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Biomedical subjects

M H Vainstein

Publications and source records attributed to M H Vainstein.

17 recordsLinked to original sources
PubMed2003

Phenotypic and molecular characterization of bovine Campylobacter fetus strains isolated in Brazil.

The objective of the present study was to characterize the phenotypic and molecular aspects of Campylobacter fetus strains isolated from bovine herds with reproductive problems. Thirty-one Brazilian field isolates, together with one reference strain of each subspecies of C. fetus, were analyzed. The strains were submitted to phenotypic identification followed by subspecies characterization using the polymerase chain reaction (PCR) and numeric evaluation of restriction fragment length polymorphism (RFLP) separated by pulsed-field gel electrophoresis (PFGE). Phenotypically, 4 isolates (12.1%) were classified as C. fetus subsp. fetus, and 29 isolates (87.9%) were classified as C. fetus subsp. venerealis. However, according to molecular analysis, only 1 isolate (3.0%) was classified as C. fetus subsp. fetus (the reference strain), whereas 32 isolates (97.0%) were considered C. fetus subsp. venerealis. SalI digestion of C. fetus genomic DNA, obtained from the 33 strains, yielded 7-10 DNA fragments ranging in size from 40 to 373kb, with 12 distinct patterns. Furthermore, the numeric analysis by neighbor-joining of the DNA from the 33 strains resulted in a dendrogram in which 2 distinct groups were identified. It was concluded that phenotypic characterization of C. fetus subspecies might lead to erroneous classification of field isolates. Although RFLP-PFGE is a powerful and reliable technique to characterize C. fetus, it has the inconvenience of being time consuming and laborious. Whereas PCR, besides providing rapid results, was found to be reliable and convenient for the characterization of field isolates of C. fetus.

Animals↗
PubMed2002

Epidemiological aspects of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul.

Cryptococcus neoformans is a pathogenic fungus that causes life-threatening meningoencephalitis in immunocompromised patients (HIV-positive patients), and lymphoproliferative disorders in patients subjected to organ transplantation and other immunosuppressive therapies. This fungus is commonly found in soil and avian excreta, mainly from pigeon and turkey. We describe the isolation and characterization of 17 clinical and 10 environmental (pigeon excreta) isolates from the Brazilian state Rio Grande do Sul. We analyzed capsule formation, carbon assimilation pattern, canavanine-glycine-bromothymol blue (CGB) reaction, and nitrate and urease tests, as well as susceptibility to antifungal drugs. The genetic variability among C. neoformans isolates was studied using randomly amplified polymorphic DNA (RAPD) analysis. Eight of 22 arbitrary polymerase chain reaction primers used confirmed genetic polymorphism among the environmental isolates tested, suggesting that it remains feasible to use RAPD analysis as a typing method. Three of the selected primers yielded 10 molecular subclasses. The majority of the clinical isolates were assigned to the molecular subclass F. The RAPD data obtained reinforce the developing consensus about the population structure of this fungus.

AIDS-Related Opportunistic Infections↗
PubMed2001

Disordered environments in spatial games.

The Prisoner's dilemma is the main game theoretical framework in which the onset and maintainance of cooperation in biological populations is studied. In the spatial version of the model, we study the robustness of cooperation in heterogeneous ecosystems in spatial evolutionary games by considering site diluted lattices. The main result is that, due to disorder, the fraction of cooperators in the population is enhanced. Moreover, the system presents a dynamical transition at rho*, separating a region with spatial chaos from one with localized, stable groups of cooperators.

Journal Article↗
PubMed2001

Influence of enrichment media and application of a PCR based method to detect Salmonella in poultry industry products and clinical samples.

To attempt the rapid detection of Salmonella enterica, we have coupled a culture procedure with PCR amplification of the genus-specific invE/invA genes. The method was applied to different kinds of samples from the poultry industry and evaluated by using hydrolyzed feather meal, meat meal, litter and viscera, all experimentally inoculated with a known number of Salmonella followed by cultivation in selenite--cystine broth prior to the PCR reaction. The expected 457bp specific DNA fragment could be amplified from dilutions containing as few as 5.7CFU, indicating that the PCR technique can be successfully coupled with culture in an enrichment broth to distinguish Salmonella species from other enteric bacteria present in samples from the poultry industry. Tetrathionate broth proved to be a much better enrichment media compared to selenite-cystine when the presence of Salmonella was evaluated by PCR in 1-day-old chicks experimentally infected with known numbers of Salmonella. Samples included cecal tonsils and viscera, collected at 48h and 7 days postinfection. The PCR technique was more sensitive in detecting infected animals than the standard microbiological procedure, which detected only 47% of all PCR positive samples.

