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Results for “differential proteomics analysis”

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At least 1,297 records · Page 72Linked to original sources
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↗
PubMed2005

Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers.

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.

Biomarkers↗
PubMed2026

The mechanism by which long-term exposure to TDCIPP promotes cognitive impairment in 3 ×Tg-AD mice: Insights from multi-omics studies.

Tri(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a commonly used organophosphate ester that has the potential to adversely affect human health. Although previous studies have closely associated TDCIPP with cognitive impairment, the underlying mechanisms remain unclear. To elucidate the neurotoxic effects of TDCIPP and its mechanistic contribution to cognitive impairment in 3 ×Tg-AD mice, a multi-omics approach incorporating proteomics, untargeted metabolomics, and 16S ribosomal RNA (rRNA) gene sequencing was employed to evaluate the impact of TDCIPP exposure on neurobehavioral function. TDCIPP exposure promoted cognitive impairment in 3 ×Tg-AD mice. Proteomic analyses revealed that this promotion is associated with disturbances in the hippocampal mitochondrial autophagy pathway. Furthermore, TDCIPP may interfere with the PINK1/Parkin-mediated mitophagy pathway at the functional level, without altering PINK1 protein abundance. Untargeted metabolomic analysis of urine samples demonstrated that TDCIPP exposure altered the metabolic profile of 3 ×Tg-AD mice, with 58 metabolites upregulated and 11 downregulated. Additionally, 16S rRNA sequencing revealed substantial modifications in gut microbiome composition following exposure to TDCIPP. Notably, significant correlations were identified between the perturbed bacterial genera and the differential metabolites. In conclusion, exposure to TDCIPP promotes cognitive impairment in 3 ×Tg-AD mice, which is associated with the interference with the PINK1/Parkin-mediated mitophagy pathway, as well as alterations in the urinary metabolome and gut microbiota. These findings suggest the potential to mitigate such cognitive impairment by targeting the microbiota-gut-brain axis.

Animals↗
PubMedDate not supplied

Proteomic studies on low- and high-grade human brain astrocytomas.

Human brain astrocytomas range from the indolent low-grade to the highly infiltrating and aggressive high-grade form, also known as glioblastoma multiforme. The extensive heterogeneity of astrocytic tumors complicates their pathological classification. In this study, we compared the protein pattern of low-grade fibrillary astrocytomas to that of glioblastoma multiforme by 2D electrophoresis. The level of most proteins remains unchanged between the different grade tumors and only few differences are reproducibly observable. Fifteen differentially expressed proteins, as well as seventy conserved spots, were identified by mass spectrometry. Western and immnunohistochemical analysis confirmed the differential expression of the identified proteins. These data provide an initial reference map for brain gliomas. Among the proteins more highly expressed in glioblastoma multiforme, we found peroxiredoxin 1 and 6, the transcription factor BTF3, and alpha-B-crystallin, whereas protein disulfide isomerase A3, the catalytic subunit of the cAMP-dependent protein kinase, and the glial fibrillary acidic protein are increased in low-grade astrocytomas. Our findings contribute to deepening our knowledge of the factors that characterize this class of tumors and, at the same time, can be applied toward the development of novel molecular biomakers potentially useful for an accurate classification of the grade of astrocytomas.

Astrocytoma↗
PubMed2006

Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.

Cell Line↗
PubMed2004

Proteome analysis for identification of target proteins of genistein in primary human endothelial cells stressed with oxidized LDL or homocysteine.

BACKGROUND: Epidemiological studies suggest that soy consumption contributes to the prevention of coronary heart disease. The proposed anti-atherogenic effects of soy appear to be carried by the soy isoflavones with genistein as the most abundant compound. AIM OF THE STUDY: To identify proteins or pathways by which genistein might exert its protective activities on atherosclerosis, we analyzed the proteomic response of primary human umbilical vein endothelial cells (HUVEC) that were exposed to the pro-atherosclerotic stressors homocysteine or oxidized low-density lipoprotein (ox-LDL). METHODS: HUVEC were incubated with physiological concentrations of homocysteine or ox-LDL in the absence and presence of genistein at concentrations that can be reached in human plasma by a diet rich in soy products (2.5 microM) or by pharmacological intervention (25 microM). Proteins from HUVEC were separated by two-dimensional polyacrylamide gel electrophoresis and those that showed altered expression level upon genistein treatment were identified by peptide mass fingerprints derived from tryptic digests of the protein spots. RESULTS: Several proteins were found to be differentially affected by genistein. The most interesting proteins that were potently decreased by homocysteine treatment were annexin V and lamin A. Annexin V is an antithrombotic molecule and mutations in nuclear lamin A have been found to result in perturbations of plasma lipids associated with hypertension. Genistein at low and high concentrations reversed the stressor-induced decrease of these anti-atherogenic proteins. Ox-LDL treatment of HUVEC resulted in an increase in ubiquitin conjugating enzyme 12, a protein involved in foam cell formation. Treatment with genistein at both doses reversed this effect. CONCLUSION: Proteome analysis allows the identification of potential interactions of dietary components in the molecular process of atherosclerosis and consequently provides a powerful tool to define biomarkers of response.

