RESUMO
The pathophysiological mechanisms associated with the effects of red blood cell (RBC) transfusion on cardiopulmonary function and inflammation are unclear. We developed an experimental model of homologous 14-days stored RBC transfusion in hypovolemic swine to evaluate the short-term effects of transfusion on cardiopulmonary system and inflammation. Sixteen healthy male anesthetized swine (68±3.3 kg) were submitted to controlled hemorrhage (25% of blood volume). Two units of non-filtered RBC from each animal were stored under blood bank conditions for 14 days. After 30 min of hypovolemia, the control group (n=8) received an infusion of lactated Ringer's solution (three times the removed volume). The transfusion group (n=8) received two units of homologous 14-days stored RBC and lactated Ringer's solution in a volume that was three times the difference between blood removed and blood transfusion infused. Both groups were followed up for 6 h after resuscitation with collection of hemodynamic and respiratory data. Cytokines and RNA expression were measured in plasma and lung tissue. Stored RBC transfusion significantly increased mixed oxygen venous saturation and arterial oxygen content. Transfusion was not associated with alterations on pulmonary function. Pulmonary concentrations of cytokines were not different between groups. Gene expression for lung cytokines demonstrated a 2-fold increase in mRNA level for inducible nitric oxide synthase and a 0.5-fold decrease in mRNA content for IL-21 in the transfused group. Thus, stored homologous RBC transfusion in a hypovolemia model improved cardiovascular parameters but did not induce significant effects on microcirculation, pulmonary inflammation and respiratory function up to 6 h after transfusion.
Assuntos
Animais , Masculino , Pneumonia/fisiopatologia , Fenômenos Fisiológicos Respiratórios , Preservação de Sangue/métodos , Fenômenos Fisiológicos Cardiovasculares , Transfusão de Eritrócitos/métodos , Hipovolemia/terapia , Suínos , Preservação de Sangue/efeitos adversos , Ensaio de Imunoadsorção Enzimática , Citocinas/sangue , Resultado do Tratamento , Transfusão de Eritrócitos/efeitos adversos , Modelos Animais de Doenças , HemodinâmicaRESUMO
The pathophysiological mechanisms associated with the effects of red blood cell (RBC) transfusion on cardiopulmonary function and inflammation are unclear. We developed an experimental model of homologous 14-days stored RBC transfusion in hypovolemic swine to evaluate the short-term effects of transfusion on cardiopulmonary system and inflammation. Sixteen healthy male anesthetized swine (68±3.3 kg) were submitted to controlled hemorrhage (25% of blood volume). Two units of non-filtered RBC from each animal were stored under blood bank conditions for 14 days. After 30 min of hypovolemia, the control group (n=8) received an infusion of lactated Ringer's solution (three times the removed volume). The transfusion group (n=8) received two units of homologous 14-days stored RBC and lactated Ringer's solution in a volume that was three times the difference between blood removed and blood transfusion infused. Both groups were followed up for 6 h after resuscitation with collection of hemodynamic and respiratory data. Cytokines and RNA expression were measured in plasma and lung tissue. Stored RBC transfusion significantly increased mixed oxygen venous saturation and arterial oxygen content. Transfusion was not associated with alterations on pulmonary function. Pulmonary concentrations of cytokines were not different between groups. Gene expression for lung cytokines demonstrated a 2-fold increase in mRNA level for inducible nitric oxide synthase and a 0.5-fold decrease in mRNA content for IL-21 in the transfused group. Thus, stored homologous RBC transfusion in a hypovolemia model improved cardiovascular parameters but did not induce significant effects on microcirculation, pulmonary inflammation and respiratory function up to 6 h after transfusion.
