Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity

Endo-alpha-D-mannosidase is an enzyme involved in N-linked oligosaccharide processing which through its capacity to cleave the internal linkage between the glucose-substituted mannose and the remainder of the polymannose carbohydrate unit can provide an alternate pathway for achieving deglucosylation and thereby make possible the continued formation of complex oligosaccharides during a glucosidase blockade. In view of the important role which has been attributed to glucose on nascent glycoproteins as a regulator of a number of biological events, we chose to further define the in vivo action of endomannosidase by focusing on the well characterized VSV envelope glycoprotein (G protein) which can be formed by the large array of cell lines susceptible to infection by this pathogen. Through an assessment of the extent to which the G protein was converted to an endo-beta-N- acetylglucosaminidase (endo H)-resistant form during a castanospermine imposed glucosidase blockade, we found that utilization of the endomannosidase-mediated deglucosylation route was clearly host cell specific, ranging from greater than 90% in HepG2 and PtK1 cells to complete absence in CHO, MDCK, and MDBK cells, with intermediate values in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of the latter group the electrophoretic pattern after endo H treatment suggested that only one of the two N-linked oligosaccharides of the G protein was processed by endomannosidase. In the presence of the specific endomannosidase inhibitor, Glcalpha1-->3(1- deoxy)mannojirimycin, the conversion of the G protein into an endo H- resistant form was completely arrested. While the lack of G protein processing by CHO cells was consistent with the absence of in vitro measured endomannosidase activity in this cell line, the failure of MDBK and MDCK cells to convert the G protein into an endo H-resistant form was surprising since these cell lines have substantial levels of the enzyme. Similarly, we observed that influenza virus hemagglutinin was not processed in castanospermine-treated MDCK cells. Our findings suggest that studies which rely on glucosidase inhibition to explore the function of glucose in controlling such critical biological phenomena as intracellular movement or quality control should be carried out in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.      

Toward rational design of bacterial genomes

The advent of genetic engineering-the ability to edit and insert DNA into living organisms-in the latter half of the 20th century created visions of a new era of synthetic biology, where novel biological functions could be designed and implemented for useful purposes. We are witnessing an exciting revolution of scale, wherein technical progresses allow for the manipulation of genetic material at the whole genome level. This will enable the manufacture of increasingly complex genetic designs to solve pressing challenges in health, energy and the environment-if and when such designs can be specified. We argue that the organized development of key common application organisms, engineered for engineerability, and attendant libraries of parts, pathways and standardized manufacturing are necessary for this genome-scale technology to realize its promise.     

Influence of language and ancestry on genetic structure of contiguous populations: A microsatellite based study on populations of Orissa

Background

Although cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the severity of disease is highly variable indicating the influence of modifier genes. The intestines of Cftr deficient mice (CF mice: Cftr ^tm1Unc ) are prone to obstruction by excessive mucus accumulation and are used as a model of meconium ileus and distal intestinal obstruction syndrome. This phenotype is strongly dependent on the genetic background of the mice. On the C57Bl/6 background, the majority of CF mice cannot survive on solid mouse chow, have inflammation of the small intestine, and are about 30% smaller than wild type littermates. In this work potential modifier loci of the CF intestinal phenotype were identified.

Results

CF mice on a mixed genetic background (95% C57Bl/6 and 5% 129Sv) were compared to CF mice congenic on the C57Bl/6 background for several parameters of the intestinal CF phenotype. CF mice on the mixed background exhibit significantly greater survival when fed dry mouse chow, have reduced intestinal inflammation as measured by quantitative RT-PCR for marker genes, have near normal body weight gain, and have reduced mucus accumulation in the intestinal crypts. There was an indication of a gender effect for body weight gain: males did not show a significant improvement at 4 weeks of age, but were of normal weight at 8 weeks, while females showed improvement at both 4 and 8 weeks. By a preliminary genome-wide PCR allele scanning, three regions were found to be potentially associated with the milder phenotype. One on chr.1, defined by marker D1Mit36, one on chr. 9 defined by marker D9Mit90, and one on chr. 10, defined by marker D10Mit14.

Conclusion

Potential modifier regions were found that have a positive impact on the inflammatory phenotype of the CF mouse small intestine and animal survival. Identification of polymorphisms in specific genes in these regions should provide important new information about genetic modifiers of the CF intestinal phenotype.     

Rationally designed families of orthogonal RNA regulators of translation

Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ~2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.     

Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574

The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L(-1)) and achieved a critical micelle concentration (CMC) value of 120 mg L(-1).     

Phylogeny of the M superhaplogroup inferred from complete mitochondrial genome sequence of Indian specific lineages

Background  

Phylogenetic analysis of human complete mitochondrial DNA sequences has largely contributed to resolving phylogenies and antiquity of different lineages belonging to the majorhaplogroups L, N and M (East-Asian lineages). In the absence of whole mtDNA sequence information of M lineages reported in India that exhibits highest diversity within the sub-continent, the present study was undertaken to provide a detailed analysis of this haplogroup to precisely characterize the lineages and unravel their intricate phylogeny.     

Peripartum Cardiomyopathy Presenting With Ventricular Tachycardia: A Rare Presentation

A 25-year-old previously asymptomatic pregnant woman at 36 weeks'' gestation was noticed to have repetitive monomorphic ventricular tachycardia. A dilated left ventricle with moderately reduced systolic function was found on echocardiographic examination. This is a very rare presentation of peripartum cardiomyopathy (PPCMP) presenting with repetitive monomorphic ventricular tachycardia.     

Glipizide matrix transdermal systems for diabetes mellitus: preparation, in vitro and preclinical studies

The aim of the present investigation was to prepare glipizide matrix transdermal systems using the combinations of ethyl cellulose/polyvinylpyrrolidone and Eudragit RL-100/Eudragit RS-100. The systems were evaluated for various in vitro (drug content, drug permeation, scanning electron microscopy and drug-polymer interactions) and in vivo (acute and long-term hypoglycemic activity, biochemical and histopathological studies, skin irritation and pharmacokinetic studies in mice) parameters. Drug content of the patches was found to be more than 98%. Variations in drug permeation profiles were observed among various formulations. The scanning electron microscopy of the patches showed the formation of pores on the surface after in vitro permeation studies. The drug-polymer interaction results suggested no interaction between drug and polymers. The in vivo results revealed that the patches successfully prevented the severe hypoglycemia in the initial hours and they were also effective on chronic application. The transdermal route exhibited negligible skin irritation and produced better improvement with all the tested in vivo parameters compared to oral administration.     

A sawdust-derived soil conditioner promotes plant growth and improves water-holding capacity of different types of soils

Sawdust, a bulky waste generated by wood processing industries, has very few profitable and ecofriendly uses and poses a problem of proper disposal. Treatment with the fungusVolvariella volvaceae and a dilute solution of urea converted sawdust from a phytoinhibitory material to a phytostimulatory soil conditioner. In different types of soils, the soil conditioner increased the moisture retention and facilitated the cohesive interaction of particles. Analyses of the major biopolymers of sawdust after fungal treatment indicated that levels of cellulose, hemicellulose and lignin decreased; however, these changes did not account for the plant growth stimulatory property attained by this material.