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PubMed2004

Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program.

When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C(2)C(12) cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C(2)C(12) myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C(2)C(12) myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C(2)C(12) myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.

Animals↗
PubMed2006

Investigation of differentially expressed proteins due to the inhibitory effects of berberine in human liver cancer cell line HepG2.

The investigation of differentially expressed proteins was used together with other techniques to study the inhibitory effects of two different doses of berberine in human liver cancer cell line HepG2. For HepG2 cells treated with 24.0 mg l(-1) of berberine, an increase in the sub G(0) phase that was indicative of cell death was observed in cell cycle analysis with flow cytometry. However, no significant increase in sub G(0) was observed in HepG2 cells treated with 4.0 mg l(-1) of berberine. Using flow cytometric analysis, significant activation of caspase 3 was not observed with HepG2 cells treated with 4.0 and 24.0 mg l(-1) of berberine. In this work, labeling of cells with stable isotope was not used in the proposed method developed. The whole cell lysates from the control and treated cells were digested with trypsin and the peptides were separated by two-dimensional (cation exchange and reversed phase) liquid chromatography and tandem mass spectrometry. Our preliminary data showed that the proposed platform provided a rapid approach to study the molecular mechanism due to the inhibitory effects of different doses of the berberine on HepG2 cell lines. This included a network of proteins involved in mitogen-activated protein kinase (MAPK) phosphorelay systems, metabolism, regulation of cell cycle and DNA damage response. The differentially expressed proteins identified using the current approach were consistent with the data obtained from cell cycle analysis with flow cytometry.

Berberine↗
PubMed2003

Sodium loading changes urinary protein excretion: a proteomic analysis.

Plasma sodium concentration is maintained even when sodium intake is altered. Sodium homeostasis may involve changes in renal tubular protein expression that are reflected in the urine. We used proteomic analysis to investigate changes in urinary protein excretion in response to acute sodium loading. Rats were given deionized water followed by hypertonic (2.7%) saline for 28 h each. Urinary protein expression was determined during the final 4 h of each treatment. Acute sodium loading increased urinary sodium excretion (4.53 +/- 1.74 vs. 1.70 +/- 0.27 mmol/day, P = 0.029). Urinary proteins were separated by two-dimensional PAGE and visualized by Sypro ruby staining. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry followed by peptide mass fingerprinting. The abundance of a total of 45 protein components was changed after acute sodium loading. Neutral endopeptidase, solute carrier family 3, meprin 1alpha, diphor-1, chaperone heat shock protein 72, vacuolar H(+)-ATPase, ezrin, ezrin/radixin/moesin-binding protein, glutamine synthetase, guanine nucleotide-binding protein, Rho GDI-1, and chloride intracellular channel protein 1 were decreased, whereas albumin and alpha-2u globulin were increased. Some of these proteins have previously been shown to be associated with tubular transport. These data indicate that alterations in the excretion of several urinary proteins occur during acute sodium loading.

Amino Acid Sequence↗
PubMed2006

Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis.

One major problem concerning the electrophoresis of mitochondria is the heterogeneity of mitochondrial appearance especially under pathological conditions. We show here the use of zone electrophoresis in a free flow electrophoresis device (ZE-FFE) as an analytical sensor to discriminate between different yeast mitochondrial populations. Impairment of the structural properties of the organelles by hyperosmotic stress resulted in broad separation profiles. Conversely untreated mitochondria gave rise to homogeneous populations reflected by sharp separation profiles. Yeast mitochondria with altered respiratory activity accompanied by a different outer membrane proteome composition could be discriminated based on electrophoretic deflection. Proteolysis of the mitochondrial surface proteome and the deletion of a single major protein species of the mitochondrial outer membrane altered the ZE-FFE deflection of these organelles. To demonstrate the usefulness of ZE-FFE for the analysis of mitochondria associated with pathological processes, we analyzed mitochondrial fractions from an apoptotic yeast strain. The cdc48(S565G) strain carries a mutation in the CDC48 gene that is an essential participant in the endoplasmic reticulum-associated protein degradation pathway. Mutant cells accumulate polyubiquitinated proteins in microsomal and mitochondrial extracts. Subsequent ZE-FFE characterization could distinguish a mitochondrial subfraction specifically enriched with polyubiquitinated proteins from the majority of non-affected mitochondria. This result demonstrates that ZE-FFE may give important information on the specific properties of subpopulations of a mitochondrial preparation allowing a further detailed functional analysis.