Assuntos
Preservação de Sangue/métodos , Fenômenos Fisiológicos Cardiovasculares , Transfusão de Eritrócitos/métodos , Hipovolemia/terapia , Pneumonia/fisiopatologia , Fenômenos Fisiológicos Respiratórios , Animais , Preservação de Sangue/efeitos adversos , Citocinas/sangue , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Transfusão de Eritrócitos/efeitos adversos , Hemodinâmica , Masculino , Oxigênio/metabolismo , Reprodutibilidade dos Testes , Ressuscitação/métodos , Suínos , Fatores de Tempo , Resultado do TratamentoRESUMO
Bovine digital dermatitis (BDD) is an infectious and contagious disease characterized by ulcerative and proliferative lesions affecting the skin on the bulbs of the heel or the interdigital cleft in dairy cattle, often associated with lameness. Evidences on the etiology of BDD indicate that it is multifactorial, involving environmental factors and multiple bacterial colonization. We isolated and identified microorganisms from BDD biopsy samples obtained from five Holstein Friesian and two Jersey cows by cultivation and molecular identification of bacterial isolates using 16S rRNA gene sequence analysis. We identified six bacterial species: Spirochetes as Treponema pedis and Leptospira broomi/L. fainei, L. licerasiae/L. wolffii; Corynebacterium appendicis, Cupriavidus gilardii and Enterococcus casseliflavus/E. gallinarum. It was quite surprising to have isolated and identified Leptospira species in three out of seven cultures, from different individual cows and two different farms. The species identified belong to the intermediate pathogenic clade, which is a group found to cause human and animal disease. Our findings indicate the need to further investigate the association of Leptospira of intermediate pathogenicity with BDD lesions and whether its presence would have any veterinary and medical significance both in Leptospirosis and with the pathogenesis of BDD lesions, especially in tropical countries.(AU)
Dermatite digital bovina (DDB) é uma doença infecciosa, contagiosa, caracterizada por lesões ulcerativas e proliferativas da região dos talões e/ou do espaço interdigital, frequentemente associada com claudicação. Evidências indicam que a etiologia da DDB é multifatorial, envolvendo fatores ambientais e colonização polimicrobiana. Relata-se aqui o isolamento e a identificação bacteriana em amostras de biópsias em lesões de DDB, obtidas de cinco vacas da raça Holandesa e duas da raça Jersey, por meio de cultivo e identificação molecular de isolados, com base na análise de sequências de genes 16S rRNA. São identificadas seis espécies bacterianas: as espiroquetas Treponema pedis e Leptospira broomi/L. fainei, L. licerasiae/L. wolffii; Corynebacterium appendicis, Cupriavidus gilardii e Enterococcus casseliflavus/E. gallinarum. O isolamento e a identificação de espécies de Leptospira surpreenderam, destacando-se sua presença em três dos sete cultivos obtidos em diferentes vacas, de duas fazendas distintas. As espécies identificadas pertencem ao grupo tipificado como de patogenicidade intermediária, causador de doenças em animais e no homem. Os resultados apresentados indicam a necessidade de maiores investigações sobre a associação entre Leptospira de patogenicidade intermediária e a patogênese das lesões DDB, investigando-se sua presença e significado nas medicinas veterinária e humana, especialmente em países tropicais.(AU)
Assuntos
Animais , Bovinos , Dermatite Digital/microbiologia , Leptospira/isolamento & purificação , RNA Ribossômico 16S/análise , Treponema/isolamento & purificação , Reação em Cadeia da Polimerase/veterináriaRESUMO
Autism spectrum disorders (ASD) is a group of behaviorally defined neurodevelopmental disabilities characterized by multiple genetic etiologies and a complex presentation. Several studies suggest the involvement of the serotonin system in the development of ASD, but only few have investigated serotonin receptors. We have performed a case-control and a family-based study with 9 polymorphisms mapped to two serotonin receptor genes (HTR1B and HTR2C) in 252 Brazilian male ASD patients of European ancestry. These analyses showed evidence of undertransmission of the HTR1B haplotypes containing alleles -161G and -261A at HTR1B gene to ASD (P=0.003), but no involvement of HTR2C to the predisposition to this disease. Considering the relatively low level of statistical significance and the power of our sample, further studies are required to confirm the association of these serotonin-related genes and ASD.
Assuntos
Transtorno Autístico/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Receptor 5-HT1B de Serotonina/genética , Receptor 5-HT2C de Serotonina/genética , Alelos , Brasil , Estudos de Casos e Controles , Família , Genótipo , Haplótipos , Humanos , Masculino , Análise de Sequência de DNARESUMO
Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.
Assuntos
Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Mutação/genética , Adolescente , Adulto , Idoso , Criança , Sondas de DNA/genética , Feminino , Transferência Ressonante de Energia de Fluorescência , Genótipo , Proteína da Hemocromatose , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.
