Classificação do diabete melito

A correta classificação do diabete melito (DM) permite o tratamento mais adequado e compreende quatro categorias: DM tipo 1; DM tipo 2; Outros tipos e Diabete Gestacional. Em alguns casos, pode ocorrer sobreposição de quadros, principalmente no DM que inicia no adulto jovem ou que se apresenta inicialmente com cetoacidose, intermediários ao DM 1 e DM 2. Assim, acréscimos ao sistema de classificação clássico têm sido propostos, avaliando a presença de autoimunidade (anticorpos) e a função de célula β (peptídeo-C) para definir mais precisamente os subtipos. O objetivo desta revisão foi de analisar o desempenho desses índices diagnósticos para a classificação do DM e descrever os subtipos em detalhe. Os anticorpos contra o pâncreas evidenciam a autoimunidade, sendo o anticorpo contra insulina o mais acurado antes dos 5 anos de idade e o anti-descarboxilase do ácido glutâmico para início da doença acima dos 20 anos, é esse o teste que permanece positivo por mais tempo. Já a medida do peptídeo-C avalia a reserva pancreática de insulina, e os métodos de estímulo mais usados são a medida após refeição ou após glucagon endovenoso. Valores de peptídeo-C < 1,5 ng/ml definem o paciente com função pancreática ausente, e acima desse valor, com função preservada. Combinando-se a presença de anticorpos (A+) dirigidos ao pâncreas e a sua capacidade secretória de insulina (β+), pode-se subdividir a classificação do DM em tipo 1A (A+β-) e 1B (A+ β-); e o DM tipo 2 em subgrupos de DM 2A (A+β+) e DM 2B (A-β+), o que permite uma classificação e tratamento mais precisos, além de abrir os horizontes para o entendimento da patogênese do DM.

Algumas exigências metabólicas de Pseudomonas denitrificans

Para estudos da bioquímica da desnitrificação os desnitrificadores são cultivados em meio parcialmente sintético, com extrato de levedura ou de carne ou ainda peptona. Procurou-se então um meio de cultura completamente sintético no qual P. denitrificans pudesse desenvolver-se e fazer desnitrificação. Para tanto, células de um "strain" dessa bactéria foram crescidas durantes, 24 e 48 horas em tubos de ensaio contendo 10 ml de meio de cultura consistindo de succinato de sódio, nitrato de potássio, extrato de levedura o tampão de fosfato 1M, valôr pH 6,8. Dêsse meio (controle) 0,1 ml foi inoculado em 10 ml do meio de cultura em estudo, mantido então em condições parcialmente anaeróbicas. Após 24 horas uma alíquota foi retirada e suspensa em água destilada e a turbidês lida em espectrofotômetro Beckman, a 420 my. Os dados obtidos após longa série de ensaios permitiram concluir que a fim de se obter em 48 horas um crescimento da mesma ordem que o obtido com o meio controle, o meio de cultura sintético deve conter micronutrientes (Zn, Fe, Mn, Cu, Co, B e Mo, em EDTA), sulfato de sódio, sulfato de magnésio e ácido glutâmico, além de KNO3, succinato e tampão de fosfato.

Aminoácidos livres do feijão 60 dias (Phaseolus vulgaris L.)

Os aminoácidos livres da nova variedade de feijão (Phaseolus vulgaris L.) de 60 Dias, foram estudados por croma tografia em papel utilizando-se o seguinte par de solventes: 1) fenol-água, 8:2 (p/v); 2) butanol-ácido acético-água, 4:1:1 (v/v). Os aminoácidos identificados e dosados nos extratos foram: ácido aspártico, ácido glutãmico, lisina, serina, asparagina, glicina, treonina, alanina, glutamina, histidina, acido gama-aminobutírico, arginina, metionina, valina e leucina. Ás determinações quantitativas foram feitas por eluição das manchas dos cromatogramas seguidas de colorimetria. Os aminoácidos mais abundantes foram lisina, ácido glutâmico e ácido aspártico.

Ação de estimulantes vegetais no desenvolvimento de pepino (Cucumis sativus L.)

Observou-se o efeito de quatro estimulantes vegetais no desenvolvimento de plantas de pepino 'Hibrido Caipira AG-207', em condições de casa de vegetação, tendo a semeadura sido realizada em vasos de cerâmica e as plantas pulverizadas sete dias após a semeadura, com Triacontanol (1-hidroxitriacontano) na dosagem de 0,5 g/l, Ergostim (L-cisteina e ácido fólico + izometilentramina) 2 ml/ l, Atonik (mononitroguaiacol sódico e outros compostos nitrogenados aromáticos) 0,5 ml/l e Agrostemin (alantoina + triptofano +ácido fólico + ácido glutâmico + ácido alantóico + arcialanina + outros aminoácidos) 1,25 g/l. Através dos resultados obtidos verificou-se que nenhum dos estimulantes vegetais estudados promoveu aumento em altura das plantas de pepino, embora tenham mostrado uma tendência em provocar um aumento no peso da matéria seca.

