Specificity and promiscuity in human glutaminase interacting protein recognition: insight from the binding of the internal and C-terminal motif

A large number of cellular processes are mediated by protein-protein interactions, often specified by particular protein binding modules. PDZ domains make up an important class of protein-protein interaction modules that typically bind to the C-terminus of target proteins. These domains act as a scaffold where signaling molecules are linked to a multiprotein complex. Human glutaminase interacting protein (GIP), also known as tax interacting protein 1, is unique among PDZ domain-containing proteins because it is composed almost exclusively of a single PDZ domain rather than one of many domains as part of a larger protein. GIP plays pivotal roles in cellular signaling, protein scaffolding, and cancer pathways via its interaction with the C-terminus of a growing list of partner proteins. We have identified novel internal motifs that are recognized by GIP through combinatorial phage library screening. Leu and Asp residues in the consensus sequence were identified to be critical for binding to GIP through site-directed mutagenesis studies. Structure-based models of GIP bound to two different surrogate peptides determined from nuclear magnetic resonance constraints revealed that the binding pocket is flexible enough to accommodate either the smaller carboxylate (COO(-)) group of a C-terminal recognition motif or the bulkier aspartate side chain (CH(2)COO(-)) of an internal motif. The noncanonical ILGF loop in GIP moves in for the C-terminal motif but moves out for the internal recognition motifs, allowing binding to different partner proteins. One of the peptides colocalizes with GIP within human glioma cells, indicating that GIP might be a potential target for anticancer therapeutics.     

Cdc6 protein activates p27KIP1-bound Cdk2 protein only after the bound p27 protein undergoes C-terminal phosphorylation

In mammalian cells Cdk2 activity during the G(1)-S transition is mainly controlled by p27(KIP1). Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21(CIP1)-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6.     

Phytoremediation potential of paragrass--an in situ approach for chromium contaminated soil

The present in situ phytoextraction approach uses paragrass (Brachiaria mutica (Forssk) Stapf) as a hyper accumulator for attenuation of chromium level in soil and mine waste water at South Kaliapani chromite mine area of Orissa. The bioconcentration factor (BCF) for Cr was maximum (0.334) in 100 days grown paragrass weeds. Transportation index (Ti) i.e. 6.16 and total accumulation rate (TAR) i.e. 8.2 mg kg(-1)day(-1) was maximum in 125 days old paragrass grown in Cr contaminated experimental cultivated plots. Cr bioaccumulation in roots was nearly 1000 times more than shoots. Paragrass showed luxuriant growth with massive fibrous roots when grown over Cr contaminated soils (11,170 mg/ kg dry soil). Cr bioaccumulation varies significantly with plant age, biomass and level of Cr contamination in irrigated mine waste water and soil. Paragrass could be used as hyperaccumulators as it showed rapid massive growth with a high tolerance to Cr.     

Unique C2V3 Sequence in HIV-1 Envelope Obtained from Broadly Neutralizing Plasma of a Slow Progressing Patient Conferred Enhanced Virus Neutralization

Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an env clone amplified from cross neutralizing plasma of an antiretroviral naïve chronically infected Indian patient (ID50 >600-fold higher compared to other autologous env clones). The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env) was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.     

Antineoplastic and Apoptotic Potential of Traditional Medicines Thymoquinone and Diosgenin in Squamous Cell Carcinoma

Thymoquinone (TQ) and diosgenin (DG), the active ingredients obtained from black cumin (Nigella sativa) and fenugreek (Trigonella foenum graecum), respectively, exert potent bioactivity, including anticancer effects. This study investigated the antineoplastic activity of these agents against squamous cell carcinoma in vitro and sarcoma 180–induced tumors in vivo. TQ and DG inhibited cell proliferation and induced cytotoxicity in A431 and Hep2 cells. These agents induced apoptosis by increasing the sub-G₁ population, LIVE/DEAD cytotoxicity, chromatin condensation, DNA laddering and TUNEL-positive cells significantly (P<0.05). Increased Bax/Bcl-2 ratio, activation of caspases and cleavage of poly ADP ribose polymerase were observed in treated cells. These drugs inhibited Akt and JNK phosphorylations, thus inhibiting cell proliferation while inducing apoptosis. In combination, TQ and DG had synergistic effects, resulting in cell viability as low as 10%. In a mouse xenograft model, a combination of TQ and DG significantly (P<0.05) reduced tumor volume, mass and increased apoptosis. TQ and DG, alone and in combination, inhibit cell proliferation and induce apoptosis in squamous cell carcinoma. The combination of TQ and DG is a potential antineoplastic therapy in this common skin cancer.     

Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.     

Genetic Diversity and Population Structure of Indian Golden Silkmoth (Antheraea assama)

Background

The Indian golden saturniid silkmoth (Antheraea assama), popularly known as muga silkmoth, is a semi-domesticated silk producing insect confined to a narrow habitat range of the northeastern region of India. Owing to the prevailing socio-political problems, the muga silkworm habitats in the northeastern region have not been accessible hampering the phylogeography studies of this rare silkmoth. Recently, we have been successful in our attempt to collect muga cocoon samples, although to a limited extent, from their natural habitats. Out of 87 microsatellite markers developed previously for A. assama, 13 informative markers were employed to genotype 97 individuals from six populations and analyzed their population structure and genetic variation.

Methodology/Principal Findings

We observed highly significant genetic diversity in one of the populations (WWS-1, a population derived from West Garo Hills region of Meghalaya state). Further analysis with and without WWS-1 population revealed that dramatic genetic differentiation (global F_ST = 0.301) was due to high genetic diversity contributed by WWS-1 population. Analysis of the remaining five populations (excluding WWS-1) showed a marked reduction in the number of alleles at all the employed loci. Structure analysis showed the presence of only two clusters: one formed by WWS-1 population and the other included the remaining five populations, inferring that there is no significant genetic diversity within and between these five populations, and suggesting that these five populations are probably derived from a single population. Patterns of recent population bottlenecks were not evident in any of the six populations studied.

Conclusions/Significance

A. assama inhabiting the WWS-1 region revealed very high genetic diversity, and was genetically divergent from the five populations studied. The efforts should be continued to identify and study such populations from this region as well as other muga silkworm habitats. The information generated will be very useful in conservation of dwindling muga culture in Northeast India.     

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line

Background

The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.

Methodology

Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.

Principal Findings

The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.

Conclusions

We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.     

Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes

The fact that malaria is still an uncontrolled disease is reflected by the genetic organization of the parasite genome. Efforts to curb malaria should begin with proper understanding of the mechanism by which the parasites evade human immune system and evolve resistance to different antimalarial drugs. We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7. We found 60 genes of various functions and lengths, majority (61.67%) of them were performing known functions. Almost all the genes have orthologs in other four species of Plasmodium, of which P. chabaudi seems to be the closest to P. falciparum. However, only two genes were found to be paralogous. Interestingly, the drug-resistant gene, pfcrt was found to be surrounded by seven genes coding for several CG proteins out of which six were reported to be responsible for providing drug resistance to P. vivax. The intergenic regions, in this specified region were generally large in size, majority (73%) of them were of more than 500 nucleotide bp length. We also designed primers for amplification of 21 noncoding DNA fragments in the whole region for estimating genetic diversity and inferring the evolutionary history of this region of P. falciparum genome.