Yeast cytosine deaminase mutants with increased thermostability impart sensitivity to 5-fluorocytosine

Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a ‘suicide’ gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil, an inhibitor of DNA synthesis and RNA function. Over 150 studies of CD-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of CD are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study, we stabilized and extended the half-life of yeast CD (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated T_m values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in stability, as well as a more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models.     

pLR: A lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters

Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site) - reporter gene system. In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression.     

Taurine supplementation restores glucose and carbachol-induced insulin secretion in islets from low-protein diet rats: involvement of Ach-M3R, Synt 1 and SNAP-25 proteins

Isolated islets from low-protein (LP) diet rats showed decreased insulin secretion in response to glucose and carbachol (Cch). Taurine (TAU) increases insulin secretion in rodent islets with a positive effect upon the cholinergic pathway. Here, we investigated the effect of TAU administration upon glucose tolerance and insulin release in rats fed on a normal protein diet (17%) without (NP) or with 2.5% of TAU in their drinking water (NPT), and LP diet fed rats (6%) without (LP) or with TAU (LPT). Glucose tolerance was found to be higher in LP, compared to NP rats. However, plasma glucose levels, during ipGTT, in LPT rats were similar to those of controls. Isolated islets from LP rats secreted less insulin in response to increasing glucose concentrations (2.8-22.2 mmol/L) and to 100 μmol/L Cch. This lower secretion was accompanied by a reduction in Cch-induced internal Ca(2+) mobilization. TAU supplementation prevents these alterations, as judged by the higher secretion induced by glucose or Cch in LPT islets. In addition, Ach-M3R, syntaxin 1 and synaptosomal associated protein of 25 kDa protein expressions in LP were lower than in NP islets. The expressions of these proteins in LPT were normalized. Finally, the sarcoendoplasmatic reticulum Ca(2+)-ATPase 3 protein expression was higher in LPT and NPT, compared with controls. In conclusion, TAU supplementation to LP rats prevented alterations in glucose tolerance as well as in insulin secretion from isolated islets. The latter effect involves the normalization of the cholinergic pathway, associated with the preservation of exocytotic proteins.     

Evidence that Hyperprolinemia Alters Glutamatergic Homeostasis in Rat Brain: Neuroprotector Effect of Guanosine

This study investigated the effects of acute and chronic hyperprolinemia on glutamate uptake, as well as some mechanisms underlying the proline effects on glutamatergic system in rat cerebral cortex. The protective role of guanosine on effects mediated by proline was also evaluated. Results showed that acute and chronic hyperprolinemia reduced glutamate uptake, Na(+), K(+)-ATPase activity, ATP levels and increased lipoperoxidation. GLAST and GLT-1 immunocontent were increased in acute, but not in chronic hyperprolinemic rats. Our data suggest that the effects of proline on glutamate uptake may be mediated by lipid peroxidation and disruption of Na(+), K(+)-ATPase activity, but not by decreasing in glutamate transporters. This probably induces excitotoxicity and subsequent energy deficit. Guanosine was effective to prevent most of the effects promoted by proline, reinforcing its modulator role in counteracting the glutamate toxicity. However, further studies are needed to assess the modulatory effects of guanosine on experimental hyperprolinemia.     

Chaperone insufficiency links TLR4 protein signaling to endoplasmic reticulum stress

Inflammation plays an important pathogenic role in a number of metabolic diseases such as obesity, type 2 diabetes, and atherosclerosis. The activation of inflammation in these diseases depends at least in part on the combined actions of TLR4 signaling and endoplasmic reticulum stress, which by acting in concert can boost the inflammatory response. Defining the mechanisms involved in this phenomenon may unveil potential targets for the treatment of metabolic/inflammatory diseases. Here we used LPS to induce endoplasmic reticulum stress in the human monocyte cell-line, THP-1. The unfolded protein response, produced after LPS, was dependent on CD14 activity but not on RNA-dependent protein kinase and could be inhibited by an exogenous chemical chaperone. The induction of the endoplasmic reticulum resident chaperones, GRP94 and GRP78, by LPS was of a much lower magnitude than the effect of LPS on TLR4 and MD-2 expression. In face of this apparent insufficiency of chaperone expression, we induced the expression of GRP94 and GRP78 by glucose deprivation. This approach completely reverted endoplasmic reticulum stress. The inhibition of either GRP94 or GRP78 with siRNA was sufficient to rescue the protective effect of glucose deprivation on LPS-induced endoplasmic reticulum stress. Thus, insufficient LPS-induced chaperone expression links TLR4 signaling to endoplasmic reticulum stress.     

