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PubMed2006

Proteomic analysis of experimentally induced azole resistance in Candida glabrata.

OBJECTIVES: The aim of the present study was to identify changes in the proteome of a laboratory-derived azole-resistant strain of Candida glabrata compared with its susceptible parent strain in an effort to identify proteins that are differentially expressed in association with azole resistance. METHODS: Soluble and membrane protein fractions were isolated from mutant strain F15 (fluconazole MIC>128 mg/L) and parent strain 66032 (fluconazole MIC=16 mg/L) grown to mid-log phase. Soluble proteins were resolved by both two-dimensional (2D) and one-dimensional (1D) polyacrylamide gel electrophoresis (GE) whereas membrane proteins were resolved by 1D GE. Spots or bands representing differentially expressed proteins were identified by matrix-assisted desorption ionization-time of flight mass spectroscopy (MALDI-TOF MS) and peptide mass fingerprinting. RESULTS: A total of 22 proteins were found to be more abundantly represented, and 3 proteins were found to be less abundantly represented, in strain F15 compared with strain 66032. These included up-regulation of the ATP-binding cassette transporter Cdr1p, the ergosterol biosynthesis enzyme Erg11p, proteins involved in glycolysis and glycerol metabolism, and proteins involved in the response to oxidative stress and cadmium exposure. CONCLUSIONS: In addition to transcriptional regulation of Cdr1p, this study identified the differential expression of several proteins that may contribute to azole resistance and suggests the possibility for a post-transcriptional mechanism for increased expression of Erg11p.

Antifungal Agents↗

PubMed2006

[Proteomic analysis of human ovarian cancer cell lines and their platinum-resistant clones].

OBJECTIVE: To perform comparative proteomic analysis of human ovarian cancer cell lines for detecting platinum-resistance associated proteins. METHODS: The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780/CBP) human ovarian cancer cell lines were separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed using image analysis software, stained with Coomassie Brilliant Blue, then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. The mRNA and protein levels of the differentially expressed protein which was most significant in all of the four resistant cell lines were validated by RT-PCR and western blotting, respectively. RESULTS: Five proteins were found to be significant in four cell lines. Annexin A3 and destrin were up-regulated and nicotinamide-adenine dinucleotide phosphate (NADP)-dependent isocitrate dehydrogenase 1 was down-regulated in all the four resistant samples. Glutathione transferase omega 1 had an increased expression in the other three resistant cell lines except for SKOV3/CBP in which its expression was not changed. However, cofilin 1 represented a different trend. In the two resistant sublines of SKOV3, cofilin 1 had a down-regulation, but it had an up-regulation in the cell lines induced from SKOV3. The expression of annexin A3 was up-regulated by 3 - 20 fold and the results of RT-PCR and western blotting showed complete consistency with that by 2-DE. CONCLUSIONS: Proteomic techniques are useful to the identification of the resistance-associated proteins in ovarian cancer platinum-resistant cell lines and five candidates have been found. The five differential proteins might become hopeful candidate biomarkers for resistance.

Annexin A3↗

PubMed2026

Gut microbiota dysbiosis and host metabolite-immune crosstalk drives the pathogenesis of neonatal lupus erythematosus: a multi-omics analysis.

