Hepatitis C virus NS5A binds to the mRNA cap-binding eukaryotic translation initiation 4F (eIF4F) complex and up-regulates host translation initiation machinery through eIF4E-binding protein 1 inactivation

Initiation, a major rate-limiting step of host protein translation, is a critical target in many viral infections. Chronic hepatitis C virus (HCV) infection results in hepatocellular carcinoma. Translation initiation, up-regulated in many cancers, plays a critical role in tumorigenesis. mTOR is a major regulator of host protein translation. Even though activation of PI3K-AKT-mTOR by HCV non-structural protein 5A (NS5A) is known, not much is understood about the regulation of host translation initiation by this virus. Here for the first time we show that HCV up-regulates host cap-dependent translation machinery in Huh7.5 cells through simultaneous activation of mTORC1 and eukaryotic translation initiation factor 4E (eIF4E) by NS5A. NS5A, interestingly, overexpressed and subsequently hyperphosphorylated 4EBP1. NS5A phosphorylated eIF4E through the p38 MAPK-MNK pathway. Both HCV infection and NS5A expression augmented eIF4F complex assembly, an indicator of cap-dependent translation efficiency. Global translation, however, was not altered by HCV NS5A. 4EBP1 phosphorylation, but not that of S6K1, was uniquely resistant to rapamycin in NS5A-Huh7.5 cells, indicative of an alternate phosphorylation mechanism of 4EBP1. Resistance of Ser-473, but not Thr-308, phosphorylation of AKT to PI3K inhibitors suggested an activation of mTORC2 by NS5A. NS5A associated with eIF4F complex and polysomes, suggesting its active involvement in host translation. This is the first report that implicates an HCV protein in the up-regulation of host translation initiation apparatus through concomitant regulation of multiple pathways. Because both mTORC1 activation and eIF4E phosphorylation are involved in tumorigenesis, we propose that their simultaneous activation by NS5A might contribute significantly to the development of hepatocellular carcinoma.     

Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration

With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use.     

A versatile and efficient gene-targeting system for Aspergillus nidulans

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.     

Role of antigen-processing machinery in the in vitro resistance of squamous cell carcinoma of the head and neck cells to recognition by CTL

Squamous cell carcinoma of the head and neck (SCCHN) cells are poorly recognized in vitro by CTL despite expressing the restricting HLA class I allele and the targeted tumor Ag (TA). Several lines of evidence indicate that the lack of SCCHN cell recognition by CTL reflects defects in targeted TA peptide presentation by HLA class I Ag to CTL because of Ag-processing machinery (APM) dysfunction. First, lack of recognition of SCCHN cells by CTL is associated with marked down-regulation of the IFN-gamma-inducible APM components low-m.w. protein 2, TAP1, TAP2, and tapasin. Second, SCCHN cell recognition by CTL is restored by pulsing cells with exogenous targeted TA peptide. Third, the restoration of CTL recognition following incubation of SCCHN cells with IFN-gamma is associated with a significant (p = 0.001) up-regulation of the APM components TAP1, TAP2, and tapasin. Lastly, and most conclusively, SCCHN cell recognition by CTL is restored by transfection with wild-type TAP1 cDNA. Our findings may explain the association between APM component down-regulation and poor clinical course of the disease in SCCHN. Furthermore, the regulatory nature of the APM defects in SCCHN cells suggests that intralesional administration of IFN-gamma may have a beneficial effect on the clinical course of the disease and on T cell-based immunotherapy of SCCHN by restoring SCCHN cell recognition by CTL.     

Light-Dependent Transformation of Aniline to Indole Esters by the Purple Bacterium Rhodobacter sphaeroides OU5

In an attempt to understand the aromatic hydrocarbon metabolism by purple bacteria that do not grow at their expense, we earlier reported 2-aminobenzoate transformation by a purple non-sulfur bacterium, Rhodobacter sphaeroides OU5 (Sunayana et al., 2005, J Ind Microbiol Biotech 32:41–45), which is extended in the present study with aniline, a major environmental pollutant. Aniline did not support photo (light anaerobic) or chemo (dark aerobic) heterotrophic growth of Rhodobacter sphaeroides OU5 either as a sole source of carbon or nitrogen. However, light-dependent aniline transformation was observed in the culture supernatants and the products were identified as indole derivatives. The transformation was dependent on a tricarboxylate intermediate, fumarate. Five intermediates of the aniline biotransformation pathway were isolated and identified as indole esters having a mass of 443, 441, 279, 189, and 167 with unstoichiometric total indole yields of 0.16 mM from 5 mM of aniline consumed. The pathway proposed based on these intermediates suggest a novel xenobiotic detoxification process in bacteria.     

