↗
PubMed2004

14-3-3 gamma associates with muscle specific kinase and regulates synaptic gene transcription at vertebrate neuromuscular synapse.

The muscle-specific receptor tyrosine kinase (MuSK) is part of a receptor complex, activated by neural agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). To gain insight into the function of the MuSK complex, we have developed a proteomic approach to identify new MuSK partners. MS analysis of MuSK crosslink products from postsynaptic membranes of the Torpedo electrocytes identified the adaptor protein 14-3-3 gamma. The 14-3-3 gamma protein was found localized at the adult rat NMJ. Cotransfection experiments in COS-7 cells showed that MuSK codistributed with the 14-3-3 gamma protein at the plasma membrane. Furthermore, 14-3-3 gamma was copurified by affinity chromatography with MuSK from transfected COS-7 cells and myotubes. The 14-3-3 gamma protein did not colocalize with agrin-elicited acetylcholine receptor (AChR) aggregates in cultured myotubes, suggesting that it is not involved in AChR clustering. Expression of 14-3-3 gamma specifically repressed the transcription of several synaptic reporter genes in cultured myotubes. This repression was potentiated by MuSK expression. Moreover, the expression of 14-3-3 gamma in muscle fibers in vivo caused both the repression of synaptic genes transcription and morphological perturbations of the NMJ. Our data extend the notion that, apart from its well documented role in AChR clustering, the MuSK complex might also be involved in the regulation of synaptic gene expression at the NMJ.

14-3-3 Proteins↗
PubMed2003

Cap G, a gelsolin family protein modulating protective effects of unidirectional shear stress.

Atherosclerosis is a progressive and complex pathophysiological process occurring in large arteries. Although it is of multifactorial origin, the disease develops at preferential sites along the vasculature in regions experiencing specific hemodynamic conditions that are predisposed to endothelial dysfunction. The exact mechanisms allowing endothelial cells to discriminate between plaque-free and plaque-prone flows remain to be explored. To investigate such mechanisms, we performed a proteomic analysis on endothelial cells exposed in vitro to these two-flow patterns. A few spots on the two-dimensional gel had an intensity that was differentially regulated by plaque-free versus plaque-prone flows. One of them was further investigated and identified as macrophage-capping protein (Cap G), a member of the gelsolin protein superfamily. A 2-fold increase of Cap G protein and a 5-fold increase of Cap G mRNA were observed in cells exposed to a plaque-free flow as compared with static cultures. This increase was not observed in cells exposed to plaque-prone flow. Plaque-free flow induced a corresponding increase in nuclear and cytoskeletal-associated Cap G. Finally, overexpression of Cap G in transfection assays increased the motility potential of endothelial cells. These observations together with the known functions of Cap G suggest that Cap G may contribute to the protective effect exerted by plaque-free flow on endothelial cells. On the contrary, in cells exposed to a plaque-prone flow, no induction of Cap G expression could be observed.

Amino Acid Sequence↗
PubMed2006

Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

Acquired Immunodeficiency Syndrome↗
PubMed2026

Distinct Effects of Rap1 Subtype A GTPase Deficiency on the Male Mouse Heart.

This study utilized a genetically engineered mouse model deficient in the small GTPase Rap1A (knockout/Rap1A-null) to understand the biological role of Rap1A in the heart. We examined differential protein expression in the left ventricle of Rap1A-null versus wild-type control C57BL/6 male mice (~5 months) using proteomics (nanoLC-MS/MS quantitative analysis), and in the whole heart of aged male mice (~16 months) using MAL-DI-TOF/TOF mass spectrometry. Additionally, we used an experimental model of acute cardiovascular stress and assessed the impact on heart tissue histology, gene expression and mortality risk. Rap1A-deficient hearts showed reduced size and reduced heart and left ventricular weights. Significantly reduced gene expression of extracellular matrix collagen type I and collagen type III was present under baseline and cardiovascular stress conditions. Assessment of the proteomic profile identified a crucial role of Rap1A in promoting healthy ventricular myocardium, as its deficiency exhibited increased impact on cytoskeletal, mitochondrial, metabolic and contractile protein expression in young and aged mice. In young Rap1A-deficient mice, overrepresentation analysis revealed markers myosin heavy chain 7 (&#x3b2;-MHC) and alpha-actinin-2 (&#x3b1;-actinin-2) associated with cardiomyopathies, and upon cardiac stress, showed mortality risk compared to controls. Altogether, these findings provide important insights into the role of Rap1A in cardiac structure and remodeling under basal and stress conditions in male mice.

Animals↗
PubMed2004

Proteome analysis of cultivar-specific deregulations of Oryza sativa indica and O. sativa japonica cellular suspensions undergoing rice yellow mottle virus infection.

