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Protein Profiles (protein + profile)
Selected AbstractsLife Cycle Stage-Specific and Encystment Protein Profiles in Pneumocystis cariniiTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2003MARK E. LASBURY No abstract is available for this article. [source] Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS): A new proteomic urinary test for patients with urolithiasisJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 3 2004Peter A. Cadieux Abstract SELDI-TOF-MS is a highly sensitive protein-analysis tool capable of detecting minute protein profile differences between biological samples. As proteins have been associated with urinary tract calculi, protein-based urinalysis may offer insights into their diagnosis. The purpose of this study was to evaluate SELDI-TOF-MS as a potential method for identifying urinary biomarkers of urolithiasis. Midstream sterile urine samples were obtained from 25 male patients with a confirmed diagnosis of urolithiasis (test group) and 25 male subjects with no known history of the disease (controls). Urinary levels of oxalate, total protein, albumin, and osteopontin were determined. Protein profiles were generated using SELDI-TOF-MS. SELDI-TOF-MS profiling revealed a relationship between protein peak intensities at 67 and 24 kDa that differed between the two groups. The ratio of p67:p24 was found to be less than 1.0 in all of the control samples (mean 0.26), while 18 out of 25 (72%) of the test group samples displayed a ratio greater than 1.0 (total group mean 4.75, P<0.001). Albumin, total protein, and oxalate levels were higher in the test group than the controls. Although SELDI-TOF-MS is not yet in widespread use in hospital and diagnostic laboratories, this system represents a promising new method for rapidly identifying patients with urolithiasis. J. Clin. Lab. Anal. 18:170,175, 2004. © 2004 Wiley-Liss, Inc. [source] Protein profiles of bovine placenta derived from somatic cell nuclear transferPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2005Hong Rye Kim Abstract Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0,7.0 and 6.0,9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT. [source] Influence of variations in sample handling on SELDI-TOF MS serum protein profiles for colorectal cancerPROTEOMICS - CLINICAL APPLICATIONS, Issue 6 2008Judith Y. M. N. Engwegen Abstract Sample handling can have a profound effect on serum protein profiles, challenging results obtained with archived sera under non-standardized sample collection. Here, we evaluate the influence of variations in sample handling on previous serum protein profiles for colorectal cancer (CRC) (Engwegen et al.,. World J. Gastroenterol. 2006, 12, 1536,1544). Sera were prospectively obtained from individuals with an indication for colonoscopy (n = 150: 65 controls, 52 adenomatous polyps, 29 CRC, 4 unknown), as well as from normal volunteers (n = 8). Protein profiles were acquired by SELDI-TOF MS on CM10 chips at pH 5. We assessed the influence of storage temperature, type of collection tube, coagulation temperature and freeze-thaw cycles on the serum protein profile. Several peptides occurred only in samples stored at ,20°C, indicating proteolytic degradation during storage. One was a previous CRC biomarker candidate, an N-terminal albumin fragment (m/z 3087), and two others complement C3f and a fragment thereof (m/z 2022 and 1863). Overall differences in protein profiles were also seen for different collection tubes, coagulation temperature and freeze-thaw cycles. However, three of five of our previously defined CRC biomarker candidates are stable to variations in the sample handling protocol, justifying their further validation in prospective studies. [source] Identification of neutrophil gelatinase-associated lipocalin (NGAL) as a discriminatory marker of the hepatocyte-secreted protein response to IL-1,: a proteomic analysisBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Arul Jayaraman Abstract The liver is the major source of proteins used throughout the body for various functions. Upon injury or infection, an acute phase response (APR) is initiated in the liver that is primarily mediated by inflammatory cytokines such as interleukin-1, (IL-1,) and interleukin-6. Among others, the APR is characterized by an altered protein synthetic profile. We used two-dimensional gel electrophoresis to study the dynamics of changes in protein synthesis in hepatocytes exposed to these inflammatory cytokines. Protein profiles were quantified using image analysis and further analyzed using multivariate statistical methods. Our results indicate that IL-1, and IL-6 each induces secreted protein responses with