Proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of Leishmania donovani promastigotes

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major parasitic disease prevalent in endemic regions of Bihar in India. In the absence of good chemotherapeutic options, there is a need to develop an effective vaccine against VL which should be dependent on the generation of a T helper type 1 (Th1) immune response. We have shown that soluble proteins from promastigote of a new clinical isolate of L. donovani (2001) ranging from 68 to 97.4 kDa (F2 fraction), induce Th1 responses in the peripheral blood mononuclear cells of cured Leishmania patients and hamsters and also showed significant prophylactic potential. To understand the nature of F2 proteins, it was further characterized using 2-DE, MALDI-TOF and MALDI-TOF/TOF-MS. In all, 63 spots were cut from a CBB stained gel for analysis and data was retrieved for 52 spots. A total of 33 proteins were identified including six hypothetical/unknown proteins. Major immunostimulatory proteins were identified as elongation factor-2, p45, heat shock protein (HSP)70, HSP83, aldolase, enolase, triosephosphate isomerase, protein disulfideisomerase and calreticulin. This study substantiates the usefulness of proteomics in characterizing a complex protein fraction (F2) map of soluble L. donovani promastigote antigen identified as Th1 stimulatory for its potential as vaccine targets against VL.     

Characterization of genetic diversity of some serovars of Bacillus thuringiensis by RAPD

RAPD based fingerprinting of 21 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 19 random decamer primers. A total of 172 polymorphic fragments, ranging in size from 161-2789 bp, were amplified from 13 of the 19 primers. Pairwise genetic similarity analysis revealed very low similarity values, ranging from 3-68%, among the serovars of Bt, indicating high genetic divergence. Nineteen serovars of Bt fell in two major clusters and remaining two formed solitary clusters in the dendogram. Clustering of Bt strains established genetic relatedness between serovars and serotypes. It has been suggested that RAPD analysis can be used for genotypic characterization of Bt to complement flagellar serotyping.     

Antifungal activity of some marine organisms from India,against food spoilage Aspergillus strains

Crude aqueous methanol extracts obtained from 31 species of various marine organisms (including flora land faunal), were screened for their antifungal activity against food poisoning strains of Aspergillus. Seventeen species exhibited mild (+ =zone of inhibition 1–2 mm) to significant (+++ =zone of inhibition 3–5 mm) activity against one or the other strain under experiment. However, extracts of 12 species were active against all the three strains. Organisms like Salicornia brachiata(obligate halophyte), Sinularia leptocladus(Soft coral), Elysia grandifolia (Mollusks),Gorgonian sp. 2 and Haliclona sp. exhibited significant (inhibition zone of 3–5 mm) antifungal activity against one or the other strains. However,extracts of A. ilicifolius, Amphiroa sp.,Poryphyra sp., Unidentified sponge, Suberites vestigium, Sinularia compressa,Sunularia sp., Sinularia maxima, Subergorgia suberosa, Echinogorgia pseudorassopo and Sabellaria cementifera were mild (inhibition zone of 1–2 mm) to moderate(inhibition zone of 2–3 mm) active against the respective strains. The growth of A. japonicuswas significantly inhibited by the extracts ofS. leptocladus (r = 0.992, p < 0.0001)and E. grandifolia (r = 0.989, p < 0.0001).This revised version was published online in October 2005 with corrections to the Cover Date.     

