Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Avaliação do teste de imunofluorescência indireta para diagnóstico da filariose bancroftiana usando a microfilária de W. bancrofti como antígeno, em Recife-PE, Brasil

Analisou-se o teste de imunofluorescência indireta com microfilárias de W. bancrofti tratadas pela papaína, como antígeno, amplamente utilizado em Recife para o imunodiagnóstico da filariose linfática. Foram testados soros de 50 pacientes portadores das diversas formas clínicas da doença, incluindo microfilaremia assintomática, eosinofilia pulmonar tropical, elefantíase de membros inferiores, linfagite aguda e quilúria. Para o grupo controle, foram selecionados 50 indivíduos vivendo pelo menos há 5 anos em área endêmica, sem nenhuma evidencia clínica e/ou laboratorial da doença, constituindo os chamados endêmicos normais. A sensibilidade e especificidade do teste, segundo diferentes pontos de corte, mostraram a impossibilidade de diferenciação entre o grupo controle e o grupo sabidamente infectado. Também não foi possível estabelecer correlação entre os títulos encontrados e as diferentes formas clínicas. Foi considerada a existência de reações cruzadas relacionadas a helmintíases intestinais, porém nenhuma relação direta foi encontrada.

Meningococcal Disease Caused by Neisseria meningitidis Serogroup B Serotype 4 in São Paulo, Brazil, 1990 to 1996

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988.21 A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

Influência da escarificação e da temperatura sobre a germinação de sementes de Samanea tubulosa (Benth.) Barneby & J.W. Grimes (sete cascas)

Samanea tubulosa (Benth.) Barneby & J.W Grimes (sete cascas) é uma planta arbórea nativa do Pantanal Matogrossense, cujas sementes possuem dormência provavelmente causada pela impermeabilidade do tegumento à água. O presente trabalho foi conduzido com o objetivo de comparar a eficiência da escarificação mecânica e química para superar a dormência tegumentar e o efeito da temperatura sobre a germinação de sementes de sete cascas. Foram empregados quatro diferentes procedimentos de escarificação: testemunha (sem escarificação); a escarificação mecânica; a escarificação com ácido sulfúrico durante cinco minutos e a escarificação com ácido sulfúrico durante dez minutos. Os níveis de temperatura empregados foram de 20, 25, 30 e 35ºC. A semeadura foi realizada em papel toalha germitest, na forma de rolo. O delineamento experimental adotado foi o inteiramente casualizado com os tratamentos em esquema fatorial 4x4. Para cada tratamento, foram utilizadas quatro repetições de 25 sementes. Foi avaliada a porcentagem de germinação das sementes, utilizado como critério emissão da raiz primária com 2 mm de comprimento. Foi observado que a espécie S. tubulosa possui dormência tegumentar causada pela impermeabilidade do tegumento a água. As escarificações química com ácido sulfúrico por cinco e dez minutos foram eficientes para superação da dormência e as combinações de escarificação com temperatura que promoveram maiores porcentagens de germinação para a espécie foram a escarificação com imersão em ácido sulfúrico durante cinco e dez minutos e as temperaturas de 25, 30 e 35ºC.

Associação entre marcadores inflamatórios e fatores de risco cardiovascular em mulheres de Kolkata, W.B, Índia

FUNDAMENTO: Recentes pesquisas tem se concentrado no uso de biomarcadores inflamatórios na previsão de risco cardiovascular. Entretanto, a informação é escassa em relação à associação entre esses marcadores inflamatórios com outros fatores de risco cardiovasculares em indianos asiáticos, particularmente em mulheres. OBJETIVO: Explorar a associação entre marcadores inflamatórios tais como proteína C-reativa de alta sensibilidade (PCR-as) e contagem de leucócitos (LEU) e fatores de risco cardiovascular tais como adiposidade geral e central, pressão arterial, variáveis lipídicas e lipoproteicas e glicemia de jejum. MÉTODOS: Conduzimos uma análise transversal de 100 mulheres com idade entre 35-80 anos. As participantes foram selecionadas através da metodologia de amostragem por cluster, de 12 distritos urbanos selecionadas ao acaso na Corporação Municipal de Kolkata, Índia. RESULTADOS: A PCR-as apresentou uma associação significante com o índice de massa corporal (IMC) (p < 0,001) e circunferência da cintura (CC) (p = 0,002). Associações significantes inversas foram observadas entre a lipoproteína de alta densidade colesterol (HDL-c) e ambos marcadores inflamatórios PCR-as (p = 0,031) e LEU (p = 0,014). A apo-lipoproteína A1 (Apo A1) também estava negativamente associada com a PCR-as. A contagem de leucócitos apresentou uma correlação significante com a glicemia de jejum e a razão colesterol total (CT) /HDL-C. Usando regressão logística ajustada para idade, IMC (odds ratio/OR, 1,186; intervalo de confiança/IC, 1,046-1,345; p=0,008) e LEU (OR, 1,045; IC, 1,005-1,087; p=0,027) foram as covariantes significantemente associadas com a PCR-as. CONCLUSÃO: No presente estudo, os fatores de risco tais como IMC, CC e HDL-c e Apo-A1 mostraram uma associação significante com PCR-as. A contagem de leucócitos estava significantemente associada com os níveis de HDL-c, glicemia de jejum, razão CT/HDL-c em mulheres.

