Distinctive electrophoretic isoenzyme profiles in Saccharomyces sensu stricto.
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Genetic variation among 35 strains representing the four currently recognized species of Saccharomyces sensu stricto (Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus/carlsbergensis and Saccharomyces paradoxus) was estimated by analysing the electrophoretic mobilities of nonspecific esterases, acid phosphatase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase isoenzymes. Twenty-two electrophoretic types were identified, a result in agreement with the phenotypic and genetic polymorphisms reported for this group of yeasts. However, the four species were clearly distinguishable based on the patterns obtained using three of the enzymes assayed, the resolving power not being improved by the introduction of data correspondent to lactate dehydrogenase. The overall diversity was higher among S. cerevisiae isolates, in contrast with S. paradoxus which showed only two patterns, one of which was common to four of the five strains studied. Concordant results from the application of the method and DNA hybridization experiments demonstrate its value for identification purposes. (+info)
The changing epidemiology of meningococcal disease in the United States, 1992-1996.
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New meningococcal vaccines are undergoing clinical trials, and changes in the epidemiologic features of meningococcal disease will affect their use. Active laboratory-based, population-based US surveillance for meningococcal disease during 1992-1996 was used to project that 2400 cases of meningococcal disease occurred annually. Incidence was highest in infants; however, 32% of cases occurred in persons >/=30 years of age. Serogroup C caused 35% of cases; serogroup B, 32%; and serogroup Y, 26%. Increasing age (relative risk [RR], 1.01 per year), having an isolate obtained from blood (RR, 4.5), and serogroup C (RR, 1.6) were associated with increased case fatality. Among serogroup B isolates, the most commonly expressed serosubtype was P1.15; 68% of isolates expressed 1 of the 6 most common serosubtypes. Compared with cases occurring in previous years, recent cases are more likely to be caused by serogroup Y and to occur among older age groups. Ongoing surveillance is necessary to determine the stability of serogroup and serosubtype distribution. (+info)
On the prokaryotic nature of red algal chloroplasts.
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The sequences of oligonucleotides released by T1 ribonuclease digestion of 32-P-labeled 16S (chloroplast) and 18S (cytoplasmic) ribosomal RNAs from a marine species of Porphyridium (Rhodophyta) have been determined. The resultant catalogs have been compared to those obtained for three prokaryotes:Escherichia coli, Bacillus subtilis, and Anacystis nidulans (a blud-green alga). There is extensive sequence homology between the Porphyridium chloroplast 16S ribosomal RNA and each of the prokaryotic 16S ribosomal RNAs, but little homology between the Porphyridium cytoplasmic 18S ribosomal RNA and any of the 16S species. These data provide a measure of the evolutionary distance separating existing chloroplasts from contemporary bacteria and blue-green algae, and are discussed in terms of the hypothesis that these organelles evolved from endosymbiotic photosynthetic prokaryotes. (+info)
Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis.
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In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guerin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-gamma by lymphocyte from subjects vaccinated with Leishvacinregister mark or target. The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 microg of each protein at 15 days interval. One hundred microg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 10(5) infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57. 14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7. 60) and 10.77UI/ml of IFN-gamma production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis. (+info)
Molecular basis for phenotypic heterogeneity in galactosaemia: prediction of clinical phenotype from genotype in Japanese patients.
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We identified 14 mutations in 15 Japanese subjects from 13 families with galactose-1-phosphate uridyltransferase (GALT) deficiency using denaturing gradient gel electrophoresis (DGGE) and direct sequence analysis. These mutations accounted for 22 (96%) of 23 mutant alleles in 15 Japanese subjects. The mutational spectrum included nine missense mutations (M142V, G179D, A199T, R231H, W249R, N314D, P325L, R333Q, and R333W), two deletions (L275fsdelT and Q317fsdelC), a nonsense mutation (W249X), and two splicing mutations (V85-N97fsdel38bp and IVS4nt+1). Ten of the 14 mutations have not been reported in Caucasians. Differences in frequency and spectrum of GALT mutations suggest that the mutations may have occurred after racial divergence of Caucasians and Asians. The Duarte variant in Japanese was associated with the N314D mutation, g.1105G > C, g.1323G > A, and g.1391G > A (SacI -) polymorphisms, as in Caucasians. The Duarte variant may have occurred before racial divergence, and was an ancient mutation. In vitro GALT activities of nine missense mutations were determined by a COS cell expression system, and indicated between 1.3% and 35% of wild-type control. Patients with R333Q (29% in vitro GALT activity) or A199T (35%) showed mild clinical phenotypes, i.e. no ovarian failure or neurological deterioration. Genotype determination is useful for predicting biochemical and clinical phenotypes in classic galactosaemia, and can be of further help in managing patients with this disorder. (+info)
The hydrophobic heptad repeat in Region III of Escherichia coli transcription factor sigma 54 is essential for core RNA polymerase binding.