We have used two-dimensional gel electrophoresis with mass spectrometry analysis to study the temporal patterns of protein expression during RYMV (Rice yellow mottle virus) infection in rice cells of two cultivars: IR64, Oryza sativa indica, susceptible, and Azucena, O. sativa japonica, partially resistant to RYMV. Proteomic analysis of nonstressed and RYMV inoculated cells showed statistically significant changes in the relative levels of 40 IR64 proteins and 24 Azucena proteins. Protein identification using mass spectrometry was attempted for all the differentially regulated proteins. This global analysis detected 32 hypothetical "new" proteins. Nineteen differentially regulated proteins were identified for IR64 cultivar, while 13 were identified for Azucena cultivar, including proteins in three functional categories: metabolism, stress-related proteins, and translation. These data revealed that a number of proteins regulated by abiotic stress response pathway were activated by RYMV in both cultivars (such as salt-induced protein, heat shock proteins (HSPs), superoxide dismutase (SOD), and others have functions consistent with the susceptibility or partially resistance trait (such as dehydrin, proteins involved in glycolysis pathway).

Heat-Shock Proteins↗
PubMedDate not supplied

Treatment of acute promyelocytic leukemia with ATRA and As2O3: a model of molecular target-based cancer therapy.

Most acute promyelocytic leukemia (APL) cases have t(15;17)(q22;q21) chromosomal translocation and PML-RARalpha chimeric gene which blocks granulocytic differentiation. The introduction of all-trans-retinoic acid (ATRA) and arsenic compounds, especially arsenic trioxide (As(2)O(3)), has provided good models to study not only differentiation and/or apoptosis therapy but also molecular target-based cancer treatment. In vivo and in vitro investigations have shown that both agents are able to induce differentiation of APL cells: ATRA tends to induce terminal differentiation, while low-dose As(2)O(3) can induce partial differentiation. Significant progress has been made in understanding the molecular mechanisms of APL pathogenesis and differentiation therapy. Pharmacological concentrations (0.1 approximately 1 microM) of ATRA derepresses transcription by releasing CoR from, and recruiting CoA to PML-RARalpha, whereas As(2)O(3) triggers a rapid degradation of PML-RARalpha. In fact, the two drugs act on the same oncoprotein through targeting different moieties and in distinct ways and thereby abrogate its dominant-negative effects on regulatory pathways necessary for granulocytic differentiation. As to apoptosis, it is clear that high-dose As(2)O(3) can induce mitochondria-mediated cell death pathway in a thiol-dependent manner, while the mechanism of ATRA-induced apoptosis needs further elucidation. Transcriptomic and proteomic analysis are also expected to find new molecular targets. It is the hope that what we have learnt from APL will benefit further developments of anti-leukemia therapy.

Animals↗
PubMed2005

Proteomic identification of activin receptor-like kinase-1 as a differentially expressed protein during hyaloid vascular system regression.

The hyaloid vascular system (HVS) is a transient network of capillaries that nourishes the embryonic lens and the primary vitreous of the developing eye. We used proteomic analysis and immunohistochemical staining to identify activin receptor-like kinase-1 (ALK1), a type I receptor for transforming growth factor-beta1, during the HVS regression phase. In addition, we overexpressed ALK1 in corneas implanted with bFGF (basic fibroblast growth factor) pellets and observed that ALK1 overexpression resulted in inhibition of bFGF-induced corneal neovascularization in vivo. Our data suggest that ALK1 may play a role in HVS regression during ocular development.

Activin Receptors, Type I↗
PubMed2004

[Identification of rabbit lens proteins by two-dimensional gel electrophoreses and mass spectrometry].

OBJECTIVE: To study the role of lens proteomics using two-dimensional electrophoresis (2-DE) and mass spectrometry in order to exploring novel effective ways for cataract prevention and therapy. METHODS: The proteins of the three-month-year old rabbit lens were separated using immobilized pH gradients 2-DE. Image analysis was carried out using Image Master 2D Elite 3.01 software package. Most of the crystallines was identified by matrix assisted laser adsorption/ionization-time of-flight-mass spectrometry (MALDI-TOF-MS). RESULTS: The maps of 2-DE showed that lens proteins were in the section of pH 5 - 9 and the relative molecular weight was 14,000 - 94,000, while relative molecular weight of more abundant crystalline was localized at 14,000 - 40,000. About 180 protein spots were detected with the similar PI, molecular weight and quantity of each spot could be acquired by image analysis software. Sixteen crystallines were identified using MALDI-TOF-MS. CONCLUSION: Proteomic analysis of lens can be accomplished and the proteins can be well separated and analyzed using 2-DE and mass spectrometry. This technique offers a new avenue for analyses of lens proteins and to assess their differential expression in cataract, and may thus provide a novel approach to cataract prevention and therapy.

Animals↗
PubMed2003

Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis.

