Purification and biochemical characterization of a highly active cysteine protease ervatamin A from the latex of Ervatamia coronaria

Ervatamia coronaria, a flowering plant (family Apocynaceae) indigenous to India, has medicinally important applications. A search for biochemical constituents of the latex of the plant yielded at least three thiol proteases with distinctly different properties. One of them, a highly active protease (ervatamin A), was purified to homogeneity by ion exchange and gel filtration chromatography. The enzyme exhibited high proteolytic activity toward natural substrates and amidolytic activity toward synthetic substrates. The pH and temperature optima for proteolytic activity were 8–8.5 and 50–55°C, respectively. Proteolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. The estimated molecular mass of the enzyme was 27.6 kDa. The extinction coefficient (1% 280) of the enzyme was estimated as 21.9, and the protein molecule consists of 8 tryptophan, 11 tyrosine and 7 cysteine residues. Isoelectric point of the purified enzyme was 8.37. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and a typical color in ELISA. The N-terminal sequence of the enzyme showed conserved amino acid residues to other plant cysteine proteases. Ervatamin A shows high activity in relation to the other thiol proteases isolated from the same source.     

Foraging ecology of aphidophagous predators against Aphis punicae Passerini

Foraging behaviour of three aphidophagous predators, viz., Cheilomenes sexmaculata (Fabricius), Scymnus sp. (Coleoptera: Coccinellidae) and Paragus serratus (Fabricius) (Diptera: Syrphidae) on prey Aphis punicae Passerini (Homoptera: Aphididae) was studied in the laboratory at Indian Institute of Horticultural Research, Bangalore (12 degrees 58' N; 77 degrees 35'E) during 2002-03 to understand the predation dynamics. The foraging studies of grubs of C. sexmaculata and Scymnus sp. on A. punicae revealed that the distance travelled to reach the food (aphids) was negatively correlated with the feeding time of prey (r = -0.434 and -0.743, respectively). The time taken for second encounter was positively correlated with feeding time in case of both C. sexmaculata (r = 0.715) and P.serratus (r = 0.641). The time taken for first encounter of prey by Scymnus sp. showed negative correlation with the feeding time (r = -0.9491) and positive correlation with the distance traveled by the predator to reach the food (r = 0.620). The foraging behavioural studies of syrphid, P. serratus on A. punicae showed that the time taken for first encounter of prey was positively correlated with both the feeding time (r = 0.467) and the distance travelled by the syrphid to reach the food (r = 0.485). The time taken for first encounter of prey also showed significant positive correlation with the search time of the prey (r = 0.384).     

Induced mutagenesis in Jatropha curcas L. using gamma rays and detection of DNA polymorphism through RAPD marker

The aim of this study is to examine the effect of different doses (control, 5, 10, 15, 20 and 25 Kr) of gamma irradiation on seed germination, flowering, fruit and seed traits of Jatropha curcas and to identify DNA polymorphism among the mutants through a Randomly Amplified Polymorphic DNA (RAPD) marker analysis. The improved agronomic traits such as flowering, fruits and seeds were recorded in 5 Kr dose and seed germination percentage in 10 Kr dose treated plants, while corresponding parameters were reduced significantly (P>0.05) in 25 Kr dose gamma rays treated plants when compared to that of control. All the twenty-three random primers used except six primers, namely OPAW16, OPAK07, OPAK15, OPS01, OPAK20 and OPAL09 were showed polymorphic bands. The primers: OPAW16, OPAK07, OPAK15, OPS01, OPAK20 and OPAL09 produced only one band each across the six mutants, while the primers: OPU13, OPAB 15, OPF01 and OPAB11 were produced with maximum number of bands (8). The number of amplicons varied from 1 to 8 with an average of 3.9 bands, of which 2.3 were polymorphic. The percentage of polymorphism per primer ranged from 0 to 100 with an average of 55.16%. The Jaccard's coefficients of dissimilarity varied from 0.324 to 0.397, indicative of the level of genetic variation among the mutants studied. The maximum dissimilarity value (0.397) was observed in 5 Kr mutant while the minimum value (0.250) was observed in 20 Kr mutant when compared to that of control. In a dendrogram constructed based on genetic similarity coefficients, the mutants were grouped into three main clusters; (a) control, 10, 15 and 20 Kr dose mutants clustered together, (b) 25 Kr dose grouped alone, (c) 5 Kr dose also grouped alone. The mutants showing the differences in morphological traits showed DNA polymorphism in PCR profile amplified by RAPD marker. It is concluded that DNA polymorphism detected by RAPD analysis offered a useful molecular marker for the identification of mutants in gamma radiation treated plants.     

