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PubMed1988

Comparison and partial characterization of the protein profiles and outer membrane antigens of Actinobacillus species isolated from ram lambs with epididymitis.

Three monoclonal antibodies (LG17, LG30, LG33) were used in the indirect fluorescent antibody test, the ELISA, and the immunoelectrotransfer blot technique to identify group-specific and strain-specific epitopes on the outer membranes of Actinobacillus seminis, A actinomycetemcomitans, and 17 field isolates of Actinobacillus spp. The field isolates had been obtained by bacteriologic culture of specimens from ram lambs with epididymitis. Only antibody LG33 consistently had specificity for an outer membrane epitope shared by most of the bacterial isolates tested. Staining of polyacrylamide gels with periodic acid-Schiff reagent, Sudan black B, and Coomassie brilliant blue R250 indicated that target antigens for antibodies LG17 and LG33 contained carbohydrate and lipoprotein components, respectively. The chemical composition of the LG30 target antigen was not determined because of its instability after exposure to sodium dodecyl sulfate. Discontinuous-gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate and spectrophotometric scans of the gels were used to analyze n-octyl-beta-D-glucopyranoside protein extracts from A seminis, A actinomycetemcomitans, and 13 representative field isolates of Actinobacillus spp. Bacterial isolates could be grouped according to their protein profiles. The first group consisted of A seminis, A actinomycetemcomitans, and 7 field isolates of Actinobacillus spp, all of which shared common protein bands with molecular masses of approximately 94 kilodaltons (kD), 64 kD, 60 kD, 52 kD, 44 kD, and 26 kD. The second group was composed of 6 field isolates, each with unique protein profiles; isolates had relatively few protein bands in common. These data suggested that members of the genus Actinobacillus cultured from ram lambs with epididymitis probably include a number of various strains.

Actinobacillus↗

PubMedDate not supplied

Multidimensional protein profiling technology and its application to human plasma proteome.

In clinical and diagnostic proteomics, it is essential to develop a comprehensive and robust system for proteome analysis. Although multidimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems have been recently developed as powerful tools especially for identification of protein complexes, these systems still some drawbacks in their application to clinical research that requires an analysis of a large number of human samples. Therefore, in this study, we have constructed a technically simple and high throughput protein profiling system comprising a two-dimensional (2D)-LC/MS/MS system which integrates both a strong cation exchange (SCX) chromatography and a microLC/MS/MS system with micro-flowing reversed-phase chromatography. Using the microLC/MS/MS system as the second dimensional chromatography, SCX separation has been optimized as an off-line first dimensional peptide fractionation. To evaluate the performance of the constructed 2D-LC/MS/MS system, the results of detection and identification of proteins were compared using digests mixtures of 6 authentic proteins with those obtained using one-dimensional microLC/MS/MS system. The number of peptide fragments detected and the coverage of protein sequence were found to be more than double through the use of our newly built 2D-LC/MS/MS system. Furthermore, this multidimensional protein profiling system has been applied to plasma proteome in order to examine its feasibility for clinical proteomics. The experimental results revealed the identification of 174 proteins from one serum sample depleted HSA and IgG which corresponds to only 1 microL of plasma, and the total analysis run time was less than half a day, indicating a fairly high possibility of practicing clinical proteomics in a high throughput manner.

Blood Proteins↗

PubMed2004

Salivary gland of Toxorhynchites splendens Wiedemann (Diptera: Culicidae): ultrastructural morphology and electrophoretic protein profiles.

The salivary glands of male and female Toxorhynchites splendens have the same morphology, and they are paired organs lying on either side of the esophagus. Each gland is composed of two identical tubular lobes, joined together at the end of the proximal region. In the gland, a salivary duct extends through the length of each lobe. The general cellular architecture of the salivary gland of this mosquito is unique. No secretory cavity was found in any cell, and the salivary materials are secreted from long microvilli and collect in a periductal space surrounding the duct. In addition, a number of mitochondria, rough endoplasmic reticulum, and a very large nucleus were observed, suggesting a high energy requirement for producing the salivary proteins involved in sugar feeding. The size of the gland is approximately 50 microm in diameter and 1.5 mm in length. These dimensions correlate with high protein content of these salivary glands (2.88+/-0.14 microg/gland pair). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic protein profiles of the male and female salivary glands were identical. No dominant major proteins were found. Compared with Aedes and Anopheles mosquitoes, the protein profile of T. splendens was similar to that observed in the males of these other species but different to that shown by the females, thus making T. splendens an excellent organism for studying the biochemistry of sugar feeding in mosquitoes.

