Cherukuri R. Babu

Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: Insight into structural and antimalarial features

A potent serine proteinase inhibitor was isolated and characterized from the seeds of the tropical legume liana, Derris trifoliata (DtTCI) by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. SDS-PAGE as well as MALDI-TOF analysis showed that DtTCI is a single polypeptide chain with a molecular mass of approximately 20 kDa. DtTCI has three isoinhibitors (pI: 4.55, 5.34 and 5.72) and, inhibited both trypsin and chymotrypsin in a 1:1 molar ratio. Both Dixon plots and Lineweaver-Burk double reciprocal plots revealed a competitive inhibition of trypsin and chymotrypsin activity, with inhibition constants (K(i)) of 1.7x10(-10) and 1.25x10(-10) M, respectively. N-terminal sequence of DtTCI showed over 50% similarity with numerous Kunitz-type inhibitors of the Papilionoideae subfamily. High pH amplitude and broad temperature optima were noted for DtTCI, and time course experiments indicated a gradual loss in inhibitory potency on treatment with dithiothreitol (DTT). Circular Dichroism (CD) spectrum of native DtTCI revealed an unordered structure whereas exposure to thermal-pH extremes, DTT and guanidine hydrochloride (Gdn HCl) suggested that an abundance of beta-sheets along with intramolecular disulfide bonds provide conformational stability to the active site of DtTCI, and that severity of denaturants cause structural modifications promoting inhibitory inactivity. Antimalarial studies of DtTCI indicate it to be a potent antiparasitic agent.

Isolation and characterization of a lectin from peanut roots

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephadex G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4-5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose.

Diversity among rhizobiophages from rhizospheres of legumes inhabiting three ecogeographical regions of India

Diversity in rhizobiophages from rhizosphere soils of different legumes inhabiting eight localities in the Delhi, Jaisalmer Plateau and Great Nicobar ecogeographical regions of India was assessed using 14 rhizobial indicator strains that nodulate three species of Sesbania (S. sesban, S. aegyptica and S. rostrata). Phage activity was detected in 47% of the soil samples tested. Most of the positive responses were observed in soils of localities inhabited by host legumes of indicator bacterial strains and their close relatives. The results suggest that rhizobiophages are maintained in the vicinity of their target host populations and are spread through the man-made introduction of host legumes and their rhizobia. Diversity among phage isolates was assessed by studying the plaque traits on their indicator bacterial strains, and variation in morphology using transmission electron microscopy. Except for plaque size, the variation in the plaque traits was rather narrow. Based on the variability pattern, the 64 phages were grouped into 11 plaque types. In contrast to plaque characteristics, the diversity in morphological characters was high and phage isolates differ from the rhizobiophages reported from Indian soils; the Indian phages of Azorhizobium caulinodans strain ORS571 are different from the Senegal isolates. Based on the morphological diversity, the phages are grouped into Bradleys basic four phage groups (A1, B1, B2 and C1) and three families, Myoviridae, Siphoviridae and Podoviridae. This pattern of grouping is reflected in cluster formation based on the variability in plaque and morphological characters. The host range of the phage isolates is rather narrow and restricted to their indicator strains and their close relatives due to marked genetic divergence among rhizobia due to legume host specialization and ecological conditions. The phages can be used for typifying closely related stem-nodulating rhizobia and other rhizobia, which are promiscuous within sesbanias.

Purification, characterization and immunolocalization of a novel protease inhibitor from hemolymph of tasar silkworm, Antheraea mylitta.

A novel serine protease inhibitor (AmPI) was purified from larval hemolymph of tasar silkworm, Antheraea mylitta by two-step process of trypsin-affinity and gel-filtration (FPLC) chromatography. AmPI was active against larval midgut and commercial bovine trypsin and chymotrypsin. The extent of purification was determined by SDS and Native PAGE. The protease inhibitor had an apparent molecular weight of approximately 14.5 kDa as determined by SDS-PAGE. Its activity was stable over a pH range of 4.5-9 and temperatures range of 4-65 degrees C. Molecular weight as determined by MALDITOF-MS was between 13241.63 and 13261.66 Da. MS profile of AmPI also suggests two isoforms of AmPI because of glycosylation by heptose (C(7)H(14)O(7)). This confirmed the result of Native PAGE showing two bands. N-terminal amino acid sequence of this protein did not show similarity to any known protease inhibitor. To study the functional implications of AmPI in insect, it was localized in insect body tissue of different larval instars by immunogold labeling technique using GAR-gold conjugate as secondary antibody. The pattern of localization suggests constitutive nature of AmPI, which may have role in insects defense mechanism.

Phage specificity and lipopolysaccarides of stem- and root-nodulating bacteria (Azorhizobium caulinodans, Sinorhizobium spp., and Rhizobium spp.) of Sesbania spp.

Phage susceptibility pattern and its correlation with lipopolysaccharide (LPS) and plasmid profiles may help in understanding the phenotypic and genotypic diversity among highly promiscuous group of rhizobia nodulating Sesbania spp.; 43 phages were from two stem-nodulating bacteria of S. rostrata and 16 phages were from root-nodulating bacteria of S. sesban, S. aegyptica and S. rostrata. Phage susceptibility pattern of 38 Sesbania nodulating bacteria was correlated with their LPS rather than plasmid profiles. Different species of bacteria (A. caulinodans- ORS571, SRS1-3 and Sinorhizobium saheli- SRR907, SRR912) showing distinct LPS subtypes were susceptible to different group of phages. Phages could also discriminate the strains of Si. saheli (SSR312, SAR610) possessing distinct LPS subtypes. Phages of Si. meliloti (SSR302) were strain-specific. All the strains of R. huautlense having incomplete LPS (insignificant O-chain) were phage-resistant. In in vitro assay, 100% of the phages were adsorbed to LPS of indicator bacterium or its closely related strain(s) only. These observations suggest the significance of LPS in phage specificity of Sesbania nodulating rhizobia. Highly specific phages may serve as biological marker for monitoring the susceptible bacterial strains in culture collections and environment.