Full analysis of cellular protein constituents is a valuable tool in the evaluation of tissues. Traditional methods of evaluation, however, are time-consuming and difficult to reproduce. Two-dimensional difference gel electrophoresis (2D-DIGE), a recently developed proteomic system, affords the ability to compare and evaluate protein extracts from multiple sources. Coupled with laser capture microdissection (LCM), this technology is a powerful tool in comparing the protein profiles of separate pure cell populations. Proteins are labeled in vitro with reactive cyanine dyes that fluoresce at differential wavelengths, and after comigration on two-dimensional gels, differing protein populations become apparent. The unique aspect of this technology is the ability to identify and quantify proteins from separate preparations without issues of gel-to-gel differences. These techniques coupled with the systems for robotic acquisition of specific spots on the gel, tryptic digestion, and MALDI mass spectrometry permit identification of proteins differentially expressed in two pure cell populations. The authors used these new technologies to analyze the protein constituents of spasmolytic polypeptide expressing metaplasia (SPEM), a gastric mucosal metaplasia (fundic antralization or pseudopyloric metaplasia) that develops in the atrophic fundus mucosa of mice infected with Helicobacter felis and in humans infected with Helicobacter pylori. In addition, SPEM has been identified in the atrophic mucosa surrounding a high percentage of gastric adenocarcinomas and may represent a precursor lineage of malignancy. This technology recognized 28 differentially expressed proteins between SPEM and surface cells. Identification of novel SPEM-related proteins would allow the development of new immunohistochemical antibodies to further study this important metaplasia.

Animals↗
PubMed2003

Proteomic analysis of mdx skeletal muscle: Great reduction of adenylate kinase 1 expression and enzymatic activity.

To investigate the pathophysiological events of Duchenne muscular dystrophy, we analyzed alterations of protein expression in hindlimb muscles of three month old mdx mice using two-dimensional gel electrophoresis and mass spectrometry. About 40 differentially expressed proteins from the cytosolic fraction and 20 from the microsomal fraction of mdx hindlimb muscles were identified. Among these altered proteins, adenylate kinase 1 (AK 1) was particular interesting because its decrease in abundance was so dramatic (> four-fold). Enzymatic assays demonstrated that AK 1 activity was also decreased in mdx mice. Furthermore, the expression and enzymatic activity of AK 1 were consistently reduced in mdx mice at one and six months of age, suggesting a direct relationship between the lack of dystrophin and alteration of AK 1. Along with AK 1, creatine kinase (CK) provides a major pathway for regulation of nucleotide ratios and energy metabolism in muscles. To gain a better understanding of mechanisms of energy metabolism, we also investigated CK activities in these mdx mice at different ages. Our results suggested that decreased AK 1 expression and activity might result in redistribution of energy flow through the alternative CK system, thus a compensatory potential might limit cellular energy failure in mdx skeletal muscle.

Adenylyl Cyclases↗
PubMed2004

Differential domain evolution and complex RNA processing in a family of paralogous EPB41 (protein 4.1) genes facilitate expression of diverse tissue-specific isoforms.

The EPB41 (protein 4.1) genes epitomize the resourcefulness of the mammalian genome to encode a complex proteome from a small number of genes. By utilizing alternative transcriptional promoters and tissue-specific alternative pre-mRNA splicing, EPB41, EPB41L2, EPB41L3, and EPB41L1 encode a diverse array of structural adapter proteins. Comparative genomic and transcript analysis of these 140- to 240-kb genes indicates several unusual features: differential evolution of highly conserved exons encoding known functional domains interspersed with unique exons whose size and sequence variations contribute substantially to intergenic diversity; alternative first exons, most of which map far upstream of the coding regions; and complex tissue-specific alternative pre-mRNA splicing that facilitates synthesis of functionally different complements of 4.1 proteins in various cells. Understanding the splicing regulatory networks that control protein 4.1 expression will be critical to a full appreciation of the many roles of 4.1 proteins in normal cell biology and their proposed roles in human cancer.

Alternative Splicing↗
PubMed2002

DNA microarray analysis of differential gene expression in Borrelia burgdorferi, the Lyme disease spirochete.

DNA microarrays were used to survey the adaptive genetic responses of Borrelia burgdorferi (Bb) B31, the Lyme disease spirochete, when grown under conditions analogous to those found in unfed ticks (UTs), fed ticks (FTs), or during mammalian host adaptation (Bb in dialysis membrane chambers implanted in rats). Microarrays contained 95.4% of the predicted B31 genes, 150 (8.6%) of which were differentially regulated (changes of > or = 1.8-fold) among the three growth conditions. A substantial proportion (46%) of the differentially regulated genes encoded proteins with predicted export signals (29% from predicted lipoproteins), emphasizing the importance to Bb of modulating its extracellular proteome. For B31 cultivated at the more restrictive UT condition, microarray data provided evidence of a bacterial stringent response and factors that restrict cell division. A large proportion of genes were responsive to the FT growth condition, wherein increased temperature and reduced pH were prominent environmental parameters. A surprising theme, supported by cluster analysis, was that many of the gene expression changes induced during the FT growth condition were transient and largely tempered as B31 adapted to the mammalian host, suggesting that once Bb gains entry and adapts to mammalian tissues, fewer differentially regulated genes are exploited. It therefore would seem that although widely dissimilar, the UT and dialysis membrane chamber growth conditions promote more static patterns of gene expression in Bb. The microarray data thus provide a basis for formulating new testable hypotheses regarding the life cycle of Bb and attaining a more complete understanding of many aspects of Bb's complex parasitic strategies.

Animals↗