Adenosine Triphosphatases↗
PubMed2004

Proteomic and metabolomic analysis of vascular smooth muscle cells: role of PKCdelta.

Recent developments of proteomic and metabolomic techniques provide powerful tools for studying molecular mechanisms of cell function. Previously, we demonstrated that neointima formation was markedly increased in vein grafts of PKCdelta-deficient mice compared with wild-type controls. To clarify the underlying mechanism, we performed a proteomic and metabolomic analysis of cultured vascular smooth muscle cells (SMCs) derived from PKCdelta+/+ and PKCdelta-/- mice. Using 2-dimensional electrophoresis and mass spectrometry, we identified >30 protein species that were altered in PKCdelta-/- SMCs, including enzymes related to glucose and lipid metabolism, glutathione recycling, chaperones, and cytoskeletal proteins. Interestingly, nuclear magnetic resonance spectroscopy confirmed marked changes in glucose metabolism in PKCdelta-/- SMCs, which were associated with a significant increase in cellular glutathione levels resulting in resistance to cell death induced by oxidative stress. Furthermore, PKCdelta-/- SMCs overexpressed RhoGDIalpha, an endogenous inhibitor of Rho signaling pathways. Inhibition of Rho signaling was associated with a loss of stress fiber formation and decreased expression of SMC differentiation markers. Thus, we performed the first combined proteomic and metabolomic study in vascular SMCs and demonstrate that PKCdelta is crucial in regulating glucose and lipid metabolism, controlling the cellular redox state, and maintaining SMC differentiation.

Animals↗
PubMed2005

Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets.

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.

Adult↗
PubMed2006

Applying proteomic methodologies to analyze the effect of methionine restriction on proliferation of human gastric cancer SGC7901 cells.

BACKGROUND: Methionine dependence is a feature unique to cancer cells, exhibited as inability to grow in a methionine-depleted environment supplemented with homocysteine, the immediate metabolic precursor of methionine. However, the molecular mechanisms by which methionine restriction inhibits cancer cells growth have not been elucidated. The effect of methionine restriction on the protein expression in gastric cancer cells was studied. METHODS: SGC7901 cells were treated with M-H+ medium for 5 days, which was followed by analysis of total cellular protein from cells by a combination of 2-DE and MS. Then the differential expressional levels of partially identified proteins were determined by Western blot analysis. RESULTS: The well-resolved, reproducible 2-DE patterns of SGC7901 cells cultured in M+H- or M-H+ medium were established. The 10 differential proteins between pairs of gastric cancer cells SGC7901 cultured either in M+H- medium or M-H+ medium, were identified by MALDI-TOF/TOF MS, and the differential expression levels of 2 identified proteins were confirmed. CONCLUSION: These data will be valuable for further study of the molecular mechanisms by which methionine restriction induces cell cycle arrest and apoptosis in human gastric cancer.

Amino Acid Sequence↗
PubMed2006

Protein expression profile in the striatum of acute methamphetamine-treated rats.

PURPOSE: Methamphetamine (MAP) is an addictive drug with psychostimulant effects. It is known that MAP induces behavioral changes, including hyperlocomotion and stereotypical movements in rodents. These behavioral changes induced by MAP have been compared with behavioral changes in patients with MAP addiction and MAP psychosis. However, little is known about the underlying mechanisms of MAPs effects on global protein expression. 2-DE proteomics allows us to examine global changes in protein expression in complex biological systems and to propose possible hypotheses of the underlying mechanisms in various pathological conditions. In the present study, we aim to identify protein expression profiles in the striatum (ST) of acute low dose MAP (1 mg/kg)-treated rats using 2-DE proteomics. MATERIALS AND METHODS: Rats were given an intraperitoneal injection of MAP (1 mg/kg) or saline. Locomotor activity was monitored. Proteins were extracted from the ST of MAP-treated and saline-treated control rats then separated and analyzed using 2-DE. RESULTS. Low dose MAP administration significantly increased locomotor activity. 2-DE analysis revealed 36 protein spots differentially regulated in the ST of acute MAP-treated rats compared to a vehicle-treated control. 26 protein spots have been identified using MALDI-TOF, including phosphoglycerate kinase 1, Dihydrolipoamide dehydrogenase, Voltage-dependent anion-selective channel protein 1, Rho GDP dissociation inhibitor alpha, peroxiredoxin 2, ubiquitin carboxy-terminal hydrolase L1, and actin beta, N-tropomodulin. DISCUSSION: These proteins could be related to underlying mechanisms of acute low dose MAP effects, indicating mitochondrial dysfunction, oxidative damages, lysosomal degradation, degenerative processes, and neuronal modification.

