Reversible Unfolding of Rhomboid Intramembrane Proteases

Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.     

Pool deconvolution approach for high-throughput gene mining from Bacillus thuringiensis

Applied Microbiology and Biotechnology - Novel genes from Bacillus thuringiensis (Bt) are required for effective deployment in agriculture, human health, and forestry. In an improvement over...     

Axenic rhizome culture and genetic fidelity assessment of Eulophia dabia (D. Don) Hochr: an endangered terrestrial orchid species

In Vitro Cellular & Developmental Biology - Plant - Eulophia dabia (D. Don) Hochr is an endemic, critically endangered, terrestrial orchid species having profound importance in traditional...     

Forest corridors maintain historical gene flow in a tiger metapopulation in the highlands of central India

Understanding the patterns of gene flow of an endangered species metapopulation occupying a fragmented habitat is crucial for landscape-level conservation planning and devising effective conservation strategies. Tigers (Panthera tigris) are globally endangered and their populations are highly fragmented and exist in a few isolated metapopulations across their range. We used multi-locus genotypic data from 273 individual tigers (Panthera tigris tigris) from four tiger populations of the Satpura–Maikal landscape of central India to determine whether the corridors in this landscape are functional. This 45 000 km² landscape contains 17% of India''s tiger population and 12% of its tiger habitat. We applied Bayesian and coalescent-based analyses to estimate contemporary and historical gene flow among these populations and to infer their evolutionary history. We found that the tiger metapopulation in central India has high rates of historical and contemporary gene flow. The tests for population history reveal that tigers populated central India about 10 000 years ago. Their population subdivision began about 1000 years ago and accelerated about 200 years ago owing to habitat fragmentation, leading to four spatially separated populations. These four populations have been in migration–drift equilibrium maintained by high gene flow. We found the highest rates of contemporary gene flow in populations that are connected by forest corridors. This information is highly relevant to conservation practitioners and policy makers, because deforestation, road widening and mining are imminent threats to these corridors.     

Membrane homoeostasis and multidrug resistance in yeast

The development of MDR (multidrug resistance) in yeast is due to a number of mechanisms. The most documented mechanism is enhanced extrusion of drugs mediated by efflux pump proteins belonging to either the ABC (ATP-binding cassette) superfamily or MFS (major facilitator superfamily). These drug-efflux pump proteins are localized on the plasma membrane, and the milieu therein affects their proper functioning. Several recent studies demonstrate that fluctuations in membrane lipid composition affect the localization and proper functioning of the MDR efflux pump proteins. Interestingly, the efflux pumps of the ABC superfamily are particularly susceptible to imbalances in membrane-raft lipid constituents. This review focuses on the importance of the membrane environment in functioning of the drug-efflux pumps and explores a correlation between MDR and membrane lipid homoeostasis.     

Modulation of anti-inflammatory response in lipopolysaccharide stimulated human THP-1 cell line and mouse model at gene expression level with indigenous putative probiotic lactobacilli

The anti-inflammatory potential of eight indigenous probiotic Lactobacillus isolates was evaluated in vitro in terms of modulating the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in human acute monocytic leukemia (THP-1) cells under inflammatory conditions. Amongst these, Lactobacillus plantarum Lp91 was the most potent anti-inflammatory strain as it evoked a significant (P < 0.001) down-regulation of TNF-α by −1.45-fold relative to the control in THP-1 cells. However, in terms of IL-6 expression, all the strains could up-regulate its expression considerably at different levels. Hence, based on in vitro expression of TNF-α, Lp91 was selected for in vivo study in lipopolysaccharide (LPS)-induced mouse model to look at the expression of TNF-α, IL-6, monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1) and E-selectin in mouse aorta. In LPS challenged (2 h) mice group fed with Lp91 for 10 days, TNF-α, IL-6, MCP-1, VCAM-1, ICAM-1 and E-selectin expressions were significantly down-regulated by 3.10-, 10.02-, 4.22-, −3.14-, 2.28- and 5.71-fold relative to control conditions. In conclusion, Lp91 could serve as a candidate probiotic strain to explore it as a possible biotherapeutic anti-inflammatory agent against inflammatory diseases including cardiovascular disease.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0347-5) contains supplementary material, which is available to authorized users.     

Structure-based virtual screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75)

HIV-1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure-based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory, and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine-Z742267384, Maybridge-HTS02400, and Specs-AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behavior and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.