Gel Electrophoretic Profiles Research Articles

Characterization of Kafirin and Zein Oligomers by Preparative Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis

Quantitative and qualitative analysis of uncooked zein and kafirin fractions were performed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic profiles. Kafirins and zeins present the same oligomer and monomer compositions with the exception of a 66 kDa oligomer that is only present in kafirins. The quantitative analysis showed differences between zein and kafirin. The composition of each oligomer was established via preparative SDS-PAGE. Part of the cooked oligomers resists reduction; the presence of those oligomers could be related to the decrease on protein digestibility with the cooking process.

Streptococcus agalactiae in a large Portuguese teaching hospital: antimicrobial susceptibility, serotype distribution, and clonal analysis of macrolide-resistant isolates.

Group B streptococci are emerging as a cause of serious infection worldwide. The capsular polysaccharides are not only important virulence factors but also the target of vaccine development efforts. Serotypes III (24.6%), V (23.4%), Ia (17.8%), and II (16.3%) were the most prevalent among 252 Streptococcus agalactiae isolates collected during 1999-2002 in the largest hospital of Lisbon, Portugal. The substantial proportion of bacteremic patients (17 neonates and 21 adults) in this period illustrates the present importance of S. agalactiae as a cause of invasive disease. All isolates were fully susceptible to penicillin (MIC(50) = 0.064 microg/ml; MIC(90) = 0.094 microg/ml, range 0.008-0.094), cefotaxime, chloramphenicol, ofloxacin, and vancomycin. Resistance was found to tetracycline (75.4%), erythromycin (10.7%), and clindamycin (9.9%). Of the 27 erythromycin-resistant isolates, 70.4% had the cMLS(B), 22.2% the iMLS(B), and 7.4% the M phenotype. All isolates presenting the M phenotype carried the mef(A) gene, whereas the erm(B) gene was found in a large fraction of MLS(B) isolates (n = 17) and only a small proportion (n = 7) the erm(A) gene [erm(TR) variant]. All isolates carried a single macrolide-resistance determinant. Macrolide resistance was not attributable to a single clone as evidenced by distinct serotype and pulsed-field gel electrophoretic profiles. Careful surveillance of S. agalactiae invasive infections in Portugal is essential, and the treatment or intrapartum prophylaxis of patients who are allergic to penicillin should be guided by contemporary resistance patterns observed in the country.

Streptococcus pneumoniae isolates with reduced susceptibility to ciprofloxacin in Spain: clonal diversity and appearance of ciprofloxacin-resistant epidemic clones.

Analysis of the pulsed-field gel electrophoretic profiles of 82 pneumococcal isolates with reduced susceptibility to ciprofloxacin (RSC) and of 90 co-occurring susceptible isolates indicates a considerable genetic diversity among isolates with RCS and points to a close relation between the two groups. This finding suggests that pneumococci with RCS emerge through independent mutational events.

Efficacy of a potentized homeopathic drug (Arsenicum-Album-30) in reducing cytotoxic effects produced by arsenic trioxide in mice: IV. Pathological changes, protein profiles, and content of DNA and RNA

Objective: To examine if the potentized homeopathic drug Arsenicum Album-30 can help restore the damage produced in protein profiles, DNA and RNA contents in liver and testis as a result of arsenic treatment in mice. Design: Sets of mice were injected with arsenic trioxide, one set was fed with Ars. Alb-30, another with Alcohol-30 and the final set was fed neither. The gel electrophoretic protein profiles and DNA and RNA contents in these three sets were studied. Methods: Protein profiles were studied by SDS-PAGE method; the DNA and RNA contents were assayed by the standard methods through diphenylamine and orcinol reactions respectively. Results: arsenic trioxide injection produced some pathological conditions, drastic changes (mainly reduction of protein bands) in protein sub-fractions, reduced DNA and RNA contents in both liver and testis; Ars. Alb-30-fed arsenic-intoxicated mice showed revival and restoration in both liver and testis as revealed by gel patterns and quantitative assay of DNA and RNA. Conclusion: Efficacy of the homeopathic drug Ars. Alb-30 in reducing arsenic-induced damage to protein and nucleic acids is substantiated and the mechanism of action of the homeopathic drug through expression of regulatory genes inferred.

