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PubMed2001

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins.

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.

Bacterial Proteins↗
PubMed1984

Demographic factors influencing psychiatric rating scales (Zung SDS and SAS).

Zung's Self-Rating Anxiety Scale (SAS) and Self-Rating Depression Scale (SDS) were completed by 489 healthy volunteers (314 men, 175 women) aged 15-84 years. The total scores were analysed in function of sex, age, occupational class, professional group, marital status, religion, foreigner status and nationality. Analysis by orthogonal polynomial regression showed no statistically significant differences between different age groups. In contrast to this, the study revealed highly significant SAS and SDS score differences between both sexes (Mann-Whitney U-test). In addition, there were statistically significant associations between the SAS scores and occupational class, professional group, religion, foreigner status and nationality; compared with significant associations between the SDS scores and marital status, foreigner status and nationality (Kruskal-Wallis test/Mann-Whitney U-test). The requirements for data analysis in clinical trials, for a differential diagnosis between transient variations of normal health and disorders, and for a differential diagnosis between anxiety and depression are discussed.

Adult↗
PubMed1976

Psychometric properties of the Self-Rating Depression Scale (SDS).

While there are data supporting the content and construct validity of the Self-Rating Depression Scale (SDS), the evidence regarding its reliability is not conclusive. For the purpose of exploring the psychometric properties of the SDS, two studies were done using Ss from two markedly different socio-cultural settings: 213 male and female drug addicts in New York City and 206 male and female undergraduates in Nigeria. Findings included high coefficient alphas and large average item-correlations (total minus item) in both studies. It was concluded that the SDS rates well in terms of internal consistency reliability and construct validity.

Depression↗
PubMed2000

The structure and dynamics of ACTH (1-10) on the surface of a sodium dodecylsulfate (SDS) micelle: a molecular dynamics simulation study.

ACTH (1-10), an adrenocorticotropin hormone fragment, was studied by molecular dynamics (MD) simulation in the NPT ensemble in an explicit sodium dodecylsulfate (SDS) micelle. Initially, distance restraints derived from NMR nuclear Overhauser enhancements were incorporated during the equilibration stage of the simulation. The analyses of the trajectories from the subsequent unrestrained MD showed that ACTH (1-10) does not conform to a helical structure at the micelle-water interface; however, the structure is amphipathic. The loss of the helical structure is due to decreased intramolecular hydrogen bonding accompanied by an increase of hydrogen bonding between the amide hydrogens of the peptide and the micelle head-groups. ACTH (1-10) was found to lie on the surface of the SDS micelle. Most of the hydrophobic interactions came from the side-chains of Met-4, Phe-7 and Trp-9. The peptide bonds were either hydrated or involved in intramolecular hydrogen bonding. Decreased hydration for the backbone of His-6 and Phe-7 was due to intermolecular hydrogen bonding with the SDS head-groups. The time correlation functions of the N-H bonds of the peptide in water and in the micelle showed that the motions of the peptide, except for the N- and C-termini, are significantly reduced when partitioned in the micelle.

Adrenocorticotropic Hormone↗
PubMed2001

Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles.

Recombinant urchin syntaxin [Xa cut], electrophoresed at pH 9.0 (25 degrees C) or 10.2 (0 degrees C) in a discontinuous Tris-chloride-glycinate buffer system in the presence of 0.03% SDS in the catholyte, exhibits a multicomponent pattern in gels of a polyacrylamide concentration of 12% and 3% crosslinking. The position in the pattern of the syntaxin band was identified by reference to electropherograms of a previous study (P. Backlund, pers. comm.). The complexity of the protein composition of the preparation was reduced by selective stacking of proteins with mobilities greater than that of syntaxin. This provides a gel pattern consisting of two bands with mobilities close to that identified as syntaxin, as well as a minor, more slowly migrating, contaminant. The two major components are designated as S1 and S2, the latter being the larger species. In the absence of SDS, the preparation exhibits two pairs of protein components. Three of the proteins are charge isomers, i.e., of equal size, differing only in net charge, assumed to be forms of S1, while the fourth component is larger and is assumed to be S2. Aliquots of the preparation, containing 150 microg of protein were loaded on a cylindrical polyacrylamide gel of 18 mm diameter, and separated S1 and S2 were excised in a position defined by their characteristic values of relative mobility (Rf). Two or three gel slices, corresponding in Rf to S1 or S2, were pooled and loaded onto a Stacking Gel (5% polyacrylamide, 20% cross-linked) of 18 mm diameter, equipped with a collection chamber of 200 microL volume. The protein was electroeluted from the gel slices and concentrated into a stack by electrophoresis. The stack, marked by bromphenolblue, was allowed to migrate into the collection chamber, was collected and analyzed by protein assay and re-electrophoresis. Re-electrophoresis of S1 shows that it consists of at least three components. Recovered S1 constitutes 47% of the preparation, based on protein assay, S2 4%. S1, isolated from SDS-PAGE, exhibits an apparent Mr of 22.7 kDa, S2 one of 34.5 kDa, similar to the value of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 degrees C and their molecular weight relationship suggest a proteolytic transformation of S2 to S1.