Ecto-5′-nucleotidase/CD73 inhibition by quercetin in the human U138MG glioma cell line

Gliomas are the most malignant of the primary brain tumors. Nucleotides represent an important class of extracellular molecules that are crucial for the normal function of the nervous system. ATP and adenosine can stimulate cell proliferation in different glioma cell lines; the events induced by extracellular adenine nucleotides are controlled by the action of ecto-nucleotidases, which hydrolyze ATP into adenosine in the extracellular space. Recent studies have shown that quercetin has an anti-proliferative effect on the U138MG glioma cell line. Since evidence suggests that purinergic signaling is involved in the growth and progression of glioma and, taking into consideration the anti-proliferative effect elicited by quercetin in this tumor type, the aim of the present study was to better investigate the extracellular metabolism of AMP and evaluate the effect of quercetin on this system in the human U138MG glioma cell line. The adenine products secreted by glioma cells were first characterized; extracellular AMP was efficiently metabolized by the glioma culture, demonstrating a very active ecto-5′-NT/CD73. Quercetin was able to inhibit the ecto-5′-NT/CD73 activity and modulate its expression. In addition, the cell treatment with APCP (α,β-methyleneadenosine-5′-diphosphate), an ecto-5′-NT/CD73 inhibitor, led to a significant reduction in glioma cell proliferation. We suggest that the inhibition of ecto-5′-NT/CD73 may result in a decrease in extracellular adenosine production with a consequent reduction in tumor progression.     

Bacterial biosurfactant increases ex situ biodiesel bioremediation in clayey soil

The contamination of soils by oily compounds has several environmental impacts, which can be reversed through bioremediation, using biosurfactants as auxiliaries in the biodegradation process. In this study, we aimed to perform ex situ bioremediation of biodiesel-contaminated soil using biosurfactants produced by Bacillus methylotrophicus. A crude biosurfactant was produced in a whey-based culture medium supplemented with nutrients and was later added to biodiesel-contaminated clayey soil. The produced lipopeptide biosurfactant could reduce the surface tension of the fermentation broth to 30.2 mN/m. An increase in the microbial population was observed in the contaminated soil; this finding can be corroborated by the finding of increased CO₂ release over days of bioremediation. Compared with natural attenuation, the addition of a lower concentration of the biosurfactant (0.5% w/w in relation to the mass of diesel oil) to the soil increased biodiesel removal by about 16% after 90 days. The added biosurfactant did not affect the retention of the contaminant in the soil, which is an important factor to be considered when applying in situ bioremediation technologies.

     

Molecular Phylogeny of Weakfish Species of the Stellifer Group (Sciaenidae,Perciformes) of the Western South Atlantic Based on Mitochondrial and Nuclear Data

The phylogenetic relationships within the Stellifer group of weakfishes (Stellifer, Odontoscion, Ophioscion, and Bairdiella) were evaluated using 2723 base pairs comprising sequences of nuclear (rhodopsin, TMO-4C4, RAG-1) and mitochondrial (16S rRNA and COI) markers obtained from specimens of nine species. Our results indicate a close relationship between Bairdiella and Odontoscion, and also that the genus Stellifer is not monophyletic, but rather that it consists of two distinct lineages, one clade containing S. microps/S. naso/S. brasiliensis and the other, S. rastrifer/S. stellifer/Stellifer sp. B, which is closer to Ophioscion than the former clade. The O. punctatissimus populations from the northern and southern Brazilian coast were also highly divergent in both nuclear (0.8% for rhodopsin and 0.9% for RAG-1) and mitochondrial sequences (2.2% for 16S rRNA and 7.3% for COI), which we conclude is consistent with the presence of two distinct species. The morphological similarities of the members of the Stellifer group is reinforced by the molecular data from both the present study and previous analyses, which have questioned the taxonomic status of the Stellifer group. If, on the one hand, the group is in fact composed of four genera (Stellifer, Ophioscion, Odontoscion, and Bairdiella), one of the two Stellifer clades should be reclassified as a new genus. However, if the close relationship and the reduced genetic divergence found within the group is confirmed in a more extensive study, including representatives of additional taxa, this, together with the morphological evidence, would support downgrading the whole group to a single genus. Obviously, these contradictory findings reinforce the need for a more systematic taxonomic revision of the Stellifer group as a whole.