Studies on Hepatic Mitochondrial Cytochrome P-450 during Plasmodium yoelii Infection and Pyrimethamine Treatment in Mice

Hepatic mitochondrial cytochrome P-450 and b5 activities were significantly depressed, whereas heme and hemozoin were increased during Plasmodium yoelii infection. Type II, aniline–HCl binding efficacy and polyacrylamide gel electrophoretic profile also depicted impairment of cytochrome P-450 during infection. However, the above alterations were more pronounced in the infected hepatic mitochondria, compared to microsomes. Oral treatment of pyrimethamine (10 mg/kg body weight×4 days) to P. yoelii-infected mice brought the altered levels of mitochondrial and microsomal cytochrome P-450 and b5 to almost normal, whereas heme and hemozoin levels remained unchanged.

Involvement of protein kinase C ɛ in thyrotropin-releasing hormone-stimulated phosphorylation of the myristoylated alanine-rich C kinase substrate in rat pituitary clonal cells

We have shown previously that novel protein kinase Cepsilon (nPKCepsilon) plays a key role in the basal and thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion in rat pituitary GH4C1 cells (Akita et al., J. Biol. Chem. 1994, 269, 4653-4660). Here we examined the region downstream of nPKCepsilon activation in order to understand the molecular mechanism by which nPKCepsilon mediates TRH-induced signal transduction. Exposure of GH4C1 cells to TRH causes a stimulation of the phosphorylation of a p80 (Mr approximately 80 000, pI approximately 4.3) and two p19 (p19a and b; Mr approximately 19 000, pI approximately 5.6 and 5.5, respectively). Phorbol ester, a potent activator of protein kinase C (PKC), also enhances these phosphorylations, whereas bisindolylmaleimide I, a specific inhibitor of PKC, clearly inhibits the phosphorylation of p80. p80 and p19 were identified as myristoylated alanine-rich C kinase substrate (MARCKS) and stathmin, respectively, as assessed by their two-dimensional gel electrophoretic profiles and their stabilities to heat and acid treatment. In nPKCepsilon-overexpressing stable clones, the phosphorylated level of MARCKS but not stathmin was high in the resting state, and enhanced and sustained upon TRH stimulation, correlating with the increased activation of nPKCepsilon. TRH stimulates the release of MARCKS from the membrane/cytoskeletal fraction to the cytosol fraction. These results, taken together with previous data concerning PRL secretion, suggest that MARCKS, a regulatory component of the cytoskeletal architecture, is the major substrate of nPKCepsilon in vivo, and that its phosphorylation may regulate TRH-stimulated PRL secretion.

Assessment of strain relatedness among Salmonella serotypes Salinatis, Duisburg, and Sandiego by biotyping, ribotyping, IS200 fingerprinting, and pulsed-field gel electrophoresis.

Salinatis (antigenic formula, 4,12:d:eh:enz15) is a rare Salmonella serotype currently designated a triphasic variant of the diphasic serotype Duisburg (1,4,12,27:d:enz15) (underlining indicates that the O antigen is determined by phage lysogenization). Salinatis could also be related to serotype Sandiego (4,[5],12:eh:enz15), from which it might have been derived by loss of H-d flagellin genes. Nineteen Salmonella strains of serotypes Salinatis, Duisburg, and Sandiego were examined by biotyping, PvuII and SmaI ribotyping, IS200 fingerprinting, and pulsed-field gel electrophoretic profiling. Results from these methods, used alone or together, indicate that serotype Salinatis is more likely to be related to serotype Sandiego than to serotype Duisburg. For future lists of serotype names, it is recommended that Salinatis be considered a variant of Sandiego.

Detection of a common genotype among strains of Taylorella equigenitalis isolated from thoroughbred horses in Japan between 1994 and 1996.

We examined whether or not the genotype J could be detected among 21 new strains of T. equigenitalis isolated between 1994 and 1996 in Japan since our previous report (MIYAZAWA et al. 1995). The respective pulsed-field gel electrophoretic profiles of the 21 Japanese strains, as well as those of an old EQ59 used as a reference strain after separate digestion with the two restriction enzymes, ApaI and NotI, were essentially identical but differed from those of T. equigenitalis NCTC11184T and KENTUCKY 188, respectively. Hence, the 21 strains and EQ59 appeared to have a common genotype J. Consequently, no strains of T. equigenitalis with any genotype other than genotype J may have reached Japan from 1980 to 1996 and strains with the genotype J have survived in Japan since the first invasion of contagious equine metritis into Japan.