Animals↗
PubMed2006

Molecular characterization of adult mouse subventricular zone progenitor cells during the onset of differentiation.

Adult mouse subventricular zone (SVZ) neural progenitor cells (NPCs) retain the capacity to generate multiple lineages in vitro and in vivo. Thus far, the mechanisms involved in the regulation of these cells have not been well elucidated. We have carried out RNA profiling of adult SVZ cell cultures undergoing differentiation, to identify pathways that regulate progenitor cell proliferation and to define a set of transcripts that can be used as molecular tools in the drug discovery process. We carried out a stepwise stratification of the results to identify transcripts specifically enriched in NPCs and validated some of these using comparative literature analysis, quantitative polymerase chain reaction and immunological techniques. The results show a set of transcription factors, secreted molecules and plasma membrane markers that are differentially regulated during differentiation. Pathway analysis highlights alterations in insulin growth factor, Wnt and transforming growth factor beta signalling cascades. Further characterization of these components could provide greater insight into the mechanisms involved in the regulation of neurogenesis in the adult brain.

Animals↗
PubMed2002

The immunoglobulinopathies: from physiopathology to diagnosis.

The diversity of immunoglobulins (Igs) results mainly from recombinations of numerous genes within the heavy (V(Heavy), D, and J(Heavy)) and within the light (V(Light), J(Light)) chain gene loci, and from somatic hypermutations occurring during the immune response of B-cells. Igs production is controlled by complex cellular and humoral mechanisms. Plasma of healthy individuals contains polyclonal Igs. Clonal expansion of cells producing antigen-specific Igs may result from physiological as well as from pathological immune events. Exquisitely sensitive and specific molecular biology techniques have been used to evaluate the clonal diversity of cells producing antigen-specific Ig. However, the application of such techniques is hampered by the necessity to collect the totality of antigen-specific B-cells for subsequent analysis, which is impossible to perform routinely in humans. In addition, these techniques do not provide quantitative information about the concentration of the circulating Igs. It is therefore necessary to use tools allowing study of the quantity of the circulating Igs, and more particularly to detect overproduction of a single homogeneous Ig resulting from the expansion of a B-cell clone secreting Igs. Here, we review the mechanisms of B-cell differentiation and Ig synthesis, discuss the diseases associated with clonal Ig production and review the methods available in the clinical laboratory for Ig analysis.

Antibodies, Monoclonal↗
PubMed2005

Proteomic analysis of the response of human lung cells to uranium.

The industrial use of uranium and particularly of depleted uranium, has pinpointed the need to review its chemical impact on human health. A proteomic approach was used to evaluate the response of a human lung cell line (A549) to uranium. We established the first 2-D reference map of the A549 cell line, identifying 87 spots corresponding to 81 major proteins. Uranium treatment triggered differential expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin (CK18) and 1 to peroxiredoxin 1. We probed several hypotheses regarding CK cleavage, and observed that it did not result from caspase or calpain activity. Furthermore, we showed that the fragments are recognised by an anti-ubiquitin antibody (KM691). These results suggest a regulatory pathway involving CK ubiquitinylation or dysfunction in the proteasome-ubiquitin system in response to uranium exposure in human lung cells.

Amino Acid Sequence↗
PubMed2006

Molecular dynamics of the Shewanella oneidensis response to chromate stress.