Annexin A5↗
PubMed2006

Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome.

Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.

Arabidopsis↗
PubMed2003

Heterogeneity analysis of the human pituitary proteome.

BACKGROUND: A human proteome is relatively dynamic compared with its corresponding genome. Our aim was to study the heterogeneity of a human pituitary proteome as a function of gender, age, and race. METHODS: Pituitary control tissues (n = 8) were used to extract proteins; each control tissue was analyzed (n = 3-5) with two-dimensional gel electrophoresis (2DGE) and PDQuest software. We obtained 30 high-resolution 2DGE gels and conducted a comparative analysis as a function of gender, age, and race. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry were used to characterize the protein in each differential spot. RESULTS: We detected approximately 1000 protein spots in each 2DGE map, and 51 differential spots (7 differing with gender, 17 with age, 15 with race, and 12 with the coeffect of age and race). Among those 51, we characterized 28 proteins [5 differing with gender, 8 with age, 6 with race, 8 with the coeffect of age and race, and 1 (somatotropin chain 1) with all of these]. Somatotropin was related to gender, age, and race, and prolactin was higher in females than males. The differentially expressed proteins that were related to age were mainly those proteins associated with cell growth, proliferation, differentiation, apoptosis, and death; those proteins showed no difference with gender and race. Age and race affected some proteins associated with hormone regulation (e.g., follistatin, thyroid hormone receptor beta-2, adenylate cyclase-inhibiting Galpha protein). CONCLUSIONS: A heterogeneity exists in the human pituitary proteome as a function of gender, age, and race. These findings will serve as a basis for our comparative proteomics studies of human pituitary adenomas.

Adult↗
PubMed2005

Transcriptional response reveals translation machinery as target for high pressure in Lactobacillus sanfranciscensis.

The effect of sublethal hydrostatic pressure on the transcriptome of Lactobacillus sanfranciscensis was determined using a shot-gun-microarray. Among the 750 spots that passed quality analysis 42 genes were induced, while six were repressed when cells were incubated at 45 MPa for 30 min. The nature of genes and their differential expression clearly indicate cellular efforts to counteract a decrease in translational capacity. The majority of high pressure affected genes were found to encode either translation factors (EF-G, EF-TU), ribosomal proteins (S2, L6, L11), genes changing translational accuracy or molecular chaperones (GroEL, ClpL). These data agree with previously reported effects observed in in vitro studies as well as with physiological and proteomic data. This study provides in vivo evidence to identify ribosomes and impaired translation among primary targets for high pressure treatment. The observed induction of heat as well as cold shock genes (e.g. hsp60, gyrA) may be explained as a result of high pressure affected protein synthesis.

Gene Expression Regulation, Bacterial↗
PubMed2004

Effect of early cold stress on the maturation of rice anthers.

Male reproductive development in rice (Oryza sativa Linnaeus is very sensitive to various forms of environmental stresses including low temperature. Here, we present our findings on the proteomic analysis of the later developmental consequences of low temperature treatment on rice anthers. Anther proteins at the trinucleate stage, with or without cold treatment for four days at 12 degrees C at the young microspore stage, were extracted, separated by two-dimensional gel electrophoresis (2-DE) and compared. More than 3000 rice anther proteins of cold-sensitive cultivar Doongara plants at the trinucleate stage were resolved on 2-DE gels over a pH range of 4-7 and detected by silver-staining. Seventy protein spots were differentially displayed after four days of cold treatment at the young microspore stage. Of these, 12 protein spots were newly-induced, 47 were up-regulated, and 11 were down-regulated by cold treatment at the early microspore stage. We identified 18 by matrix-assisted laser desorption/ionization mass spectrometry time of flight (MALDI-TOF) analysis. Of the identified proteins, seven were observed as breakdown (cleavage) products by a combination of 2-DE and MALDI-TOF analysis, thus demonstrating for the first time that cold temperature stress at the young microspore stage enhances and induces partial degradation of proteins in the rice anthers at the trinucleate stage.

Cold Temperature↗
PubMed2005

Large-scale analysis of human alternative protein isoforms: pattern classification and correlation with subcellular localization signals.