Immunolocalization and functional role of Sclerotium rolfsii lectin in development of fungus by interaction with its endogenous receptor

Many fungi are known to secrete lectins, but their functional roles are not clearly understood. Sclerotium rolfsii, a soilborne plant pathogenic fungus capable of forming fruiting bodies called sclerotial bodies, secrete a cell wall-associated Thomsen-Friedenreich antigen-specific lectin. To understand the functional role of this lectin, we examined its occurrence and expression during development of the fungus. Furthermore, putative endogenous receptors of the lectin were examined to substantiate the functional role of the lectin. Immunolocalization studies using FITC-labeled lectin antibodies revealed discrete distribution of lectin sites at the branching points of the developing mycelia and uniformly occurring lectin sites on the mature sclerotial bodies. During development of the fungus the lectin is expressed in small amounts on the vegetative mycelia and reaching very high levels in mature sclerotial bodies with a sudden spurt in secretion at the maturation stage. Capping of the lectin sites on the sclerotial bodies by lectin antibodies or haptens inhibit strongly the germination of these bodies, indicating functional significance of the lectin. At the maturation stage the lectin interacts with the cell wall-associated putative endogenous receptor leading to the aggregation of mycelium to form sclerotial bodies. The lectin-receptor complex probably acts as signaling molecule in the germination process of sclerotial bodies. Using biotinylated lectin, the receptors were identified by determining the specific lectin binding to lipid components, extracted from sclerotial bodies, and separated on thin-layer chromatograms. Preliminary characterization studies indicated that the receptors are glycosphingolipids and resemble inositolphosphoceramides. These findings together demonstrate the importance of lectin-receptor interactions to explain hitherto speculated functional role of the lectins and also the glycosphingolipids of fungi.     

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.     

High-frequency plant regeneration by <Emphasis Type="Italic">in vitro</Emphasis> shoot proliferation in cotyledonary node explants of grasspea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.     

Heme oxygenase-mediated vasodilation involves vascular smooth muscle cell hyperpolarization

Chronic hypoxia is associated with both blunted agonist-induced and myogenic vascular reactivity and is possibly due to an enhanced production of heme oxygenase (HO)-derived carbon monoxide (CO). However, the mechanism of endogenous CO-meditated vasodilation remains unclear. Isolated pressurized mesenteric arterioles from chronically hypoxic rats were administered the HO substrate heme-l-lysinate (HLL) in the presence or absence of iberiotoxin, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), ryanodine, or free radical spin traps (N-tert-butyl-alpha-phenylnitrone and 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt). The effects of HLL administration on vascular smooth muscle (VSM) membrane potential were assessed in superior mesenteric artery strips in the presence and absence of zinc protoporphyrin IX or iberiotoxin. The vasodilatory responses to exogenous CO were assessed in the presence and absence of ODQ or iberiotoxin. HLL administration produced a dose-dependent vasodilatory response that was nearly eliminated in the presence of iberiotoxin. Neither ODQ, spin traps, nor ryanodine altered the vasodilatory response to HLL, although ODQ abolished the vasodilatory response to S-nitroso-N-acetyl-penicillamine. HLL administration produced a zinc protoporphyrin IX- and iberiotoxin-sensitive VSM cell hyperpolarization. Iberiotoxin and ODQ inhibited the vasodilatory response to exogenous CO. Thus the vasodilatory response to endogenous CO involves cGMP-independent activation of VSM large-conductance Ca2+-activated K+ channels and does not likely involve the formation of Ca2+ sparks emanating from ryanodine-sensitive stores.     

Parameterization and classification of the protein universe via geometric techniques

We present a scheme for the classification of 3487 non-redundant protein structures into 1207 non-hierarchical clusters by using recurring structural patterns of three to six amino acids as keys of classification. This results in several signature patterns, which seem to decide membership of a protein in a functional category. The patterns provide clues to the key residues involved in functional sites as well as in protein-protein interaction. The discovered patterns include a "glutamate double bridge" of superoxide dismutase, the functional interface of the serine protease and inhibitor, interface of homo/hetero dimers, and functional sites of several enzyme families. We use geometric invariants to decide superimposability of structural patterns. This allows the parameterization of patterns and discovery of recurring patterns via clustering. The geometric invariant-based approach eliminates the computationally explosive step of pair-wise comparison of structures. The results provide a vast resource for the biologists for experimental validation of the proposed functional sites, and for the design of synthetic enzymes, inhibitors and drugs.