Can endometrial arylsulfatase A activity predict the onset of endometrial polyps over the years?

PURPOSE: To assess if arylsulfatase A activity (ASA) and sulfatide (SL) concentration in the human endometrium can be predictive of the development of endometrial polyps over the years, since ASA activity reflects the endometrial sensitivity to hormones. METHODS: ASA activity and SL concentration were determined by biochemical procedures on endometrial samples collected between 1990 and 1994 in non-menopausal women. These women underwent a new endometrial sampling following the clinical indication some years after the first endometrial sampling. The histological assessment of the second endometrial specimens found four patients with normal endometrial pattern and 10 patients with one or more endometrial polyps. ASA activity/years elapsed and SL concentration/years elapsed were compared using two tailed Mann-Whitney test for unpaired data between patients with normal pattern and patients with endometrial polyps. RESULTS: Median ASA activities were 2.62 (normal pattern) versus 1.85 (endometrial polyps) nmol hydrolized substrate/min. Median activity/years elapsed is higher in patients with second endometrial sample presenting normal pattern (p=0.006) and median SL concentration/years elapsed does not differ significantly among groups, even if median SL concentration seems to be higher in patients who subsequently developed polyps (1031 µg/g of fresh tissue versus 341,5 µg/g of fresh tissue). CONCLUSIONS: ASA activity can predict the onset of endometrial polyps over the years.

Enxofre disponível para a soja e atividade de arilsulfatase em solo tratado com gesso agrícola

O constante uso de fertilizantes que não contêm S, aliado ao baixo teor de matéria orgânica de alguns solos, pode resultar em limitação desse nutriente para as culturas. Graças à alta mobilidade do íon sulfato no perfil da maioria dos solos, é importante conhecer o efeito residual da aplicação do gesso agrícola feita com a finalidade de fornecimento de S, visando estimar sua freqüência de realização. em dois anos agrícolas, foram efetuados experimentos em campo para avaliar o efeito da aplicação de gesso agrícola nos teores de S-sulfato, S-reserva e atividade da arilsulfatase de um Latossolo Vermelho distrófico cultivado com soja, bem como nos teores de macronutrientes nas folhas e na produtividade de grãos da cultura. Os tratamentos consistiram na aplicação de: 0, 67, 133, 267, 533 e 1.067 kg ha-1 de gesso agrícola, em delineamento inteiramente casualizado, com quatro repetições. Realizaram-se amostragens de solo nas profundidades de 0-20 e 20-40 cm a cada 21 dias durante o ciclo da cultura, totalizando seis por ano. A primeira amostragem de cada ano foi feita antes da aplicação do gesso. No segundo ano, a distribuição dos tratamentos foi feita sobre as mesmas parcelas do ano anterior. O gesso agrícola aumentou os teores de S-sulfato no solo nas épocas próximas à aplicação, os quais diminuíram com o tempo, em decorrência da precipitação pluvial. No primeiro ano, o S-sulfato foi lixiviado para além da profundidade de 20-40 cm, sem efeito residual de um ano para o outro. A atividade da arilsulfatase foi maior a 0-20 cm, de forma semelhante ao observado para o S-reserva, havendo correlação positiva entre essas variáveis. A aplicação de gesso agrícola não influenciou a produção de grãos e, à exceção do teor de S no segundo ano, não alterou os teores de macronutrientes nas folhas de soja.

Biomassa microbiana e atividade enzimática em solos do Estado de São Paulo sob vegetação nativa e cultivados

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri

Epigenetic silencing of foreign genes introduced into plants poses an unsolved problem for transgenic technology. Here we have used the simple multicellular green alga Volvox carteri as a model to analyse the relation of DNA methylation to transgenic silencing. Volvox DNA contains on average 1.1% 5-methylcytosine and 0.3% N6-methyladenine, as revealed by electrospray mass spectrometry and phosphoimaging of chromatographically separated 32P-labelled nucleotides. In two nuclear transformants of V.carteri, produced in 1993 by biolistic bombardment with a foreign arylsulphatase gene (C-ars), the transgene is still expressed in one (Hill 181), but not in the other (Hill 183), after an estimated 500–1000 generations. Each transformant clone contains multiple intact copies of C-ars, most of them integrated into the genome as tandem repeats. When the bisulphite genomic sequencing protocol was applied to examine two select regions of transgenic C-ars, we found that the inactivated copies (Hill 183) exhibited a high-level methylation (40%) of CpG dinucleotides, whereas the active copies (Hill 181) displayed low-level (7%) CpG methylation. These are average values from 40 PCR clones sequenced from each DNA strand in the two portions of C-ars. The observed correlation of CpG methylation and transgene inactivation in a green alga will be discussed in the light of transcriptional silencing.