Compositional changes during commercial processing of flaxseed

Compositional changes occurring during processing of flaxseed are of primary importance in adding value to flaxseed products. The physicochemical characteristics of flaxseed products at four stages of commercial processing, cleaning, flaking, pressing and solvent extraction, were determined to improve the functional properties of flaxseed products. The effect of industrial oil extraction on proximate composition, protein extractability, gel electrophoretic profiles, in vitro protein digestibility and viscosity of eight flaxseed products was assessed. The processes of flaking, heating, solvent extraction and solvent removal significantly increased the contents of protein, ash and soluble carbohydrate, and decreased oil content, total phenolics and protein solubility. Pepsin digestibility ranged from 11 to 72%, and generally increased with processing. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of solubilized proteins showed the presence of four major polypeptide components, with only minimal changes in the protein profiles among the samples as a result of processing. The viscosity of the slurry was largely unaffected but shear thinning indices increased with processing. Multiple-regression analysis showed that protein content was related to carbohydrate content, cyanogenic content, and viscosity, but not associated with protein solubility and digestibility.

In vitro genotypic variation of Campylobacter coli documented by pulsed-field gel electrophoretic DNA profiling: implications for epidemiological studies

Six isolates of Campylobacter coli from different pig herds were subcultured up to 50 times over a 6-month period and DNA samples suitable for pulsed-field gel electrophoretic (PFGE) profiling prepared at regular (1, 20, 40 and 50 passages) intervals. In 5/6 strains, changes in the banding patterns of Sma1, Sal1 and/or BamH1 digests were observed. In one such strain the differences were considered artifactual. However, significant alterations in PFGE profiles between subcultures of four strains were seen, irrespective of the restriction enzyme used. Spontaneous intramolecular genomic rearrangements were considered the most likely mechanism for the changes observed. A numerical analysis based upon the combined distribution of Sma1- and Sal1-derived fragments clustered most strain subcultures together, with the exception of those from one isolate which were divided into two clusters. The effect of spontaneous genetic change on PFGE profiles must be considered when evaluating strain relationships. Numerical techniques may aid data interpretation but results must be evaluated cautiously.

In vitro genotypic variation of Campylobacter coli documented by pulsed-field gel electrophoretic DNA profiling: implications for epidemiological studies.

Six isolates of Campylobacter coli from different pig herds were subcultured up to 50 times over a 6-month period and DNA samples suitable for pulsed-field gel electrophoretic (PFGE) profiling prepared at regular (1, 20, 40 and 50 passages) intervals. In 5/6 strains, changes in the banding patterns of Sma1, Sal1 and/or BamH1 digests were observed. In one such strain the differences were considered artifactual. However, significant alterations in PFGE profiles between subcultures of four strains were seen, irrespective of the restriction enzyme used. Spontaneous intramolecular genomic rearrangements were considered the most likely mechanism for the changes observed. A numerical analysis based upon the combined distribution of Sma1- and sal1-derived fragments clustered most strain subcultures together, with the exception of those from one isolate which were divided into two clusters. The effect of spontaneous genetic change on PFGE profiles must be considered when evaluating strain relationships. Numerical techniques may aid data interpretation but results must be evaluated cautiously.

TGF-beta1 modifications in nuclear matrix proteins of osteoblasts during differentiation.

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-beta1 treatment of osteoblasts to gain insight into the effects of TGF-beta on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-beta1 for 48 h (day 4-6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-beta-treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-beta-treated cultures. These findings support the concept that TGF-beta1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-beta-dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-beta in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-beta signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization.

Subtyping of Campylobacter jejuni Penner heat-stable (HS) serotype 11 isolates from human infections.

High resolution molecular subtyping was applied to Campylobacter jejuni Penner heat-stable (HS) 11 isolates from human infections and other sources. Strains were genotyped by restriction fragment length polymorphism analysis involving PCR-based flagellin gene (flaA) profiling with HinfI and DdeI, and pulsed-field gel electrophoretic (PFGE) profiling with SmaI and KpnI. Fla-genes of the strains were highly conserved as most (95%) had the same fla-profile. PFGE analysis of SmaI digests was more discriminatory with 15 profile subtypes identified, although 36% of isolates had a common profile. The study showed that strains of C. jejuni HS11, unlike those of HS1 and the HS4 complex, were relatively homogeneous at the genomic level and that high resolution molecular techniques were essential for detailed epidemiological subtyping.

