The effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M-catalyzed hydrolysis of enkephalins.

High performance liquid chromatography and high performance liquid chromatography/electrospray ionization-mass spectrometry were used to study the effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M (EC 3.4.11.2)-catalyzed hydrolysis of methionine (Met-enk) and leucine enkephalins (Leu-enk). Acetylation imparts a significant enhancement in the proteolytic stability of these two peptides. After 30 min of the reaction, < 10% of both acetylated enkephalins was hydrolyzed. In an 8-h incubation period, only a maximum of 54% acetylated (Ac)-Met-enk and 38% Ac-Leu-enk was hydrolyzed. Vmax and Km [infil] for the degradation of Ac-Met-enk were 1.4 nmol/min/50 ng and 2.2 mM, respectively. The corresponding values for the reaction of Ac-Leu-enk were 0.5 nmol/min/50 ng and 0.9 mM. Also, the aminopeptidase M activity on Met-enk can be inhibited in the presence of Ac-Met-enk, which acts as a mixed-type inhibitor with the inhibition constant (K(i)) of I x 10(-3) M.     

Biophysical delivery of peptides: applicability for cancer therapy

There is a current trend towards evaluation of molecular agents for treatment of a variety of ailments, including cancer. One class of such biomolecules is proteins, and their shortened versions, peptides. Use of peptidic entities has been hindered by poor bioavailability in vivo and the high cost involved in mass-producing these macromolecular drugs. The need for localized delivery is being met with the development of various biophysical means, which include devices and aids, mainly transdermal and invasive implants. In addition, various cell-based delivery modalities, which include the use of spore-forming bacteria and stem cells, are being explored. This review discusses these methods in turn, and examines ways by which these can be enhanced for peptide delivery to tumors.     

Insilico modeling and molecular dynamic simulation of claudin-1 point mutations in HCV infection

Claudin-1 (CLDN1) in association with envelope glycoprotein (CD81) mediates the fusion of HCV into the cytosol. Recent studies have indicated that point mutations in CLDN1 are important for the entry of hepatitis C virus (HCV). To validate these findings, we employed a computational platform to investigate the structural effect of two point mutations (I32M and E48K). Initially, three-dimensional co-ordinates for CLDN1 receptor sequence were generated. Then, three mutant models were built using the point mutation including a double mutant (I32M/E48K) model from the native model structure. Finally, all the four model structures including the native and three mutant models were subjected to molecular dynamics (MD) simulation for a period of 25?ns to appreciate their dynamic behavior. The MD trajectory files were analyzed using cluster and principal component method. The analysis suggested that either of the single mutation has negligible effect on the overall structure of CLDN1 compared to the double mutant form. However, the double mutant model of CLDN1 shows significant negative impact through the impairment of H-bonds and the simultaneous increase in solvent accessible surface area. Our simulation results are visibly consistent with the experimental report suggesting that the CLDN1 receptor distortion is prominent due to the double mutation with large surface accessibility. This increase in accessible surface area due to the coexistence of double mutation may be presumed as one of the key factor that results in permissive action of HCV attachment and infection.     

Presence of gamma-glutamyltransferase in the renal microvascular compartment

The association between the brush border enzyme alkaline phosphatase and gamma-glutamyltransferase was determined by sucrose density gradient analysis of crude kidney homogenates, isolated glomeruli, and isolated microvessels. As previously established there is an overlap of these enzyme activities in the crude homogenate corresponding to a density of 1.17 g.cm-3. In contrast, isolated glomeruli sedimented with a peak of 1.25 g. cm-3 and exhibited gamma-glutamyltransferase activity but little alkaline phosphatase activity; homogenizing isolated glomeruli shifted the fragments to a density coincident with that observed for the crude homogenate gamma-glutamyltransferase peak. A second population of capillaries, isolated microvessels, were homogenized and analysed on the sucrose density gradient. These fragments sedimented over the same range as crude homogenate gamma-glutamyltransferase peak but were devoid of alkaline phosphatase activity and yet exhibited remarkable gamma-glutamyltransferase activity. The results indicate homogenization of renal cortex results in a heterogeneous collection of particles from both tubular and microvascular locations exhibiting gamma-glutamyltransferase activity which overlap with the brush border alkaline phosphatase containing membranes. However, isolation of microvessels and glomeruli prior to homogenization allows separation of gamma-glutamyltransferase from alkaline phosphatase activity; between 10 and 20% of the total homogenate gamma-glutamyltransferase activity is estimated to be associated with the microvascular compartment.     