We investigated human alternative protein isoforms of >2600 genes based on full-length cDNA clones and SwissProt. We classified the isoforms and examined their co-occurrence for each gene. Further, we investigated potential relationships between these changes and differential subcellular localization. The two most abundant patterns were the one with different C-terminal regions and the one with an internal insertion, which together account for 43% of the total. Although changes of the N-terminal region are less common than those of the C-terminal region, extension of the C-terminal region is much less common than that of the N-terminal region, probably because of the difficulty of removing stop codons in one isoform. We also found that there are some frequently used combinations of co-occurrence in alternative isoforms. We interpret this as evidence that there is some structural relationship which produces a repertoire of isoformal patterns. Finally, many terminal changes are predicted to cause differential subcellular localization, especially in targeting either peroxisomes or mitochondria. Our study sheds new light on the enrichment of the human proteome through alternative splicing and related events. Our database of alternative protein isoforms is available through the internet.

Alternative Splicing↗
PubMed1999

Proteomics in human disease: cancer, heart and infectious diseases.

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease-associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma-derived aldose reductase-like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two-dimensional electrophoresis (2-DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2-DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2-DE approach. The comparison of 2-DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). A human myocardial 2-DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.

Animals↗
PubMed2005

Optimisation of a 2-D gel electrophoresis protocol for the human-pathogenic fungus Aspergillus fumigatus.

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. One of the important questions concerning A. fumigatus is the identification of pathogenicity determinants. To obtain a comprehensive overview about the proteins produced at different physiological conditions that are related to the infectious process a proteomic approach has been applied. Here, 2-D gel electrophoresis for filamentous fungi was optimised concerning removal of interfering compounds, protein extraction and separation methods. A trichloroacetic acid-based precipitation method of proteins with their subsequent solubilisation by the use of a combination of CHAPS with a second sulfobetaine detergent gave the best results. The optimised protocol was evaluated by the analysis of the proteomes of A. fumigatus grown on two different carbon sources, i.e., glucose and ethanol. Carbon catabolite repression has not been studied in detail at the protein level in A. fumigatus yet. In addition, growth on ethanol leads to activation of the glyoxylate cycle which was shown to be essential for pathogenesis in bacteria and fungi. In A. fumigatus, differential patterns of enzymes of the gluconeogenesis, glyoxylate cycle and ethanol degradation pathway during growth on glucose and ethanol were observed.

Aspergillus fumigatus↗
PubMed2004

Large scale gene expression profiling of metabolic shift of mammalian cells in culture.

The metabolic state of hybridoma cells in continuous culture varies with the cultivation condition from which the culture is initiated. At a metabolically shifted state, cells have markedly reduced glucose and other nutrient consumption and lactate production as compared to cells in batch culture or in continuous culture without a metabolic shift. Taking a combined genomics and proteomics approach, we investigated the molecular mechanism of metabolic shift. Cells from continuous cultures at two different steady states with a glucose consumption to lactate production molar ratio (DeltaL/DeltaG) of 0.08 and 1.4 were studied. Affymetrix GeneChips as well as cDNA microarrays were employed to identify differentially expressed mRNA transcripts, and the differentially expressed proteins were identified using the 2D gel electrophoresis-mass spectrometry approach. The decrease in glucose metabolism upon metabolic shift is accompanied by a decrease in gene expression of a number of genes involved in its metabolism. However, the number of genes differentially expressed and the extent of differential expression upon metabolic shift are relatively moderate. The change in the expression of metabolic genes at the transcriptional level was confirmed by real time PCR. The results suggest that metabolic shift is a combined effect of both biochemical events at reaction level and gene expression at transcription and translation level. This approach of integrating transcriptional profiling, proteomic techniques and biochemical analysis provides a more global view of the metabolism of mammalian cells in culture.

Animals↗
PubMed2005

The transcriptome's drugable frequenters.

Microarray studies are widely employed in the exploratory phase of the drug discovery process. Expectations raised by the genomics revolution led to the belief that they would rapidly lead to the identification of novel drug targets. However, a few basic questions were often overlooked. Are members of drugable gene families properly represented in the transcriptome? Or are they poorly expressed and below the detection limit of the microarray technology? This review explores the representation of drug targets and components of downstream cellular signaling pathways in the transcriptome. It appears that members of drugable gene families are underrepresented in the transcriptomes of non-pathological human tissues. But, they are represented at or above the expected frequency in the differential transcriptome (i.e. the set of genes that changes expression upon a change in cellular environment). Analysis of differential gene expression on a genome-wide scale will therefore give a comprehensive overview of cellular pathways and possible drug targets.

Drug Design↗