Assuntos
Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Mutação/genética , Sondas de DNA/genética , Transferência Ressonante de Energia de Fluorescência , Genótipo , Proteínas de Membrana/genética , Fenótipo , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Propolis samples collected in the dry and rainy seasons, from an experimental apiary located in a cerrado vegetation region in Brazil were used in this study. Microscopic analysis showed the presence of 31 pollen types, secretory hairs (genus Baccharis) and fragments of plant epidermis. The oxidation rates and the wax content of the samples after physicochemical analyses were in agreement with the Cuban Guideline NRAG 870-88. A high performance liquid chromatography analysis showed a similar pattern of chromatograms, characterized by the presence of ten phenolic compounds. There was no significant difference in the pro fi le of phenolic compounds and also in the total flavonoid concentration in propolis samples collected in different seasons. Antibacterial assays were performed by the method of dilution of an ethanol extract of propolis (EEP) in agar (v/v%) and showed that all 16 A. actinomycetemcomitans strains tested were inhibited by propolis concentrations of 0.1% to 0.25%, and did not grow at all at 0.5%. The growth inhibition of six Fusobacterium spp. and 16 black-pigmented anaerobes was observed at concentrations of 0.05% to 0.1%, and no growth was observed at 0.25%. There was no effect of seasonality on the inhibitory activity of propolis. The antibiotics tetracycline and meropenem were used as positive controls.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Fitoterapia , Própole/farmacologia , Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Brasil , Cromatografia Líquida de Alta Pressão , Fusobacterium/efeitos dos fármacos , Humanos , Medicina Tradicional , Meropeném , Testes de Sensibilidade Microbiana , Porphyromonas gingivalis/efeitos dos fármacos , Prevotella/efeitos dos fármacos , Própole/administração & dosagem , Própole/química , Própole/uso terapêutico , Estações do Ano , Tetraciclina/farmacologia , Tienamicinas/farmacologiaAssuntos
Proteínas do Citoesqueleto/genética , Glicoproteínas de Membrana/genética , Distrofias Musculares/genética , Adolescente , Adulto , Brasil , Criança , Pré-Escolar , Proteínas do Citoesqueleto/metabolismo , Distroglicanas , Distrofina/genética , Distrofina/metabolismo , Saúde da Família , Feminino , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Glicoproteínas de Membrana/metabolismo , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Mutação , Fenótipo , SarcoglicanasRESUMO
Specific clonal types of Actinobacillus actinomycetemcomitans, a major human periodontal pathogen, may be responsible for clinical manifestations and the production of leukotoxin virulence factors. Leukotoxicity is associated with genetic polymorphism at the promoter region of the leukotoxin (lItx) gene. Here, we describe the use of arbitrarily primed polymerase chain reaction (AP-PCR) and ltx promoter PCR to molecularly characterise 35 A. actinomycetemcomitans Brazilian isolates: 21 of human origin and 14 from captive marmosets (Callitrix spp., primates commonly used as animal models for periodontal research). The discriminative capacity of each of 12 arbitrary primers was found to be variable, yielding between 3 and 24 PCR amplitypes. Combination of the results for all primers led to characterisation of 14 genotypes that grouped into four major clusters based on genetic similarity. Clusters 2, 3, and 4 were discriminative to host origin. A correlation with periodontal disease was suggested for strains belonging to clusters 3 and 4. The JP2-like PCR amplification pattern, associated with highly leukotoxic strains, was exclusive to human isolates and present in 29% of human isolates where it occurred in close relationship with AP genotypes L and J (cluster 3).
Assuntos
Aggregatibacter actinomycetemcomitans/genética , Callithrix/microbiologia , Aggregatibacter actinomycetemcomitans/classificação , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Aggregatibacter actinomycetemcomitans/patogenicidade , Animais , Toxinas Bacterianas/genética , Sequência de Bases , Brasil , DNA Bacteriano/genética , Exotoxinas/genética , Genes Bacterianos , Variação Genética , Genótipo , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Virulência/genéticaRESUMO
Propolis collected from a cerrado area in Minas Gerais State, Brazil, was subjected to chromatography on silica gel column and to partition between immiscible solvents. Propolis aqueous-ethanolic extract and fractions obtained were tested for inhibitory activity against periodontitis-causing bacteria. All of the assayed bacterium species were susceptible to propolis extract. The two fractionation methodologies yielded fractions which were active against bacteria, with minimum inhibitory concentrations (MIC) ranging from 64 to 1024 microg/ml. TLC and HPLC analyses of the extract and of active fractions showed the presence of phenolic compounds of varied polarity. None of the assayed fractions was more active than the extract, suggesting that the antibacterial activity is probably due to the synergistic effect of several compounds.
