Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean

Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.     

Synthesis of a new 5'-O-triphosphate analog of 5-methyl 2'-O-deoxycytidine. Preliminary in vitro labelling for non-radioactive detection of DNA

We report the synthesis of the triphosphate of 5-methyl 4-N-[6-(p-bromobenzamido)hex-1-yl]-2'-O-deoxycytidine 3A. We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-[n-[6-(p-bromobenzamido) caproyl amino]alk-1-yl]-2'-deoxycytidine compounds, and confirmed their presence by 1H-NMR studies. In vitro DNA labeling with modified nucleotides is preliminarily evaluated.     

The anti-HIV pentameric pseudopeptide HB-19 binds the C-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells

The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process.     

Fecundity of the Hypostomus affinis (Siluriformes,Loricariidae) in the Lajes Reservoir,Rio de Janeiro,Brazil

The armored catfish Hypostomus affinis is a widespread Loricariidae fish in the Lajes Reservoir (22 degrees 42'-22 degrees 50' S; 43 degrees 53'-44 degrees 05' W), the largest lentic environment in the Rio de Janeiro State, but little is known about their reproductive biology. One hundred and twenty five females, captured from January 1996 to December 1997, were used to assess ovarian development (29 were used to determine fecundity). The oocyte distribution by size-diameter classes revealed the occurrence of two modes suggesting an asynchronic development of the ovaries. The total fecundity ranged from 1235 to 4304, averaging 2374. An exponential relationship between fecundity and total length was determined, and a direct linear relationship was found between fecundity and total weight and gonad weight.     

Pancreatic hepatocytes in hamsters (Mesocricetus auratus) infected with Trypanosoma cruzi

Hepatocytic metaplasia may be induced in hamsters by carcinogens, and associated with aging, diabetes or chronic pancreatitis. By means of histopathologic and immunohistochemic studies, we observed pancreatic hepatocytes in hamsters infected and reinfected with Trypanosoma cruzi. The change was seen in 18 (19%) out of 94 infected animals, and was not found among 53 controls, Normal islet cells were immunoreactive for neuron-specific enolase and not reactive for NCL-HAS. Metaplastic cells were immunoreactive for NCL-HAS and not reactive for islet hormones and enolase. No relationship was observed between number of inoculations and metaplasia; however, the intensity of the inflammatory process and sequels seems to favor the development of metaplastic cells. Hamsters infected with T. cruzi may be useful to study hepatocytic metaplasia, and contribute to clarify aspects of Chagas' disease and pancreatic changes. Our data indicate that aging, in addition to inflammation and atrophy, plays a role in this change.     

Ethnicity and type 2 diabetes in Rio de Janeiro,Brazil, with a review of the prevalence of the disease in Amerindians

To what extent can ethnic factors contribute to the prevalence of type 2 diabetes and impaired glucose tolerance (IGT) in an urban Brazilian population? Conversely, how can environmental factors such as diet change these prevalences in a given ethnic group, in this case Brazilian Indians? To answer these questions estimates of ethnic admixture in Afro- and Euro-Brazilians from Rio de Janeiro, Brazil, were established using eight genetic systems and compared with the prevalences of these conditions obtained previously. This information was integrated with results obtained inside and outside of Brazil. The similarity of prevalences for type 2 diabetes and IGT in Afro- and Euro-Brazilians may be related to the extensive gene flow that occurred between them and to similar socioeconomic levels in the samples investigated. On the other hand, changes in the traditional diet are probably conditioning the appearance of diabetes among Brazilian and other South American Indians.     

Multiple virus resistance in transgenic plants conferred by the human dsRNA-dependent protein kinase

We have developed a new strategy for engineering resistance to multipleviruses in plants. The strategy exploits the human double stranded (ds)RNA-dependent protein kinase (PKR). PKR is one of theinterferon-induced enzymes. It confers viral resistance in mammals byinhibitingviral replication through the inactivation of the translational initiationfactor, eIF-2, upon activation by dsRNA. The humanPKR gene was fused to the promoter of theArabidopsis blue copper binding protein gene(BCB) that is induced rapidly in response to wounding. Thechimeric gene cassette was introduced into tobacco plants. Expression of thePKR gene in transgenic tobacco plants was demonstrated byRNA gel blot analysis and autophosphorylation assay of anM _r 68,000 protein. The transgenic plantsexpressing the PKR gene showed significantly reduced viralsymptoms or no viral symptoms at all, when challenged by different plant RNAviruses, such as Cucumber mosaic virus, Tobaccoetch virus, or Potato virus Y. Thus, expressionof a single component in the human interferon pathway, thePKR gene, can effectively confer resistance to multipleviruses in transgenic plants.     

Influence of a low background radiation environment on biochemical and biological responses in V79 cells

We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma-rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanità (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma-rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive.