Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (<Emphasis Type="Italic">Ct</Emphasis>CBM11) of cellulosomal bifunctional cellulase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.     

Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography

A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K _m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN₃, NaF, and HgCl₂.     

SEC14-like protein 1 interacts with cholinergic transporters

Trafficking of the vesicular acetylcholine transporter (VAChT) to synaptic vesicles has the potential to regulate storage and release of acetylcholine. We used the C-terminal tail of the vesicular acetylcholine transporter as bait for the screening of a brain cDNA library by yeast-two hybrids. Here we report an interaction uncovered in this screening with SEC14L1, a mammalian SEC14-like protein that may function as a phospholipid transfer protein. The interaction of VAChT and SEC14L1 occurred through the GOLD domain found in the latter and was confirmed in mammalian cells. In addition, we also found that SEC14L1 co-immunoprecipitates with the high affinity choline transporter (CHT1), but not with synaptophysin or synaptotagmin. In cultured cells SEC14L1 was predominantly found in the cytosol with little or no localization in defined organelles. In contrast, overexpression of VAChT or CHT1 with SEC14L1 recruited the latter to large intracellular organelles similar to vesicles or vesicle aggregates. Finally, we find that overexpression of SEC14L1 modestly decreases high affinity choline transport activity. We suggest that interaction of cholinergic transporters with proteins containing the GOLD domain may be relevant for transporter function.     

Reduction of heavy metal contents in liquid effluents by vermicomposts and the use of the metal-enriched vermicomposts in lettuce cultivation

The removal of Cu, Ni and Zn from electroplating effluents by adsorption in cattle manure vermicompost has been discussed. A glass column 38 cm long and 7 cm i.d. was loaded with cattle manure vermicompost and effluents were passed through it. The metal concentrations were measured in the elutant. The experiments on adding effluent aliquots into the columns were continued until the metal concentrations in the elutant reached the maximum values established for effluent discharges in water courses by the Brazilian quality criteria, i.e., Cu=1.0 mg L(-1), Ni=2.0 mg L(-1), and Zn=5.0 mg L(-1). The amount of Cu retention by the vermicompost was determined at the natural effluent pH (2.0). The Zn and Ni retentions were evaluated at the natural effluent pH (6.9 and 7.4, respectively) as well pH 2.0. Vermicompost residues obtained from this process were used for lettuce cultivation. The vermicompost was found to be efficient in removing metals from the electroplating wastes, as well as in the increase of its pH values. Metal retention values were close to 100%. The Cu concentrations in lettuce leaves from the treatment with vermicompost enriched with this metal were below the range of critical toxicity level to plants, i.e., from 20 to 100 mg L(-1). However, the estimated Cu concentrations in the roots from the treatment with vermicompost enriched with Cu were much larger than that of the treatment with the natural vermicompost, reaching 246.3 mg L(-1). The Ni and Zn concentrations in lettuce leaves from the treatments, with vermicomposts enriched with the respective metals, were above the range of critical toxicity levels to plants, i.e., from 10 to 50 mg kg(-1) and from 15 to 30 mg kg(-1), respectively. However, no symptom of toxicity was found visually. Larger accumulations of Cu, Ni and Zn were found in the lettuce leaves than in the roots after the treatments with the uncontaminated vermicompost. A greater absorption of Cu and Ni by roots was found in treatments with vermicompost enriched with these elements, whereas Zn was found preferentially in the leaves. The statistical analysis was done by analyses of variance and regression.     

Length–weight relationships of four fish species from the Curimatidae family,Patos‐Mirim system,southern Brazil

Determined were the length–weight relationships (LWRs) of four fish species of the Curimatidae family from the Patos‐Mirim lagoon system. Estimates for three of the species are reported here for the first time.     

BMI and Metabolic Profile in Patients With Prolactinoma Before and After Treatment With Dopamine Agonists

Hyperprolactinemia might be related to weight gain, metabolic syndrome (MS), and insulin resistance (IR). Treatment with dopamine agonist (DA) has been shown to reduce body weight and improve metabolic parameters. The objectives of this study were to determine the prevalence of obesity, overweight, MS, and IR in patients with prolactinoma before and after therapy with DA and to evaluate the relation between prolactin (PRL), body weight, fat distribution, leptin levels, IR, and lipid profile before treatment. In addition, we investigated the correlation of the reduction in PRL levels with weight loss and metabolic profile improvement. Twenty‐two patients with prolactinoma completed 6 months of treatment with DA. These patients were submitted to clinical (BMI, waist circumference, blood pressure (BP)), laboratory evaluation (leptin, glucose, low‐density lipoprotein (LDL)‐cholesterol, and triglyceride (TG) levels) and abdominal computed tomography (CT) before and after treatment. The statistical analyses were done by nonparametric tests. At the beginning of the study, the prevalence of obesity, overweight, MS, and IR was 45, 27, 27, and 18%, respectively. After 6 months of treatment with DA, PRL levels normalized, but no significant difference in BMI was observed. However, there was a significant decrease on homeostasis model assessment of insulin resistance (HOMA_IR) index, glucose, LDL‐cholesterol, and TG levels. This study suggests a possible involvement of prolactinoma on the prevalence of obesity. We should consider that DA may be effective on improving metabolic parameters, and we speculate that a period longer than 6 months of treatment is necessary to conclude whether this drug can interfere in the body weight of patients with prolactinoma.     

A method for testing the host specificity of ectoparasites: give them the opportunity to choose

Host-choice experiments were carried out with rodent and bat ectoparasites on Ilha Grande, state of Rio de Janeiro, Brazil. We constructed experimental chambers that enclosed three different rodent or bat host species, and then introduced a selected set of ectoparasitic arthropods. When given the opportunity to choose among host species, the ectoparasites showed a strong tendency to select their primary hosts, and reject novel host species. These kinds of simple experiments can be valuable tools for assessing the ability of ectoparasites to locate and discern differences between host species, and make choices about which hosts to infest, and which hosts to avoid.     

Expression of Sara2 human gene in erythroid progenitors

A human homologue of Sar1, named Sara2, was shown to be preferentially expressed during erythropoiesis in a culture stimulated by EPO. Previous studies, in yeast, have shown that secretion-associated and Ras-related protein (Sar1p) plays an essential role in protein transport from the endoplasmic reticulum to the Golgi apparatus. Here, we report the molecular analysis of Sara2 in erythroid cell culture. A 1250 bp long cDNA, encoding a 198 amino-acid protein very similar to Sar1 proteins from other organisms, was obtained. Furthermore, we also report a functional study of Sara2 with Real-time quantitative PCR analysis, demonstrating that expression of Sara2 mRNA increases during the initial stages of erythroid differentiation with EPO and that a two-fold increase in expression occurs following the addition of hydroxyurea (HU). In K562 cells, Sara2 mRNA was observed to have a constant expression and the addition of HU also up-regulated the expression in these cells. Our results suggest that Sara2 is an important gene in processes involving proliferation and differentiation and could be valuable for understanding the vesicular transport system during erythropoiesis.