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.     

Design,synthesis and evaluations of acridone derivatives using Candida albicans—Search for MDR modulators led to the identification of an anti-candidiasis agent

In order to search for MDR modulators, rationally designed acridone derivatives were investigated for their effect on influx or efflux of Rhodamine6G (R6G) in CAI4 cells. Results of these investigations indicate that in presence of compound 12, inhibition of growth of CAI4 cells and also an increased influx/efflux of R6G in CAI4 cells have been observed. This seems to be occurring due to the cell wall rupturing of Candida albicans. Compound 12 may be a suitable candidate for candidiasis therapy.     

XomAnnotate: Analysis of Heterogeneous and Complex Exome- A Step towards Translational Medicine

In translational cancer medicine, implicated pathways and the relevant master genes are of focus. Exome''s specificity, processing-time, and cost advantage makes it a compelling tool for this purpose. However, analysis of exome lacks reliable combinatory analysis tools and techniques. In this paper we present XomAnnotate – a meta- and functional-analysis software for exome. We compared UnifiedGenotyper, Freebayes, Delly, and Lumpy algorithms that were designed for whole-genome and combined their strengths in XomAnnotate for exome data through meta-analysis to identify comprehensive mutation profile (SNPs/SNVs, short inserts/deletes, and SVs) of patients. The mutation profile is annotated followed by functional analysis through pathway enrichment and network analysis to identify most critical genes and pathways implicated in the disease genesis. The efficacy of the software is verified through MDS and clustering and tested with available 11 familial non-BRCA1/BRCA2 breast cancer exome data. The results showed that the most significantly affected pathways across all samples are cell communication and antigen processing and presentation. ESCO1, HYAL1, RAF1 and PRKCA emerged as the key genes. Network analysis further showed the purine and propanotate metabolism pathways along with RAF1 and PRKCA genes to be master regulators in these patients. Therefore, XomAnnotate is able to use exome data to identify entire mutation landscape, pathways, and the master genes accurately with wide concordance from earlier microarray and whole-genome studies -- making it a suitable biomedical software for using exome in next-generation translational medicine.

Availability

http://www.iomics.in/research/XomAnnotate     

Novel Trichoderma polysporum Strain for the Biocontrol of Pseudogymnoascus destructans,the Fungal Etiologic Agent of Bat White Nose Syndrome

White-nose syndrome (WNS), an emerging disease of hibernating bats, has rapidly spread across eastern North America killing millions of bats. Pseudogymnoascus destructans (Pd), the sole etiologic agent of WNS, is widespread and persistent in bat hibernacula. Control of Pd in the affected sites is urgently needed to break the transmission cycle while minimizing any adverse impact on the native organisms. We isolated a novel strain of Trichoderma polysporum (Tp) from one of the caves at the epicenter of WNS zoonotic. Detailed experimental studies revealed: (1) Tp WPM 39143 was highly adapted to grow at temperatures simulating the cave environment (6°C-15°C), (2) Tp WPM 39143 restricted Pd colony growth in dual culture challenges, (3) Tp WPM 39143 caused four logs reduction of Pd colony forming units and genome copies in autoclaved soil samples from one of the WNS affected caves, (4) Tp WPM 39143 extract showed specific fungicidal activity against Pd in disk diffusion assay, but not against closely related fungus P. pannorum (Pp), (5) Tp WPM 39143 extract retained inhibitory activity after exposure to high temperatures, light and proteinase K, and (6) Inhibitory metabolites in Tp WPM 39143 extract comprised of water-soluble, high polarity compounds. These results suggest that Tp WPM 39143 is a promising candidate for further evaluation as a biocontrol agent of Pd in WNS affected sites.     