BACKGROUND: Neonatal lupus erythematosus (NLE) is a rare autoimmune condition triggered by the transplacental transfer of maternal antibodies. Despite its recognized clinical manifestations, the underlying pathogenesis remains incompletely understood. This study seeks to explore the disruption of the gut microbiota-host metabolism-immune axis in anti-Ro/La-positive neonates, and to assess its potential role in the development of NLE. METHODS: This multicenter, cross-sectional study included 90 neonates, divided into three groups: 30 with neonatal lupus erythematosus (NLE), 30 with positive antibodies but without clinical manifestations (No-NLE), and 30 healthy controls. We performed 16 S rRNA sequencing to analyze gut microbiota composition, untargeted plasma metabolomic profiling, and proteomic analysis to identify alterations associated with the pathogenesis of NLE. RESULTS: We identified significant alterations in the gut microbiota, plasma metabolome, and proteome profiles of anti-Ro/La-positive neonates. NLE infants exhibited marked enrichment of Enterobacteriaceae and depletion of Bifidobacterium and Clostridium butyricum. Metabolomic analysis revealed hyperactivation of β-alanine and purine metabolism, along with impaired α-linolenic acid metabolism and endocannabinoid signaling. Proteomic profiling indicated aberrant protein expression that modulated IFN signaling, particularly within the C-type lectin receptor pathway. Dysregulation of the spleen tyrosine kinase (SYK) and high-affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) decoupling was observed, correlating with elevated IFN-α and NF-κB p65 levels. Integrated correlation analysis revealed significant associations among differential microbial taxa, plasma metabolites, and proteins. Notably, E. coli-associated metabolites and proteins displayed inverse relationships with those associated with C. butyricum. CONCLUSIONS: These findings represent comprehensive evidence of dysregulation along the "gut microbiota-host metabolism-immune" axis in neonatal lupus erythematosus (NLE), providing novel insights into the disease's underlying heterogeneity.

Humans↗

PubMed2005

Protein expression pattern distinguishes different lymphoid neoplasms.

To identify proteins associated with the histological subtypes of lymphoid neoplasms, we studied the proteomes of 42 cell lines from human lymphoid neoplasms including Hodgkin's lymphoma (HL; four cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell lymphoma (three cell lines). The protein spots were sequentially selected by (i) Wilcoxon or Kruskal-Wallis tests to find the spots whose intensity was significantly (p <0.05) different among the cell line groups, (ii) by statistical-learning methods to prioritize the spots according to their contribution to the classification, and (iii) by unsupervised classification methods to validate the classification robustness by the selected spots. The selected spots discriminated (i) between HL cells and other cells, (ii) between the cells from B cell malignancies, T cell malignancies, and NK cell lymphoma cells, and (iii) between HL cells and anaplastic large cell lymphoma cells. Among the 31 informative protein spots, MS identified 24 proteins corresponding to 23 spots. Previous reports did not correlate these proteins to lymphocyte differentiation, suggesting that a proteomic study would identify the novel mechanisms responsible for the histogenesis of lymphoid neoplasms. These proteins may have potential as differential diagnostic markers for lymphoid neoplasms.

Amino Acid Sequence↗

PubMed2026

Proteomic and phosphoproteomic profiles of time-dependent dynamic changes in LPS-induced macrophage polarization.

The temporal proteomic and phosphoproteomic reprogramming during early M1 macrophage polarization (0-6 h) remains poorly understood. We performed time-resolved proteomic and phosphoproteomic analyses of LPS-stimulated RAW264.7 macrophages at seven time points within 6 h. Time-clustering of differentially expressed molecules revealed two patterns: initial change with partial recovery, and sustained dysregulation. Upregulated proteins and phosphorylation sites were enriched in the Rho GTPase signaling pathway, T-cell receptor signaling pathway, NF-κB cascade, osteoclast differentiation pathway, and antiviral immune pathway. Downregulated pathways were associated with cell cycle regulation, chromatin remodeling, RNA metabolism, and mRNA processing, indicating resource reallocation to prioritize acute inflammatory responses. Kinase-substrate network analysis confirmed the mitogen-activated protein kinase (MAPK), cyclin-dependent kinase (CDK), protein kinase B (AKT), and ribosomal S6 kinase (RSK) families as core upstream phosphorylation regulators. Integrated analysis revealed synergistic and antagonistic relationships between proteomic and phosphoproteomic changes. This study provides a temporal molecular atlas of M1 polarization, delineating inflammatory signaling dynamics and offering a basis for therapeutic target discovery in inflammatory diseases. SIGNIFICANCE: Macrophage M1 polarization is a central event in innate immune defense against pathogenic invasion, yet its dysregulation is a pivotal driver of the onset and progression of a broad spectrum of inflammation-associated disorders, spanning autoimmune diseases, infectious conditions and inflammatory bone diseases, making the dissection of its molecular regulatory mechanisms an urgent research priority in immunology and translational medicine. Dynamic molecular events within 0-6 h after LPS stimulation are critical for initiating and shaping M1 inflammatory activation, yet systematic time-resolved proteomic and phosphoproteomic profiling remains insufficient.In this study, we comprehensively characterized temporal proteome and phosphoproteome changes at seven consecutive time points during macrophage polarization, clarified two distinct dynamic molecular patterns, identified core signaling pathways and key kinase regulators involved in inflammatory reprogramming, and uncovered the leading role of post-translational phosphorylation modifications in initiating polarization. This work delineates the time-series molecular atlas of early macrophage activation, provides novel insights into the temporal regulatory mechanism of inflammatory signaling networks, and lays a solid experimental foundation for exploring new intervention targets and regulatory nodes in clinical translational research.

Lipopolysaccharides↗

PubMed2005

Proteome analysis in the clinical chemistry laboratory: myth or reality?

BACKGROUND: We review here modern aspects of proteomic analysis, as displayed via orthogonal mass/charge analysis (isoelectric focusing in the first dimension, followed by sodium dodecyl sulphate electrophoresis in polyacrylamide gels, SDS-PAGE, at right angles, in the second dimension). METHODS: This technique is capable of displaying a few thousand polypeptide chains, characterized by a single pI and M(r) value as coordinates, and recognized via elution, digestion and mass spectrometry analysis. Although, up to the present, this technique has been used mostly for advanced research, with no immediate applications in the clinical chemistry laboratory, there are hints that such applications will soon become a reality. RESULTS AND CONCLUSIONS: In the field of cancer research, it is here shown that stathmin (Op18) becomes heavily phosphorylated in cancerous mantle cell lymphomas and that the progression of the disease can be followed by the progression of phosphorylation of Op18 and by the appearance of additional phosphorylated spots. Also chemoresistance of different tumors has been evaluated via 2D-PAGE through quantitative, differential proteomics: among up- and down-regulated proteins in a human cervix squamous cell carcinoma cell line (A431), rendered resistant to cisplatin, one particular protein was found to appear in large quantities by de novo synthesis: 14-3-3, a protein known to impart resistance to apoptosis to cells. In the field of brain disorders, we could set up an easy test for detecting pathological prions in sporadic Creutzfeldt-Jakob disease (sCJD), by simply searching for those pathological forms in the olfactory mucosa (up to this finding, diagnosis could only be confirmed post-mortem). We are currently working on a test for differentiating sCJD from all the other degenerative dementias. Upon 2D mapping of cerebrospinal fluid (CSF) and immunoblot analysis, we could identify a major spot (pI 4.8, M(r) 30 kDa) followed by some two-three minor spots (pIs 5.0-6.0, same M(r) value) of the same 14-3-3 anti-apoptotic protein involved in chemoresistance. By this test, sCJD could be differentiated from all the other degenerative dementias, which are 14-3-3 negative (in sCJD, the rapid and massive brain cell damage releases large quantities of 14-3-3 in the cerebrospinal fluid). Another protein that appears very promising as a marker for sCJD is cystatin C, that is strongly up-regulated in this pathology. Human sera should also be mined for discovery of many more markers for disease. Up to the present, no one could be found, but this was due to the presence of several major proteins, obscuring all rare ones. Via several immuno-subtraction steps, followed by ion exchange and size exclusion chromatography, one can now detect proteins and peptides present in sera at levels below 10 ng/mL, highlighting the road to discovery of novel markers of disease. Another technique that could revolutionize biomarker discovery in biological fluids consists in the use of combinatorial beads to reduce the dynamic range. They consist in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of amino acids, resulting in millions different structures. When these beads are impregnated with complex proteomes (e.g., human sera, CSF, urines) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility of evidencing low-abundance species.