distinct dynamics and dose-dependence. Parallel stimulation by IL-1, and IL-6 results in a protein pattern indistinguishable from the IL-1, pattern, indicating a dominant effect of IL-1, over IL-6 at the doses tested. Multidimensional scaling (MDS) of correlation distances between protein secretion levels revealed two protein pairs that are robustly co-secreted across the various cytokine stimulation conditions, suggesting shared regulatory pathways. Finally, we also used multivariate alternating conditional expectation (MACE) to identify transformation functions that discriminated the cytokine-stimulated and untreated hepatocyte-secreted protein profiles. Our analysis indicates that the expression of neutrophil gelatinase-associated lipocalin (NGAL) was sufficient to discriminate between IL-1, and IL-6 stimulation. The combination of proteomics and multivariate analysis is expected to provide new information on the cellular regulatory networks involved in generating specific cellular responses. © 2005 Wiley Periodicals, Inc. [source] Protein profile study in European eel (Anguilla anguilla) seminal plasma and its correlation with sperm qualityJOURNAL OF APPLIED ICHTHYOLOGY, Issue 5 2010D. S. Peñaranda Summary Along with sperm quality parameters, the protein profile of European eel seminal plasma was analyzed during induced spermiation (n = 56 samples). Motility, Percentage of live cells, spermatozoa head morphometry and concentration showed low values during the initial weeks of spermiation and maintained high levels throughout the rest of the experiment. The protein profile gradient by SDS-PAGE (4,15%) registered four important electrophoretic bands around 80, 40, 26 and 12 KDa. Three of them showed significant differences in concentration during treatment (80, 40 and 12 KDa), and all of them showed the highest value on the 8th week. Both 80 and 12 KDa bands increased until the 8th week, followed by a progressive decline. One possible explanation for these profiles is that, in the first weeks of treatment, proteins originated from blood plasma are accumulated in the seminal plasma, and from the 8th week some of these proteins are incorporated into the spermatic membranes. The 40 KDa protein band also increased during the first 8 weeks, but maintained high concentrations in the seminal plasma for the rest of the experiment. One result confirms the theory that the presence of proteins in the seminal plasma having a molecular weight lower than 50 KDa increased spermatozoa motility, since the 40 KDa band displayed significantly higher values coinciding with the high percentages of spermatozoa motility. Seminal plasma proteins seem to have an important role in spermatogenesis and spermatozoa movement, but further studies are necessary to discover the identity of these proteins and their precise functions. [source] Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrolJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2006Karel Bezstarosti Abstract Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardiopro-tection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apopto-sis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and precondi-tioning-related proteins were identified that were all upregulated by resveratrol: MAPKK, two different aB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxire-doxin-2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -aB-crys-tallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofila-ment regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins. [source] Characterization and application of monoclonal antibodies against white spot syndrome virusJOURNAL OF FISH DISEASES, Issue 3 2001Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north-eastern Taiwan. By sodium dodecyl sulphate,polyacrylamide gel electrophoresis (SDS,PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG1 class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non-target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies. [source] A further investigation on a MALDI-based method for evaluation of markers of renal damageJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2009Annunziata Lapolla Abstract The validity of the urinary protein profile to characterize the pathological states of diabetic, nephropathic and diabetic,nephropathic patients was considered on the basis of previously obtained results by MALDI/MS, showing a different abundance ratio of the collagen ,1 and ,5 chain precursor fragments at m/z 1219 and 2049 and of the uromodulin precursor fragment at m/z 1912 observed in healthy subjects and patients; a larger number of subjects was examined and the obtained results were statistically evaluated. The p values related to the observed