Animals↗

PubMed2005

A proteomics approach to identify changes in protein profiles in pre-cancerous colon.

The development of colon cancer is characterised by alterations in multiple genetic and epigenetic pathways in colon tissue leading ultimately to deregulation of colon epithelial cells. Early detection is an important factor in decreasing colon cancer deaths. Proteomic techniques were used to identify potential early markers in colon tissue exhibiting pre-cancerous activity that may characterise pathological changes in a chemically induced colon cancer rat model. Protein profiles were assessed in soluble and insoluble fractions prepared from distal colon of rats treated with the colonotropic carcinogen, dimethylhydrazine. Alterations in protein profiles were associated with the presence of aberrant crypt foci, hyperplasia and dysplasia, microanatomical changes, and metabolic changes in rat colon. These changes may have a potential role in the identification of pre-pathological features preceding colon tumorigenesis.

Animals↗

PubMedDate not supplied

Protein profile of Helicobacter pylori strains by isoelectrofocusing in patients affected by chronic gastritis.

BACKGROUND: To understand if relapse, following antimicrobial treatment was due to re-infection or to recrudescence. METHODS: Fifty patients with dyspepsia were studied prospectively. They were followed up by endoscopy and biopsy of antral mucosa before and after treatment with anti-microbial therapy. Gel isoelectrofocusing was used to characterize protein profile of Hp. RESULTS: At baseline 40 patients were affected by chronic gastritis associated with Hp. At the end of treatment 75% patients given omeprazole, amoxicillin and clarithromycin were Hp infected: 43% showed the same protein profile and 57% different. CONCLUSIONS: Our data suggest that the relapse is due to recrudescence or to reinfection.

Adult↗

PubMed2004

Protein profiling of microdissected prostate tissue links growth differentiation factor 15 to prostate carcinogenesis.

Identification of proteomic alterations associated with early stages in the development of prostate cancer may facilitate understanding of progression of this highly variable disease. Matched normal, high-grade prostatic intraepithelial neoplasia (hPIN) and prostate cancer cells of predominantly Gleason grade 3 were procured by laser capture microdissection from serial sections obtained from snap-frozen samples dissected from 22 radical prostatectomy specimens. From these cells, protein profiles were generated by surface-enhanced laser desorption/ionization-time of flight mass spectrometry. A 24-kDa peak was observed at low or high intensity in profiles of prostate cancer cells in 19 of 27 lesions and at low intensity in 3 of 8 hPIN lesions but was not detectable in matched normal cells. SDS-PAGE analysis of prostate cancer and matched normal epithelium confirmed expression of a prostate cancer-specific 24-kDa protein. Mass spectrometry and protein data-based analysis identified the protein as the dimeric form of mature growth differentiation factor 15 (GDF15). The increased expression of mature GDF15 protein in prostate cancer cells cannot be explained on the basis of up-regulation of GDF15 mRNA because reverse transcription-PCR analysis showed similar amounts of transcript in normal, hPIN, and prostate cancer cells that were obtained by laser capture microdissection in the same set of serial sections from which the protein profiles were obtained. Our findings suggest that early prostate carcinogenesis is associated with expression of mature GDF15 protein.

Adenocarcinoma↗

PubMed2005

Gene expression and protein profiling of AGS gastric epithelial cells upon infection with Helicobacter pylori.

Helicobacter pylori, one of the most common bacterial pathogens, colonizes the human stomach and causes a variety of gastric diseases. This pathogen elicits a range of phenotypic responses in infected cultured AGS gastric epithelial cells, including expression of proinflammatory genes and changes in the actin cytoskeleton. Some of these responses are mediated by the type IV secretion system (T4SS) encoded by the cag pathogenicity island. We have used two global approaches, namely 2-DE combined with PMF and cDNA expression array analyses, to study in both a comprehensive and quantitative manner the protein profile and the temporal patterns of mRNA accumulation in AGS cells upon infection with H. pylori and isogenic T4SS mutants. We identified 140 transcripts and detected 190 protein species that were differentially regulated upon infection. Infection with wild-type H. pylori induced expression of a variety of host genes and changes in protein pattern involved in transcriptional responses, cell shape regulation and signal transduction. Among them, some were differentially regulated in a cag PAI-dependent manner, as shown by both the proteomic and cDNA expression array approaches. While 2-DE and PMF allowed us to examine the protein profiles in the infected host, array analysis enabled us to demonstrate dynamic temporal changes in host gene expression profile. In conclusion, our combined application of the two global approaches provides further molecular details on how the host cell responds to infection by H. pylori and its isogenic T4SS mutants on both transcriptional and protein levels. The findings pinpoint host proteins such as serine/threonine and tyrosine kinases, transcription factors, cell cycle related components and actin cytoskeletal signaling molecules as potential targets of individual H. pylori virulence determinants. This study serves as a basis for future work on transcription and proteome analyses of the H. pylori infection model.