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Escherichia coli transcription factor sigma 54 contains motifs that resemble closely those used for RNA polymerase II in mammalian cells, including two hydrophobic heptad repeats, a very acidic region and a glutamine-rich region. Triple changes in hydrophobic or multiple changes in acidic residues in Region III are known to severely impair core-binding ability. To investigate whether all the changes in triple mutants are necessary for core binding, site-directed mutagenesis was performed to create single and double mutants in the leucine or isoleucine residues in the heptad repeat in Region III. Single mutants showed no discernible loss of function. Double mutants showed partial protection of the -12 promoter element of the glnAp2 promoter due to the partial loss of their ability to bind core RNA polymerase. These mutations were deleterious to the function of sigma 54, which retained only 30-40% of wild-type mRNA levels. However, double mutants retained nearly normal ability to form open complexes. Two triple mutants created during previous work lost most, if not all, of their ability to bind core RNA polymerase, to protect the -12 promoter element of the glnAp2 promoter and to open the transcription start site. The two triple mutants produced about 20% or less than 10% of the wild-type transcripts from the glnAp2 promoter. These results demonstrate that the hydrophobic heptad repeat in Region III is essential for core RNA polymerase binding. Progressive loss of hydrophobicity of the hydrophobic heptad repeat in Region III of sigma 54 resulted in a progressive loss of core-binding ability, leading to the loss of -12 promoter element recognition and mRNA production. (+info)
Changes in myosin heavy chain mRNA and protein isoforms in single fibers of unloaded rat soleus muscle.
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Changes in myosin heavy chain (MHC) mRNA and protein isoforms were investigated in single fibers from rat soleus muscle unloaded by hindlimb suspension for 4 and 7 days. Dramatic changes were seen after 4 days, when all fibers co-expressed slow and fast MHC mRNAs. Most fibers contained mRNAs for MHCIbeta, MHCIIa, MHCIId(x), and MHCIIb. The up-regulation of the fast isoforms was only partially transmitted to the protein level. Atypical combinations of MHC mRNA isoforms, which deviated from the 'next-neighbor rule', were frequent in fibers from unloaded soleus. These atypical combinations increased with time and were not observed in the controls. The results suggest that hindlimb suspension elicits in soleus muscle pronounced perturbations in the expression of MHC isoforms by disrupting transcriptional and translational activities. (+info)
Biochemical characterization of different conformational states of the Sf9 cell-purified p53His175 mutant protein.
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In this study, we expressed and purified the p53 mutant encoded by the His175 allele (p53His175) in a baculovirus expression system in order to study the folding and the DNA binding activity of the protein. A two-site ELISA revealed that purified p53His175 protein preferentially displayed a PAb1620 conformation, which appeared to be not sufficient to interact specifically with DNA. The cryptic DNA binding activity of this mutant was then investigated by electrophoretic mobility shift assay in the presence of anti-p53 antibodies, and shown to be refractory to significant activation by PAb421 (a potent allosteric activator of wild-type p53's DNA binding activity). Nevertheless, p53His175 DNA binding was regulated by antibodies targeting the N-terminal region of the protein. Furthermore, while the protein preferentially displayed a PAb1620 conformation, our data suggested the existence of an equilibrium between at least two folding states of the protein (PAb1620 and PAb240 conformations). A model rationalizing the conformation, antibody-interacting ability and DNA binding regulation potential of p53His175 is presented. (+info)