Resolvin D1 prevents TNF-α-mediated disruption of salivary epithelial formation

Sj?gren's syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sj?gren's syndrome.     

Carbohydrate Hydrolysis and Transport in the Extreme Thermoacidophile Sulfolobus solfataricus

Extremely thermoacidophilic microbes, such as Sulfolobus solfataricus, are strict chemoheterotrophs despite their geologic niche. To clarify their ecophysiology, the overlapping roles of endoglucanases and carbohydrate transporters were examined during growth on soluble cellodextrins as the sole carbon and energy source. Strain-specific differences in genome structure implied a unique role for one of three endogenous endoglucanases. Plasmid-based endoglucanase expression promoted the consumption of oligosaccharides, including cellohexaose (G6) through cellonanaose (G9). Protein transporters required for cellodextrin uptake were identified through mutagenesis and complementation of an ABC transporter cassette, including a putative oligosaccharide binding protein. In addition, ablation of the binding protein compromised growth on glucose and alpha-linked oligosaccharides while inactivation of a previously described glucose transporter had no apparent impact. These data demonstrate that S. solfataricus employs a redundant mechanism for soluble cellodextrin catabolism having both substrate uptake and extracytoplasmic hydrolytic components.     

Bio-Fuel Crops Research for Energy Security and Rural Development in Developing Countries

Soaring prices of fossil fuels, geo-political issues and environmental pollution associated with fossil fuel use has led to worldwide interest in the production and use of bio-fuels. Both the developed and developing countries have developed a range of policies to encourage production of combustible fuels from plants that triggered public and private investments in bio-fuel crop research and development, and bio-fuels production. In this article, we discuss the potential benefits of bio-fuels in increasing the farmers’ incomes, reducing environment pollution, the crop options and research and development interventions required to generate feedstocks to produce bio-fuels to meet projected demand without compromising food/fodder security in developing countries.     

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU,a regulatory switch involved in type III secretion

Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single‐wavelength anomolous dispersion and refined with X‐ray diffraction data extending up to atomic resolution (1.13 Å). These crystallographic studies provide structural insights into the conformational changes induced upon auto‐cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263‐P264 peptide bond cleavage is found on a β‐turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N‐terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto‐cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU.     

Production and partial purification of cellulase from a novel fungus,Aspergillus flavus BS1

This study describes the isolation and characterization of a novel fungus, Aspergillus flavus BS1 and its cellulolytic activities with special emphasis on endoglucanase production. Preliminary screening studies showed that A. flavus BS1 was a potent strain for the production of cellulase. To study the cellulolytic activities in detail by submerged fermentation (SmF), productions of endoglucanase, exoglucanase, and β-glucosidase were estimated from the basal salt medium (BSM) supplemented with 1 % carboxy methyl cellulose (CMC). CMC medium supported the maximum yield of endoglucanase (2,793 U/ml) on day 5 of incubation at 28 °C and 150 rpm, which was higher than that obtained with naturally available supplements (flour) from banana, tapioca, potato, or banana peel. During cellulase production by solid-state fermentation, 10 % (w/w) tapioca flour in sawdust (teak wood) moisturized with BSM (1:2, w/v) supported maximum cellulase yield (5,408 U/g dry substrate) on day 3 at 28 °C, which was 2-fold higher than that obtained during SmF. The active cellulase was qualitatively estimated by polyacrylamide gel electrophoresis (PAGE). Native-PAGE (0.25 % CMC impregnated on the 10 % gel) activity staining with congo-red showed a clear zone for CMCase activity, whereas SDS-PAGE showed a distinct band. In conclusion, this study showed that A. flavus strain BS1 is a potent strain for the production of cellulase on lignocellulosic media, the hot enzyme for bioethanol production from the lignocellulosic biomass by SSF.