differences indicate that they are statistically significant when comparing all patients versus healthy controls, diabetic with normo or microalbuminuria versus nephropathic with advanced renal disease patients and diabetic with normo or microalbuminuria versus diabetic with advanced nephropathy patients. The scatter plot matrix gives evidence of the strict inverse relationship between the abundances of ions at m/z 1912 and 1219, the correlation coefficient being particularly high (r = 0.921, p < 0.001). The relationship between the true positive rate (sensitivity) and false positive rate (1,specificity) for every possible cutoff value in abundance of the considered ionic species was investigated through the receiver-operating characteristic (ROC) curve. The obtained data indicate that a good differentiation of nephropathic patients with advanced renal disease and diabetic patients with advanced nephropathy versus healthy subjects can be easily obtained by this approach. Copyright © 2009 John Wiley & Sons, Ltd. [source] Multivariate analysis approach to the plasma protein profile of patients with advanced colorectal cancer,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2006Eugenio Ragazzi Abstract The aim of the present study was to identify the pattern of plasma protein species of interest as markers of colorectal cancer (CRC). Using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), the plasma protein profile was determined in nine stage IV CRC patients (study group) and nine clean-colon healthy subjects (control group). Multivariate analysis methods were employed to identify distinctive disease patterns at protein spectrum. In the study and control groups, cluster analysis (CA) on the complete MALDI-MS spectra plasma protein profile showed a distinction between CRC patients and healthy subjects, thus allowing the identification of the most discriminating ionic species. Principal component analysis (PCA) and linear discriminant analysis (LDA) yielded similar grouping results. LDA with leave-one-out cross validation achieved a correct classification rate of 89% in both the patients and the healthy subjects. Copyright © 2006 John Wiley & Sons, Ltd. [source] Isozyme Analysis and Soluble Mycelial Protein Pattern in Iranian Isolates of Several formae speciales of Fusarium oxysporumJOURNAL OF PHYTOPATHOLOGY, Issue 5 2004M. Mohammadi Abstract A total of 13 representative isolates of Fusarium oxysporum f. sp. melonis (FOM) from Iran, USA and France, eight isolates of seven formae speciales from Iran and one isolate of F. oxysporum f. sp. niveum from the USA were compared based on isozyme analysis and soluble mycelial protein pattern. Isozyme analyses of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD) and xanthine dehydrogenase (XDH) revealed polymorphism among the F. oxysporum isolates in which 22 electrophoretic phenotypes (EP) were determined. At least 10 putative loci for these six enzymes were detected and they were all polymorphic. Maximum genetic diversity was observed in CAT, EST and XDH loci. Using UPGMA, the 22 isolates were separated into three main groups with one of the groups divided into two subgroups. Group I included isolates belonging to five formae speciales from Iran, whereas group II that included FOM isolates from both Iran and the USA was divided into two subgroups each containing the vast majority of the respective isolates from either country. Group III constituted FOM isolates from France and one pathogenic isolate on pepper from Iran. FOM isolates representing five different geographical regions from Iran belonged to two different races of 1 and 1,2Y and one vegetative compatibility group (VCG)0134 and thus were genetically homologous. Isozyme polymorphism in these isolates was highly correlated with VCG and geographical origins and to a lesser extent with races. Variations in soluble protein profile in FOM isolates were correlated with genetic distances determined in isozyme analysis. This study suggests that isozyme analysis could be a useful tool for identifying genetic diversity not only in FOM but also several formae speciales of F. oxysporum. [source] Molecular and Cellular Events in Alcohol-Induced Muscle DiseaseALCOHOLISM, Issue 12 2007Joaquim Fernandez-Solà Alcohol consumption induces a dose-dependent noxious effect on skeletal muscle, leading to progressive functional and structural damage of myocytes, with concomitant reductions in lean body mass. Nearly half of high-dose chronic alcohol consumers develop alcoholic skeletal myopathy. The pathogenic mechanisms that lie between alcohol intake and loss of muscle tissue involve multiple pathways, making the elucidation of the disease somewhat difficult. This review discusses