Production of Recombinant Human Bile Salt Stimulated Lipase and Its Variant inPichia pastoris

hBSSL and its truncated variant hBSSL-C cDNA clones were expressed inPichia pastorisusing two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45–50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized inP. pastorisand was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile.     

Survivability of parthenogenetic embryos following in vivo transfer in naturally synchronized Capra hircus

The present study was conducted to see the in vivo developmental potency of caprine parthenogenetic embryos generated in a modified way. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and then activated by 7% ethanol followed by 2 mM 6-dimethyl amino purine (6-DMAP) and embryo development was recorded. Early stage parthenogenetic embryos (two to four cells) were surgically transferred in recipients (10). The pregnancy diagnosis was done by nonreturn to oestrus, ultrasonography (USG), and progesterone estimation. The levels of progesterone were above normal values (1 ng/ml) of pregnancy and fall below the level of pregnancy just before retuned to oestrus. Progesterone profile revealed that out of ten recipients (G1–G10), four goats (G1, G2, G3, and G5) returned to oestrus after 43?±?7.29 (Mean?±?SE) d of embryo transfer and six goats (G4, G6, G7, G8, G9, and G10) did not return to cycle even after 70 d of embryo transfer. In three recipients (G4, G5, and G6), the USG on day 40 revealed that there was fluid filled uterine body with solid fetus-like structure. These might be dead fetus and had started resorption. The progesterone profile also corroborated the assumption of pregnancy in these animals. Authors believe that this may be the first report on in vivo diploid parthenogenetic embryo development in caprine species.     

High level expression of soybean trypsin inhibitor gene in transgenic tobacco plants failed to confer resistance against damage caused byHelicoverpa armigera

Helicoverpa armigera is a major pest of many tropical crop plants. Soybean trypsin inhibitor (SBTI) was highly effective against the proteolytic activity of gut extract of the insect. SBTI was also inhibitory to insect growth when present in artificial diet. The gene coding for SBTI was cloned from soybean (Glycine max, CVBirsa) and transferred to tobacco plants for constitutive expression. Young larvae ofH. armigera, fed on the leaves of the transgenic tobacco plants expressing high level of SBTI, however, maintained normal growth and development. The results suggest that in certain cases the trypsin inhibitor gene(s) may not be suitable candidates for developing insect resistant transgenic plants.     

New record of nucleopolyhedroviruses in tea looper caterpillars in India

Tea production in North-East India hit a record loss due to the widespread severe outbreak of a mixed brood of three species of looper caterpillar pests of geometrid moths (Lepidoptera) in 2008-2010. In addition to Buzura suppressaria, two newly recorded geometrids, viz., Hyposidra infixaria and Hyposidra talaca have caused widespread severe damage in recent years. In the present study we report the nucleopolyhedroviruses (NPV) isolated from the tea looper caterpillar from North-East India. We identified and characterized the NPV by cloning and sequencing a partial segment of polyhedrin gene of virus infected larvae of B. suppressaria, H. talaca and H. infixaria. A comparison of deduced amino acids of polyhedrin gene among H. talaca, H. infixaria and B. suppressaria showed that same strain was found to infect all the three loopers in India, which show high sequence identity with B. suppressaria Chinese isolates. Based on the polyhedrin sequence homology, it is predicted that a variant of B. suppressaria Chinese isolate of NPV found to infect H. talaca, H. infixaria and B. suppressaria in India.     

A New Target for Amyloid Beta Toxicity Validated by Standard and High-Throughput Electrophysiology

Background

Soluble oligomers of amyloid beta (Aβ) are considered to be one of the major contributing factors to the development of Alzheimer''s disease. Most therapeutic development studies have focused on toxicity directly at the synapse.

Methodology/Principal Findings

Patch clamp studies detailed here have demonstrated that soluble Aβ can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of the synapse as well, but may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of the temporal electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs) at low concentrations of Aβ (1–42). We have also shown that this functional deficit can be reversed through use of curcumin, an inhibitor of Aβ oligomerization, using both analysis methods.

Conclusions/Significance

The MEA recording method utilized here is non-invasive, thus long term chronic measurements are possible and it does not require precise positioning of electrodes, thus it is ideal for functional screens. Even more significantly, we believe we have now identified a new target for drug development for AD based on functional toxicity of hippocampal neurons that could treat neurodegenerative diseases prior to the development of mild cognitive impairment.     

AFLP-Based Genetic Diversity Assessment of Commercially Important Tea Germplasm in India

India has a large repository of important tea accessions and, therefore, plays a major role in improving production and quality of tea across the world. Using seven AFLP primer combinations, we analyzed 123 commercially important tea accessions representing major populations in India. The overall genetic similarity recorded was 51%. No significant differences were recorded in average genetic similarity among tea populations cultivated in various geographic regions (northwest 0.60, northeast and south both 0.59). UPGMA cluster analysis grouped the tea accessions according to geographic locations, with a bias toward China or Assam/Cambod types. Cluster analysis results were congruent with principal component analysis. Further, analysis of molecular variance detected a high level of genetic variation (85%) within and limited genetic variation (15%) among the populations, suggesting their origin from a similar genetic pool.     

The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress

The 29-kDa surface antigen (thiol-dependent peroxidase; Eh29) of Entamoeba histolytica exhibits peroxidative and protective antioxidant activities. During tissue invasion, the trophozoites are exposed to oxidative stress and need to deal with highly toxic reactive oxygen species (ROS). In this investigation, attempts have been made to understand the role of the 29-kDa peroxidase gene in parasite survival and pathogenesis. Inhibition of eh29 gene expression by antisense RNA technology has shown approximately 55% inhibition in eh29 expression, maximum ROS accumulation, and significantly lower viability in 29-kDa downregulated trophozoites during oxidative stress. The cytopathic and cytotoxic activities were also found to decrease effectively in the 29-kDa downregulated trophozoites. Size of liver abscesses was substantially lower in hamsters inoculated with 29-kDa downregulated trophozoites compared to the normal HM1:IMSS. These findings clearly suggest that the 29-kDa protein of E. histolytica has a role in both survival of trophozoites in the presence of ROS and pathogenesis of amoebiasis.     

Latex extractables of Calotropis gigantea

The hexane and methanol soluble extract of the latex coagulum of Calotropis gigantea afforded two new triterpene esters, viz. 3′-methylbutanoates of α-amyrin and ψ-taraxasterol, besides the known 3′-methylbutanoates of three triterpene alcohols. The compositions of the alkane fraction, total triterpene alcohol fraction, and free, acetyl and 3′-methylbutanoyl triterpene alcohol fractions of the extract were determined.     

Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.