Temporal genomic profiling and whole-cell proteomic analyses were performed to characterize the dynamic molecular response of the metal-reducing bacterium Shewanella oneidensis MR-1 to an acute chromate shock. The complex dynamics of cellular processes demand the integration of methodologies that describe biological systems at the levels of regulation, gene and protein expression, and metabolite production. Genomic microarray analysis of the transcriptome dynamics of midexponential phase cells subjected to 1 mm potassium chromate (K(2)CrO(4)) at exposure time intervals of 5, 30, 60, and 90 min revealed 910 genes that were differentially expressed at one or more time points. Strongly induced genes included those encoding components of a TonB1 iron transport system (tonB1-exbB1-exbD1), hemin ATP-binding cassette transporters (hmuTUV), TonB-dependent receptors as well as sulfate transporters (cysP, cysW-2, and cysA-2), and enzymes involved in assimilative sulfur metabolism (cysC, cysN, cysD, cysH, cysI, and cysJ). Transcript levels for genes with annotated functions in DNA repair (lexA, recX, recA, recN, dinP, and umuD), cellular detoxification (so1756, so3585, and so3586), and two-component signal transduction systems (so2426) were also significantly up-regulated (p < 0.05) in Cr(VI)-exposed cells relative to untreated cells. By contrast, genes with functions linked to energy metabolism, particularly electron transport (e.g. so0902-03-04, mtrA, omcA, and omcB), showed dramatic temporal alterations in expression with the majority exhibiting repression. Differential proteomics based on multidimensional HPLC-MS/MS was used to complement the transcriptome data, resulting in comparable induction and repression patterns for a subset of corresponding proteins. In total, expression of 2,370 proteins were confidently verified with 624 (26%) of these annotated as hypothetical or conserved hypothetical proteins. The initial response of S. oneidensis to chromate shock appears to require a combination of different regulatory networks that involve genes with annotated functions in oxidative stress protection, detoxification, protein stress protection, iron and sulfur acquisition, and SOS-controlled DNA repair mechanisms.

Anion Transport Proteins↗
PubMed2001

Proteomic analysis of arabidopsis seed germination and priming.

To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-regulation) of 74 proteins were observed during germination sensu stricto (i.e. prior to radicle emergence) and the radicle protrusion step. This approach was also used to analyze protein changes occurring during industrial seed pretreatments such as priming that accelerate seed germination and improve seedling uniformity. Several proteins were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Some of them had previously been shown to play a role during germination and/or priming in several plant species, a finding that underlines the usefulness of using Arabidopsis as a model system for molecular analysis of seed quality. Furthermore, the present study, carried out at the protein level, validates previous results obtained at the level of gene expression (e.g. from quantitation of differentially expressed mRNAs or analyses of promoter/reporter constructs). Finally, this approach revealed new proteins associated with the different phases of seed germination and priming. Some of them are involved either in the imbibition process of the seeds (such as an actin isoform or a WD-40 repeat protein) or in the seed dehydration process (e.g. cytosolic glyceraldehyde-3-phosphate dehydrogenase). These facts highlight the power of proteomics to unravel specific features of complex developmental processes such as germination and to detect protein markers that can be used to characterize seed vigor of commercial seed lots and to develop and monitor priming treatments.

Arabidopsis↗
PubMed2005

Inhibitory effects of a chemically standardized extract from Scutellaria barbata in human colon cancer cell lines, LoVo.

Scutellaria barbata (SB) is a medicinal plant that contains flavonone compounds such as scutellarein, scutellarin, carthamidin, isocarthamidin, and wogonin. A functional proteomic approach was used to study the inhibitory effects of a chemically standardized extract from SB in human colon adrencarcinoma, LoVo. In this work, a stable isotope was not used in the proposed method developed. The whole cell lysates from the control and treated cells were digested with trypsin, and the peptides were separated by two-dimensional (cation-exchange and reversed-phase) liquid chromatography and tandem mass spectrometry. The differentially expressed proteins identified using the current approach supported the data obtained from cell-cycle analysis with flow cytometry. With flow-cytometry analysis, a significant increase in the sub G1 phase was observed with a higher dose of extract from SB. Our results suggest that the chemically standardized extract from SB can induce cell death in the human colon cancer cell line. Our current work showed that the proposed platform provided a rapid approach to study the molecular mechanism because of the inhibitory effects of different doses of the botanical extracts on LoVo cell lines. This included a network of proteins involved in metabolism, regulation of the cell cycle, and transcription-factor activity.