Physa Marmorata Guilding, 1828 (Pulmonata: Physidae)

A description of Physa marmorata Guilding, 1828, based on material collected at its type-locality, the Caribbean island of Saint Vincent, is presented. The shell is thin, horn-colored, surface very glossy, diaphanous. Spire acute, elevated; protoconch distinct, rounded-conical, reddish-brown; five not shouldered, broadly convex whorls with subobsolete spiral lines and thin growth lines. Aperture elongated, 1.4-2.0 times as long as the remaining shell length, narrow obovate-lunate; upper half acute-angled,lower half oval,narrowly rounded at the base, outer lip sharp, inner lip completely closing the umbilical region; a very distinct callus on the parietal wall; columellar lip with a low ridge gradually merging into the callus. ratios: shell width/shell length = 0.44 - 0.52 (mean 0.47); spire length /shell lenght = 0.33-0.41 (mean 0.39); aperture length/shell lenght = 0.59-0.67 (mean 0.62). Oral lappets laterally mucronate, foot spatulate with deeply pigmented acuminate tail. Mantle reflection with 6-10 short triangular dentations covering nearly half the right surface of the body whorl, and 4-6 covering a part of the ventral wall. Body surface with tiny dots of greenish-yellow pigment besides melanin. Renal tube tightly folded in toa zigzag course. Ovotestis diverticula acinous, laterally pressed against each other around a collecting canal. Ovispermiduct with well-developed seminal vesicle. oviduct highly convoluted, merging into a less convoluted nidamental gland which narrows to a funnel-shaped uterus and a short vagina. Spermathecal body oblong, more or less constricted in the middle and somewhat curved; spermathecal duct uniformly narrow, a little longer than be body. About 20 prostatic diverticula, simple, bifurcate or divided into a few short branches, distalmost ones assembled into a cluster. Penis long, nearly uniformly narrow; penial canal with lateral opening about the junction of its middle and lower thirds. Penial sheath with a bulbous terminal expasion the tip of which isinserted into the caudal end of the prepuce. Prepuce shouldered, much wider than the narrow portion of the penial sheath. Penial sheath/prepuce ratio about 2.08 (1.45-2.75). The main extrinsic muscles of the penial complex are a retractor, with a branch attached to the bulb, and another to the caudal end of the penial sheath; and a protractor, with a branch attached to the shoulder of the prepuce and adjoining area of the penial sheath, and another to the caudal end of the penial sheath. Egg capsule C-shaped, with 10-30 elliptical eggs (snails 10mm long) measuring about 1.10 mm (0.90-1.32) through the long axis and surrounded by an inner and an outer lamellate membranes. Jaw a simple obtusely V-shaped plate. radula will be described separately.

Physa cubensis Pfeiffer, 1839 (Pulmonata: Physidae)

A description of Physa cubensis Pfeiffer, 1839, based on 15 speciments collected in Havana, Cuba, is presented. The shell, measuring 9.0 x 4,8mm to 12.3 x 6.4mm, is ovate-oblong, thin, diaphanous, horncolored, shining. Spire elevated, broadly conical; protoconch distinct, roundish, reddish-brown. About five moderately shouldered, roundly convex whorls, penultimate whorl expanded; spiral striation subobsolete; growth line faint on the intermediate whorls, clearly visible on the body whorl, crowded here and there. Suture well impressed. Aperture elongated 2.05 - 2.67 (mean 2.27) times as long as the remaining length of the shell, narrow obovulate-lunate; upper half acute-angled, lower half oval, narrowly rounded at the base; outer lip sharp, inner lip completely closing the umbilical region; a thick callus on the parietal wall; columellar plait well marked. Ratios: shell width/shell length - 0.52-0.61 (mean 0.55); spire length/shell length = 0.27 - 0.33 (mean 0.31); aperture length/shell length = 0.67 - 0.73 (mean 0.69). Oral lappets laterally mucronate; foot spatulate with acuminate tail. Mantle relection with 6 - 8 short triangular dentations in the right lobe (columellar side) and 4 - 6 in the left lobe (near the pneumostome). Renal tube tightly folded into a zigzag course. Ovotestis, ovispermiduct, seminal vesicle, oviduct, nidamental gland, uterus and vagina as in Physa marmorata (see Paraense, 1986, Mem. Inst. Oswaldo Cruz, 81: 459-469). Spermathecal body egg-shaped or pear-shaped; spermathecal ducta uniformly narrow with expanded base, a little longer than the body. Spermiduct, prostate and vas deferens as in P. marmorata (Paraense, loc. cit.). Penis wide proximally, narrowing gradually apicad; penial canal with subterminal outlet. Penial sheath following the width of the penis and ending up by a bulbous expansion somewhat narrower than the proximal portion. Penaial sheath/prepuce ration = 1,25 - 1,83 (mean 1.49). Prepuce much wider than the bulb of the penial shealth, moderately shouldered owing to the intromission of the bulb, and with a large gland in one side of its proximal half occupating about a third of its length. Extrinsic muscles of the penial complex as in P. marmorata. Jaw a simple obtusely V-shaped plate. Radula to be described separetely.