Acth e enzimas na supra-renal

Estudou-se a atividade da ceruloplasmina, adenosina desaminase (AD) e transaminases glutâmico oxalacética (TGO) e glutâmico pirúvica (TGP) na supra-renal de ratos normais e injetados previamente (24 horas antes) com uma injeção única, por via intraperitoneal, de ACTH em doses de 0,5 e 1,2 un. internacionais. observou-se uma elevação estatisticamente significativa das TGO e ceruloplasmina enquanto a AD sofria diminuição sensível em sua atividade.

Nonhemocyte sources of selected lysosomal enzymes in Biomphalaria glabrata, B. tenagophila and B. straminea (Mollusca: Pulmonata)

The specific activities of acid phosphatase, alkaline phosphatase, β-glucuronidase, lysozymes, glutamate-oxalacetate transaminase and glutamate-pyruvate transaminate were determined in the head-foot and digestive gland of Brazilian Biomphalaria glabrata (Touros), B. tenagophila (Caçapava) and B. straminea (Monsenhor Gil). All six enzymes were detected inthe 3000g supernatant. Both cytoplasmic enzymes, glutamate-oxalacetate and glutamate-pyruvate transaminase exhibited the highest specific activities. In the case of the four hydrolytic enzymes assayed, β-glucuronidase exhibited the highest specific activity while lysozyme showed the lowest activity. All six enzymes are thought to be produced by cells within the head-foot and digestive gland of B. glabrata, B. tenagophila and B. straminea.

Bioactivity of neolignans from fructus Schizandrae

Fructus Schisandrae sinensis Baill, a traditional Chinese medicine, used as tonic and sedative, has been shown at the beginning of 70's to lower the elevated serum glutamic-pyruvic transaminase (SGPT) levels of patients suffering from chronic viral hepatitis. Durign past 20 years, a series of neolignans have been isolated and identified as effective principles. Pharmacological studies revealed that they increased liver protein and glicogen synthesis, antagonized liver injuries from CCl4 and thioacetamide. The mechanism of SGPT lowering was considered as a hepato-protective and membrane stabilize action, although inhibition of the activity of liver GPT may also be existed. It was found that some principles of Schisandrae have an inducing effect on hepatic microsomal drug-metabolizing enzynme system P-450, thus explained their anti-toxic, anti-carcinogenic and anti-mutagenic effects. A synthetic derivative compound of Schisandrin called DDB has most of the above mentioned actions now used widely in Chine as a hepato-protective drug with high effectiveness in normalizing liver functions and very low side effects. From Schisandrin to synthesized DDB, pointed out a successful way in the development of new drugs from natural products.

Aggregate cultures of foetal rat liver cells: development and maintenance of liver gene expression.

Rotation-mediated aggregate cultures of foetal rat liver cells were prepared and grown in a chemically defined medium. Their capacity for cellular organisation and maturation was studied over a culture period of 3 wk by using both morphologic and biochemical criteria. It was found that within each aggregate, distinct liver cell types were present and attained their normal, differentiated phenotype. Parenchymal cells formed small acini with a central lumen. Within the first 2 wk in culture, albumin and ferritin mRNA levels were maintained, while the alpha-fetoprotein mRNA levels decreased, and tyrosine aminotransferase (TAT) gene expression increased. No significant response to glucocorticoids was observed in early cultures, whereas after 3 wk a marked increase in TAT mRNA levels was elicited by dexamethasone and glucagon (additive stimulatory effects). The results show that foetal rat liver cells cultured in a chemically defined medium are able to rearrange themselves into histotypic structures, and display a developmental pattern of gene expression comparable to that of perinatal rat liver in vivo. This culture system offers therefore a useful model to study the development and function of liver cells.

Development of a coculture model of encapsulated cells.