Animals↗

PubMed2004

Proteomic analysis of mouse liver for the evaluation of effects of Scutellariae radix by liquid chromatography with tandem mass spectrometry.

Scutellariae radix or Scutellaria baicalensis is a medicinal plant that contains major flavonoids such as baicalein, baicalin, wogonin and wogonosides. The present work describes the development of an approach using proteomic analysis of mouse liver to study the effects of prolonged exposure to substances present in chemically standardized Scutellariae radix extracts. Histopathological examination of the mouse liver was compared with the proteome data. The botanical extracts were prepared using pressurized liquid extraction (PLE). A method without isotope labeling was developed, using proteolytic digestion with one- and two-dimensional liquid chromatography with tandem mass spectrometry, and was used to characterize the extent of differential protein expression in mouse liver in response to external factors such as extracts from Scutellariae radix. From the histopathological examination and proteome data, significant changes in the mouse livers were not observed for the low-dose group. The Scutellariae radix extracts at high dose were observed to cause damage at the bile duct and expression change of a number of proteins including some involved in catabolism of triglyceride-rich particles, carbohydrate metabolism, regulators of cell signaling processes, and enzymes involved in biotransformation. Thus, proteomic analysis of liver samples from mice treated with botanical extracts is a promising approach to provide information on any potential toxicity effects of the extracts. The present method also provides another means for comparing proteomes in biological samples such as liver lysates from mice subjected to different treatments.

Animals↗

PubMed2026

Spatial Proteomics of the Human Atherosclerotic Microenvironment Reveals Heterogeneity in Intraplaque Proteomes and Extracellular Matrix Remodeling.

Plaque heterogeneity underlies the propensity of atherosclerotic lesions to rupture and trigger cardiovascular events. Most proteomic studies examine bulk changes, obscuring key spatial differences in protein abundance. We report a high-resolution spatial proteomics workflow exploring the molecular landscape of human plaques and a murine myocardium. By combining laser capture microdissection with high-sensitivity ion-mobility mass spectrometry, spatial profiling of cellular and extracellular matrix (ECM) proteomes was achieved. Over 2700 proteins were detected from 50,000 μm2 areas, revealing substantial intraplaque heterogeneity across distinct regions (lipid-rich, media, shoulder, necrotic core, intima) and distance from the artery lumen. Inverse correlations between proteases (cathepsin B) and core structural ECM proteins (perlecan, HSPG2) indicated active ECM remodeling. Analysis of media layers indicated distinct protein signatures associated with smooth muscle contraction and cell-cell communication. Blood coagulation signatures, including platelet degranulation and fibrin formation, were enriched at the intima. Inflammatory (clusters of differentiation 4/68, CD4/CD68; vascular cell adhesion molecule 1, VCAM1) and vascular damage markers (tenascin-C, TNC) were enriched in shoulder regions. The necrotic core was dominated by blood proteins, consistent with intraplaque hemorrhage. This workflow resolves proteomic changes over ∼200 μm distances, providing unprecedented insights into plaque morphology and offers a powerful tool for elucidating plaque biology.

Humans↗

PubMed2004

Differential expression of the skeletal muscle proteome in mdx mice at different ages.

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy (DMD), a disease caused by the absence of dystrophin. Although much has been done to elucidate the structure and function of dystrophin and the dystrophin-associated glycoprotein complex (DGC), little is known about the cascade of molecular events triggered by the absence of dystrophin that lead to muscle degeneration. To study the molecular basis of DMD, we decided to systematically study the skeletal muscle proteome in mdx mice at different ages. By using two-dimensional (2-D) gel electrophoresis, we defined changes in the protein expression pattern between mdx and control muscles. Approximately 46 differentially expressed proteins from the cytosolic fraction of mdx hindlimb muscles at three months of age were detected by 2-D gel analysis, of which 24 were identified by matrix assisted laser desorption/ionization- mass spectrometry. Most of the proteins fell into five groups of functionally related proteins. These functional categories are (i) metabolism and energy production, (ii) serine protease inhibitor family, (iii) growth and differentiation, (iv) calcium homeostasis, and (v) cytoskeletal reorganization and biogenesis. The potential roles of the differentially expressed proteins are discussed in the context of the mdx phenotype. Finally, we analyzed alterations of protein expression in mdx mice at one and six months of age to determine how protein expression changes with disease progression.