Adenocarcinoma↗

PubMed1999

Matrix-assisted laser desorption/ionization mass spectrometry in evaluation of protein profiles of infant formulae.

Breast-feeding is the nutritional model in humans, and is continued after birth for variable periods. Milk represents an essential source of growth from both nutritional and functional points of view. When nursing is not possible, indicated or sufficient, artificial feeding becomes essential. Substitutes for mother's milk are usually obtained by modifying the composition of cow's milk either qualitatively or quantitatively. The changes usually involve enzymatic and/or thermal treatment, and for this reason a description of the protein profiles of milk formulae is of great interest. After examination of the results obtained by matrix-assisted laser desorption/ionization (MALDI) in the characterization of protein profiles of cow's milk after different thermal treatments, the application of this analytical technique in the above context appeared interesting. MALDI data for eleven milk formulae, directly acquired from the market, are described and discussed. Results indicate that MALDI mass spectrometry is a particularly powerful method which may be employed either during the production stages of milk formulae or to study the relationships between milk protein content and clinical formula evaluation.

Humans↗

PubMed1993

Characterization of mesophilic Aeromonas from clinical specimens by computerized analysis of SDS-PAGE protein profiles and by enzymatic activity.

Mesophilic Aeromonas (Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae) have recently been considered important aetiological agents of human diseases, mainly gastrointestinal infections. Although several findings have pointed out the significance of this group of microorganisms as enteric pathogens and suggested the presence of virulence factors, epidemiological and clinical studies are limited by the difficulty of correctly identifying mesophilic Aeromonas at the species level. SDS-PAGE of radiolabelled total protein profiles and bacterial enzymatic activities were used to type 31 clinical isolates (6 A. hydrophila, 7 A. sobria and 18 A. caviae) from patients with gastroenteritis and from healthy controls. Analysis of SDS-PAGE protein patterns, reinforced by the UPGMA-grouping system (AMBIS software) provided a good characterization of A. caviae strains as a homogeneous group of microorganisms, possessing significant differences from the other two species of mesophilic Aeromonas, in good agreement with biochemical and enzymatic tests. Data obtained in analyzing A. sobria protein profiles clearly showed two groups, with a correlation coefficient (CC) = 0.70, which in our experience is a doubtful value for assigning two strains to the same species. Strains biochemically identified as A. hydrophila showed a CC = 0.64, which is equally not acceptable for species assignment. Inter-species comparison highlighted this heterogeneity, showing two mixed subgroups, both containing strains that were assigned to A. sobria and A. hydrophila species on the basis of biochemical features.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2006

Activity-based protein profiling implicates urokinase activation as a key step in human fibrosarcoma intravasation.

Entry of malignant cells into the vasculature (i.e. intravasation) requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they extravasate and establish metastatic lesions. This study utilizes activity-based protein profiling to compare the active serine hydrolase repertoire in high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line to determine which enzyme(s) play a role in intravasation. Activity-based protein profiling revealed multiple serine hydrolases with altered activity between HT-hi/diss and HT-lo/diss cells, with the largest difference being the activity of urokinase-type plasminogen activator (uPA). Levels of inactive uPA zymogen were similar between the two cell variants, but only HT-hi/diss conditioned medium contained active uPA, suggesting that uPA activation may contribute to the enhanced intravasation of HT-hi/diss cells. To analyze the role of uPA activity specifically in the process of intravasation, we grafted cells from the two HT-1080 variants onto the chorioallantoic membrane of chick embryos and measured levels of tumor cell intravasation in the distal chorioallantoic membrane using quantitative human-specific Alu PCR. Inhibition of uPA activity with natural (plasminogen activator inhibitor-1) or synthetic (amiloride) inhibitors diminished HT-hi/diss Matrigel invasion in vitro and intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression.

Animals↗

PubMed2003

Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues.

Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.

Antibodies↗

PubMed2000

Measurement of nine human milk proteins by nephelometric immunoassays: application to the determination of mature milk protein profile.

OBJECTIVES: Microparticle-enhanced nephelometric immunoassays for six human milk proteins (beta-casein, kappa-casein, alpha-lactalbumin, serum albumin, lactoferrin, and lysozyme) and conventional immunonephelometry assays for immunoglobulin A, C3, and C4 complement proteins were developed and characterized. DESIGN AND METHODS: Microparticle-enhanced nephelometric immunoassays are competitive assays based on the nephelometric quantification of the inhibition of microparticle-protein conjugates immunoagglutination by the proteins to be assayed. RESULTS: High precision (CVs ranged from 1% to 14% in within- and between-assays) and recovery (linear recovery in dilution-overloading assay) ensure a reliable determination of the main human milk proteins by single-step homogeneous nephelometric immunoassays, accurate over wide ranges of concentration. These immunoassays were easily applied to a large number of mature human milk samples (between 373 and 503 according to the proteins tested). CONCLUSIONS: The immunoassays developed could be applied to the fast determination of human milk protein profile usable for nursery milk bank and fortification.

Calibration↗

PubMed1977

Developmental changes in the protein profile of cornifying lingual epithelium.

Epithelia from the tongue dorsum of 14- to 21-day embryos, 21-day embryos, 3-week-old, and adult rats were separated from their connective tissues by incubation in balanced salt solution containing EDTA. Aliquots from total extracts of these tissues were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Scans of gels stained with fast green (FG) revealed more than 20 peaks. Ten major peaks ranging from apparent molecular weights (MW) of 120,000 to 14,000 daltons comprised about 70% of the total protein on each gel. This report focuses primarily on two pairs of peaks, arbitrarily numbered 2 and 3 (MWs 71,000 and 69,000) and 9 and 10 (MWs 17,500 and 14,000). Peaks 9 and 10 predominated in the 15-day embryos where they comprised about 30% of the total protein. As development proceeded, there was a gradual shift in the protein profile in favor of peaks 2 and 3 until on the 20th day the relative amounts of these peaks reached a maximum and peaks 9 and 10 decreased in relative amounts. The protein profile on the 20th fetal day resembled that of the 3-week-old rats and the adults. The rise in the relative amounts of peaks 2 and 3 coincided with the morphologic appearance of large numbers of tonofilaments and the onset of cornification. When the gel was stained by a procedure specific for sulfhydryl groups, peaks 9 and 10 were especially reactive after the 18th day; plainmetric analysis revealed that these had twice the relative affinity for this stain than for FG whereas other peaks had equal or less affinity. The incorporation of [3H]cystine into peaks 9 and 10 was relatively greater than into the other proteins.

Animals↗

PubMed1983

Urinary protein profiles after burn injury.

A study of the change of urinary protein excretion has been made in 26 patients followed from the time of admission to time of discharge from the Yorkshire Regional Burn Centre (7-200 days). Total protein, IgG and albumin (as indicators of glomerular function) and alpha 1-microglobulin, beta 2-microglobulin, retinol binding protein and N-acetyl-beta-D-glucosaminidase (as indicators of proximal tubular function) as well as the patterns on SDS polyacrylamide gel electrophoresis were examined. Patients were divided into three groups according to their urinary protein profiles. The first group was 3 patients who showed little or no evidence of proteinuria; the second group consisted of 7 patients who showed a mild and transient tubular proteinuria, all of whom recovered normal function within one week. The third group, 16 patients, developed moderate to severe proteinuria usually started as a mixed pattern of glomerular and tubular proteinuria, characterized by the excretion of a high level of total protein. After a few days, this pattern changed to a typical tubular proteinuria and the latter could last for as long as 200 days; in some of these patients a secondary increase of low molecular weight protein excretion occurred either due to a latent renal complication or induced by the administration of aminoglycosides. There is a close correlation between the intensity and patterns of the proteinuria and the severity and clinical progress of the burn.

Acetylglucosaminidase↗

PubMed1987

Cell envelope protein profiles of Acinetobacter calcoaceticus strains isolated in hospitals.

