Horizontal reactor for the continuous production of ethanol by yeasts immobilized in pectin

Summary Yeast cells (Saccharomyces cerevisiae) were immobilized in pectin gel, incubated 12 h at 30°C and then used for the continuous production of ethanol employing a wedge-shaped horizontal reactor and sugar cane molasses as the carbon source. Under steady state conditions the mean residence time was 1.6 h and the volumetric productivity 40 g EtOH/hl. The gas evolved was easily released. Successive batch incubation in a synthetic medium substantially restored the fermentative capacity of the beads already used in the continuous assay.Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, México D.F.Member of the Scientific Researcher's Career of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

Glycosylation-independent Lysosomal Targeting of Acid α-Glucosidase Enhances Muscle Glycogen Clearance in Pompe Mice

We have used a peptide-based targeting system to improve lysosomal delivery of acid α-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients.     

Structural Determinants of HIV-1 Reverse Transcriptase Stereoselectivity Towards (β)-L-Deoxy- and Dideoxy-Pyrimidine Nucleoside Triphosphates: Molecular Basis for the Combination of L-Dideoxynucleoside Analogs with Non-nucleoside Inhibitors in Anti HIV Chemotherapy

Abstract

We have compared the HIV-1¹ RT mutants containing the single substitutions L100I, K103N, V106A, V179D, Y181I and Y188L, known to confere NNI-resistance in treated patients, to HIV-1 RT wt for their sensitivity towards inhibition by D- and L-deoxy- and dideoxy-nucleoside tiphosphates. The results showed a differential effect of the substitutions on the affinity for both D- and L-enantiomers of deoxy- and dideoxy-nucleoside triphosphates and provide a rationale for the utilization of L-dideoxynucleoside analogs with NNI in combination chemotherapy.     

The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD‐box RNA helicases are nucleolar‐ and chromocenter‐localized proteins that undergo stress‐mediated relocalization and are involved in epigenetic gene silencing

DEAD‐box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD‐box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up‐regulated stress‐responsive gene expression. Here, we show that Arabidopsis STRS‐overexpressing lines displayed a less tolerant phenotype and reduced expression of stress‐induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP–STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis‐localization in specific gene‐silencing mutants and exhibited RNA‐dependent ATPase and RNA‐unwinding activities. In particular, STRS2 showed mis‐localization in three out of four mutants of the RNA‐directed DNA methylation (RdDM) pathway while STRS1 was mis‐localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.     

Assessing unintended effects of a mammary-specific transgene at the whole animal level in host and non-target animals

Risk assessment in transgenic plants is intrinsically different than that for transgenic animals; however both require the verification of proper transgene function and in conjunction, an estimate of any unintended effects caused by expression of the transgene. This work was designed to gather data regarding methodologies to detect pleiotropic effects at the whole animal level using a line of transgenic goats that produce the antimicrobial protein human lysozyme (hLZ) in their milk with the goal of using the milk to treat childhood diarrhea. Metabolomics was used to determine the serum metabolite profile of both the host (lactating does) and non-target organism (kid goats raised on control or hLZ milk) prior to weaning (60 days), at weaning (90 days) and 1 month post-weaning (120 days). In addition, intestinal histology of the kid goats was also carried out. Histological analysis of intestinal segments of the pre-weaning group revealed significantly wider duodenal villi (p = 0.014) and significantly longer villi (p = 0.028) and deeper crypts (p = 0.030) in the ileum of kid goats consuming hLZ milk. Serum metabolomics was capable of detecting differences over time but revealed no significant differences in metabolites between control and hLZ fed kids after correction for false discovery rate. Serum metabolomics of control or hLZ lactating does showed only one significant difference in an unknown metabolite (q = 0.0422). The results as a whole indicate that consumption of hLZ milk results in positive or insignificant intestinal morphology and metabolic changes. This work contributes to the establishment of the safety and durability of the hLZ mammary-specific transgene.     

Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells

The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell’s genome is important to advance strategies for biotechnology and genetic medicine.     

Molecular characterization of c-Abl/c-Src kinase inhibitors targeted against murine tumour progenitor cells that express stem cell markers

Background

The non-receptor tyrosine kinases c-Abl and c-Src are overexpressed in various solid human tumours. Inhibition of their hyperactivity represents a molecular rationale in the combat of cancerous diseases. Here we examined the effects of a new family of pyrazolo [3,4-d] pyrimidines on a panel of 11 different murine lung tumour progenitor cell lines, that express stem cell markers, as well as on the human lung adenocarcinoma cell line A549, the human hepatoma cell line HepG2 and the human colon cancer cell line CaCo2 to obtain insight into the mode of action of these experimental drugs.

Methodology/Principal Findings

Treatment with the dual kinase inhibitors blocked c-Abl and c-Src kinase activity efficiently in the nanomolar range, induced apoptosis, reduced cell viability and caused cell cycle arrest predominantly at G0/G1 phase while western blot analysis confirmed repressed protein expression of c-Abl and c-Src as well as the interacting partners p38 mitogen activated protein kinase, heterogenous ribonucleoprotein K, cyclin dependent kinase 1 and further proteins that are crucial for tumour progression. Importantly, a significant repression of the epidermal growth factor receptor was observed while whole genome gene expression analysis evidenced regulation of many cell cycle regulated genes as well integrin and focal adhesion kinase (FAK) signalling to impact cytoskeleton dynamics, migration, invasion and metastasis.

Conclusions/Significance

Our experiments and recently published in vivo engraftment studies with various tumour cell lines revealed the dual kinase inhibitors to be efficient in their antitumour activity.