Animals↗

PubMed2005

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform.

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2005

Transcriptome and proteome analysis of soleus muscle of hormone-sensitive lipase-null mice.

Hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization in adipocytes, has been demonstrated also in skeletal muscle. To gain further insight into the role and importance of HSL in skeletal muscle, a transcriptome analysis of soleus muscle of HSL-null mice was performed. A total of 161 transcripts were found to be differentially expressed. Increased mRNA levels of fructose-1,6-bisphosphatase, fructose-2,6-bisphosphatase, and phosphorylase kinase gamma1A suggest a higher glycogen flux in soleus muscle of HSL-null mice. An observed increase in the utilization of glycogen stores supports this finding. Moreover, an increased amount of intramyocellular lipid droplets, observed by transmission electron microscopy, suggests decreased mobilization of lipid stores in HSL-null mice. To complement the transcriptome data, protein expression analysis was performed. Five spots were found to be differentially expressed: pyruvate dehydrogenase E1alpha, creatine kinase (CK), ankyrin-repeat domain 2, glyceraldehyde-3-phosphate dehydrogenase, and one protein yet to be identified. The increased protein level of CK indicates creatine phosphate degradation to be of increased importance in HSL-null mice. The results of this study suggest that in the absence of HSL, a metabolic switch from reliance on lipid to carbohydrate energy substrates takes place, supporting an important role of HSL in soleus muscle lipid metabolism.

Animals↗

PubMed2005

Differential protein expression in anatomical zones of the prostate.

The prostate has three anatomical zones: the peripheral (PZ), the transition (TZ), and the central (CZ) zone. It is proposed that the CZ may be of mesodermal origin, whereas the other two are of endodermal origin. Proteome patterns in the zones were characterized to test for differences. Cells were scraped from macroscopically normal areas of PZ, TZ, and CZ in radical prostatectomy specimens. After exclusion of samples with cancer or prostatic intraepithelial neoplasia, 18 cases remained for analysis. Cells were collected in a medium with protease inhibitors, and the protein material was prepared for two-dimensional gel electrophoresis. The proteins in spots that differed quantitatively between regions were identified via mass spectrometric fingerprinting of tryptic fragments and selected tandem mass spectrometry sequence analysis. Ten proteins with significant zonal differential expression were identified, eight with underexpression in the CZ versus the PZ and the TZ (arginase II, ATP synthase, cytokeratin 8, lamin A/C, peroxiredoxin 4, protein disulfide isomerase A3, tropomyosin, and vimentin), and two with overexpression in the CZ (peroxiredoxin 2 and creatine kinase B). The PZ and TZ, although differing in terms of incidence of cancer and hyperplasia, have epithelium with highly similar major protein expression profiles. However, the protein profile of the CZ differs from that of the other regions, suggesting functional differences.

Aged↗

PubMed2001

Proteome analysis of cultivar-specific interactions between Rhizobium leguminosarum biovar trifolii and subterranean clover cultivar Woogenellup.