Animals↗
PubMed1992

A simple method for the release of asparagine-linked oligosaccharides from a glycoprotein purified by SDS-polyacrylamide gel electrophoresis.

A simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-alpha 1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9 +/- 2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5 micrograms. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2 +/- 1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.

Animals↗
PubMed1992

Characterisation of hospital isolates of Moraxella (Branhamella) catarrhalis by SDS-PAGE of whole-cell proteins, immunoblotting and restriction-endonuclease analysis.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins (WCP), immunoblot analysis and DNA restriction-endonuclease analysis (REA) were applied as potential typing methods to 31 clinically significant strains of Moraxella (Branhamella) catarrhalis, five of which came from a suspected outbreak of nosocomial infection in a respiratory-diseases ward. Twelve of 31 isolates were placed in four groups, each of which contained strains indistinguishable by the three typing techniques used. Each of a further two groups contained two strains, and they were similar by at least one technique; the remaining 15 strains were unique by all three methods. Four of five strains from the suspected outbreak were indistinguishable by SDS-PAGE of WCP, immunoblotting and REA. Results show that SDS-PAGE of WCP, immunoblotting and REA are suitable techniques for characterising M. catarrhalis and that there is a considerable degree of strain heterogeneity. Nosocomial infection with M. catarrhalis may be relatively common and further epidemiological studies with a combination of typing techniques are indicated.

Aged↗
PubMed2006

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS.

Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.

Dimerization↗
PubMed1992

Comparison of Mycoplasma bovis strains based on SDS-PAGE and immunoblot protein patterns.

Whole-cell protein patterns generated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were compared for 34 isolates of Mycoplasma bovis. A high degree of similarity between most of the strains was established with strain-to-strain differences mainly confined to quantitative variations of certain protein bands, particularly in the molecular weight regions of 64-68, 55 and 26 kD. Two of the isolates provided more deviating patterns. Hydrophobic membrane protein fractions of the strains as prepared by Triton X-114 phase partitioning were compared by SDS-PAGE, which confirmed some of the characteristic strain features found with whole-cell proteins. The immunoblot analysis revealed that up to 20 of the 50-55 discrete protein bands detected in SDS-PAGE patterns were recognized to be antigenic by rabbit and bovine hyperimmune sera. It is concluded that the same set of major antigens is present in all strains investigated, but amounts of individual constituents may be differing.

Animals↗
PubMed1993

Molecular characterization of South American bovine herpesvirus-1 isolates with monoclonal antibodies and SDS-PAGE.

Antigenic patterns of BHV-1 isolates from Argentina, Chile and Brazil were examined with a panel of monoclonal antibodies (MAbs) generated with BHV-1.1 and BHV-1.3 strains. Among the Argentinian isolates, 7/8 neurological cases showed BHV-1.3 characteristics and 10/12 non-neurological cases from all countries exhibited BHV-1.1/2 pattern. All viral isolates from genital infections showed BHV-1.1/2 characteristics. Chilean isolates exhibited BHV-1.1/2 pattern and one among 3 Brazilian viruses was defined as belonging to BHV-1.3. Results of the characterization using MAbs, were confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of representative isolates. A total correlation was observed between the reactivity with MAbs and the SDS-polyacrylamide analysis. Based on the data presented, it is concluded that the type 1.3, most frequently obtained from the brain of neurological cases, is present in Argentina and Brazil and coexists with BHV-1.1/2.

Animals↗
PubMed1996

An investigation into the use of SDS-PAGE of cell surface extracts and proteolytic activity to differentiate Prevotella nigrescens and Prevotella intermedia.