The cell envelope protein patterns of 78 strains of Acinetobacter calcoaceticus, mainly isolated in hospitals, were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns were stable and reproducible. Comparison of the protein profiles made possible differentiation between two groups of strains. The patterns of the first group could be classified on the basis of concordance. Some profiles appeared to be associated with the epidemiological origin of the strains. The second group consisted of strains with unique patterns which could not be classified. Comparison of SDS-PAGE patterns appears to be a suitable method for the relative classification of A. calcoaceticus strains of nosocomial origin.

Acinetobacter↗

PubMedDate not supplied

[The protein profile. Statistical treatment and use of the results, principles and applications].

The serum protein profile is a biological multi parametric analysis. Its validity will be tested by multidimensional statistical treatment to help the medical decisions and the practical exploitation of the results. The mean and the range are given by classical methods which also allow the correlations of some proteins. The factorial analysis of the tables of correlations by showing the proximities between the proteins and the pathological classes gives a first indication of the structure of the data. The multidimensional approach introduces the notion of distance between two profiles, gives an automatic classification of the profiles in distinct groups and an evaluation of these groups as real pathological entities. A table of distance between the profiles can also discriminate between the groups of patients. However the biological model implies some hypotheses: does any disease imply a specific profile and the reverse? Are the variations of each protein compatible and do they require some pondering? Therefore the best in the present time is to try a multidimensional approach as an help for medical decision rather than an automatic mean of diagnosis.

Blood Proteins↗

PubMed2000

Epidemiology of ovine Campylobacter infection determined by numerical analysis of electrophoretic protein profiles.

The relationship of 50 Campylobacter strains isolated from aborted ovine foetuses, and the faeces of sheep, cattle and chickens were determined by numerical analysis of electrophoretic (SDS-PAGE) protein profiles. Comparison of protein patterns by numerical methods revealed differences between C. fetus ssp. fetus, C. jejuni, and C. coli strains as well as heterogeneity among isolates from different outbreaks. Isolates from each farm produced a distinct cluster and flocks from different locations were found to be infected with relatively different strains. In most cases, protein patterns of ovine foetal isolates were very similar to those of ovine faecal isolates. Ovine isolates of C. fetus ssp. fetus, C. jejuni and C. coli gave similar protein patterns to the corresponding Campylobacter species isolated from cattle or chicken, on the same farm. Thus, it was concluded that certain protein types of ovine Campylobacter strains were more likely associated with local areas, and Campylobacter strains causing ovine abortions are distributed in the environment more widely than assumed to date.

Animals↗

PubMed2004

Ethanol-mediated variations in cellular fatty acid composition and protein profiles of two genotypically different strains of Escherichia coli O157:H7.

Two strains of Escherichia coli O157:H7 were grown in tryptic soy broth (TSB, pH 7.1) supplemented with 0, 2.5, 5.0, 7.5, and 10% ethanol at 30 degrees C for up to 54 h. Growth rates in TSB supplemented with 0, 2.5, and 5.0% ethanol decreased with an increase in ethanol concentration. Growth was not observed in TSB supplemented with 7.5 or 10% ethanol. The pH of TSB containing 5.0% ethanol decreased to 5.8 within 12 h and then increased to 7.0 at 54 h. The ethanol content in TSB supplemented with 2.5 or 5.0% ethanol did not change substantially during the first 36 h of incubation but decreased slightly thereafter, indicating utilization or degradation of ethanol by both strains. Glucose was depleted in TSB supplemented with 0, 2.5, or 5.0% ethanol within 12 h. Cells grown under ethanol stress contained a higher amount of fatty acids. With the exceptions of cis-oleic acid and nonadecanoic acid, larger amounts of fatty acid were present in stationary-phase cells of the two strains grown in TSB supplemented with 5.0% ethanol for 30 h than in cells grown in TSB without ethanol for 22 h. The trans-oleic acid content was 10-fold higher in the cells grown in TSB with 5.0% ethanol than those grown in TSB without ethanol. In contrast, cis-oleic acid was not detected in ethanol-stressed cells but was present at concentrations of 0.32 and 0.36 mg/g of cells of the two strains grown in TSB without ethanol. Protein content was higher in ethanol-stressed cells than in nonstressed cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles varied qualitatively as affected by the strain and the presence of ethanol in TSB. An ethanol-mediated protein (28 kDa) was observed in the ethanol-stressed cells but not in control cells. It is concluded that the two test strains of E. coli O157:H7 underwent phenotypic modifications in cellular fatty acid composition and protein profiles in response to ethanol stress. The potential for cross protection against subsequent stresses applied in food preservation technologies as a result of these changes is under investigation.

DNA, Bacterial↗