the recent advances in basic and clinical research on the molecular and cellular events involved in the development of alcohol-induced muscle disease. The main areas of recent research interest on this field are as follows: (i) molecular mechanisms in alcohol exposed muscle in the rat model; (ii) gene expression changes in alcohol exposed muscle; (iii) the role of trace elements and oxidative stress in alcoholic myopathy; and (iv) the role of apoptosis and preapoptotic pathways in alcoholic myopathy. These aforementioned areas are crucial in understanding the pathogenesis of this disease. For example, there is overwhelming evidence that both chronic alcohol ingestion and acute alcohol intoxication impair the rate of protein synthesis of myofibrillar proteins, in particular, under both postabsorptive and postprandial conditions. Perturbations in gene expression are contributory factors to the development of alcoholic myopathy, as ethanol-induced alterations are detected in over 400 genes and the protein profile (i.e., the proteome) of muscle is also affected. There is supportive evidence that oxidative damage is involved in the pathogenesis of alcoholic myopathy. Increased lipid peroxidation is related to muscle fibre atrophy, and reduced serum levels of some antioxidants may be related to loss of muscle mass and muscle strength. Finally, ethanol induces skeletal muscle apoptosis and increases both pro- and antiapoptotic regulatory mechanisms. [source] A gel-free quantitative proteomics approach to investigate temperature adaptation of the food-borne pathogen Cronobacter turicensis 3032PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2010Paula Carranza Abstract The opportunistic food-borne pathogen Cronobacter sp. causes rare but significant illness in neonates and is capable to grow at a remarkably wide range of temperatures from 5.5 to 47°C. A gel-free quantitative proteomics approach was employed to investigate the molecular basis of the Cronobacter sp. adaptation to heat and cold-stress. To this end the model strain Cronobacter turicensis 3032 was grown at 25, 37, 44, and 47°C, and whole-cell and secreted proteins were iTRAQ-labelled and identified/quantified by 2-D-LC-MALDI-TOF/TOF-MS. While 44°C caused only minor changes in C. turicensis growth rate and protein profile, 47°C affected the expression of about 20% of all 891 identified proteins and resulted in a reduced growth rate and rendered the strain non-motile and filamentous. Among the heat-induced proteins were heat shock factors, transcriptional and translational proteins, whereas proteins affecting cellular morphology, proteins involved in motility, central metabolism and energy production were down-regulated. Notably, numerous potential virulence factors were found to be up-regulated at higher temperatures, suggesting an elevated pathogenic potential of Cronobacter sp. under these growth conditions. Significant alterations in the protein expression profile and growth rate of C. turicensis exposed to 25°C indicate that at this temperature the organism is cold-stressed. Up-regulated gene products comprised cold-shock, DNA-binding and ribosomal proteins, factors that support protein folding and proteins opposing cold-induced decrease in membrane fluidity, whereas down-regulated proteins were mainly involved in central metabolism. [source] A comparative proteomic analysis of HepG2 cells incubated by S(,) and R(+) enantiomers of anti-coagulating drug warfarinPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2010Jing Bai Abstract Warfarin is a commonly prescribed oral anti-coagulant with narrow therapeutic index. It interferes with vitamin K cycle to achieve anti-coagulating effects. Warfarin has two enantiomers, S(,) and R(+) and undergoes stereoselective metabolism, with the S(,) enantiomer being more effective. We reported that the intracellular protein profile in HepG2 cells incubated with S(,) and R(+) warfarin, using iTRAQ-coupled 2-D LC-MS/MS. In samples incubated with S(,) and R(+) warfarin alone, the multi-task protein Protein SET showed significant elevation in cells incubated with S(,) warfarin but not in those incubated with R(+) warfarin. In cells incubated with individual enantiomers of warfarin in the presence of vitamin K, protein disulfide isomerase A3 which is known as a glucose-regulated protein, in cells incubated with S(,) warfarin was found to be down-regulated compared to those incubated with R(+) warfarin. In addition, Protein DJ-1 and 14-3-3 Protein, were down-regulated in cells incubated with either S(,) or R(+) warfarin regardless of the presence of vitamin K. Our results indicated that Protein DJ-1 may act as an enzyme for expression of essential enzymes in vitamin K cycle. Taken