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Protein profiles as an aid to taxonomy in the genus Diphyllobothrium.

Specific and distinct protein profiles were demonstrated by isoelectric focusing performed on soluble whole-body proteins of four Diphyllobothrium species. The protein patterns differed in number, position and relative density of the fractions separated. Altogether 31-36 protein bands per species were separated by isoelectric focusing in thin-layer polyacrylamide gels. The results show that sensitive chemotaxonomic methods can be used to help characterize and delimit species in the troublesome Diphyllobothrium group.

Animals↗

Protein profiles of Spodoptera litura (F.) male accessory reproductive gland and its secretions.

Protein profiles of the male accessory reproductive glands of a polyphagous pest, Spodoptera litura (Lepidoptera: Noctuidae) and their glandular secretions were analyzed by electrophoresis under denaturing conditions. The study revealed the presence of 23 proteins in the glands and 14 in the secretions. The molecular weights of the gland proteins ranged from 163 to 3.8 KD whereas that of secretions ranged from 100 to 3.8 KD. The 9 low molecular weight proteins of the secretions are a characteristic feature. It is suggested that the secretions may contain a sex peptide that has a role in the fertility and fecundity of the females.

Age Factors↗

Toward a protein profile of Escherichia coli: comparison to its transcription profile.

High-pressure liquid chromatography-tandem mass spectrometry was used to obtain a protein profile of Escherichia coli strain MG1655 grown in minimal medium with glycerol as the carbon source. By using cell lysate from only 3 x 108 cells, at least four different tryptic peptides were detected for each of 404 proteins in a short 4-h experiment. At least one peptide with a high reliability score was detected for 986 proteins. Because membrane proteins were underrepresented, a second experiment was performed with a preparation enriched in membranes. An additional 161 proteins were detected, of which from half to two-thirds were membrane proteins. Overall, 1,147 different E. coli proteins were identified, almost 4 times as many as had been identified previously by using other tools. The protein list was compared with the transcription profile obtained on Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein products were found was detected at the mRNA level. The arithmetic mean mRNA signal intensity for these genes was 3-fold higher than that for all 4,300 protein-coding genes of E. coli. Thus, GeneChip data confirmed the high reliability of the protein list, which contains about one-fourth of the proteins of E. coli. Detection of even those membrane proteins and proteins of undefined function that are encoded by the same operons (transcriptional units) encoding proteins on the list remained low.

Cell Membrane↗

Windows to the ward: graphically oriented report forms. Presentation of complex, interrelated laboratory data for electrophoresis/immunofixation, cerebrospinal fluid, and urinary protein profiles.

BACKGROUND: Automated laboratory analyzers that mass produce data have been linked to information systems for more than two decades, but little progress has been made in developing more comprehensible report forms. Results are still reported in computer-generated printouts containing hundreds of numbers crowded into columns on each printed page. METHODS: We developed three software applications focusing on the graphic presentation of laboratory results. RESULTS: The first application summarizes data for a patient with a monoclonal gammopathy. The report provides a cumulative graphic presentation of immunofixation/electrophoresis data without any additional interpretation, focuses on a color-coded electrophoresis scan, and records up to 5 years on a single page. The second application deals with cerebrospinal fluid analysis. The report calculates relevant data and graphs the complex relationship between albumin and immunoglobulin results from paired serum and cerebrospinal fluid samples. Manually added interpretive text assures an output comprehensible to clinicians in all specialties. The third application produces a report summarizing quantitatively measured urinary marker protein profiles. The report form is generated by a flexible, completely user-definable knowledge-based system. It calculates numerous ratios and formulae, supports reflex testing, supplies an automated interpretation, and generates a specific graphic signature pattern of the results (MDI LabLink proteinuria differentiation). CONCLUSIONS: Increased clinical demand for graphically oriented report forms 5 years after their introduction has provided evidence that these reports transfer complex laboratory data and results to the clinician more effectively. The highest (more than threefold) increase in demand has been for reports for urinary marker protein profiles that feature a largely self-explanatory graphic signature pattern.

Autoanalysis↗

A strong association between body fat mass and protein profiles in nipple aspirate fluid of healthy premenopausal non-lactating women.