Exposure to chronic hypoxia induces qualitative changes of collagen in the walls of peripheral pulmonary arteries

Qualitative changes of vascular wall matrix collagens in chronic hypoxic pulmonary hypertension were studied by gel electrophoresis. Male adult rats (n = 12) were exposed to hypoxia (F i02 = 0.1, 3 wks). Control rats (n = 13) were kept in air. Samples of peripheral pulmonary arteries (PPA, diam. 100 – 400 μm), main branches of pulmonary artery, and aorta were dissected. Arterial samples were treated with 4M guanidine-HCl to remove noncollagenous moieties and the collagenous stroma was dissolved by limited pepsin digestion at low pH. Low molecular mass peptides (M. W. approx. 76 and 66 kD) were detected in the gel electrophoretic profile of collagen peptides of PPA of the chronically hypoxic animals and in aorta of both hypoxic and normoxic groups. These peptides were absent in the PPA of normoxic rats. Since the 76 kD peptide bound anticollagen type I antibodies, it appears to be of collagenous nature and it may be the result of collagenolytic activity in PPA isolated from hypoxic lungs. This was confirmed by zymography. We conclude that exposure of rats to chronic hypoxia results in the presence of low molecular mass peptides in the wall matrix of PPA which resemble those found in aorta of normoxic animals. Collagenolytic activity in the walls of peripheral pulmonary arteries may participate in the mechanism of lung vascular remodelling in chronic hypoxia.

Lineages within Campylobacter jejuni defined by numerical analysis of pulsed-field gel electrophoretic DNA profiles.

Forty-seven Penner heat-stable (HS) serotype reference strains for Campylobacter jejuni and 47 serologically non-typable strains were examined by pulsed field gel electrophoresis (PFGE) DNA restriction analysis. The SmaI and KpnI digest profiles were compared by numerical analysis. Most strains grouped differently in the two analyses but strain lineages were inferred where the two agreed. Genetic relationships between reference strains in the cross-reacting HS4 complex were examined. Three clonal lines were evident and comprised: (i) HS4, HS13 and HS16; (ii) HS50 and HS65; (iii) HS43. The majority of those C. jejuni expressing HS antigens not recognised by currently available antisera had > 50% PFGE DNA digest similarity to one or more Penner scheme reference strain(s) and so did not necessarily represent distinct genetic lineages. PFGE analysis provided a high level of discrimination amongst strains of C. jejuni but overall similarity estimates for defining types must be based on the analysis of more than one restriction pattern.

Flagellin gene profiling of Campylobacter jejuni heat-stable serotype 1 and 4 complex

Flagellin gene ( flaA) sequence polymorphisms were used to discriminate amongst 167 strains of Campylobacter jejuni serotype HS1 and the HS4 complex. Direct PCR of cell suspensions provided a rapid method for analysing DNase-negative strains, whereas purified DNA was necessary for the DNase-positive strains. Nine different PCR-RFLP patterns (genotypes) were identified by analysis with HinΛ and 12 with Ddel, giving a total of 19 combined flaA profile types. The most common combined fla types were H1D1 (35%) and H1D2 (20%) for serotype HS1, and H1D2 (23%) and H4D7 (43%) for serotype HS4. Comparison of flaA typing with other key subtyping methods for C. jejuni showed it to be less discriminatory than pulsed field gel electrophoretic (PFGE) profiling, but more so than ribotyping. Fla types provided a useful indication of strain diversity, but as some were conserved across different serotypes, ribotypes and PFGE types, the same fla type could not be used as the sole basis for grouping strains. We provide evidence for several distinct subgroups based on conserved multiple genomic criteria within the HS1 and HS4 strains, and conclude that monitoring of such subgroups could provide a novel basis for future epidemiological surveillance of C. jejuni.

