Oxidative stress and diabetes in pregnant rats

A considerable amount of clinical and experimental evidence now exists suggesting the involvement of free radical-mediated oxidative processes in the pathogenesis of diabetic complications. If the diabetic state is associated with a generalized increase in oxidative stress, it might well be reflected in the alterations in embryonic and fetal development during pregnancy. In the present study, incidence of the malformed fetuses, biochemical parameters and antioxidant system activity of streptozotocin (STZ)-induced diabetic pregnant rats was investigated and the results obtained were compared with those of the control group (non-diabetic). Virgin female Wistar rats were injected with 40 mg/kg streptozotocin (STZ) before mating. All the females were killed on Day 21 of pregnancy and the fetuses were analyzed. A maternal blood sample was collected by venous puncture and the maternal liver was removed for biochemical measurement. The diabetic dams presented hyperglycemia, hyperlipemia, hypertriglyceridemia, hypercholesterolemia, hyperuricemia, decreased reduced glutathione (GSH), hepatic glycogen and superoxide dismutase (SOD) determinations. There was an increased incidence of skeletal and visceral malformation in fetuses from diabetic rats. Our findings suggest that oxidative stress occurs in the diabetic pregnant state, which might promote maternal homeostasis alterations. These diabetic complications might be a contributory factor to conceptus damage causing embryonic death (abortion/miscarriage) or the appearance of malformations in the fetuses of diabetic dams. Antioxidant treatment of women with diabetes may be important in future attempts to prevent congenital malformations.     

Frequency of Strongyloides stercoralis infection in alcoholics

Several studies have shown that chronic alcoholics have increased susceptibility to infections due to higher exposure to infectious agents as well as breakdown in their immune defenses. As Strongyloides stercoralis infection is usually more relevant in immunocompromised patients, the aim of this study was to evaluate the frequency of S. stercoralis infection in alcoholics. Thus, coproparasitological examination was carried out in 145 subjects, from which 45 were chronic alcoholics (mean age of 45.7 +/- 11.0 years), 10 were nonalcoholic cirrhotic patients (mean age of 50.2 +/- 13.1 years), and 90 were asymptomatic nonalcoholic subjects (mean age of 46.7 +/- 10.1 years), which served as controls. From the alcoholics, 9 had hepatic cirrhosis, 9 had chronic pancreatitis and 27 had neither cirrhosis nor pancreatitis. For the diagnosis of strongyloidiasis, the Baermann-Moraes and Lutz methods were used in three fecal samples from each subject. Samples were collected at alternated days, and three slides of each sample were analyzed for each method, thus totalizing 2,610 slides examined. The frequency of strongloidiasis in the total alcoholic group (33.3%) and in the subgroups of alcoholics, i.e., patients with hepatic cirrhosis (44.4%), with chronic pancreatitis (33.3%), and those with no cirrhosis or pancreatitis (29.6%) was statistically higher than that found in the control group (5.5%). None of the individuals with nonalcoholic hepatic cirrhosis had S. stercoralis infection. Our results showed that the chronic alcoholism itself is an important factor that predisposes to strongyloidiasis.     

Seasonal change in the proportion of bacterial and phytoplankton production along a salinity gradient in a shallow estuary

We intended to evaluate the relative contribution of primary production versus allochthonous carbon in the production of bacterial biomass in a mesotrophic estuary. Different spatial and temporal ranges were observed in the values of bacterioplankton biomass (31–273 g C l^–1) and production (0.1–16.0 g C l^–1 h^–1, 1.5–36.8 mg C m^–2 h^–1) as well as in phytoplankton abundance (50–1700 g C l^–1) and primary production (0.1–512.9 g C l^–1 h^–1, 1.5–512.9 mg C m^–2 h^–1). Bacterial specific growth rate (0.10–1.68 d^–1) during the year did not fluctuate as much as phytoplankton specific growth rate (0.02–0.74 d^–1). Along the salinity gradient and towards the inner estuary, bacterio- and phytoplankton biomass and production increased steadily both in the warm and cold seasons. The maximum geographical increase observed in these variables was 12 times more for the bacterial community and 8 times more for the phytoplankton community. The warm to cold season ratios of the biological variables varied geographically and according to these variables. The increase at the warm season achieved its maximum in the biomass production, particularly in the marine zone and at high tide (20 and 112 times higher in bacterial and phytoplankton production, respectively). The seasonal variation in specific growth rate was most noticeable in phytoplankton, with seasonal ratios of 3–26. The bacterial community of the marine zone responded positively – generating seasonal ratios of 1–13 in bacterial specific growth rate – to the strong warm season increment in phytoplankton growth rate in this zone. In the brackish water zone where even during the warm season allochthonous carbon accounted for 41% (on average) of the bacterial carbon demand, the seasonal ratio of bacterial specific growth rate varied from about 1 to 2. During the warm season, an average of 21% of the primary production was potentially sufficient to support the whole bacterial production. During the cold months, however, the total primary production would be either required or even insufficient to support bacterial production. The estuary turned then into a mostly heterotrophic system. However, the calculated annual production of biomass by bacterio- and phytoplankton in the whole ecosystem showed that auto- and heterotrophic production was balanced in this estuary.     