Fluid can be aspirated from the nipples of most non-lactating women. This nipple aspirate fluid (NAF) is a potential source for the discovery of new breast cancer biomarkers. NAF has two distinct protein profiles. Type I NAF is similar to the fluid associated with cystic disease of the breast, whereas type II NAF is enriched in milk-associated proteins. The prevalence of these two profiles differs in healthy women and in breast cancer patients. This study investigated the relationship of these two NAF profiles to reproductive history, body composition, diet, and levels of lipids, steroids and thyroid hormones in healthy premenopausal women (age 30-40 years) who had regular menstrual cycles and normal mammograms and were not taking contraceptive medications. On average, women with the type I NAF profile were older, had more years since last childbirth, were less likely to have breastfed their babies and had higher dietary saturated fat intake, body mass index, body fat mass, and levels of plasma low density lipoproteins than women with the type II profile (P <0.05). Using multiple logistic regression, type I NAF was predicted independently (P <0.05) by higher body fat mass [Odds Ratio (OR) = 3.0; 95% Confidence Interval (CI): 1.5-6.1], more years since last childbirth (OR = 2.6; 95% CI: 1.3-5.2) and a higher percentage of calories from saturated fat (OR = 4.1; 95% CI: 1.1-14.6). These results suggest that protein profiles of NAF might be influenced by amounts or types of dietary and body fat, but further study of the relationship of the two profiles to breast cancer risk is needed.

Adipose Tissue↗

An assessment of protein profiles from the marine oligotrophic ultramicrobacterium, Sphingomonas sp. strain RB2256.

The protein expression profile of a novel marine oligotrophic ultramicrobacterium, Sphingomonas sp. strain RB2256, was investigated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Analytical reference maps were generated from mid-log phase batches and steady-state chemostat cultures with pH 4-8 immobilised pH gradients (IPGs) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were detected by two different methods: radioactive labeling and silver staining. Protein profiles generated from analytical 2-D PAGE gels were compared and differential analysis was performed using Melanie II software. Both methods (radioactive labeling and silver staining) resulted in reproducible, high resolution gels (up to 1600 protein spots). This approach is proving to be a powerful tool for investigating the molecular basis of the unique physiology of this model oligotrophic microorganism.

Bacterial Proteins↗

Distinguishing between respiratory syncytial virus subgroups by protein profile analysis.

We subgrouped 75 strains of respiratory syncytial virus by a protein profile method (PPM) which relies on different mobilities of the phosphoprotein in one-dimensional polyacrylamide gel electrophoresis and does not require monoclonal antibodies. When compared with enzyme immunoassay, PPM correctly subgrouped 54 of 56 subgroup A and all 19 subgroup B strains.

Antigens, Viral↗

Nephelometric urinary protein profile as an index of renal involvement in hypertensive disorders of pregnancy.

Protein analysis was performed on the urine of 62 pregnant women with the use of a nephelometric technique. Thirty-seven women were classified as having mild or severe pregnancy-induced hypertension and/or chronic hypertension. The protein analysis was performed on urine samples obtained prior to delivery and was compared to the degree of proteinuria as determined by the dipstick technique. The degree of immunoglobulinuria increases with increasing severity of pregnancy-induced hypertension while the urine concentration of albumin and transferrin becomes nondetectable. The urinary protein profile appears to be extremely sensitive and accurate in predicting severity of disease.

Adult↗

Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge.

The protein content of bronchoalveolar lavage fluid (BALF) from actively sensitised Brown Norway (BN) rats challenged with allergen (ovalbumin, OA) and from naïve Brown Norway rats challenged with endotoxin (lipopolysaccharide, LPS) was analyzed and compared to healthy controls treated with vehicle only. BALF proteins were analyzed by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis and identified by peptide mass fingerprinting matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or nanoliquid chromatography-tandem MS (nanoLC-MS/MS) after in-gel trypsin digestion of selected 2-D gel spots. Our study shows that the BALF protein profile is significantly different in animals after allergen (OA) or endotoxin (LPS) challenge as compared to controls, concerning the content of proteins derived from plasma or produced locally in the lung. In both challenges the following proteins presented patterns which differed qualitatively compared to control: T-kininogen I and II, alpha-1-antitrypsin, calgranulin A, fetuin A and B, and haptoglobin. Other proteins were diminished in both challenges, such as Clara cell 10 kDa secretory protein (CC10) and pulmonary surfactant associated protein B (SP-B); c-reactive protein increased in the OA-challenge and decreased in the LPS-challenge, and pulmonary surfactant associated protein A (SP-A) was decreased in the OA-challenge and was not significantly changed in the LPS-challenge. The identified proteins could be important not only for the diagnosis but have also interesting implications for medical treatment of lung inflammatory conditions. Furthermore, even if based on a limited number of animals, our results are of interest for the identification of lung protein markers and a better understanding of the mechanisms involved in the pathogenesis of lung diseases.