Polyamines and jasmonic acid induce plasma membrane potential variations in lima bean

Exogenous polyamines [cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm)] elicit the production of volatiles in Lima bean (Phaseolus lunatus). Among the tested PAs, Spm induces the production of some volatile terpenoids that are known to be induced by the spider mite Tetranychus urticae. Spm treatment elicits the biosynthesis of Jasmonic acid (JA), a phytohormone known to regulate the production of the volatile terpenoids. The treatment with JA together with Spm resulted in the increased volatile emission, and predatory mites Phytoseiulus persimilis preferred JA and Spm-treated leaves over those treated with JA alone.⁵ JA and Spm treatment has no effects on polyamine oxidase (PAO) and Cu-amine oxidase (CuAO) but has a significant induction of calcium influx, ROS production, enzyme activities for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, and gene expressions except for NADPH-oxidase complex.⁵ Here, we report that a plasma membrane potential (V_m) depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd. JA perfusion did not alter V_m but the perfusion of JA and the polyamines significantly increased Cad and Put V_m depolarization. When JA was perfused with polyamines, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Key words: polyamines, lima bean, herbivore-induced volatile organic compounds, calcium and ROS signalling, jasmonic acid, quantitative gene expression, transmembrane potentialPolyamines are involved in plants’ stress responses and growth. By activating biosynthesis of nucleic acids, polyamines concern the plant growth and differentiation.^1–3 Furthermore, it has been reported that polyamines are involved in the response against environmental stress and plant disease.^1–4 We recently reported that exogenously applied polyamines ∼diamines [cadaverine (Cad), putrescine (Put)], triamine [spermidine (Spd)] and tetraamine ]spermine (Spm)]∽ induce volatile emission in Lima bean leaves.⁵ Membrane potentials (V_m) and intracellular calcium variations were also studied in Lima bean leaves after perfusion with the polyamines and with these addition of JA and here we report on these additional results.The primary candidate for intercellular signaling in higher plants is the stimulus-induced change in V_m.⁶ The plasma membrane potential (V_m), which lies in the range of −50 to −200 mV in Lima bean leaves,⁷ may be shifted either to more negative (hyperpolarization) or to more positive values (depolarization) in response to various biotic or abiotic stresses.Measurement of V_m were performed and data statistically treated as previously described (ANOVA and Tukey-Kramer’s HSD test).⁷ Perfusion with the polyamines (Fig. 1 single arrow) shows a specific response of the leaf tissues with a different V_m depolarization, depending on the polyamine. In general, a V_m depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd (Fig. 1). Spm and Spd V_m depolarization values were significantly different (p < 0.05) from all other polyamines, whereas no significant difference was found between Put and Cad V_m depolarization (p = 0.435). In all cases, V_m depolarization was reversed by washing polyamine-treated leaves with a fresh buffer solution (Fig. 1 double arrow); however, a full recovery of the V_m was observed only for Put (Fig. 1). The linearization of the data from Figure 1 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines which was higher for Spd (6.0 mV min⁻¹; R = 0.96), equal for Put and Cad (4.8 mV min⁻¹; Put R = 0.95; Cad R = 0.97) and lower for Spm (3.0 mV min⁻¹; R = 0.96).Open in a separate windowFigure 1Effect of 1 mM polyamines (arrow) on the V_m of Lima bean palisade cells. Spermine (Spm) caused the lowest V_m depolarization, whereas spermidine (Spd) showed the highest values of V_m depolarization. intermediate values were found when putrescine (Put) and cadaverine (cad) were perfused. after washing the tissues with fresh buffer (double arrow) V_m was always hyperpolarized, however the initial potential was recovered only for Put, while for all other polyamines the V_m never reached the initial values. Metric bars indicate standard deviation.Perfusion with JA caused a slight and not significant (p = 0.332) V_m depolarization (Fig. 2) with respect to control. The addition of JA caused a significant increase (p < 0.01) in V_m depolarization when perfused with Cad, with respect to the sole perfusion with Cad (Fig. 1). The same was observed when JA was perfused with Put, whereas not significant differences were observed when Spm (p = 0.513) and Spd (p = 0.107) were perfused with JA (Fig. 2), with respect to the sole perfusion with Spm and Spd (Fig. 1). The linearization of the data from Figure 2 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines + JA, which was higher for Cad (24.40 mV min⁻¹; R = 0.99), almost equal for Put and Spd (Put: 14.21 mV min⁻¹, R = 0.99; Spd: 13.49 mV min⁻¹, R = 0.99) and lower for Spm (1.34 mV min⁻¹; R = 0.93). For JA the rate of V_m depolarization was 0.19 mV min⁻¹ (R = 0.96). With the addition of JA, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Open in a separate windowFigure 2Effect of 1 mM polyamines + 0.1 mMJA (arrow) on the V_m of Lima bean palisade cells. the perfusion with Ja did not cause any variation in the V_m. addition of JA to Spm and Spd caused the same V_m depolarization observed in the absence of JA, whereas when JA was added to Put and Cad a stronger and significantly different V_m depolarization was observed. even in this case washing the tissues with fresh buffer (double arrow) caused a V_m hyperpolarized, however in this case Spd reached V_m values significantly more negative that the initial V_m. Metric bars indicate standard deviation. For abbreviations see Figure 1.Since ion fluxes through channels directly influence V_m, it seems reasonable to assume that molecules able to act on channel activity might be considered as important factors inducing electrical signals. Among the various channels, calcium and potassium channels are predominantly involved in cell signaling.⁸ In the present study, rapid and reversible V_m depolarization observed upon perfusion of Lima bean mesophyll cells with polyamines was found to be significantly increased when JA was added to Cad and Put. The reversibility of the V_m may be linked to the overall physico-chemical amphiphilic properties of polyamines, probably depending on non covalent interaction with plasma membrane molecules, as polyamines occur in plants in free form, bound electrostatically to negatively charged molecules, and conjugated to small molecules and proteins.⁹ Liu et al.¹⁰ showed that Spm, Spd, Cad and Put strongly inhibited opening and closing of stomata in Vicia faba, suggesting that polyamines target inward potassium channels in guard cells and modulate stomatal movements, so providing a link between abiotic stress, polyamine levels and stomatal regulation. Moreover, the transport of polyamines across the plasma membrane of plant cells is energy-dependent and calcium is involved in the uptake mechanism.^1,11 Both mechanisms can be correlated to the observed V_m depolarization, and the positive correlation between intracellular Ca²⁺ concentration⁵ and V_m depolarizing activity of polyamines confirms the involvement of Ca²⁺ during polyamine uptake.¹¹     

Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana

Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.Key words: LORELEI, glycosylphosphatidylinositol (GPI)-anchored protein, embryogenesis, DD45, seed germination, primary and lateral root growth, seedling developmentDouble fertilization is unique to flowering plants. Upon female gametophyte reception of a pollen tube, the egg and central cells of the female gametophyte fuse with the two released sperm cells to form zygote and endosperm, respectively and initiate seed development.¹ The female gametophyte controls seed development by (1) repressing premature central cell or egg cell proliferation until double fertilization is completed,^1–3 (2) supplying factors that mediate early stages of embryo and endosperm development^1,4,5 and (3) regulating imprinting of genes required for seed development.^1,6The molecular mechanisms underlying female gametophyte control of early seed development are poorly understood. Although much progress has been made in identifying genes and mechanisms by which the female gametophyte represses premature central cell or egg cell proliferation until double fertilization is completed and regulates imprinting of genes required for seed development,^1,6 only a handful of female gametophyte-expressed genes that affect early embryo^7,8 and endosperm⁹ development after fertilization have been characterized. This is particularly important given that a large number of female gametophyte-expressed genes likely regulate early seed development.⁵We recently reported on a mutant screen for plants with reduced fertility and identification of a mutant that contained a large number of undeveloped ovules and very few viable seeds.¹⁰ TAIL-PCR revealed that this mutant is a new allele of LORELEI(LRE) [At4g26466].^10,11 Four lre alleles have been reported;¹¹ so, this mutant was designated lre-5.¹⁰ The Arabidopsis LORELEI protein contains 165 amino acids and possesses sequence features indicative of a glycosylphosphatidylinositol (GPI)-anchor containing cell surface protein. GPI-anchors serve as an alternative to transmembrane domains for anchoring proteins in cell membranes and GPI-anchored proteins participate in many functions including cell-cell signaling.¹²     

MVA recombinants expressing the fusion and hemagglutinin genes of PPRV protects goats against virulent challenge

Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.     

Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: A RPE specific phenomenon

SMCT1 is a Na⁺-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na⁺-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, β-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.