Thiosulfate Oxidation by <Emphasis Type="Italic">Comamonas</Emphasis> sp. S23 Isolated from a Sulfur Spring

A bacterial isolate S23 capable of oxidizing thiosulfate was isolated from a sulfur spring. Strain S23 is gram-negative, aerobic, and motile. The G + C content of DNA is 61.4 mol%. The fatty acid composition and phylogenetic analysis of the 16S rRNA gene sequence of strain S23 showed that it is related to the members of the genus Comamonas, and most closely related to Comamonas testosteroni (99.9% sequence similarity). The isolate S23 exhibited thiosulfate oxidation under a mixotrophic growth condition. Polymerase chain reaction (PCR) using soxB-specific primers and DNA sequencing showed the presence of the soxB gene. This is the first report in Comamonas sp. showing thiosulfate oxidation under a mixotrophic growth condition.     

Osmolyte controlled fibrillation kinetics of insulin: New insight into fibrillation using the preferential exclusion principle

Amyloid proteins are converted from their native‐fold to long β‐sheet‐rich fibrils in a typical sigmoidal time‐dependent protein aggregation curve. This reaction process from monomer or dimer to oligomer to nuclei and then to fibrils is the subject of intense study. The main results of this work are based on the use of a well‐studied model amyloid protein, insulin, which has been used in vitro by others. Nine osmolyte molecules, added during the protein aggregation process for the production of amyloid fibrils, slow‐down or speed up the process depending on the molecular structure of each osmolyte. Of these, all stabilizing osmolytes (sugars) slow down the aggregation process in the following order: tri > di > monosaccharides, whereas destabilizing osmolytes (urea, guanidium hydrochloride) speed up the aggregation process in a predictable way that fits the trend of all osmolytes. With respect to kinetics, we illustrate, by adapting our earlier reaction model to the insulin system, that the intermediates (trimers, tetramers, pentamers, etc.) are at very low concentrations and that nucleation is orders of magnitude slower than fibril growth. The results are then collated into a cogent explanation using the preferential exclusion and accumulation of osmolytes away from and at the protein surface during nucleation, respectively. Both the heat of solution and the neutral molecular surface area of the osmolytes correlate linearly with two fitting parameters of the kinetic rate model, that is, the lag time and the nucleation rate prior to fibril formation. These kinetic and thermodynamic results support the preferential exclusion model and the existence of oligomers including nuclei and larger structures that could induce toxicity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Carboxylated N‐glycans on RAGE promote S100A12 binding and signaling

The receptor for advanced glycation end products (RAGE) is a signaling receptor protein of the immunoglobulin superfamily implicated in multiple pathologies. It binds a diverse repertoire of ligands, but the structural basis for the interaction of different ligands is not well understood. We earlier showed that carboxylated glycans on the V‐domain of RAGE promote the binding of HMGB1 and S100A8/A9. Here we study the role of these glycans on the binding and intracellular signaling mediated by another RAGE ligand, S100A12. S100A12 binds carboxylated glycans, and a subpopulation of RAGE enriched for carboxylated glycans shows more than 10‐fold higher binding potential for S100A12 than total RAGE. When expressed in mammalian cells, RAGE is modified by complex glycans predominantly at the first glycosylation site (N25IT) that retains S100A12 binding. Glycosylation of RAGE and maximum binding sites for S100A12 on RAGE are also cell type dependent. Carboxylated glycan‐enriched population of RAGE forms higher order multimeric complexes with S100A12, and this ability to multimerize is reduced upon deglycosylation or by using non‐glycosylated sRAGE expressed in E. coli. mAbGB3.1, an antibody against carboxylated glycans, blocks S100A12‐mediated NF‐κB signaling in HeLa cells expressing full‐length RAGE. These results demonstrate that carboxylated N‐glycans on RAGE enhance binding potential and promote receptor clustering and subsequent signaling events following oligomeric S100A12 binding. J. Cell. Biochem. 110: 645–659, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis of chitosan–polycaprolactone blend for control delivery of ofloxacin drug

In the present research work chitosan has been blended with different amounts of polycaprolactone (PCL) (80:20, 75:25, 60:40 and 50:50) for using them for control delivery of ofloxacin. The blends were characterized by Fourier transmission infra red spectroscopy (FTIR), UV–visible spectroscopy (UV), scanning electron microscopy (SEM), X-ray diffraction (XRD) analysis. From the FTIR spectra the various groups present in chitosan and PCL blend were monitored. The homogeneity, morphology and crystallinity of the blends were ascertained from SEM and XRD data, respectively. The swelling studies have been measured at different drug loading. The kinetics of the drug delivery system has been systematically studied. Drug release kinetics was analyzed by plotting the cumulative release data vs. time by fitting to an exponential equation which indicated the non-Fickian type of kinetics. The drug release was investigated at different pH medium and it was found that the drug release depends upon the pH medium as well as the nature of matrix.