UDP-Gal: <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

1H, 13C, 15N assignments of the dimeric regulatory subunit (ilvN) of the E. coli AHAS I

Acetohydroxyacid synthase (AHAS) is an enzyme involved in the biosynthesis of the branched chain amino acids viz, valine, leucine and isoleucine. The activity of this enzyme is regulated through feedback inhibition by the end products of the pathway. Here we report the backbone and side-chain assignments of ilvN, the 22 kDa dimeric regulatory subunit of E. coli AHAS isoenzyme I, in the valine bound form. Detailed analysis of the structure of ilvN and its interactions with the catalytic subunit of E. coli AHAS I will help in understanding the mechanism of activation and regulation of the branched chain amino acid biosynthesis.     

In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring

Selaginella, an extant genus of primitive vascular plants, has survived over 400 million years of evolution. In vitro morphogenesis in Selaginella microphylla is considered for the first time to establish a well-documented aseptic culture on half- strength Murashige and Skoog’s basal medium with 2ip (4.92–49.21 μM), or Kn (4.65–46.47 μM) or GA₃ (2.89–28.90 μM) for shoot multiplication, and with different concentrations of IBA (4.9–49 μm) to initiate root cultures. GA₃ was instrumental for shoot multiplication as well as induction of reproductive structures in each and every leaf axil. On the other hand, it is observed that IBA alone in S. microphylla can act as signal molecules for induction of enormous numbers of root masses from a few existing roots. An interesting pattern of re-differentiation has also been observed where apical portions of large numbers of roots were converted to green shoot apical meristems. Further differentiation produced tiny green shoots. Distinct bipolarity was noted in shoots when they were isolated from root masses and appeared as embryo-like structures. Chromosome analysis from in vitro sporophytic plants revealed 2n = 16 chromosomes, indicating chromosomal stability. The interesting in vitro pattern of morphogenesis obtained in S. microphylla may provide new insights into totipotency of plants.     

Comparative physico-kinetic properties of a homogenous purified β-glucosidase from Withania somnifera leaf

β-glucosidase from Withania somnifera (Solanaceae) leaf has been purified to homogeneity and characterized for its physico-kinetic properties. The enzyme purification was achieved through a sequence of gel filtration and ion-exchange column chromatography, and PAGE revealed the homogeneity purification status of the enzyme. The properties of the enzyme included an acidic pH optima (4.8), alkaline pI (8.7), meso-thermostabity, monomeric structure with subunit molecular weight of about 50 kDa, high affinity for substrate (K _m) for pNPG (0.19 mM) and high (105,263 M^?1 s^?1) catalytic efficiency (K _cat/K _m). The mesostable enzyme had a stringent substrate specificity restricted only to β-linked gluco-conjugate. The enzyme is optimally active at 40 °C with 12.4 kcal Mol^?1 activation energy, and was highly sensitive to d-gluconic acid lactone inhibition (94 % at 1 mM) with an apparent K _i 0.21 mM. The enzyme could catalyze transglucosylation of geraniol with pNPG as glucosyl donor, but not with cellobiose. Some of the physico-kinetic properties were noted to be novel when comprehensively compared with its counterparts from plant, animal and microbial counterparts. Nevertheless, the catalytic and other features of the enzyme were relatively closer to Oryza sativa among plants and Talaromyces thermophillus among fungi. Significance of building-up of a library of novel plant β-glucosidases for structural investigation to understand naturally evolved mechanistics of catalysis has been indicated.     

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional ‘single target–single drug’ approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3’-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC₅₀ and EC₅₀ values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.     

Switching DNA-binding specificity by unnatural amino acid substitution

The specificity of protein–nucleic acid recognition is believed to originate largely from hydrogen bonding between protein polar atoms, primarily side-chain and polar atoms of nucleic acid bases. One way to design new nucleic acid binding proteins of novel specificity is by structure-guided alterations of the hydrogen bonding patterns of a nucleic acid–protein complex. We have used cI repressor of bacteriophage λ as a model system. In the λ-repressor–DNA complex, the -NH₂ group (hydrogen bond donor) of lysine-4 of λ-repressor forms hydrogen bonds with the amide carbonyl atom of asparagine-55 (acceptor) and the O6 (acceptor) of CG6 of operator site O_L1. Substitution of lysine-4 (two donors) by iso-steric S-(2-hydroxyethyl)-cysteine (one donor and one acceptor), by site-directed mutagenesis and chemical modification, leads to switch of binding specificity of λ-repressor from C:G to T:A at position 6 of O_L1. This suggests that unnatural amino acid substitutions could be a simple way of generating nucleic acid binding proteins of altered specificity.     

A review on fisheries and conservation status of Asian horseshoe crabs

Horseshoe crabs are the only extant xiphosurans and are believed to be morphologically unchanged for more than 200 million years. Of the four extant species namely, Limulus polyphemus, Tachypleus tridentatus, Tapinauchenius gigas and Carcinoscorpius rotundicauda, the latter three are found in Asian waters. Recent evidences showed that Asian horseshoe crabs are facing serious threats such as degradation of their spawning grounds and habitat, environmental pollution, overexploitation as a culinary delicacy and biomedical bleeding practices. Baseline data on the distribution and existing population of the wild horseshoe crabs remain poorly known in several Asian regions. Several studies have clearly revealed that pressure due to over-fishing of wild stock has increased tremendously in the last decade. Due to an increase in demand for Tachypleus Amebocyte Lysate (TAL) analogous to Limulus Amebocyte Lysate (LAL) in the United States, there is an urgent need to comprehensively address their fishing and conservation measures in the Asian region. This review addresses the overall studies on three species of Asian horseshoe crabs in relation to their fishing practices, local exploitation of their wild stock either for human consumption (or) by biomedical industries. The authors have structured the discussion on an international scale to address the existing problems in fishing and conservation of horseshoe crabs. Since no specific regulatory force or legislative protection act or a policy to preserve their natural stock are available to this date, this paper strongly recommends representative countries to include horseshoe crabs under their wildlife protection act to avoid further unsustainable exploitation of their wild populations.