Allergens↗

Protein profiles in rabbit pancreatic juice analyzed by HPLC after stimulation of secretion by secretin and cerulein.

This study analyzed the secretory pattern of pancreatic proteins released from the rabbit pancreas after acute stimulation of secretion by the cholecystokinin analog cerulein. To facilitate this, a new analytical approach utilizing high performance liquid chromatography (HPLC) was considered. Secretin (0.1 CU/kg x h) was intravenously infused in anaesthetized rabbits in combination with cerulein (0.05, 0.2 or 0.05 followed by 0.2 ug/kg x h) over 3 hours. Pancreatic juice was collected from the main pancreatic duct. The release of protein, amylase, trypsin and chymotrypsin was measured by conventional photometric methods, and the protein profiles were analyzed by reversed phase HPLC. Separation of pancreatic juice proteins by HPLC (Nucleosil 300-7 RP column; injection of 50 ul aliquots of samples normalized to 10 mg/ml protein concentration) resulted in a resolution of up to 16 peaks. Peaks representing amylase, prolipase, prophospholipase A2, procarboxypeptidases, chymotrypsinogen, trypsinogen, and glycoproteins were identified with some certainty by SDS-gel electrophoresis. Secretin infusion produced a small and short lasting rise in total protein secretion but lead to a persistent increase of fluid flow. The release of enzymes followed a mainly parallel pattern according to the photometric measurements. The resolution of the whole profile of pancreatic juice proteins by HPLC demonstrated only minor variations without a consistent or increasing tendency towards a preferential release of individual enzymes. Since even microheterogenities in the samples became apparent after HPLC, this approach would be sensitive enough to mirror effects like nonparallel release of enzymes.

Animals↗

The discrimination between different blood-CSF barrier dysfunctions and inflammatory reactions of the CNS by a recent evaluation graph for the protein profile of cerebrospinal fluid.

A graph for the evaluation of the CSF-protein profile is presented as a basic program for the clinical-neurochemical laboratory. The graph has the following advantages: Simultaneous information on the functional state of the blood-CSF barrier and the inflammatory response of the CNS; maximal sensitivity for the determination of a pathological local IgG production in CNS--with the possibility of calculating the IgG fraction in CSF originating from the CNS; minimal number of protein assays necessary (albumin and IgG in serum and CSF); suitable for the demonstration of the course of the disease in a single patient as well as for demonstration of a group of cases for statistical purposes. Complementary chemical investigations and the correspondence with the clinical diagnosis are discussed for a number of typical cases. Two different types of blood-CSF barrier dysfunctions are discriminated by a proportional and a dis-proportionate increase of the CSF/serum protein concentration gradients.

Albumins↗

CSF protein profile in infantile spasms. Influence of etiology and ACTH or dexamethasone treatment.

CSF proteins in 107 children ranging from 3 to 24 months of age were analyzed by means of quantitative zone electrophoresis on agarose gel. Subjects included 50 children with infantile spasms, 41 children without CNS disease serving as controls, and 16 infants with acute aseptic meningitis who demonstrated the protein pattern of blood-CSF barrier disturbance. Children with infantile spasms were subdivided into several groups according to etiological categories: symptomatic (pre-, peri-, and postnatal), doubtful, and cryptogenetic. Before any treatment was started, these children showed the protein profile of increased permeability of the blood-CSF barrier, especially for albumin. There was an association between the severity of the changes and the etiological category. Changes were most marked in the symptomatic group, intermediate in the doubtful group, and slight in the cryptogenetic group. No child with infantile spasms of doubtful or unknown etiology revealed changes of the immunoglobulin-containing gamma fractions. Ten children who had received adrenocorticotropic hormone (ACTH) or dexamethasone for 2-11 weeks no longer showed any protein leakage into the CSF. The period of ACTH or dexamethasone treatment was characterized by the following findings: the disappearance or reduction of hypsarrhythmia; the reappearance of normal cerebrovascular permeability for protein; and the occurrence of reversible dilatation of the subarachnoid and intraventricular spaces.

Acute Disease↗

Protein profiling of bladder cancer using the 2D-PAGE and SELDI-TOF-MS technique.