Renal dopamine and salt-retaining states

Although generally regarded as a neurotransmitter, dopamine is also known to be secreted by the kidney whereby it promotes sodium excretion in its role as a natriuretic honnone. Peripheral dopamine may be formed by two alternative pathways; the decarboxylation of circulating L-Dopa by L-aromatic amino acid decarboxylase (LAAAD), and the desulphation of dopamine sulphate by arylsulphatase A (ASA), the latter being poorly represented in the literature. In many conditions and diseases with which sodium retention is associated, a reduced urinary excretion of dopamine has been noted implicating the involvement of dopamine in the maintenance of sodium homeostasis.This study investigates renal dopamine production via the desulphation of dopamine sulphate in a sample cohort during normal unregulated dietary sodium intake and following a low sodium regimen. After dietary salt restriction urinary dopamine sulphate levels were significantly increased, indicating that dopamine sulphate is indeed a physiological reservoir of active free dopamine, the necessity for which is reduced during self depletion. This confirmed the dopamine/dopamine sulphate pathway as one which may be relevant to the maintenance of sodium homeostasis. The activity of urinary ASA was investigated in diabetes mellitus as an example of a sodium-retaining state, and compared with that in a matched normal control group. A decreased ASA activity was anticipated, given the blunted dopamine excretion observed in many sodium-retaining states, however an unexpected increase in activity in the diabetic group was observed. Enzyme kinetic analysis of ASA showed that this was not due to the existence of an isoform having an altered affinity for dopamine sulphate. This rather paradoxical situation, that urinary-dopamine is decreased while ASA activity is increased, may be explained by the sequestering of free dopamine by autoxidation to 6-hydroxydopamine as has been hypothesised recently to occur in diabetes mellitus. To confirm the homogeneity of ASA in the normal and diabetic groups, four amplicons spanning the 3637bp intronic and exonic regions of the gene were generated by PCR. These were sequence utilising a fluorescent-dye terminator reaction using the forward PCR primer as sequencing primer. Although single nucleotide polymorphisms were observed between the two groups these occurred either in intronic regions or, when exonic, generated silent mutations, supporting the enzyme kinetic data. The expression of ASA was investigated to determine the basis of the increased activity observed in diabetes mellitus. Although a validated comparative RT-PCR assay was developed for amplification of arsa transcripts from fresh blood samples, expression analysis from archived paraffin-embedded renal tissue was complicated by the low yield and degradation of unprotected mRNA. Suggestions for the development of this work using renal cell-culture are discussed.

Phenotypic and genotypic analysis of intestinal spirochacies

Pulsed field gel electrophoresis of 82 intestinal spirochaete isolates showed specific differentiation of Serpulina pilosicoli and Serpulina hyodysenteriae although considerable heterogeneity was observed, especially amongst S. pilosicoli isolates. In several cases genotypically similar isolates originated from different animals suggesting that cross-species transmission may have occurred. The Caco-2 and Caco-21HT29 cell models have been proposed as potentially realistic models of intestinal infection. Quantitation of adhesion to the cells showed isolate 3 82/91 (from a bacteraemia) to adhere at significantly greater numbers than any other isolate tested. This isolate produced a PFGE profile which differed from other S. pilosicoli isolates and so would be of interest for further study. Comparison of bacteraemic and other S. pilosicoli isolates suggested that bacteraemic isolates were not more specifically adapted for adhesion to, or invasion of the epithelial cell layer than other S. pilosicoli isolates. Genotypically similar isolates from differing animal origins adhered to the Caco-2 model at similar levels. Generation of a random genomic library of S. pilosicoli and screening with species specific monoclonal antibody has enabled the identification of a gene sequence encoding a protein which showed significant homology with an ancestral form of the enzyme pyruvate oxidoreductase. Immunoscreening with polyclonal serum identified the sequences of two gene clusters and a probable arylsulphatase. One gene cluster represented a ribosomal gene cluster which has a similar molecular arrangement to Borrelia burgdorjeri, Treponema pallidum and Thermatoga maritima. The other gene cluster contained an ABC transporter protein, sorbitol dehydrogenase and phosphomannose isomerase. An ELISA type assay was used to demonstrate that isolates of S. pilosicoli could adhere to components of the extracellular matrix such as collagen (type 1), fibronectin, laminin, and porcine gastric mucin.