Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Arbitrarily primed PCR fingerprinting of RNA and DNA in Entamoeba histolytica

Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Polymerase chain reaction and restriction fragment length polymorphism analysis of the ITS2 region for differatiation of Brazilian Biomphalaria intermediate hosts of the Schistosoma mansoni

We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the three Schistosoma mansoni intermediate hosts in Brazil: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. Analysis of a restriction map from those sequences allowed us to select putative restriction enzymes able to identify the snail species under study. Four restriction enzymes were used and HpaII provided simple species-specific profiles easily visualized in polyacrylamide gels. The use of ITS2 is advantageous as it provides a small fragment of 460 bp which may be easily amplified by PCR. In the current work, we showed that the amplification of ITS2-DNAr together with HpaII enzyme restriction is an auxiliary molecular tool for the morphological identification of such snails as well as for taxonomic and phylogenetic studies of neotropical planorbids.

Invasive aspergillosis in a user of inhaled cocaine: rhinosinusitis with bone and cartilage destruction

Aspergillosis is an infection caused by saprophytic fungi of the genus Aspergillus, which typically occurs in immunosuppressed individuals, but has also been reported in immunocompetent patients. The main routes of entry are the respiratory tract, skin, cornea, and ear, and the infection may be localized or disseminated by contiguity or vascular invasion. We report a severe case of rhinosinusitis with cutaneous involvement, caused by invasive aspergillosis, in an immunocompetent user of inhaled cocaine. Invasive aspergillosis related to cocaine abuse has not yet been reported in the literature. After itraconazole treatment and surgical debridement, complete clinical remission was achieved. Nasal reconstruction with a skin graft over a silicone prosthesis resulted in a satisfactory esthetic outcome.

Biology of amazonian mosquitoes. III. Esterase isozymes in Anopheles darlingi.

Six esterase isozymes were studied during the development of Anopheles darlingi by using polyacrylamide gel electrophoresis and two different substrates, a-naphthylcelate and a-naphthylpropionate. Esterases 5 and 6'were detected in all developmental stages esterases 1 and 2 were more intensively stained if larvae, while esterases 3 and 4 were better visualized in pupae and adults. Strong differences in intensity of some of the isozymes were observed during the pupal stage.Four out of the six isozymes showed variation in the electrophoretic mobility. Esterase-2 was choosed for genetic studies, because was the best stained isozyme in the gels. Two codominant alleles {Est2*S and Est2*F) code for this polymorphic system, with the Est*S frequency equal to 0.521. Phenotypic distribution is in agreement with hardy-Weinberg expectations.

Variação temporal de nutrientes na água escorrida pelo caule em floresta primária explorada no nordeste do Pará

Foi enfocada a contribuição de nutrientes da água escorrida pelo caule (CE), na ciclagem hidroquímica em floresta primária explorada, em Benevides, PA. A concentração de nutrientes na CE foi avaliada de dezembro de 1993 a abril de 1995, mediante coletores tipo colarinho, construídos com espuma de silicone, acoplados por tubos a recipientes plásticos. O monitoramento de CE se restringiu a doze árvores, nas quais foram coletadas mensalmente amostras para análise química de K+, Na+, Ca2+ e Mg2+ por espectrofotometria de absorção atômica, o N-NH4+, N-NO3 - e P-PO4 3- por colorimetria, em espectofotômetro de fluxo contínuo, o N-total por micro Kjeldahl e pH por potenciometria. A quantidade de nutrientes trazidos pela CE foi maior no início da época chuvosa, exibindo marcante variabilidade temporal para K+, Na+, Ca2+, Mg2+, N-total, SO4 2-, enquanto que o oposto aconteceu com PO4 3-. A magnitude na concentração dos nutrientes decresceu na seguinte ordem: K+ > Na+ > Ca2+ > N-t > SO4 2- > Mg2+ > PO4 3-. A distribuição e a intensidade de chuva não influenciam marcantemente o pH na CE.

Patologia do bloqueio atrioventricular na cardiomiopatia por depósito de desmina

Geralmente, a cardiomiopatia restritiva por deposição de desmina é caracterizada pela restrição ao enchimento diastólico ventricular e por diferentes graus de bloqueio atrioventricular (BAV). Neste relato, são descritas as alterações anatomopatológicas do sistema de condução cardíaco relacionadas ao BAV. O nó sinusal, o nó compacto e o feixe penetrante (feixe de His) não apresentavam anormalidades, entretanto, havia extensa fibrose das porções terminais do feixe ramificante e do início dos feixes esquerdo e direito, no topo do septo ventricular. A patogenia dessa substituição fibrosa é provavelmente a mesma que origina a extensa fibrose do miocárdio ventricular contrátil, e permanece por ser elucidada.