Analyses of genetic diversity among maize inbred lines differing for resistance to pink borer and post-flowering stalk rot

Identification of the diverse sources of resistance is an important issue among the breeders for developing pest and disease free hybrids, to reduce the inoculum load, to prolong the life of inbred lines/hybrids and to reduce the cost of cultivation. Molecular diversity analysis was carried out among 23 maize inbred lines with respect to post flowering stalk rot and pink borer. Forty six SSR markers were employed among eight post flowering stalk rot (PFSR) and seven pink borer resistant lines along with eight other inbred lines to identify diverse resistant sources for developing resistant heterotic combinations to above pests and diseases. Number of alleles per SSR marker ranged from 2 to 9 averaging 4.11. The polymorphism information content (PIC) ranged from 0.272 to 0.839 with an average of 0.568. Discrimination rate (DR) of the markers ranged from 0.095 to 0.861 with a mean of 0.618. Number of alleles was highly correlated with PIC and DR. The pair-wise genetic dissimilarity values ranged from 0.05 to 0.84 with an overall mean of 0.64. Un-weighted neighbour joining clustering put 23 genotypes in two main clusters, which were further subdivided into 5 and 6 sub-clusters, respectively. We obtained 56 rare and 26 unique alleles in specific inbred lines, which can be used for identification of these lines. The present study has revealed considerable diversity among inbred lines differing for resistance against PFSR and pink borer; and provided ample scope for selection of parents for utilization in heterosis breeding     

Studies on the uptake of trivalent and hexavelent chromium by onion (Allium cepa)

Abstract

Experiments were conducted to examine the uptake and translocation of root-absorbed trivalent and hexavalent state of chromium in the onion plant (Allium cepa) grown in soil and sand culture. Chromium content in plant tissues increased with increasing amount of added chromium. Distribution of chromium in the plant in general, found to be in the order: root>>bulb>shoot. Higher uptake in the plants grown in sand from both the sources of chromium was observed as compared with the corresponding values for soil culture. Morphological and growth effects of the treatments of different oxidation state of chromium indicated that higher doses of Cr(VI) [150 and 300 μg mL^?1] were more toxic to the onion plants compared to equivalent doses of Cr(III).     

A multifaceted computational report on the variants effect on <Emphasis Type="Italic">KIR2DL3</Emphasis> and <Emphasis Type="Italic">IFNL3</Emphasis> candidate gene in HCV clearance

HCV infection causes acute and chronic liver diseases including, cirrhosis and hepatocellular carcinoma. Following HCV infection, spontaneous clearance occurs in approximately 20 % of the population dependant upon HCV genotype. In this study, functional and non-functional variant analysis was executed for the classical and the latest HCV clearance candidate genes namely, KIR2DL3 and IFNL3. Initially, the functional effects of non-synonymous SNPs were assigned on exposing to homology based tools, SIFT, PolyPhen-2 and PROVEAN. Further, UTR and splice sites variants were scanned for the gene expression and regulation changes. Subsequently, the haplotype and CNV were also identified. The mutation H77Y of KIR2DL3 and R157Q, H156Y, S63L, R157W, F179V, H128R, T101M, R180C, and F176I of IFNL3 results in conservation, RMSD, total energy, stability, and secondary structures revealed a negative impact on the structural fitness. UTRscan and the splice site result indicate functional change, which may affect gene regulation and expression. The graphical display of selected population shows alleles like rs270779, rs2296370, rs10423751, rs12982559, rs9797797, and rs35987710 of KIR2DL3 and rs12972991, rs12980275, rs4803217, rs8109886, and rs8099917 of IFNL3 are in high LD with a measure of \({\text{r}}^{2 }\ge 0.8\) broadcasting its protective effect in HCV clearance. Similarly, CNV report suggests major DNA fragment loss that could have a profound impact on the gene expression affecting the overall phenotype. This roundup report specifies the effect of NK cell receptor, KIR2DL3 and IFNL3 variants that can have a better prospect in GWAS and immunogenetic studies leading to better understanding of HCV clearance and progression.     

Gene Expression Prediction and Hierarchical Clustering Analysis of Plant <Emphasis Type="Italic">CCD</Emphasis> genes

Plant carotenoid cleavage dioxygenase (CCD) catalyses the formation of industrially important apocarotenoids. Here, we applied codon-based classification for 72 CCD genes from 35 plant species using hierarchical clustering analysis. The codon adaptation index (CAI) and relative codon bias (RCB) were utilized to estimate the level of gene expression. The codon-based cluster tree result shows neatly clustered subclass of CCD genes except BoCCD1 gene of Bixa orellana. Correlation analysis of CAI values with RCB indicates an overall low-level expression of CCD across different species. Similarly, the closeness in the codon cluster with same CAI values was not reflected in 3-D structural report of selected CCD genes. These finding not only enhances our insights into the classification of CCD gene across the species but also identifies the critical factors responsible for this variation, which could aid in prediction of gene expression and function for newly reported CCD genes.     

GFP expression alters osteosarcoma cell biology

Green fluorescent protein (GFP) expression is widely used for tracking of cells in vivo. Three human osteosarcoma cell lines were transfected with GFP and analyzed for expression of tissue markers and physiology. GFP-mediated increased tumor aggression in vivo in one cell line was corroborated by altered expression of bone markers and osteoblast-like cell biology in vitro as a result of transfection. The use of GFP for cell tracking in osteosarcoma animal models should therefore be used with caution.