Cell Death↗
PubMedDate not supplied

Application of isotope coded affinity tag (ICAT) analysis for the identification of differentially expressed proteins following infection of atlantic salmon (Salmo salar) with infectious hematopoietic necrosis virus (IHNV) or Renibacterium salmoninarum (BKD).

Aquaculture and commercial fisheries worldwide suffer from significant economic loss due to diseases of net-pen reared fish. In British Columbia, infectious hematopoietic necrosis (IHN) and bacterial kidney disease (BKD) epidemics occur because there are currently no commercially available drugs or fully licensed vaccines to treat these diseases. With a better understanding of the molecular mechanisms underlying these diseases, this circumstance might be significantly improved. In the present study, we have used a proteomics approach in an effort to identify and quantitate differentially expressed proteins in the liver and kidneys of diseased and healthy Atlantic salmon (Salmo salar). Isotope coded affinity tagging (ICAT), 2D gel electrophoresis, and multidimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC MS/MS) were used to identify hundreds of differentially expressed proteins. While the direct significance of changes in expression levels of many proteins remains to be elucidated, others appear to be more clearly related to the infectious process. Examples of the latter are discussed here and include, a natural killer cell enhancement factor (NKEF), procathepsin L, superoxide-producing NADPH oxidase and interferon-induced viral resistance protein Mx (IFI-Mx).

Affinity Labels↗
PubMed2003

Gene expression profiling and functional proteomic analysis reveal perturbed kinase-mediated signaling in genetic stroke susceptibility.

The stroke-prone spontaneously hypertensive rat (SHRSP) is a model of heritable hypertension-associated cerebrovascular injury. This study sought to compare SHRSP to the stroke-resistant SHR strain to identify genes and protein pathways whose expression and/or function was significantly altered between the strains prior to the onset of stroke. Cerebral cortex gene expression profiles from male SHRSPs and matched SHRs were examined by Affymetrix microarray analysis. mRNAs encoding the brain-derived neurotrophic factor receptor (TrkB) and multiple kinases of the MAPK/AKT signaling pathways, including JNK2, AKT2, and PI3K, were differentially expressed between SHRSP and SHR. Because these data suggest altered function in pathways involving MAP and AKT kinase activity, we performed Western blot using phosphorylation state-specific antibodies to characterize activity of MAP kinase and PI3K/AKT pathways. Changes in the levels of the phosphorylated forms of these kinases paralleled the changes in transcript levels observed between the strains. Two-dimensional gel electrophoresis and peptide fragment mass fingerprinting were used to identify altered protein substrates of these kinases. Protein profiling of kinase substrates further supported the notion of perturbed kinase-mediated signaling in SHRSP and identified adenylyl cyclase associated protein 2, TOAD-64, propionyl CoA carboxylase, APG-1, and valosin-containing protein as kinase targets whose phosphorylation state is altered between these strains. Altered gene and protein expression patterns in SHRSP are consistent with increased vulnerability of this strain to cerebrovascular injury.

Animals↗
PubMed2006

Proteomic profiles of induced hepatotoxicity at the subcellular level.

In the present study proteomes of liver samples were analyzed after administration of phenobarbital (PB) or 3-methylcholantrene (3-MC) to mice. Liver cell homogenates were subfractionated by differential ultracentrifugation into cytosol and microsomes, which were subjected to 2-DE to generate the proteomic maps of these fractions. 2-DE yielded 1100 and 800 protein spots for microsomes and cytosol, respectively. General trends of the fraction-specific alterations after 3-MC or PB treatment were evaluated using the Student's t-test and the principal component analysis (PCA). According to the PCA-derived data, the microsomal changes after 3-MC and PB treatment were quite similar. However, in the case of the cytosol data, the specificities of 3-MC- and PB-induced responses could be clearly distinguished from each other. Protein spots, whose expression levels differed from control, were identified by MALDI-TOF PMF. Proteomic studies such as those reported herein can be useful in identifying the molecular-based toxicity of lead drug candidates.

Animals↗