By comparison of the cell surface proteins derived from the outer membrane and fibrils from 14 Prevotella intermedia and 19 Prevotella nigrescens strains using SDS and analysed by SDS-PAGE, it was possible to distinguish the two species. A polypeptide of approx. 21 kDa distinguished P. intermedia strains, whereas two polypeptides of approx. 18 and 22 kDa could be used to identify P. nigrescens strains. Four other human oral black pigmented bacterial species (Porphyromonas gingivalis, Prevotella denticola, Prevotella loescheii and Prevotella melaninogenica) did not have the 18-, 21- or 22-kDa polypeptides shown by P. intermedia or P. nigrescens. The cell-associated proteolytic activity of eight strains of P. intermedia, 14 strains of P. nigrescens and one strain of P. gingivalis (W50) was assessed using four chromogenic substrates. The hydrolysis of the substrate GPPNA (indicative of dipeptidyl peptidase IV-like activity) and SAAPPNA (elastase-like activity) by P. intermedia strains varied from 32 to 114 units and 0.5 to 12.6 units of activity respectively, where one unit was defined as the amount of protease enzyme catalysing the formation of 1 nmol of p-nitroaniline under experimental conditions. 37.5% (3 of 8) of P. intermedia strains hydrolysed SAAPPNA (chymotrypsin-like enzyme activity) with activities of between 7 and 12 units. The hydrolysis of GPPNA and SAAAPNA by P. nigrescens strains was 32-149 and 3-16 units, respectively. 57% (8 of 14) of P. nigrescens strains hydrolysed SAAPPPNA with activities ranging from 3 to 8 units. None of the P. intermedia or P. nigrescens strains examined were found to have trypsin-like enzyme activity (BAPNA hydrolysis). The GPPNA and SAAAPNA hydrolytic activity associated with the proteases from Porphyromonas gingivalis W50 was at least twice that of P. intermedia and P. nigrescens strains. The similar peptidase activities of P. intermedia and P. nigrescens against chromogenic substrates cannot be used to differentiate the species, but SDS-PAGE of cell surface protein extracts allowed unambiguous speciation between P. intermedia and P. nigrescens. This simple technique of cell surface protein analysis can be performed in most laboratories and offers a convenient way by which to differentiate the two species.

Bacterial Outer Membrane Proteins↗
PubMed2005

Involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing intestinal IEC-6 cell death.

Monomeric 14-kDa bovine alpha-lactalbumin was purified with a preparation of lower molecular weight whey protein concentrate from Holstein cow normal milk followed by size exclusion chromatography. The protein showed a stimulatory rather than an inhibitory effect on the proliferation of a cultured IEC-6 cell line from the rat small intestine. But incubation in 30% trifluoroethanol/acetate buffer (pH 5.5) at 37 degrees C for 5 d in a slowly rotating test tube rendered it highly cytotoxic with concomitant appearance of SDS-stable 20- and 30-kDa forms of alpha-lactalbumin on electrophoresis. Furthermore, alpha-lactalbumin obtained by a one-step purification procedure by affinity chromatography on an anti-alpha-lactalbumin antibody column from the lower molecular weight whey protein concentrate, which had been found to contain several SDS-stable higher M(r) forms of alpha-lactalbumin, exhibited potent inhibitory activity on IEC-6 cell growth. These results indicate the involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing cell death on the intestinal IEC-6 cell line.

Animals↗
PubMed1999

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp.

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37 degrees C, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

Basement Membrane↗
PubMed2004

Evaluation of biochemical and serological methods to identify and clustering yeast cells of oral Candida species by CHROMagar test, SDS-PAGE and ELISA.

The purpose of this work was to evaluate biochemical and serological methods to characterize and identify Candida species from the oral cavity. The strains used were five Candida species previously identified: C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. tropicalis, and Kluyveromyces marxianus, as a negative control. The analyses were conducted through the SDS-PAGE associated with statistical analysis using software, chromogenic medium, and CHROMagar Candida (CA), as a differential medium for the isolation and presumptive identification of clinically important yeasts and an enzyme-linked immunoabsorbent assay (ELISA), using antisera produced against antigens from two C. albicans strains. This method enabled the screening of the three Candida species: C. albicans, C. tropicalis, and C. krusei, with 100% of specificity. The ELISA using purified immunoglobulin G showed a high level of cross-reaction against protein extracts of Candida species. The SDS-PAGE method allowed the clustering of species-specific isolates using the Simple Matching coefficient, S(SM) = 1.0. The protein profile analysis by SDS-PAGE increases what is known about the taxonomic relationships among oral yeasts. This methodology showed good reproducibility and allows collection of useful information for numerical analysis on information relevant to clinical application, and epidemiological and systematical studies.