Proteome analysis was used to identify proteins that are involved in the early stages of nodulation between the subterranean clover cultivar Woogenellup and the Rhizobium leguminosarum bv. trifolii strains ANU843 and ANU794. Strain ANU843 induces nitrogen-fixing nodules whereas strain ANU794 forms aberrant nodules on the roots of cv. Woogenellup that fail to develop beyond an early stage. Our aim was to identify proteins that might be involved in the early stages of nodulation over a 48 h period and to identify proteins that are differentially displayed during the interactions between the host and the two microbes. Proteome maps from control Woogenellup roots and inoculated roots were generated and compared at 24 and 48 h post inoculation. Over 1500 spots were resolved on all gels. Of the 16 protein spots that were differentally displayed or developmentally regulated, 10 were assigned putative identities. These included an alpha-fucosidase, several ethylene-induced proteins, a Cu/Zn superoxide dismutase, a hypothetical 16.5 kDa protein, tubulin alpha-chain, chaperonin 21 precursor and triosephosphate isomerase. Of the 22 constitutively expressed proteins spots examined, eight spots were assigned putative protein homologies through N-terminal sequencing and included several pathogenesis and stress-related proteins. The result may suggest that ethylene levels are upregulated during the early stages of infection but that this does not result in the induction of common pathogenesis-related proteins. The specific induction of alpha-fucosidase by ANU794 may be important in the nodulation failure phenotype of strain ANU794.

Amino Acid Sequence↗

PubMed2004

A proteomic analysis of malaria biology: integration of old literature and new technologies.

The genomic revolution has brought a new vitality into research on Plasmodium, its insect and vertebrate hosts. At the cellular level nowhere is the impact greater than in the analysis of protein expression and the 'assembly' of the supramolecular machines that together comprise the functional cell. The repetitive phases of invasion and replication that typify the malaria life cycle, together with the unique phase of sexual differentiation provide a powerful platform on which to investigate the 'molecular machines' that underpin parasite strategy and stage-specific functions. This approach is illustrated here in an analysis of the ookinete of Plasmodium berghei. Such analyses are useful only if conducted with a secure understanding of parasite biology. The importance of carefully searching the older literature to reach this understanding cannot be over-emphasised. When viewed together, the old and new data can give rapid and penetrating insights into what some might now term the 'Systems-Biology' of Plasmodium.

Animals↗

PubMed2006

Identification of alkaline proteins that are differentially expressed in an overgrowth-mediated growth arrest and cell death of Escherichia coli by proteomic methodologies.

The available Escherichia coli genome sequences offer an opportunity to further expand our understanding of this bacterium. In the current study, we present a rapid method for the isolation of bacterial alkaline proteins using acid incubation, purification and protein array by 2-DE, followed by protein identification using MS. Fifty-seven proteins were randomly chosen, in which 55 were identified by a database searching of MS data. The searching results showed that most of these alkaline proteins were involved in special functions within the cell, suggesting that alkaline proteome is an ideal fraction for an understanding of their special functions. Furthermore, alkaline proteomes were compared between the period of majority live bacteria (18-h culture), the period of similar amount of live and dead bacteria (30-h culture) and the period of majority dead bacteria (48-h culture). Six proteins were identified as differentially expressed targets, in which putative transcriptional regulator and superoxide dismutase genes were cloned and expressed for antiserum preparations. The antisera were applied for the confirmation of results obtained from 2-DE. The presented data clearly reveal that alkaline proteome analysis by 2-DE with MS plays an important role in the understanding of protein functions within the cell, and six alkaline proteins are determined as key ones in an overgrowth-mediated growth cycle of E. coli.

Cell Death↗

PubMed2004

Calcyclin, a Ca2+ ion-binding protein, contributes to the anabolic effects of simvastatin on bone.