Animals↗
PubMed2001

Molecular cloning and characterization of a gene encoding the 29-kDa proteasome subunit from Trypanosoma cruzi.

Antiserum raised against purified Trypanosoma cruzi proteasomes was used to isolate two cDNA clones, tcpr29 and tcpr29B, and the corresponding genomic sequence, termed tcpr29A. Both cDNAs and the gene contain a 798-bp ORF, coding for a 266-amino acid protein, with a predicted molecular mass of 29 kDa. Sequence comparisons show that the protein encoded by tcpr29 belongs to the alpha6 subfamily of proteasome subunits. Southern analysis indicated that tcpr29 subunit is encoded by a single-copy gene which maps to chromosome 20 of the CL Brener clone. Allelic variants were found in other T. cruzi isolates, suggesting heterozygosity for the gene in some and homozygosity in other strains. A spliced-leader addition site was identified 123 bp upstream from the start codon, generating a stable 1.5-kb transcript. Western analysis revealed that tcpr29A is constitutively expressed during the life cycle of the parasite.

5' Untranslated Regions↗
PubMed2000

In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus.

Metarhizium anisopliae is a filamentous fungus used for tick control. The in vitro effects of 12 M. anisopliae isolates on engorged Boophilus microplus females were analysed. The most pathogenic isolate (E6S1) caused a 100% death rate when 10(7) spores/ml were used to infect ticks. Isolates of M. anisopliae taken from experimentally infected ticks proved to be more pathogenic than fungus maintained on culture media. A comparison between dsRNA mycovirus-free and infected M. anisopliae isolates suggested that, in general, virus free isolates were more infective. The results showed that the biological control of B. microplus by M. anisopliae infection might constitute an additional method to integrated tick control management.

Animals↗
PubMed1998

Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance.

Germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved using lithium acetate treatment or electroporation. The efficiency of transformation was up to 32 and 25 transformants per microgram of plasmid DNA with the two methods, respectively. Transformants obtained by the lithium acetate method were more stable and showed a high copy number of the hph gene integrated into their genome. The other transformants, from the electroporation procedure, were stable, but unable to grow in the presence of high levels of hygromycin, and detection of the hph gene was only possible by polymerase chain reaction analysis.

Dose-Response Relationship, Drug↗
PubMed1998

A chitinase encoding gene (chit1 gene) from the entomopathogen Metarhizium anisopliae: isolation and characterization of genomic and full-length cDNA.

There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome.

Amino Acid Sequence↗
PubMed1997

High frequency gene transfer by microprojectile bombardment of intact conidia from the entomopathogenic fungus Paecilomyces fumosoroseus.

Two different methods, (i) PEG and (ii) biolistic, were employed to transform protoplasts and conidia of Paecilomyces fumosoroseus using hygromycin resistance as selectable marker. Transformation frequencies varied from 1.9 to 2.5 transformants microgram-1 of DNA by the PEG method, and from 33 to 153 transformants microgram-1 of DNA by the biolistic procedure.

Animals↗
PubMed1996

High frequency gene conversion among benomyl resistant transformants in the entomopathogenic fungus Metarhizium anisopliae.

Three different methods, (i) PEG, (ii) electroporation and (iii) biolistic, were employed to transform Metarhizium anisopliae using benomyl resistance as a selectable marker. Transformation frequencies and mitotic stability varied for each method, from 0.8 to 6.9 transformants micrograms-1 of DNA and 46%, respectively, by the PEG method; 1.3 to 1.8 transformants micrograms-1 of DNA and 67% by electroporation; and 32 to 201 transformants micrograms-1 of DNA and 90% by biolistic. We demonstrate by PCR that 60% of the transformants were generated by gene conversion.

Animals↗
PubMed1995

Transformation of Aspergillus nidulans by microprojectile bombardment on intact conidia.

This paper describes transformation of intact conidia of Aspergillus nidulans, auxotrophic for arginine, by using the biolistic process. The plasmid employed was pFB39, carrying the argB gene. The transformation frequency obtained was 81 transformants/microgram of DNA. Classical genetics and molecular analysis were conducted to analyse transformants and to determine in which chromosome integration took place.