together, our findings provided molecular evidence on a comprehensive protein profile on warfarin,cell interaction, which may shed new lights on future improvement of warfarin therapy. [source] Subcellular proteomics of Trypanosoma cruzi reservosomesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2009Celso Sant'Anna Abstract Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC-MS/MS analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi -specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles. [source] Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stagePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2008Frédérique Baruthio Abstract The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression. [source] Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2007Nicolai Juul Abstract The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage. [source] Proteomic analysis of rabbit tear fluid: Defensin levels after an experimental corneal wound are correlated to wound closurePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2007Lei Zhou Abstract The cornea is the major refracting optical element of the eye and therefore critical for forming a retinal image. The exposed surface of the eye is protected from pathogens by the innate immune system whose components include defensins, naturally occurring peptides with antimicrobial properties, and the physical barrier formed by the outer epithelial layer of the cornea. The proteomic approach has revealed that tear levels of defensins are correlated with the course of healing of an experimental corneal wound. Tears were collected from New Zealand White rabbits prior to (day 0) and daily for 5 days (days 1,5) following a standard unilateral 6,mm diameter corneal epithelial abrasion. Tear protein profiles obtained from wounded and contra-lateral control eyes were compared using SELDI ProteinChip technology. Peptides and proteins of interest were purified by RP-HPLC and characterized by nanoESI-MS/MS. Mass spectra of tears on post-wound day 1, revealed 13,peaks whose level decreased and five that increased. During wound healing the tear protein profile correlated with wound closure. An important finding was that the levels of rabbit defensins (NP-1 and NP-2), which were elevated after wounding returned to normal levels by the time the corneal abrasion healed. Relative quantification of NP-2 in tear fluid prior to (day 0) and after corneal wounding (days 1, 3) was determined using iTRAQ technology. A corneal wound eliminates the barrier function of innate immunity and puts the cornea at risk from microbial attack until the epithelial cells restore the surface barrier. The increased availability of defensins in the tears during healing suggests that these peptides could protect the cornea from microbial attack during a period of increased vulnerability. [source] Proteomic analysis of the E2F1 response in p53-negative cancer cells: New aspects in the regulation of cell survival and deathPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 21 2006Zhenpeng Li Abstract E2F1 is an essential transcription factor that regulates cell-cycle progression and apoptosis. Overexpression of E2F1 sensitizes neoplastic cells to apoptosis and leads to tumor growth suppression, making it an interesting target for anticancer therapy. Use of E2F1 as a therapeutic, however, requires a detailed knowledge of the mechanisms by which it controls cellular proliferation and apoptosis, and of other potential E2F1 activities. In this study, a differential proteome analysis was performed to identify proteins associated with E2F1 activity in inducible p53-deficient Saos-2ERE2F1 osteosarcoma cells. 2-DE revealed a distinct protein profile at 32,h after E2F1 activation. Thirty-three proteins were reproducibly identified as either up-regulated or down-regulated. Proteins were identified by MALDI-MS. They included hitherto unknown E2F1 target proteins of cytoskeletal origin, chaperones, enzymes, proteasomal proteins, and several heterogeneous nuclear ribonucleoproteins, suggesting its role in the ER-stress response, protein degradation, and modulation of pre-mRNA splicing. Protein analysis-derived results were verified by Western blot using representative protein candidates. Thirteen identified proteins were the products of genes known to be cancer related. Thus, proteome analysis provides new information about the complexity of E2F1 activities in human cancer cells that may be considered when using E2F1 as a drug. [source] Inflammatory protein profile during systemic high dose interleukin-2 administrationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2006Leonardo Rossi Abstract Systemic interleukin-2 (IL-2) administration induces