Ecological Restoration of Lantana-Invaded Landscapes in Corbett Tiger Reserve, India

Lantana (Lantana camara), one of the worlds most troublesome invasive weeds, has become a menace in most of the protected areas located in tropical and subtropical belt of India. The lantana-infested landscapes not only are impoverished as habitats of wildlife but also contribute to human-wildlife conflicts owing to diminished ecosystem services. This paper is a case study of successful eradication and restoration of two lantana-invaded sites in Corbett Tiger Reserve, India. A method for eradicating lantana was developed using knowledge about its ecology, and, subsequently, weed-free landscapes were restored to productive grasslands and mixed woodlands using native species. The restoration of these areas to grassland communities has successfully prevented secondary invasions by lantana and other weeds and has enhanced the habitat quality for herbivores whose populations are vital for the survival of top carnivores such as tiger (Panthera tigris corbetti).

Structure of a novel ribosome-inactivating protein from a hemi-parasitic plant inhabiting the northwestern Himalayas.

This is the first report of the structural studies of a novel ribosome-inactivating protein (RIP) obtained from the Himalayan mistletoe (Viscum album) (HmRip). HmRip is a type II heterodimeric protein consisting of a toxic enzyme (A-chain) with an active site for ribosome inactivation and a lectin subunit (B-chain) with well defined sugar-binding sites. The crystal structure of HmRip has been determined at 3.8 A resolution and refined to a crystallographic R factor of 0.228 (R(free) = 0.271). A comparison of this structure with other type II RIPs reveals the presence of distinct structural features in the active site of the A-chain and in the 2gamma sugar-binding site of the B-chain. The conformation of the side chain of Tyr110, which is a conserved active-site residue in the A subunit, is strikingly different from those observed in other mistletoe RIPs, indicating its unique substrate-binding preference. The deletion of two important residues from the kink region after Ala231 in the 2gamma subdomain of the B-chain results in a significantly different conformation of the sugar-binding pocket. A ribosome-recognition site has also been identified in HmRip. The site is a shallow cavity, with the conserved residues Arg51, Asp70, Thr72 and Asn73 involved in the binding. The conformations of the antigenic epitopes of residues 1-20, 85-103 and 206-223 differ from those observed in other type II RIPs, resulting in the distinct antigenicity and pharmacological properties of HmRip.

Antifungal activity of some Himalayan medicinal plants and cultivated ornamental species

Extracts of roots of Rumex nepalensis, Berberis aristata, Arnebia benthamii, bark of Taxus wallichiana, Juglans regia and petals of Jacquinia ruscifolia were tested for their antifungal activity against twelve different fungal pathogens. Ethanolic extracts of R. nepalensis and J. ruscifolia extracts showed a broad spectrum of activity.

Nutritional potential ofVigna minima (Roxb.) Ohwi and Ohashi

The seed protein content and amino acid composition of 14 natural populations and their three-generation progenies (grown in different locations) belonging toVigna minima (Roxb.) Ohwi & Ohashi and ofV. umbellata cv IC 1568 have been investigated. The populations ofV. minima were sampled from different ecozones of Western Ghats of Kerala and Tamil Nadu (India). The range of variation in protein levels is narrow, but the protein content of the coastal population is higher than the rice bean suggesting its breeding potential for high protein and salt tolerant lines of rice bean. Although the seed protein content shows genotype × environment interaction, there is a substantial genetic variability among the populations. The tenuous relationship between protein content and yield conponents suggest the presence of correlation breakers which can be utilized in breeding programmes of rice bean. There is a broad genetic base in the levels of essential amino acids, and the range of variation observed is higher than that recorded for different species ofVigna andPhaseolus. The wild relative is nutritionally as good as or superior to the cultigen.

Variability in Foliar Essential Oils among Different Morphotypes of Lantana Species Complexes, and Its Taxonomic and Ecological Significance

The genus Lantana has many species complexes, and L. camara is one of the aggressive alien weedy species complexes; species delimitation in these complexes is a nightmare for taxonomists. We examined the diversity in the chemical composition of foliar essential oils among morphotypes of Lantana species complexes inhabiting the same ecological gradient, and its taxonomic and ecological significance. The yields of essential oils varied from 0.1 to 0.79% in foliar hydrodistillates of eleven morphotypes, and a total of 39 chemical constituents were detected by GC/MS. The quantitative and qualitative variability in the composition of essential oils among morphotypes was very high, and hence they represent chemotypes. The diversity observed in the composition of essential oils appears to be of genetic origin and thus of taxonomic value. The formation of distinct clusters and sub‐clusters at high distance cluster combine values also substantiates that the patterns of distribution of chemical constituents among morphotypes can be used in delimiting species and infraspecific taxa within the species complexes. The presence of β‐caryophyllene and other such compounds, which are known to prevent herbivory, in morphotypes of Lantana species complexes suggest that these compounds may provide selective advantage to Lantana over native species in the invasion of new and disturbed habitats.