Assuntos
Antibacterianos/farmacologia , Bactérias Anaeróbias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/microbiologia , Periodontite/microbiologia , Própole/farmacologia , Antibacterianos/química , Brasil , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Própole/química , Solventes/químicaRESUMO
The cell surface plays an important role in the interaction of parasites with their hosts. Drug resistance in the protozoan Leishmania may involve changes in cell-surface composition, although it is not known whether infectivity is also affected. One sensitive and two glucantime-resistant lines of Leishmania (Viannia) guyanensis previously isolated were inoculated into hamsters. The sensitive line caused the disease to manifest earlier than the resistant lines. Imprinting analyses of infected macrophages showed that the sensitive line was more infective than the resistant cell lines. In vitro drug resistance was evaluated and the comparative analyses of dose-response curves showed that the susceptibility pattern of the sensitive line did not change after passage in animals, but a decrease in drug resistance was observed in resistant cell lines recovered from the mammalian host. Cell surface carbohydrates of sensitive and resistant cell lines were analysed before and after passage in animals by agglutination tests with several plant lectins. Passage in animals changed the agglutination pattern for many lectins from all three cell lines. Loss of reactivity to lectins seemed to be correlated with a decrease in infectivity of the parasite-resistant cell lines. This study opens possibilities for exploring the relationship between drug susceptibility, infectivity and surface carbohydrate composition of protozoan parasites.
Assuntos
Antiprotozoários/farmacologia , Glicoesfingolipídeos/metabolismo , Leishmania guyanensis/efeitos dos fármacos , Leishmaniose Mucocutânea/parasitologia , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antimônio/farmacologia , Cricetinae , Modelos Animais de Doenças , Resistência a Medicamentos , Lectinas/metabolismo , Leishmania guyanensis/metabolismo , Leishmania guyanensis/patogenicidade , Leishmaniose Mucocutânea/metabolismo , Masculino , Antimoniato de Meglumina , Mesocricetus , Fatores de TempoRESUMO
We have performed association studies between a novel coding single nucleotide polymorphism (D104N) in endostatin, one of the most potent inhibitors of angiogenesis, and prostate cancer. We observed that heterozygous N104 individuals have a 2.5 times increased chance of developing prostate cancer as compared with homozygous D104 subjects (odds ratio, 2.4; 95% confidence interval, 1.4-4.16). Modeling of the endostatin mutant showed that the N104 protein is stable. These results together with the observation that residue 104 is evolutionary conserved lead us to propose that: (a) the DNA segment containing this residue might contain a novel interaction site to a yet unknown receptor; and (b) the presence of N104 impairs the function of endostatin.
Assuntos
Adenocarcinoma/genética , Inibidores da Angiogênese/genética , Colágeno/genética , Fragmentos de Peptídeos/genética , Polimorfismo Genético , Neoplasias da Próstata/genética , Idoso , Inibidores da Angiogênese/química , Inibidores da Angiogênese/fisiologia , Colágeno/química , Colágeno/fisiologia , Endostatinas , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD21 (19q13.3). We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency. In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B. Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients. Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle. Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin. Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (approximately 14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.
Assuntos
Proteínas de Membrana , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiopatologia , Distrofias Musculares/metabolismo , Adulto , Idade de Início , Proteínas de Ligação ao Cálcio , Calpaína/genética , Calpaína/metabolismo , Criança , Conectina , Disferlina , Distrofina/genética , Distrofina/metabolismo , Feminino , Ligação Genética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Músculo Esquelético/patologia , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Mutação , Polissacarídeos/genética , Polissacarídeos/metabolismoAssuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Glicoproteínas , Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , Proteínas de Protozoários , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Doença de Chagas/parasitologia , Resistência a Medicamentos , Amplificação de Genes , Humanos , Cariotipagem , Camundongos , Testes de Sensibilidade Parasitária , Polimorfismo de Fragmento de Restrição , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMO
AIMS: Characterization of yeast populations and genetic polymorphism of Saccharomyces cerevisiae strains collected during the short fermentative cycles from the spontaneous fermentations during the artisanal cachaça production. METHODS AND RESULTS: The prevalent S. cerevisiae strains were analysed by PFG and RAPD-PCR using primers EI1 and M13. The molecular analysis have showed a high degree of genetic polymorphism among the strains within a 24 h fermentative cycle. CONCLUSION: The genetic diversity observed in the S. cerevisiae strains may be occurring due to the existence of a large number of individual genotypes within the species. The unique characteristics of the cachaça fermentation process probably allows for a faster detection of molecular polymorphisms of yeast strains than other types of fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: Spontaneous fermentations to produce cachaça, due to their characteristics, are an excellent model for the study of molecular diversity of S. cerevisiae strains during the production of fermented beverages.
Assuntos
Bebidas Alcoólicas/microbiologia , Poaceae/microbiologia , Polimorfismo Genético/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Brasil , Fermentação , Genótipo , Técnicas de Tipagem Micológica , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Leveduras/genética , Leveduras/crescimento & desenvolvimento , Leveduras/isolamento & purificação , Leveduras/metabolismoRESUMO
In patients with sarcoglycan (SG) deficiency, a primary defect in any one of the four SG proteins usually leads to reduced expression of the whole SG complex. We report a limb-girdle muscular dystrophy type 2D family (LGMD2D), with variable phenotype, where a mutation in the alpha-SG gene resulted in the partial deficiency of alpha-SG alone. The normal expression of the other three SG proteins suggests that mutations close to the alpha-SG transmembrane domain might be less critical for complex integrity, and that weakness may occur despite its retention.
Assuntos
Proteínas do Citoesqueleto/genética , Distrofina/genética , Saúde da Família , Glicoproteínas/genética , Glicoproteínas de Membrana/genética , Distrofias Musculares/genética , Adulto , Biópsia , Distrofina/análise , Glicoproteínas/análise , Humanos , Masculino , Músculo Esquelético/química , Músculo Esquelético/patologia , Distrofias Musculares/patologia , Mutação , Núcleo Familiar , Linhagem , Polimorfismo Conformacional de Fita Simples , SarcoglicanasRESUMO
Autosomal recessive limb-girdle muscular dystrophies (AR LGMDs) are a genetically heterogeneous group of disorders that affect mainly the proximal musculature. There are eight genetically distinct forms of AR LGMD, LGMD 2A-H (refs 2-10), and the genetic lesions underlying these forms, except for LGMD 2G and 2H, have been identified. LGMD 2A and LGMD 2B are caused by mutations in the genes encoding calpain 3 (ref. 11) and dysferlin, respectively, and are usually associated with a mild phenotype. Mutations in the genes encoding gamma-(ref. 14), alpha-(ref. 5), beta-(refs 6,7) and delta (ref. 15)-sarcoglycans are responsible for LGMD 2C to 2F, respectively. Sarcoglycans, together with sarcospan, dystroglycans, syntrophins and dystrobrevin, constitute the dystrophin-glycoprotein complex (DGC). Patients with LGMD 2C-F predominantly have a severe clinical course. The LGMD 2G locus maps to a 3-cM interval in 17q11-12 in two Brazilian families with a relatively mild form of AR LGMD (ref. 9). To positionally clone the LGMD 2G gene, we constructed a physical map of the 17q11-12 region and refined its localization to an interval of 1.2 Mb. The gene encoding telethonin, a sarcomeric protein, lies within this candidate region. We have found that mutations in the telethonin gene cause LGMD 2G, identifying a new molecular mechanism for AR LGMD.