an assortment of downstream effects whose biological and therapeutic significance remains unexplored mostly because of the methodological inability to globally address their complexity. Protein array analysis of sera from patients with renal cell carcinoma obtained prior and during high-dose IL-2 therapy had previously revealed extensive alterations in expression of the soluble factors analyzed, whose discovery was limited by the number of capture antibodies selected for protein detection. Here, we expanded the analysis to SELDI-TOF-MS and quantitative protein analysis (nephelometry). All cytokines/chemokines detected by protein arrays were below the SELDI detection limit, while novel IL-2-specific changes in expression of acute-phase reactants and high-density lipoprotein metabolites could be identified. Serum amyloid protein,A (SAA) and C-reactive protein expression were consistently up-regulated after four doses of IL-2, while other proteins were down-regulated. These findings were confirmed by SELDI immunoaffinity capture and nephelometry. Immunoaffinity capture revealed different, otherwise undetectable, isoforms of SAA. A linear correlation between peak area by SELDI and protein concentration by nephelometry was observed. Overall distinct yet complementary information was obtained using different platforms, which may better illustrate complex phenomena such as the systemic response to biological response modifiers. [source] Plasma protein profiling: Unique and stable features of individualsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2005Gary L. Nelsestuen Dr. Abstract Carefully controlled ZipTip extraction of diluted human plasma or serum was combined with MALDI-TOF-MS to produce highly reproducible protein profiles. Components detected included apolipoproteins CI, CII and CIII as well as transthyretin and several isoforms of each protein that are created by glycosylation or other modification and by proteolytic processing. Profiles of healthy individuals all contained the same 15,components. Others were found in plasma from individuals with disease. Profiles were analyzed by peak ratios within the same spectrum. Reproducibility for multiple assays was generally 4 to 10%. Within the healthy population, a given peak ratio occurred with a range of about fourfold. However, peak ratios of multiple samples from the same individual showed a much lower range, typically ±10%. In fact, each individual displayed a personal protein profile that changed very little over time. Because of the stability of protein profiles over time within individuals, these results suggest further studies may discover that certain profile characteristics or changes in an individual's profile may be a sign of current or future disease, even when the altered profile remains within the range for healthy individuals. [source] Differential protein expression in anatomical zones of the prostatePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2005Helena Lexander Abstract 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. [source] Protein profiling in pathology: Analysis and evaluation of 239 frozen tissue biopsies for diagnosis of B-cell lymphomasPROTEOMICS - CLINICAL APPLICATIONS, Issue 5 2010Corine Jansen Abstract Purpose: We determined the potential value of protein profiling of tissue samples by assessing how precise this approach enables discrimination of B-cell lymphoma from reactive lymph nodes, and how well the profiles can be used for lymphoma classification. Experimental design: Protein lysates from lymph nodes (n=239) from patients with the diagnosis of reactive hyperplasia (n=44), follicular lymphoma (n=63), diffuse large B-cell lymphoma (n=43), mantle cell lymphoma (n=47), and chronic lymphocytic leukemia/small lymphocytic B-cell lymphoma (n=42) were analysed by SELDI-TOF MS. Data analysis was performed by (i) classification and regression tree-based analysis and (ii) binary and polytomous logistic regression analysis. Results: After internal validation by the leave-one-out principle, both the classification and regression tree and logistic regression classification correctly identified the majority of the malignant (87 and 96%, respectively) and benign cases (73 and 75%, respectively). Classification was less successful since approximately one-third of the cases of each group were misclassified according to the histological classification. However, an additional mantle cell lymphoma case that was misclassified as chronic lymphocytic leukemia/small lymphocytic B-cell lymphoma initially was identified based on the protein profile. Conclusions and clinical relevance: SELDI-TOF MS protein profiling allows for reliable identification of the majority of malignant lymphoma