Nitric oxide synthase-mediated phytoalexin accumulation in soybean cotyledons in response to the Diaporthe phaseolorum f. sp. meridionalis elicitor

Phytoalexin biosynthesis is part of the defense mechanism of soybean (Glycine max) plants against attack by the fungus Diaporthe phaseolorum f. sp. meridionalis (Dpm), the causal agent of stem canker disease. The treatment of soybean cotyledons with Dpm elicitor or with sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in a high accumulation of phytoalexins. This response did not occur when SNP was replaced by ferricyanide, a structural analog of SNP devoid of the NO moiety. Phytoalexin accumulation induced by the fungal elicitor, but not by SNP, was prevented when cotyledons were pretreated with NO synthase (NOS) inhibitors. The Dpm elicitor also induced NOS activity in soybean tissues proximal to the site of inoculation. The induced NOS activity was Ca(2+)- and NADPH-dependent and was sensitive to the NOS inhibitors N(G)-nitro-L-arginine methyl ester, aminoguanidine, and L-N(6)-(iminoethyl) lysine. NOS activity was not observed in SNP-elicited tissues. An antibody to brain NOS labeled a 166-kD protein in elicited and nonelicited cotyledons. Isoflavones (daidzein and genistein), pterocarpans (glyceollins), and flavones (apigenin and luteolin) were identified after exposure to the elicitor or SNP, although the accumulation of glyceollins and apigenin was limited in SNP-elicited compared with fungal-elicited cotyledons. NOS activity preceded the accumulation of these flavonoids in tissues treated with the Dpm elicitor. The accumulation of these metabolites was faster in SNP-elicited than in fungal-elicited cotyledons. We conclude that the response of soybean cotyledons to Dpm elicitor involves NO formation via a constitutive NOS-like enzyme that triggers the biosynthesis of antimicrobial flavonoids.     

Melanophore appearance in wild and red tilapia embryos

Red tilapia has aroused interest in many countries for its commercial potential. This tilapia strain combines a desirable coloration and appearance with other advantageous farming characteristics. To study the early appearance of melanophore pigmentation in tilapia, a red tilapia strain originating from Thailand and a wild type coloration of Oreochromis niloticus were used as broodstock to produce artificially wild x wild and red x red progenies. The larvae were assessed periodically up to the first feeding and were recorded. Wild type fish showed a regular appearance of stellate melanophores. In the red strain, the pattern of chromatophores varies from total absence of black spotting to different degrees of macromelanophore distribution. Comparison between red and wild types showed that these two tilapia can be easily scored at day 7. Further, we present indications that the pigmentation over the body develops independently of the initial degree of pigmentation.     

Topographical analysis of Schizolobium parahyba chymotrypsin inhibitor (SPCI) by atomic force microscopy

Atomic Force Microscopy (AFM) has been a useful tool for molecular surface analysis and to estimate topographical properties of proteins. Here we report a topographical study of a chymotrypsin inhibitor from Schizolobium parahyba seeds (SPCI) by AFM. The underlying structure of SPCI oligomers has been resolved in nanometer order resolution. SPCI oligomerize in hexagonal, ellipsoid, comet, pyramidal, and "Z" shaped. The hexagonal was the most observed oligomer shape.     

Expression and purification of the recombinant ConBr (Canavalia brasiliensis lectin) produced in Escherichia coli cells

ConBr, a D-glucose/D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a (c)DNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.     

The role of smooth muscle in regulating prostatic induction

We have examined the role that smooth muscle plays during prostatic organogenesis and propose that differentiation of a smooth muscle layer regulates prostatic induction by controlling mesenchymal/epithelial interactions. During development of the rat reproductive tract, an area of condensed mesenchyme involved in prostatic organogenesis is formed. This mesenchyme (the ventral mesenchymal pad, VMP) is found in both males and females, yet only males develop a prostate. We demonstrate that a layer of smooth muscle differentiates between the VMP and the urethral epithelium, and that there is a sexually dimorphic difference in the development of this layer. Serial section reconstruction showed that the layer formed at approximately embryonic day 20.5 in females, but did not form in males. In cultures of female reproductive tracts, testosterone was able to regulate the thickness of this layer resulting in a 2.4-fold reduction in thickness. We observed that prostatic buds were present in some female reproductive tracts, and determined that testosterone was able to stimulate prostatic organogenesis, depending upon the bud position relative to the smooth muscle layer. In vitro recombination experiments demonstrated that direct contact with the VMP led to the induction of very few epithelial buds, and that androgens dramatically increased bud development. Taken together, our data suggest that differentiation of a smooth muscle layer regulates signalling between mesenchyme and epithelium, and comprises part of the mechanism regulating prostatic induction.     

Is bacterioplankton production in the Ria de Aveiro influenced by salt marshes and bed sediment?

The contribution of potential export of materials from bottom sedimentsand salt marshes into the water column of a shallow estuarine system of Ria deAveiro to the observed high bacterial productivity in the mid section of thisestuary was evaluated. Vertical profiles of physical, chemical and bacterialvariables were studied in the marine and brackish water zones, and oftransversal profiles in the brackish zone only. Although the concentrations ofseston (17–241 mg l^–1), particulate organiccarbon (3–15.5 mg l^–1) and chlorophyll(1.2–7.0 g l^–1) varied widely, thevertical and transversal profiles were without much variation. Total bacterialnumber (0.2–8.5 × 10⁹ cellsl^–1) and the number of particle-attached bacteria(0.02–2.50 × 10⁹ cellsl^–1)along vertical and transversal profiles did not differ much, but the rate ofbacterial production (0.05–14.2 g C l^–1h^–1) and dissolved organic carbon concentration(6.0–69.2 mg l^–1) were frequently highernear the salt marsh margin at the brackish water transect. The increase inproductivity could not be associated with runoff of particulate matter butcoincided with the inputs of dissolved organic carbon. The results of verticaland transversal profiles point to a minor role of particulate matter additionsfrom the salt marsh area or from bed sediments.     

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.