In the whole animal, metabolic regulations are set by reciprocal interactions between various organs, via the blood circulation. At present, analyses of such interactions require numerous and uneasily controlled in vivo experiments. In a search for an alternative to in vivo experiments, our work aims at developing a coculture system in which different cell types are isolated in polymer capsules and grown in a common environment. The signals exchanged between cells from various origins are, thus, reproducing the in vivo intertissular communications. With this perspective, we evaluated a new encapsulation system as an artificial housing for liver cells on the one hand and adipocytes on the other hand. Murine hepatocytes were encapsulated with specially designed multicomponent capsules formed by polyelectrolyte complexation between sodium alginate, cellulose sulphate and poly(methylene-coguanidine) hydrochloride, of which the permeability has been characterized. We demonstrated the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Encapsulated hepatocytes retain their specific functions--transaminase activity, urea synthesis, and protein secretion--during the first four days of culture in minimum medium. Mature adipocytes, isolated from mouse epidydimal fat, were embedded in alginate beads. Measurement of protein secretion shows an identical profile between free and embedded adipocytes. We finally assessed the properties of encapsulated hepatocytes, cryopreserved over a periods of up to four months. The perspective of using encapsulated cells in coculture are discussed, since this system may represent a promising tool for fundamental research, such as analyses of drug metabolism, intercellular regulations, and metabolic pathways, as well as for the establishment of a tissue bank for storage and supply of murine hepatocytes.

Establishment of HEV RNA reverse transcriptase PCR assays for the diagnosis of acute and chronic hepatitis E

Background and aim: H epatitis E v irus (HEV) infection has emerged as a c ause o f travel-related a nd autochthonous a cute hepatitis as well as chronic hepatitis in immunosuppressed patients. While t ravel-related cases a re c aused primarily b y infections w ith HEV of g enotype 1 ( HEV-1), autochthonous c ases a nd chronic cases a re d ue t o genotype 3 (HEV-3), which is s hared between humans and diverse animal species. The aim of this study was to establish HEV RNA detection assays f or q uantitative v iral load testing and genotyping. Methods: V iral RNA was p urified from plasma or s erum a nd converted to cDNA prior to (1) multiplex real-time PCR for HEV RNA quantification and (2) multiplex PCR coupled to DNA sequencing for HEV genotype determination. Real-time PCR was d esigned to match a ll known HEV genotypes available i n Genbank while PCR was designed using conserved primers flanking a variable region of the HEV RNA. Results: In a validation panel, the newly developed assays allowed for the reliable detection and genotyping of HEV-1 or HEV-3. Cases of t ravel-related and a utochthonous a cute h epatitis E a s well a s chronic hepatitis E i n immunosuppressed patients have b een identified using t hese a ssays a nd will be p resented in detail. Anti- HEV antibodies were n egative i n three well-characterized patients with chronic hepatitis E after organ transplantation. Conclusions: We developed and validated a quantitative HEV RNA detection assay that c an now be o ffered on a r outine basis (www.chuv.ch/imul/imu-collaborations-viral_hepatitis). Genotyping can also be offered on selected cases. HEV RNA detection is key in diagnosing chronic hepatitis E i n immunosuppressed patients with unexplained transaminase elevations, as serology can be negative in these patients.

First record of human trichinosis in Chile associated with consumption of wild boar (Sus scrofa)

The first South American case of human trichinosis, resulting from the consumption of roast wild boar (Sus scrofa) is reported in Chile. The patient presented fever, diarrhea, myalgias, facial edema, sub-conjunctival reddening, photophobia, eosinophilia, and elevated glutamic oxalacetic transaminase. The diagnosis was confirmed by two immunoenzymatic tests (ELISA) using somatic and excretion-secretion antigens.

Morphological studies in a model for dengue-2 virus infection in mice

One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2). Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.

Enhanced production of indole-3-acetic acid by a genetically modified strain of Pseudomonas fluorescens CHA0 affects root growth of cucumber but does not improve protection of the plant against Pythium root rot

The biocontrol strain CHA0 of Pseudomonas fluorescens produces small amounts of indole-3-acetic acid via the tryptophan side chain oxidase and the tryptophan transaminase pathways. A recombinant plasmid (pME3468) expressing the tryptophan monooxygenase pathway was introduced into strain CHA0; this resulted in elevated synthesis of indole-3-acetic acid in vitro, especially after addition of -tryptophan. In natural soil, strain CHA0/pME3468 increased fresh root weight of cucumber by 17-36%, compared to the effect of strain CHA0; root colonization was about 106 cells per g of root. However, both strains gave similar protection of cucumber against Pythium ultimum. In autoclaved soil, at 6×107 cells per g of root, strain CHA0 stimulated growth of roots and shoots, whereas strain CHA0/pME3468 caused root stunting and strong reduction of plant weight. These results are in agreement with the known effects of exogenous indole-3-acetic acid on plant roots and suggest that in the system examined, indole-3-acetic acid does not contribute to the biocontrol properties of strain CHA0.

Candidate gene study of TRAIL and TRAIL receptors: association with response to interferon beta therapy in multiple sclerosis patients.

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10(-4), pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.