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page). Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted.

Biomphalaria obstructa (Morelet, 1849): a study of topotypic specimens (Mollusca: pulmonata: planorbidae)

A description of Biomphalaria obstructa (Morelet, 1849), based on specimens collected at its type locality - isla del carmen, state of Campeche, Mexico - is presented. The Shell is small, 13 mm in diameter, 3.5 mm in width and with 5.75 whorls in the largest specimen, thin, moderately lustrous and translucent, horn-colored. Whorls increasing regularly (neither slowly nor rapidly) in diameter, rounded on the periphery side, bluntly angular on the left. Suture well-marked, deeper on the left. Right side widely concave, with first whorl deeply situated and partly hidden by the next. Left side shallower than right one, largely flattened, with first whorl plaintly visible. Aperture roundly heart-shaped, usually in the same plane as the body whorl but somewhat deflected to the left (less frequently to the right) in some specimens. Peristome sharp, seldom blunt; a distinct callus on the parietal wall. A number of young shells develop one set (seldom more) of apertural lamellae which tend to be resorbed as the shell grows. Absence of renal ridge. Ovotestis with about 70 mostly unbrached diverticula. Seminal vesicle beset with well-developed knoblike to fingerlike diverticula. Vaginal pouch more or less developed. Spermatheca club-shaped when empty, egg-shaped when full, and with intermediate forms between those extremes. Spermathecal body usually somewhat longer than the duct. Prostate with 7 to 20 (mean 12.06 ± 2.51) usually short diverticula which give off plumpish branches spreading out in a fan shape and overlapping to some extent their immediate neighbors. Foremost prostatic diverticulum nearly always partially or completely inserted between the spermathecal body and the uterine wall. Penial sheath consistently narrower and shorter than the prepuce. Muscular coat of the penis consisting of an inner longitudinal and an outer circular layers. Ratios between organ lengths: caudal to cephalic parts of female duct = 0.55 to 1.37 (mean 0.85 +- 0.17); cephalic parte of female duct to penial complex = 1.36 to 2.81 ((mean 1.90 +- 0.33); penial sheath to prepuce = 042 to 0.96 (mean 0.67 +- 0.13). Comparison with Morelet’s type specimens of Planorbis orbiculus and P. retusus points to the identity of those nominal species with B. obstructa.

Planorbis meridaensis Preston, 1907 (Gastropoda: Planorbidae), a synonym of Biomphalaria prona (Martens, 1873)

Biomphalaria prona from Lake Valencia, Venezuela (type locality) has a polymorphic shell wich in the great majority of specimens is wider, with fewer rapidly expanding whorls, the outer one subcarinate on the left side and more or less strongly deflected leftward. Besides those modal forms there are little frequent variants characterized by narrower shell with less rapidly expanding, regularly curved whorls directed forward. Recent studies have shown that such variants constitute the predominant shell phenotype in extralacustrine populations, but are anatomically and biochemically indistinguishable from the modal class of the Lake. In the present paper it is demonstrated that the nominal species Planorbis meridaensis Preston, 1907, from Mérida, Venezuela, is identical with B. prona (Martens, 1873) of wich it must be considered a junior synonym.

N-terminal amino acid sequences of the major outer membrane proteins from a Neisseria meningitidis group B strain isolated in Brazil

The four dominant outer membrane proteins (46, 38, 33 and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a semi-purified preparation of vesicle membranes of a Neisseria meningitidis (N44/89, B:4:P1.15:P5.5,7) strain isolated in Brazil. The N-terminal amino acid sequence for the 46 kDa and 28 kDa proteins matched that reported by others for class 1 and 5 proteins respectively, whereas the sequence (25 amino acids) for the 38 kDa (class 3) protein was similar to class 1 meningococcal proteins. The sequence for the 33 kDa (class 4) was unique and not homologous to any known protein.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 µg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.