Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.     

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 is functional in Agrobacterium tumefaciens

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors β-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high β-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens.     

A new genus and species of Coenosiini from Costa Rica (Diptera,Muscidae, Coenosiinae)

Palpilongus gen. n. is herein described for one species – Palpilongus bifurcus sp. n., from Costa Rica, based on male and females. The striking morphological characters of the species – palpus very long, about as long as prementum; upper calypter truncate and very short and setae of male sternite 5 bifurcated, confirm that this new species is also a new genus in the tribe Coenosiini. Male and female terminalia were dissected and illustrated.     

Concentration by ultrafiltration and stabilization of phytase produced by solid-state fermentation

Ultrafiltration is an attractive downstream processing technique for concentrating enzymes and could be considered the primary step of purification. However, the efficiency of this process is often limited by protein fouling and shear-induced enzyme inactivation, which decreases permeate flux and results in the loss of enzyme activity. Although the rejection of phytase was higher than 99%, the loss of the enzyme activity was 14% during operation, indicating that the shear forces generated in the filter have significant influences on the enzyme activity. Two preparations using glycerol (25% and 35%, v/v) as a cryo-protecting agent at different temperatures were studied. The preparation containing 35% glycerol retained 70% of the initial enzyme activity at 70 °C after 1 h and had more than 3 and 6 months storage half-life at 29 °C and 4 °C, respectively.     

Inhibition of Mitochondrial Complex III Blocks Neuronal Differentiation and Maintains Embryonic Stem Cell Pluripotency

The mitochondrion is emerging as a key organelle in stem cell biology, acting as a regulator of stem cell pluripotency and differentiation. In this study we sought to understand the effect of mitochondrial complex III inhibition during neuronal differentiation of mouse embryonic stem cells. When exposed to antimycin A, a specific complex III inhibitor, embryonic stem cells failed to differentiate into dopaminergic neurons, maintaining high Oct4 levels even when subjected to a specific differentiation protocol. Mitochondrial inhibition affected distinct populations of cells present in culture, inducing cell loss in differentiated cells, but not inducing apoptosis in mouse embryonic stem cells. A reduction in overall proliferation rate was observed, corresponding to a slight arrest in S phase. Moreover, antimycin A treatment induced a consistent increase in HIF-1α protein levels. The present work demonstrates that mitochondrial metabolism is critical for neuronal differentiation and emphasizes that modulation of mitochondrial functions through pharmacological approaches can be useful in the context of controlling stem cell maintenance/differentiation.     

Predictors of Attrition and Immunological Failure in HIV-1 Patients on Highly Active Antiretroviral Therapy from Different Healthcare Settings in Mozambique

In Mozambique, the evaluation of retention in HIV care and ART programmes is limited. To assess rate and predictors of attrition (no retention in care) and HAART effectiveness in HIV-1 infected patients who pay for medication and laboratory testing in Mozambique, we conducted a multicenter survey of HIV-1-infected patients who started HAART during 2002–2006. Cox proportional hazard models were used to assess risk of attrition and of therapy failure. Overall, 142 patients from 16 healthcare centers located in the capital city Maputo were followed-up for 22.2 months (12.1–46.7). The retention rate was 75%, 48% and 37% after one, two and three years, respectively. Risk of attrition was lower in patients with higher baseline CD4 count (P = 0.022) and attending healthcare center 1 (HCC1) (P = 0.013). The proportion of individuals with CD4 count ≤200 cells/µL was 55% (78/142) at baseline and decreased to 6% (3/52) at 36 months. Among the patients with available VL, 86% (64/74) achieved undetectable VL levels. The rate of immunologic failure was 17.2% (95% CI: 12.6–22.9) per 100 person-years. Risk of failure was associated to higher baseline CD4 count (P = 0.002), likely reflecting low adherence levels, and decreased with baseline VL ≥10,000 copies/mL (P = 0.033). These results suggest that HAART can be effective in HIV-1 infected patients from Mozambique that pay for their medication and laboratory testing. Further studies are required to identify the causes for low retention rates in patients with low CD4 counts and to better understand the association between healthcare setting and attrition rate.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.