I diretriz de ressuscitação cardiopulmonar e cuidados cardiovasculares de emergência da Sociedade Brasileira de Cardiologia: resumo executivo

Apesar de avanços nos últimos anos relacionados à prevenção e a tratamento, muitas são as vidas perdidas anualmente no Brasil relacionado à parada cardíaca e a eventos cardiovasculares em geral. O Suporte Básico de Vida envolve o atendimento às emergências cardiovasculares principalmente em ambiente pré-hospitalar, enfatizando reconhecimento e realização precoces das manobras de ressuscitação cardiopulmonar com foco na realização de compressões torácicas de boa qualidade, assim como na rápida desfibrilação, por meio da implementação dos programas de acesso público à desfibrilação. Esses aspectos são de fundamental importância e podem fazer diferença no desfecho dos casos como sobrevida hospitalar sem sequelas neurológicas. O início precoce do Suporte Avançado de Vida em Cardiologia também possui papel essencial, mantendo, durante todo o atendimento, a qualidade das compressões torácicas, adequado manejo da via aérea, tratamento específico dos diferentes ritmos de parada, desfibrilação, avaliação e tratamento das possíveis causas. Mais recentemente dá-se ênfase a cuidados pós-ressuscitação, visando reduzir a mortalidade por meio do reconhecimento precoce e tratamento da síndrome pós-parada cardíaca. A hipotermia terapêutica tem demonstrado melhora significativa da lesão neurológica e deve ser realizada em indivíduos comatosos pós-parada cardíaca. Para os médicos que trabalham na emergência ou unidade de terapia intensiva é de grande importância o aperfeiçoamento no tratamento desses pacientes por meio de treinamentos específicos, possibilitando maiores chances de sucesso e maior sobrevida.

Random amplified polymorphic DNA analysis of DNA extracted from Trichuris trichiura (Linnaeus, 1771) eggs and its prospective application to paleoparasitological studies

Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.

Controle de Rhyzopertha dominica pela atmosfera controlada com CO2, em trigo

A utilização de gases inertes como fumigantes no controle de pragas é uma alternativa ao uso de fosfina. O objetivo deste trabalho foi avaliar a eficiência de uma atmosfera com CO2 no controle de Rhyzoperta dominica (Fabr.) (Coleoptera: Bostrichidae) em grãos de trigo armazenado. O trabalho constou de cinco concentrações de CO2 (0, 30 , 40, 50 e 60%, completadas com N2), três períodos de exposição (5, 10, 15 dias), três populações de R. dominica (Fabr.) (Coleoptera: Bostrichidae) (Campo Mourão, PR, Sete Lagoas, MG e Santa Rosa, RS) e sete fases de desenvolvimento do inseto (ovo, larva de 1º, 2º, 3º e 4º ínstar, pupa e adulto) com três repetições. As diferentes fases da R. dominica foram acondicionadas em tecido organza e levadas para câmaras de expurgo de 200 litros com 75% deste volume repletos de grãos. As câmaras foram vedadas com borracha de silicone para garantir a hermeticidade. Após a vedação das câmaras injetavam-se os gases contendo diferentes teores de CO2. Os resultados mostraram que todos os teores de CO2 causaram 100% de mortalidade de adultos das três populações nos três períodos de exposição utilizados. Em pupas a mortalidade atingiu 100% no teor de 60% de CO2 para as três populações no período de 15 dias de exposição; porém, todos os teores de CO2 utilizados no período de 15 dias de exposição causaram 100% de mortalidade das pupas da população de Santa Rosa. Para o adequado controle de larvas de diferentes ínstares são necessários teores de CO2 iguais ou acima de 50%. Nos períodos de 10 e 15 dias de exposição, todos os teores de CO2 causaram 100% de mortalidade dos ovos das três populações avaliadas.

Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins

In order to detect fluctuations in ruminal microbial populations due to forage tannins using 16S ribosomal RNA (rRNA) probes, recovery of intact rRNA is required. The objective of this work was to evaluate the effect of polyethylene glycol (PEG) and polyvinylpirrolidone (PVP) on extraction of bacterial rRNA, in the presence of tannins from tropical legume forages and other sources, that hybridize with oligonucleotide probes. Ruminococcus albus 8 cells were exposed to 8 g/L tannic acid or 1 g/L condensed tannins extracted from Acacia angustissima, banana (Musa sp.) skin, Desmodium ovalifolium, red grape (Vitis vinifera) skin and Inga edulis, or no tannins. Cells were rinsed with Tris buffer pH 7 containing either 8% PEG or 6% PVP prior to cell lysis. Total RNA samples rinsed with either PEG or PVP migrated through denaturing agarose gels. The 16S rRNA bands successfully hybridized with a R. albus species-specific oligonucleotide probe, regardless of tannin source. The effect of rinsing buffers on the density of 16S rRNA bands, as well as on the hybridization signals was compared. There were significant effects (P<0.01) when the controls were compared to either buffer treatments due to tannin type, buffer used and the interaction of tannin type and buffer. The significant interaction indicates the influence of tannin type on the parameters evaluated.

Subunit composition of seed storage proteins in high-protein soybean genotypes

The objective of this work was to quantify the accumulation of the major seed storage protein subunits, β-conglycinin and glycinin, and how they influence yield and protein and oil contents in high-protein soybean genotypes. The relative accumulation of subunits was calculated by scanning SDS-PAGE gels using densitometry. The protein content of the tested genotypes was higher than control cultivar in the same maturity group. Several genotypes with improved protein content and with unchanged yield or oil content were developed as a result of new breeding initiatives. This research confirmed that high-protein cultivars accumulate higher amounts of glycinin and β-conglycinin. Genotypes KO5427, KO5428, and KO5429, which accumulated lower quantities of all subunits of glycinin and β-conglycinin, were the only exceptions. Attention should be given to genotypes KO5314 and KO5317, which accumulated significantly higher amounts of both subunits of glycinin, and to genotypes KO5425, KO5319, KO539 and KO536, which accumulated significantly higher amounts of β-conglycinin subunits. These findings suggest that some of the tested genotypes could be beneficial in different breeding programs aimed at the production of agronomically viable plants, yielding high-protein seed with specific composition of storage proteins for specific food applications.

Molecular characterization of soybean cultivars by microsatellite markers with universal tail sequence

The objective of this work was to standardize a semiautomated method for genotyping soybean, based on universal tail sequence primers (UTSP), and to compare it with the conventional genotyping method that uses electrophoresis in polyacrylamide gels. Thirty soybean cultivars were genotypically characterized by both methods, using 13 microsatellite loci. For the UTSP method, the number of alleles (NA) was 50 (2-7 per marker) and the polymorphic information content (PIC) ranged from 0.40 to 0.74. For the conventional method, the NA was 38 (2-5 per marker) and the PIC varied from 0.39 to 0.67. The genetic dissimilarity matrices obtained by the two methods were highly correlated with each other (0.8026), and the formed groups were coherent with the phenotypic data used for varietal registration. The 13 markers allowed the distinction of all analyzed cultivars. The low cost of the UTSP method, associated with its high accuracy, makes it ideal for the characterization of soybean cultivars and for the determination of genetic purity.

Determinação por cromatografia gasosa de açúcares em frutíferas de clima temperado

As frutíferas de clima temperado apresentam o fenômeno da dormência. Na saída da dormência, há a conversão do amido para açúcares solúveis, como substrato para a retomada de crescimento na primavera. Visando à maior compreensão da fisiologia das plantas em respostas a eventos, como as variações climáticas, estresses e problemas de adaptação, desenvolveu-se este trabalho, no Laboratório de Fisiologia Vegetal da Embrapa Clima Temperado, com o objetivo de descrever uma metodologia para a determinação das concentrações dos açúcares solúveis (frutose, sorbitol, alfa-glicose, beta-glicose e sacarose), em tecidos vegetais de frutíferas, via cromatografia gasosa. O cromatógrafo utilizado para as análises dos açúcares por essa metodologia é o GAS CHROMATOGRAPH e a coluna do tipo Packed Column J. K. de 3,2mm de diâmetro por 2m de comprimento, empacotada com Silicone SE-52 Uniport HP 80/100 mesh. Através da cromatografia gasosa, obtêm-se eficiência e resolução cromatográfica, para análises de açúcares solúveis, sendo, desta forma, vantajoso e executável esse tipo de análise pelo método descrito.