Classification and Identification of Campylobacter sputorum using Numerical Analyses of Phenotypic Tests and of One-dimensional Electrophoretic Protein Profiles

Summary The application of numerical analyses of phenotypic tests and sodium-dodecyl sulphate polyacrylamide gel electrophoretic protein profiles in the classification and identification of Campylobacter sputorum was examined. Thirty-eight strains received, or isolated as, representatives of each of the defined biovars of Campylobacter sputorum (i.e. bubulus, fecalis, and sputorum), along with seventeen type or reference strains of other Campylobacter, Arcobacter , and Helicobacter species, were studied. The results from both analyses indicated, (i) that thirty-two strains formed a distinct cluster which included the type strain of C. sputorum , confirming their identity as members of this species, (ii) that catalase-positive strains of C. sputorum are associated with faeces, (iii) that the normal subdivision of catalase-negative C. sputorum strains into two source-specific biovars is invalid. Thus, catalase-positive strains of C. sputorum should remain as biovar fecalis and catalase-negative strains (i.e. biovars sputorum and bubulus) should be unified as a single biovar sputorum and the biovar bubulus discarded. Five oral isolates initially believed to be C. sputorum biovar sputorum were identified as potentially novel campylobacters. Numerical analysis of both types of data used were shown to be effective methods of classifying and identifying campylobacters and allied taxa.

Hemorrhagic principles in the venom of Bitis arietans, a viperous snake. II. Enzymatic properties with special reference to substrate specificity

The optimal pH of the proteinase activity of hemorrhagins, BHRa and BHRb, isolated from the venom of Bitis arietans (puff adder) is pH 9. The activity was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline and 8-hydroxyquinoline, but not by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, suggesting that they are metalloproteinases. The hemorrhagins hydrolyzed all gelatin preparations derived from types I, II, III and IV collagen. On the other hand, only type IV native collagen was hydrolyzed. Gel electrophoretic profiles of type IV collagen hydrolysates suggested that the hemorrhagins affect the collagen helical chains at different cleavage sites. The hemorrhagins hydrolyzed several synthetic peptides such as angiotensin I and luteinizing hormone-releasing hormone, but not synthetic substrates for bacterial and animal collagenases. The hydrolysis of various peptides indicated that the hemorrhagins are endopeptidases. The insulin B chain is cleaved by BHRa and BHRb at 11 and 10 positions, respectively. The substrate specificity of the hemorrhagins was compared with those of known hemorrhagic and nonhemorrhagic venom proteinases.

Electrophoretic Karyotypes of Authentic Strains of the Sensu Stricto Group of the Genus Saccharomyces

A comparative electrophoretic karyotyping study was performed with several certified authentic strains of the four species that could be distinguished by nuclear DNA (nDNA)-nDNA reassociation data within the sensu stricto group of the genus Saccharomyces. A multivariate analysis of the polymorphisms observed in pulsed-field gel electrophoretic profiles (numbers and molecular weights of separated units) revealed that the strains could be separated into four clusters that corresponded to the taxa that were distinguished on the basis of nDNA comparisons. Discrepancies between nDNA reassociation data and membership in the corresponding clusters were observed only with two strains of Saccharomyces paradoxus. Blind tests carried out with additional industrial strains confirmed the general validity of the statistical model created for comparison of karyotypes within the species included in Saccharomyces sensu stricto.

The effect of cooking on proteins from acha (Digitaria exilis) and durum wheat

AbstractThe effect of cooking on proteins from acha and durum wheat was assessed from an analysis of protein extractability, gel electrophoretic profiles, in‐vitro protein digestibility (IVPD) and the amino acid compositions of wholemeal flour and residue proteins. Heating wholemeal flour samples at 100–140°C (t = 10–40 min) resulted in 0–30% and 45–55% decreases in acha and durum protein solubility, respectively. In general, high molecular weight (30–70 k Da) protein subunits were more susceptible to heat damage. For both cereals, sodium dodecyl sulphate (SDS; 10 g litre−1) and/or dithiothrcitol (DTT; 10 mM) increased protein solubility in unheated and heated samples. The IVPD index was 90–91% and was not significantly altered by cooking (100–120°C, t = 40 min). Cooking at extreme temperatures (140°C, t = 40 min) reduced the IVPD by 8% (P = 0.05). Osborne fractionation resulted in a durum or acha residue level of 7.8% or 55.2%. Treatment with solvent containing propanol, SDS and/or DTT at room temperature followed by SDS‐polyacrylamide gel electrophoresis of non‐solubilised proteins showed that the glutelin fraction of acha, with the exception of a 65 kDa subunit, was insoluble owing to strong inter‐subunit hydrophobic and disulphide interactions. Wholemeal acha flour and residue protein showed a significantly greater level of hydrophobic and sulphur amino acids as well as glutamine which is associated with H‐bonding. The possibility that cereal protein solubility is also dependent on protein‐carbohydrate links is discussed.