Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by <Emphasis Type="Italic">Humicola grisea</Emphasis> var. <Emphasis Type="Italic">thermoidea</Emphasis> grown on sugarcane bagasse

The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively. The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-Dgalactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.     

Evolutionism and the Teaching of Science: How Portugal Has Been “Playing with the Big Tree of Evolution”

The concept of evolution is fundamental to the teaching of biological sciences. Nevertheless, it seems frequently neglected and/or forgotten in our classrooms and absent from the school syllabus. These difficulties are present today in the Portuguese educational system, especially concerning the issue of human evolution. To overcome this difficulty, a multidisciplinary pilot project entitled Playing with the Big Tree of Evolution was developed by a nonprofit association called Group of Studies in Human Evolution in Portuguese schools and in other public and private organizations. Combining non-formal and informal apprenticeship, the project is composed of a set of pedagogical and experimental activities that aim to promote the broad concept of human evolution as well as to demystify the anthropocentric perspective that places humans at the top of the chain of life.     

Fluorescence cross-correlation spectroscopy as a valuable tool to characterize cationic liposome-DNA nanoparticle assembly

The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross-correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)-DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.     

The Pivotal Role of 5-Lipoxygenase-Derived LTB4 in Controlling Pulmonary Paracoccidioidomycosis

Leukotrienes (LTs) produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. However, studies published in the literature regarding these mediators are contradictory and it remains uncertain whether these lipid mediators play a role in host defense against the fungal pathogen Paracoccidioides brasiliensis. To determine the involvement of LTs in the host response to pulmonary infection, wild-type and LT-deficient mice by targeted disruption of the 5-lipoxygenase gene (knockout mice) were studied following intratracheal challenge with P. brasiliensis yeasts. The results showed that infection is uniformly fatal in 5-LO-deficient mice and the mechanisms that account for this phenotype are an exacerbated lung injury and higher fungal pulmonary burden. Genetic ablation or pharmacological inhibition of LTs resulted in lower phagocytosis and fungicidal activity of macrophages in vitro, suggesting that deficiency in fungal clearance seems to be secondary to the absence of activation in 5-LO^−/− macrophages. Exogenous LTB₄ restored phagocytosis and fungicidal activity of 5-LO^−/− macrophages. Moreover, P. brasiliensis killing promoted by LTB₄ was dependent on nitric oxide (NO) production by macrophages. Taken together, these results reveal a fundamental role for 5-LO-derived LTB₄ in the protective response to P. brasiliensis infection and identify relevant mechanisms for the control of fungal infection during the early stages of the host immune response.     

Serum cytokines and CXCR2: potential tumour markers in ovarian neoplasms

Abstract

Purpose

The aim was to investigate the systemic levels of cytokines and the expression of the chemokine receptor CXCR2 in circulating neutrophils in patients with non-neoplastic ovarian lesions, benign neoplasia or malignant neoplasia.     

Phytoremediation: green technology for the removal of mixed contaminants of a water supply reservoir

The Iraí Reservoir, a water supply in Brazil, is constantly impacted by anthropogenic activities such as waste inputs from agriculture, hospitals and urbanization, resulting toxic cyanobacterial blooms causing economic, social and environmental problems. This study assessed the concentration of some common contaminants of the Iraí Reservoir, namely paracetamol, diclofenac and microcystin-LR and tested whether a laboratory scale Green Liver System^® would serve as a suitable technology to remove these contaminants. Further, the study investigated whether the pollutants caused adverse effects to the macrophytes using catalase as a biomarker for oxidative stress and investigated whether biotransformation (glutathione S-transferase) was a main route for detoxification. Egeria densa, Ceratophyllum demersum and Myriophyllum aquaticum were exposed to a mixture of the three contaminants for 14?days in a concentration range similar to those detected in the reservoir. The plants removed 93% of diclofenac and 100% of MC-LR after 14?days. Paracetamol could not be detected. Catalase and glutathione S-transferase enzyme activities remained unaltered after the 14-day exposure, indicating that the mixture did not cause oxidative stress. The study showed that the aquatic macrophytes used are suitable tools to apply in a Green Liver System^® for the remediation of mixed pollutants.     

Chitooligosaccharides enzymatic production by Metarhizium anisopliae

The products of chitosan hydrolysis are chitooligosaccharides and are used mainly for medical applications due to their specific biological activities. The objective of this study was to detect and identify the products of enzymatic hydrolysis of chitosan (dimers to hexamers) using a crude extract of chitosanolytic enzymes produced by the fungus Metarhizium anisopliae. These fungus was able to produce, during 48 h cultivation in a medium containing chitosan, chitooligosaccharides ranging from dimers, trimers, tetramers and pentamers at concentrations 0.2, 0.19, 0.06, 0.04 mg/mL, respectively, and the enzymatic activity was 2.5 U/L. Using the crude enzyme extract for chitosan hydrolysis, we detected the presence of dimers to hexamers at hydrolysis times of 10, 20, 30, 40, 50 and 60 min of enzymatic reaction, but the yields were higher at 10 min (54%). The hexamers was obtained only with 30 min of reaction with concentration of 0.004 mg/mL.     

CagA phosphorylation EPIYA-C motifs and the vacA i genotype in Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric diseases. Virulence factors such as VacA and CagA have been shown to increase the risk of these diseases. Studies have suggested a causal role of CagA EPIYA-C in gastric carcinogenesis and this factor has been shown to be geographically diverse. We investigated the number of CagA EPIYA motifs and the vacA i genotypes in H. pylori strains from asymptomatic children. We included samples from 40 infected children (18 females and 22 males), extracted DNA directly from the gastric mucus/juice (obtained using the string procedure) and analysed the DNA using polymerase chain reaction and DNA sequencing. The vacA i1 genotype was present in 30 (75%) samples, the i2 allele was present in nine (22.5%) samples and both alleles were present in one (2.5%) sample. The cagA-positive samples showed distinct patterns in the 3’ variable region of cagA and 18 of the 30 (60%) strains contained 1 EPIYA-C motif, whereas 12 (40%) strains contained two EPIYA-C motifs. We confirmed that the studied population was colonised early by the most virulent H. pylori strains, as demonstrated by the high frequency of the vacA i1 allele and the high number of EPIYA-C motifs. Therefore, asymptomatic children from an urban community in Fortaleza in northeastern Brazil are frequently colonised with the most virulent H. pylori strains.     

Nitrogen and phosphorus concentration of different structures of the aquatic macrophytes Eichhornia azurea Kunth and Scirpus cubensis Poepp & Kunth in relation to water level variation in Lagoa Infernão (São Paulo, Brazil)

We evaluated the nitrogen and phosphorus concentration of floating meadows of Eichhornia azurea and Scirpus cubensis in Lagoa do Infernao (State of Sao Paulo, Brazil). The chemical composition of these plants and of the water (littoral and limnetic zones) was determined during the period March 1987 to march 1988. During high water, the water becomes enriched by soluble nutrients and particulates washed from the flooded areas adjacent to the lake, especially from areas situated between the river and Lagoa do Infernão. The soluble allochthonous material is rapidly assimilated and stocked in the plant tissues, which results in an increase in its concentration. Besides this, it was established that the aquatic macrophytes studied utilize the nutrients dissolved in the water of the floating meadow itself, these nutrients being derived principally from their own decomposition.Since these are floating stands, the sediment does not function as a direct source of nutrients, and since concentrations in the water are low, the nutrients excreted by the aquatic macrophytes during growth and liberated during decomposition do not remain available in the water, but are rapidly absorbed by the young plants.This closed nutrient system makes possible the maintenance of the high biomass of the stands during the entire hydrological cycle.