Assuntos
Cromossomos Humanos Par 17 , Proteínas Musculares/genética , Distrofias Musculares/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Conectina , Éxons , Feminino , Genes Recessivos , Marcadores Genéticos , Humanos , Íntrons , Masculino , Repetições de Microssatélites , Dados de Sequência Molecular , Proteínas Musculares/química , Distrofias Musculares/classificação , Núcleo Familiar , Linhagem , Regiões Promotoras Genéticas , Sarcômeros/genética , Sarcômeros/metabolismo , Alinhamento de SequênciaRESUMO
We demonstrated the existence of three transport activities in promastigotes of Leishmania braziliensis, Leishmania guyanensis, and Leishmania mexicana. The first activity, an energy-dependent efflux of pirarubicin, was observed in all Leishmania species and inhibited by verapamil, by 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-py ridinecarboxylate P oxide (PAK104P) and by the phenothiazine derivatives: thioridazine, prochlorperazine, trifluoperazine, chlorpromazine and trifluoropromazine. The second activity, an energy-dependent efflux of calcein acetoxymethylester, was observed in all Leishmania species and inhibited by PAK104P and the same phenothiazine derivatives, but not by verapamil. The third activity, an energy-dependent efflux of calcein, was clearly detected in L. braziliensis and guyanensis and inhibited only by prochlorperazine and trifluoperazine. The fact that prochlorperazine and trifluoperazine inhibited the energy-dependent efflux of the three substrates suggests that these activities are mediated by the same transport system. It is noteworthy that the transport system identified in this study shares several properties with the mammalian multidrug resistance pump, MRP1. Pirarubicin, calcein acetoxymethylester and calcein are well known substrates of the MRP. Furthermore, the three types of inhibitors are also inhibitors of the MRP function.
Assuntos
Doxorrubicina/análogos & derivados , Resistência a Múltiplos Medicamentos , Fluoresceínas/metabolismo , Leishmania/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Óxidos P-Cíclicos/farmacologia , Doxorrubicina/metabolismo , Metabolismo Energético , Corantes Fluorescentes/metabolismo , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Ácidos Nicotínicos/farmacologia , Fenotiazinas/farmacologia , Células Tumorais Cultivadas , Verapamil/farmacologiaRESUMO
Based on the pattern of distribution of the SG proteins in patients with LGMD2C and 2D, and on the observed decreased abundance of dystrophin through WB in some sarcoglycans (SG) patients, we have recently suggested that alpha, beta and delta subunits of sarcoglycan complex might be more closely associated and that gamma-SG might interact more directly with dystrophin. Two additional SG patients here reported give further support to these suggestions: an LGMD2F patient showed patchy labelling for gamma-SG, despite the lack of staining of the other three SG proteins; an LGMD2C boy showed deficiency in dystrophin by means of WB and IF, comparable with an DMD manifesting carrier. These two patients represent further evidence of a closer relation of alpha, beta and delta-SG than of gamma-SG and of the possible association of gamma-SG with dystrophin. In addition the LGMD2C patient illustrates the potential risk of misdiagnosis using only dystrophin analysis, in cases with no positive family history, or when DNA analysis is not informative.
Assuntos
Proteínas do Citoesqueleto/análise , Distrofina/análise , Glicoproteínas de Membrana/análise , Adolescente , Criança , Distroglicanas , Feminino , Humanos , Masculino , SarcoglicanasRESUMO
The autosomal recessive limb-girdle muscular dystrophies (AR-LGMDs) are a heterogeneous group of disorders of progressive weakness of the pelvic and shoulder girdle musculature. The clinical course is characterized by great variability, ranging from severe forms with onset in the first decade and rapid progression resembling clinically Xp21 Duchenne muscular dystrophy (DMD) to milder forms with later onset and slower course. Eight genes are mapped for the AR-LGMDs; they are: LGMD2A (CAPN3) at 15q, LGMD2B (dysferlin) at 2p, LGMD2C (gamma-SG) at 13q, LGMD2D (alpha-SG) at 17q, LGMD2E (beta-SG) at 4q, LGMD2F (6-SG) at 5q, LGMD2G at 17q, and more recently LGMD2H at 9q. The LGMD2F (delta-SG) and LGMD2G genes were mapped in Brazilian AR-LGMD families. Linkage analysis in two unlinked families excluded the eight AR-LGMD genes, indicating that there is at least one more gene responsible for AR-LGMD. We have analyzed 140 patients (from 40 families) affected with one of seven autosomal recessive LGMD loci, that is, from LGMD2A to LGMD2G. The main observations were: 1) all LGMD2E and LGMD2F patients had a severe condition, but considerable inter- and intra-familial clinical variability was observed among patients from all other groups; 2) serum CK activities showed the highest values in LGMD2D (alpha-SG) patients among sarcoglycanopathies and LGMD2B (dysferlin) patients among nonsarcoglycanopathies; 3) comparison between LGMD2A (CAPN3) and LGMD2B (dysferlin) showed that the first have on average a more severe course and have calf hypertrophy more frequently (86% versus 13%); and 4) inability to walk on toes was observed in approximately 70% of LGMD2B patients.