Protein profiling is a promising tool for tumor characterization and the detection of tumor markers in bladder cancer. Techniques for 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and surface-enhanced laser desorption/ionization with time-of-flight mass spectrometry (SELDI-TOF-MS) have improved; both were evaluated using bladder tumor tissue. Normal urothelium and pTa G2, pT1 G3, and >or=pT3 G3 tissues were obtained from the operating room and, after macrodissection, subjected to 2D-PAGE and to SELDI-TOF-MS ProteinChip. 2D-PAGE gels expressed significantly different protein patterns for pTa G2 and pT3 G3 tumors. pT1 G3 tumors showed expression profiles similar to those of the invasive tumors, with upregulation of galectin 3, gelsolin, villin 2, moesin, and annexin 6. Similarly, distinct protein peaks were detected for superficial and muscle-invasive urothelial cancers by SELDI-TOF-MS. Six of seven superficial pTa G2 tumors showed an intense peak at 6.7 and 10.1 kD, while invasive carcinomas showed an intense peak near 9.5 kD. No disturbing influence of surrounding tissue on the results was detected. It was shown that both techniques (2D-PAGE and ProteinChip) work well, and especially ProteinChip analysis seems promising for clinical application.

Biomarkers, Tumor↗

Protein profile analysis from advanced third-stage larvae (AdvL3) and adult worms of Gnathostoma binucleatum (Nematoda: Spirurida).

Proteins from crude extracts of advanced third-stage larvae and adult Gnathostoma binucleatum nematode worms showed protein profiles in SDS-PAGE analysis similar to Echinococcus granulosus, Trichinella spiralis, Dipylidium caninum, Ancylostoma caninum, Ascaris lumbricoides and Toxocara canis. The immunoblot analysis of the human serum infected or suspected to be infected with G. binucleatum using the total larvae extract recognized the 40, 60, 80 and 115 kDa proteins and using the total adult worm extract recognized only the 80 and 115 kDa proteins. However, the 115 kDa protein showed cross-reactions with A. caninum, A. lumbricoides, T. canis and D. caninum with human serum positive to gnathostomosis, while the 40 kDa protein was only recognized with the G. binucleatum total larvae extract. The results obtained suggest that the use of antigens from the advanced third-stage larvae of the parasite were best recognized for immunodiagnosis of gnathostomosis.

Animals↗

Protein profiles of Streptomyces aureofaciens producing tetracyclines: reappraisal of the effect of benzyl thiocyanate.

Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 microM benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.

Bacterial Proteins↗

Discovery of biomarker candidates within disease by protein profiling: principles and concepts.

Proteins and peptides present within clinical samples represent a valuable library of information regarding the ongoing processes within cells and tissues in health and disease. We have developed and validated novel technology applications that can be used to characterize the patterns of global protein expression in tissue and biofluids in either gel-based systems or by automated multidimensional nanocapillary liquid chromatography. Mass spectrophotometry platforms using MALDI MS and MS/MS or LTQ ion trap MS were capable of delivering sensitive and accurate identifications of hundreds of proteins contained in individual samples including individual forms of processing intermediates such as phospho peptides. The Systems Biology approach of integrating protein expression data with clinical data such as histopathology, clinical functional measurements, medical imaging scores, patient demographics, and clinical outcome provides a powerful tool for linking biomarker expression with biological processes that can be segmented and linked to disease presentation.

Animals↗

Protein profile in leprosy.

Serum proteins and plasma fibrinogen were estimated in 103 patients in various groups of leprosy and 52 patients of reactional leprosy. Total proteins, serum globulin and fibrinogen showed significant rise while serum albumin showed fall over the immunological spectrum from TT to LL. Type II reactional leprosy similarly revealed significant rise in globulin and fibrinogen. The comparison of these parameters between most of the comparable groups of leprosy was statistically significant. ENL patients after complete subsidence of reaction and after steroid treatment showed significant decrease in these protein fractions, thus conferring some prognostic implication on these tests.

Blood Proteins↗

Serum protein profiles in coccidioidomycosis.

Serum protein analysis is a valuable addition to the present methods for evaluating the status of the individual patient with coccidioidomycosis. The albumin protein and albumin glycoprotein decrease and gamma protein increases in relation to severity of infection. In 40 patients with coccidioidomycosis, changes in individual protein fractions could be significantly correlated with conventional laboratory tests, such as the complement fixation test, erythrocyte sedimentation rate and hematocrit. Changes in the alpha, glycoprotein concentration, the erythrocyte sedimentation rate and the hematocrit value appear to be related to the degree of inflammation, while the changes in the gamma protein and the beta, glycoprotein appear to be related to the specific antibody response.

Blood Proteins↗