cases; however, further validation and testing robustness in a diagnostic setting is needed. [source] Comparative proteomic analysis of differentially expressed proteins in primary retinoblastoma tumorsPROTEOMICS - CLINICAL APPLICATIONS, Issue 4 2010Kandalam Mallikarjuna Abstract Purpose: To understand the disease mechanism and to identify the potential tumor markers that would help in therapeutics, comparative proteomic analysis of 29 retinoblastoma (RB) tumors was performed using 14 non-neoplastic retinas (age ranged from 45 to 89 years) as control tissues. Experimental design: 2-DE and MALDI-TOF-TOF MS/MS were used to identify differentially expressed proteins. Results: Twenty-seven distinct differentially expressed proteins were identified, including 16 upregulated 11 downregulated proteins. Significantly, higher mRNA levels of apolipoprotein A1 (p<0.001), transferrin (TF; p<0.001), CRABP2 (p<0.001), ,-crystallin A (CRYAA; p<0.001) were observed in RBs when compared with normal retinas and hence are consistent with the proteomic data. Immunohistochemistry was also performed for selected proteins on paraffin RB blocks to confirm protein expression. RB with invasion showed significantly higher expression by 2-DE-MS/MS analysis of CRABP2 (p<0.001), peroxiredoxin 6 (p=0.025), apolipoprotein A1 (p<0.001), recoverin (p<0.001). Conclusions and clinical relevance: Thus, this study provides a dynamic protein profile of RB tumors, which could provide clues to study the mechanisms of RB oncogenesis and possibly be developed as potential biomarkers for prognosis and therapy. [source] Is GRP78/BiP a potential salivary biomarker in patients with rheumatoid arthritis?PROTEOMICS - CLINICAL APPLICATIONS, Issue 3 2010Laura Giusti Abstract Purpose: In the last few years, serum and joint synovial fluid have been extensively analyzed for the proteomic research of rheumatoid arthritis (RA) biomarkers. Nonetheless, to date, there have been no studies investigating salivary biomarkers in this condition. Therefore, aim of this study is to investigate the presence of potential biomarkers of RA in human whole saliva. Experimental design: We combined 2-DE and MS to analyze the whole saliva protein profile of 20 RA patients in comparison with 20 sex- and age-matched healthy subjects. Results: Eight salivary proteins resulted differentially expressed, namely calgranulin A, calgranulin B, apolipoprotein A-1, 6-phosphogluconate dehydrogenase, peroxiredoxin 5, epidermal fatty acid-binding protein, 78,kDa glucose-regulated protein precursor (GRP78/BiP), and 14-3-3 proteins. It is particularly interesting that chaperone GRP78/BiP showed the greatest increase in RA patients. This finding was validated by Western Blot analysis and the over-expression of GRP78/BiP appear to be distinctive of RA and drugs treatment independent. Conclusions and clinical relevance: This study provides a rationale for further studies aimed at evaluating any correlation between GRP78/BiP and different clinical/serological aspects of the disease in order to improve the diagnostic algorithms of RA. [source] Influence of variations in sample handling on SELDI-TOF MS serum protein profiles for colorectal cancerPROTEOMICS - CLINICAL APPLICATIONS, Issue 6 2008Judith Y. M. N. Engwegen Abstract Sample handling can have a profound effect on serum protein profiles, challenging results obtained with archived sera under non-standardized sample collection. Here, we evaluate the influence of variations in sample handling on previous serum protein profiles for colorectal cancer (CRC) (Engwegen et al.,. World J. Gastroenterol. 2006, 12, 1536,1544). Sera were prospectively obtained from individuals with an indication for colonoscopy (n = 150: 65 controls, 52 adenomatous polyps, 29 CRC, 4 unknown), as well as from normal volunteers (n = 8). Protein profiles were acquired by SELDI-TOF MS on CM10 chips at pH 5. We assessed the influence of storage temperature, type of collection tube, coagulation temperature and freeze-thaw cycles on the serum protein profile. Several peptides occurred only in samples stored at ,20°C, indicating proteolytic degradation during storage. One was a previous CRC biomarker candidate, an N-terminal albumin fragment (m/z 3087), and two others complement C3f and a fragment thereof (m/z 2022 and 1863). Overall differences in protein profiles were also seen for different collection tubes, coagulation temperature and freeze-thaw cycles. However, three of five of our previously defined CRC biomarker candidates are stable to variations in the sample handling protocol, justifying their further validation in prospective studies. [source] iTRAQ-coupled two-dimensional liquid chromatography/tandem mass spectrometric analysis of protein profile in Escherichia coli incubated with human neutrophil peptide 1 , potential in antimicrobial strategyRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2010Yu Si Zhou First page of article [source] Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patientsAPMIS, Issue 3 2010TADAO HASEGAWA Hasegawa T, Okamoto A, Kamimura T, Tatsuno I, Hashikawa S-N, Yabutani M, Matsumoto M, Yamada K, Isaka M, Minami M, Ohta M. Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients. APMIS 2010; 118: 167,78. Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in Japan and many other developed countries. Epidemiological studies have revealed that the M1 serotype of Streptococcus pyogenes is the most dominant causative isolate of STSS. Recent characterization of M1 isolates revealed that the mutation of covS, one of the two-component regulatory systems, plays an important role in STSS by altering protein expression. We analyzed the M1 S. pyogenes clinical isolates before or after 1990 in Japan, using two-dimensional gel electrophoresis (2-DE) and pulsed-field gel electrophoresis (PFGE). PFGE profiles were different between the isolates before and after 1990. Markedly different profiles among isolates after 1990 from STSS and pharyngitis patients were detected. Sequence analysis of two-component regulatory systems showed that covS mutations were detected not only in STSS but also in three pharyngitis isolates, in which proteins from the culture supernatant displayed the invasive type. The mutated CovS detected in the pharyngitis isolates had impaired function on the production of streptococcal pyrogenic exotoxin B (SpeB) analyzed by 2-DE. These results suggest that several covS mutations that lead to the malfunction of the CovS protein occurred even in pharyngeal infection. [source] A study of the antigenicity of Rickettsia helvetica proteins using two-dimensional gel electrophoresisAPMIS, Issue 4 2009NEDAA HAJEM Rickettsia helvetica is an obligate intracellular Gram-negative microorganism found in Ixodes ricinus ticks. When R. helvetica was first discovered in 1979, little was known about its physiology and it fell into oblivion until it recently was suspected of being pathogenic to humans. However, all efforts to isolate R. helvetica from patients have been unsuccessful, although serological responses against R. helvetica can be demonstrated. The aim of our study was to investigate the protein profile of R. helvetica and study the antigenicity of its proteins using two-dimensional (2D) immunoblot in order to characterize the immunological response against R. helvetica infection. Our results show that in addition to the known PS120 and OmpB antigenic R. helvetica proteins, three other antigens exist: a 60 kDa GroEL protein, a 10 kDa GroES protein and a hitherto unknown 35 kDa hypothetical protein that has similarities with ORF-RC0799 of Rickettsia conorii. Furthermore, the lipopolysaccharide showed strong antigenicity. In this study, we present the first proteome map and the first 2D immunoblot profile of R. helvetica and finally we present the 35 kDa R. helvetica as an additional antigen to the previously known rickettsial antigens. [source] Virulence status, viral accommodation and structural protein profiles of white spot syndrome virus isolates in farmed Penaeus monodon from the southeast coast of IndiaAQUACULTURE RESEARCH, Issue 2 2009Victor Stalinraj Abstract The objective of this study was to investigate the reason for variation in the virulence of white spot syndrome virus (WSSV) from different shrimp farms in the Southeast coast of India. Six isolates of WSSV from farms experiencing outbreaks (virulent WSSV; vWSSV) and three isolates of WSSV from farms that had infected shrimps but no outbreaks (non-virulent WSSV; nvWSSV) were collected from different farms in the Southeast coast of India. The sampled animals were all positive for WSSV by first-step PCR. The viral isolates were compared using histopathology, electron microscopy, SDS-PAGE analysis of viral structural proteins, an in vivo infectivity experiment and sequence comparison of major structural protein VP28; there were no differences between isolates in these analyses. A significant observation was that the haemolymph protein profile of nvWSSV-infected shrimps showed three extra polypeptide bands at 41, 33 and 24 kDa that were not found in the haemolymph protein profile of vWSSV-infected shrimps. The data obtained in this study suggest that the observed difference in the virulence of WSSV may not be due to any change in the virus, rather it could be due to the shrimp defence system producing certain factors that help it to accommodate the virus without causing any mortality. [source] | |||||||||||||||||||