In vitro treatment with a pharmacological dose of simvastatin, a potent pro-drug of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, stimulates bone formation. In our study, simvastatin stimulated differentiation of osteoblasts remarkably in a dose-dependent manner, with minimal effect on proliferation. To identify the mediators of the anabolic effects of simvastatin on osteoblasts, we tried to identify and characterize simvastatin-induced proteins by using proteomic analysis. Calcyclin was significantly up-regulated by more than 10 times, and annexin I was also up-regulated by simvastatin. However, annexin III, vimentin, and tropomyosin were down-regulated. Up-regulated calcyclin mRNA by simvastatin was validated by reverse transcription in mouse calvarial cells. In confocal microscope analysis, green fluorescence protein-calcyclin fusion protein was ubiquitously observed in the of MC3T3-E1 cells transfected with green fluorescence protein-calcyclin cDNA containing plasmid and was quickly concentrated in the nucleus 20 min after simvastatin treatment. Overexpression of calcyclin cDNA stimulated both the proliferation and expression of alkaline phosphatase mRNA significantly, without exposure to simvastatin in MC3T3-E1 cells. However, both the rate of proliferation of the osteoblasts and the expression of alkaline phosphatase mRNA were suppressed significantly 1 day after treatment with the calcyclin-specific small interference RNA, and furthermore, simvastatin did not overcome this suppression in the small interference RNA-pretreated MC3T3-E1 cells. In conclusion, calcyclin is one of the candidate proteins that plays a role in osteoblastogenesis in response to simvastatin, although the precise functions of calcyclin in osteoblast remain to be verified.

Animals↗

PubMed2003

Western array analysis of human atherosclerotic plaques: downregulation of apoptosis-linked gene 2.

OBJECTIVE: In recent years, microarray techniques have been used to characterize differences in mRNA populations between atherosclerotic plaques and normal arterial tissue. Because proteomics provide an attractive complementary approach to genomics, we used Western array technology as a global protein profiling method to identify differentially expressed proteins with potential pathobiological relevance in human atherosclerotic plaques. METHODS: Cell lysates from human carotid endarterectomy specimens and non-atherosclerotic mammary arteries were screened with monoclonal antibodies (823 in total) that were combined into unique cocktails. Hits were verified with traditional Western blotting. RESULTS: Seven proteins with a >5-fold relative expression difference were identified. One of the most apparent changes in human plaques was the downregulation of apoptosis-linked gene 2 (ALG-2), a positive mediator of apoptotic cell death. Differential expression of ALG-2 in human plaques relative to mammary arteries was not confirmed by real-time quantitative RT-PCR, suggesting post-transcriptional regulation. Uptake of aggregated LDL (agLDL) downregulated ALG-2 protein expression in THP-1 macrophages, but not in smooth muscle cells (SMCs). Transfection of THP-1 cells with ALG-2-specific small interfering RNA (siRNA) caused ALG-2 depletion and inhibited the execution phase of apoptosis (DNA fragmentation) but did not affect caspase-3 activation, annexin-V labeling and necrotic cell death. CONCLUSION: Western array screening of carotid endarterectomy specimens revealed a strong downregulation of ALG-2 protein. Because ALG-2 has pro-apoptotic potential, our results point to a novel survival mechanism against cell death in human atherosclerotic plaques.

Aged↗

PubMed2006

Proteomic analysis of glutamine-treated human intestinal epithelial HCT-8 cells under basal and inflammatory conditions.

Glutamine (Gln) promotes intestinal growth and maintains gut structure and function, especially in situations of injury and during inflammation. Several mechanisms could contribute to Gln protective effects on gut. Proteomics enable us to characterize differentially expressed proteins in tissues in response to modifications of the biological or nutritional environment. Gln effects on the human intestinal epithelial HCT-8 cell line proteome were assessed under basal and proinflammatory conditions. The 2-DE gels were obtained and compared. Proteins were identified by MS and using databases. About 1200 spots were detected in both 2- and 10-mM Gln concentrations. Under basal conditions, 24 proteins were differentially expressed in response to Gln. Half of these proteins were implicated in protein biosynthesis or proteolysis and 20% in membrane trafficking. Under proinflammatory conditions, 27 proteins were up- or down-regulated by Gln 10 mM. From these proteins, 40% were involved in protein biosynthesis or proteolysis, 16% in membrane trafficking, 8% in cell cycle and apoptosis mechanisms and 8% in nucleic acid metabolism. This study provides the first holistic picture of proteome modulation by Gln in a human enterocytic cell line under basal and proinflammatory conditions, and supports further evaluation of nutritional modulation of intestinal proteome in humans.

Cell Line↗