Animals↗
PubMed1990

Sweat protein components tested by SDS-polyacrylamide gel electrophoresis followed by immunoblotting.

Sweat samples were collected in a sauna from 74 healthy volunteers (72 men and 2 women) and concentrated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the individual samples revealed, in general, five main proteins and four PAS positive components. In pooled sweat, a method of SDS-PAGE followed by immunoblotting with specific antisera or antibodies against 24 human serum components was applied, and three out of the five main proteins showed the same molecular weights and antigenicities corresponding to serum albumin (67,000 Da), Zn-alpha 2-glycoprotein (42,000 Da) and lysozyme (14,000 Da). Moreover, orosomucoid, transferrin, IgG and IgA were demonstrated in the pooled sweat. Although alpha 1-antitrypsin was probably in the pooled sweat, other serum components could not be detected. On the pooled and individual sweat samples, anti-carcinoembryonic antigen (CEA) formed three bands at 42,000, 19,000 and 18,000 Da, but the antibody did not react with normal serum. It might be considered from these molecular weights that those sweat components are CEA-related antigens.

Adult↗
PubMed2001

Protein aggregation mediated by cysteine oxidation during the stacking phase of discontinuous buffer SDS-PAGE.

The resolution of complex protein mixtures by discontinuous buffer SDS-PAGE is accomplished by their concentration into thin bands in the stacking gel, followed by their separation during migration through the resolving gel. Recombinant human interferon-inducible protein-10 (IP-10), a 10-kDa C-X-C chemokine with four cysteines, aggregated during the stacking phase of SDS-PAGE and generated a band with an apparent molecular mass of 18 kDa. This aggregation depended on the presence of reduced sulfhydryl residues on IP-10, on the amount of loaded protein, and on the concentration of the ammonium persulfate used to polymerize the stacking gel. The aggregation of IP-10 could be prevented by reduction of its sulfhydryls with dithiothreitol followed by irreversible blockade with iodoacetamide. These methods may be useful in the prevention of aggregation of sulfhydryl-containing proteins during SDS-PAGE, especially when large quantities are analyzed to assess their purity.

Chemokine CXCL10↗
PubMed1991

Biologically active basic fibroblast growth factor migrates at 27 kD in "non-denaturing" SDS-polyacrylamide gel electrophoresis.

Using mild conditions of SDS-PAGE, i.e. no heating of the sample, and the PhastSystem (Pharmacia), we found that bFGF, either natural bovine or recombinant human migrated at a 27 kD position in addition to the classical 18 kD one. By the cell-blot technique, we found that the biological activity toward rat astroblasts and 3T3 mouse fibroblasts was always restricted to the 27 kD band. Partial heat denaturation experiments revealed a close correlation between the remaining biological activity of bFGF in solution and the ratio of the 27 kD band versus the 18 kD band seen on SDS gels. These observations suggest that the bFGF which is biologically active in solution migrates at an apparent Mr of 27 kD in our conditions of electrophoresis, keeping its biological activity after electrophoresis, and the molecules which are inactive (denatured) in solution migrate at 18 kD and remain inactive. These experimental conditions, in which the biological activity appears to be preserved, could be referred to as "non-denaturing SDS-polyacrylamide gel electrophoresis" and could be useful, associated to cell-blot, for the search and characterization of new growth factors active on cells in culture.

3T3 Cells↗
PubMed1991

Changes of IgM and IgG antibody levels in experimental rabbit anisakiasis as observed by ELISA and SDS-PAGE/immunoblot.

Antibody changes in experimental anisakiasis were observed in 10 rabbits which were infected each with 10 Anisakis simplex larvae. The sera were collected before and on the 6th to the 95th day after the infection. Using crude saline extract of Anisakis larvae as antigen, specific IgM and IgG antibody levels were observed by ELISA and SDS-polyacrylamide gel electrophoresis/immunoblot. Levels of specific-IgM antibody were elevated from the 6th day, reached their peaks on the 11th day after the infection, and dropped thereafter. Serum levels of IgG antibody increased from the 6th day and reached their peak on the 26th day after the infection, and decreased gradually thereafter. When SDS-PAGE of the crude extract was done, at least forty-one SDS-polypeptide bands were recognized. Of them, IgM antibody reacted mainly to the bands of 168, 95, 74, 64, 51, 47 and 34 kDa while IgG antibody reacted strongly to 168, 92, 85, 64, 58, 52, 42 and 40 kDa bands. The crude extract showed negligible cross reactions with sera of other parasitic diseases and normal control.

Animals↗