Arginine↗
PubMed1995

Free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic Escherichia coli.

The non-immunoglobulin component of human milk responsible for the inhibition of Escherichia coli cell adhesion (haemagglutination) mediated by colonisation factor antigen 1 (CFA1) was determined by chromatographic fractionation of human whey proteins with Sephadex G-200, DEAE cellulose and heparin-sepharose. Pure free secretory component (fSC) and pure lactoferrin (Lf) were isolated and both compounds inhibited the haemagglutination induced by E. coli CFA1+. The lowest concentrations of purified fSC and Lf able to inhibit the haemagglutination induced by E. coli strain TR50/3 CFA1+ were 0.06 mg/ml and 0.1 mg/ml respectively. Commercially available lactoferrin from human milk and transferrin from human serum, which has a great structural analogy to lactoferrin, also inhibited the haemagglutination. The lowest concentrations of the commercial lactoferrin and transferrin able to inhibit the haemagglutination induced by E. coli TR50/3 CFA1+ were 0.03 mg/ml and 0.4 mg/ml, respectively. These results indicate that fSC and Lf may be important non-specific defence factors against enterotoxigenic E. coli infections.

Bacterial Adhesion↗
PubMed1991

Regulation of invertase in Aspergillus nidulans: effect of different carbon sources.

Aspergillus nidulans produces an extracellular beta-D-fructofuranoside fructohydrolase (invertase) when grown on a medium containing the beta-fructofuranosides sucrose or raffinose, indicating that synthesis is subject to induction by the substrate. On a growth medium containing sucrose, production was maximal at 15 h in cultures incubated at 28 C degrees. After this time the level of detectable invertase in the cultures declined. A proportion of the enzyme was secreted during the linear growth phase of the fungus. Various sugars were investigated for induction of invertase, but only the two beta-fructofuranosides induced high production levels; with the other sugars, the enzyme was produced only at a low constitutive level. Mycelium grown under repressive conditions (1% glucose), rapidly produced invertase when transferred to sucrose-containing medium. After 80 min the invertase level in these cultures was 26-fold higher than the constitutive level. The repressive effect of other sugars, e.g. glucose and xylose, on invertase production was also demonstrated in this experimental system.

Aspergillus nidulans↗
PubMed1990

Solubilisation of a cell wall bound invertase in Aspergillus nidulans.

Aspergillus nidulans produces an extracellular invertase when incubated in the presence of sucrose and about half of the activity produced was found to be associated with the mycelium. Sixty percent of this mycelial invertase could be solubilised by simple mechanical disruption. Among the agents tested for solubilisation of invertase, proteinase K and dithiothreitol were the most effective.

Aspergillus nidulans↗
PubMedDate not supplied

Purification and characterisation of an Aspergillus niger invertase and its DNA sequence.

A secreted invertase was purified 23-fold by ultrafiltration, ion-exchange, and gel filtration chromatography from the culture supernatant of 18 h sucrose-grown cultures of Aspergillus niger. The purified enzyme hydrolysed sucrose and raffinose but there was no detectable hydrolysis of inulin, melezitose or PNPG. Invertase activity was optimal at pH 5.5 and 50 degrees C. The molecular mass of reduced invertase was 115 kDa, as determined by SDS gel electrophoresis. The native molecular weight of between 225 kDa and 250 kDa, estimated by electrophoresis under non-denaturing conditions, suggests that the protein is a dimer of identical subunits. The suc1 gene encoding this protein was completely-sequenced. The translated sequence yields a protein of 566 amino acids with a calculated molecular mass of 61 kDa, suggesting that carbohydrates represent about 50% of the mass of the protein.

Amino Acid Sequence↗
PubMedDate not supplied

In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcus neoformans isolated in Southern of Brazil.

The purpose of the present study was to compare the susceptibility to four antifungal agents of 69 Cryptococcus neoformans strains isolated from AIDS patients with that of 13 C. neoformans strains isolated from the environment. Based on the NCCLS M27-A methodology the Minimal Inhibitory Concentrations (MICs) obtained for amphotericin B, itraconazole and ketoconazole were very similar for clinical and environmental isolates. Clinical isolates were less susceptible to fluconazole than environmental isolates. The significance of these findings and aspects concerning the importance, role and difficulties of C. neoformans susceptibility testing are also discussed.

AIDS-Related Opportunistic Infections↗