Purification and characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera

Phenoloxidases are oxidative enzymes, which play an important role in both cell mediated and humoral immunity. Purification and biochemical characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera (Hubner) were carried out to study its biochemical properties. Prophenoloxidase consists of a single polypeptide chain with a relative molecular weight of 85 kDa as determined by SDSPAGE, MALDITOF MS and LCESI MS. After the final step, the enzyme showed 71.7 fold of purification with a recovery of 49.2%. Purified prophenoloxidase showed high specific activity and homology with phenoloxidase subunit-1 of Bombyx mori and the conserved regions of copper binding (B) site of phenoloxidase. Purified prophenoloxidase has pH optima of 6.8 and has high catalytic efficiency towards the dopamine as a substrate in comparison to catechol and L-Dopa. The PO activity was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and kojic acid.

Phyllosphere of Cotton as a Habitat for Diazotrophic Microorganisms

Positive nitrogenase activities ranging from 0.18 to 0.78 nmol of C2H4 cm−2 h−1 were detected on the leaf surfaces of different varieties of cotton (Gossypium hirsutum L. and G. herbaceum L.) plants. Beijerinckia sp. was observed to be the predominant nitrogen-fixing microorganism in the phyllosphere of these varieties. A higher level of phyllosphere nitrogen-fixing activity was recorded in the variety Varalaxmi despite a low C/N ratio in the leaf leachates. Leaf surfaces of the above variety possessed the largest number of hairy outgrowths (trichomes) which entrapped a majority of microbes. Immersion of plant roots in nutrient medium containing 32Pi led to the accumulation of label in the trichome-borne microorganisms, thereby indicating a possible transfer of nutrients from leaf to microbes via trichomes. Extrapolation of acetylene reduction values suggested that 1.6 to 3.2 kg of N ha−1 might be contributed by diazotrophs in the phyllosphere of the variety Varalaxmi during the entire growth period.

Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA

Gemini viral assembly and transport of viral DNA into nucleus for replication, ssentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton LeafCtirl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and Surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K-A, of 2.6 +/- 0.29 x 10(8) M-1 in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes. The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a Crucial role ill Virus assembly and in nuclear transport. (C) 2009 Elsevier Inc.

Role of juvenile hormone in the synthesis and sequestration of vitellogenins in the red cotton stainer, Dysdercus koenigii (Heteroptera : Pyrrhocoridae)

Investigations were carried out to determine the role of juvenile hormone (JH) and 20-hydroxy ecdysone in the synthesis and uptake of vitellogenins, which were earlier identified, purified and characterised, in Dysdercus koenigii. The concentration(s) of vitellogenin(s) in fat body, haemolymph and that of vitellin(s) in ovary were significantly lower after chemical allatectomy at eclosion. In addition, at 70 h after emergence, chemical allatectomy reduced ovarian vitellin concentration, but vitellogenin levels remained normal in the fat body and haemolymph. The haemolymph vitellogenins were not incorporated into oocytes in such insects. Administration of JH-III at 20 h after allatectomy restored vitellogenin levels in the fat body and haemolymph, but the ovary failed to incorporate the available vitellogenins from haemolymph in such insects. However, when JH-III was administered twice, one at 20 h and then at 70 h after allatectomy, vitellogenin concentrations in fat body and haemolymph and also vitellin concentrations in ovary approached control levels. It is suggested that JH has two separate roles, one in vitellogenin synthesis and the other in uptake. 20-hydroxy ecdysone had no apparent role in either vitellogenin synthesis or uptake in D. koenigii. (C) 2000 Elsevier Science Inc. All rights reserved.

Biochemical characterization of C4 protein of cotton leaf curl Kokhran virus-Dabawali

Background: Cotton leaf curl Kokhran Virus-Dabawali (CLCuKV-Dab) is a monopartite begomovirus encoding two proteins V1 and V2 in the virion sense and four proteins Cl, C2, C3 and C4 in the complementary sense. The C4 protein of monopartite begomoviruses has been implicated to play a role in symptom determination and virus movement. The present work aims at the biochemical characterization of this protein. Methods: The C4 protein of CLCuKV-Dab was purified in fusion with GST and tested for the ability to hydrolyze ATP and other phosphate containing compounds. ATPase activity was assayed by using radiolabeled gamma-32P]-ATP and separating the product of reaction by thin layer chromatography. The hydrolysis of other compounds was monitored by the formation of a blue colored phosphomolybdate complex which was estimated by measuring the absorbance at 655 nm. Results: The purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Deletion of a sequence resembling the catalytic motif present in phosphotyrosine phosphatases resulted in 70% reduction in both the activities. Mutational analysis suggested arginine 13 to be catalytically important for the ATPase and cysteine 8 for the pyrophosphatase activity of GST-C4. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4. Conclusions: The residues important for the enzymatic activities of GST-C4 are present in a motif different from the classical Walker motifs and the non-classical ATP binding motifs reported so far. General significance: The C4 protein of CLCuKV-Dab, a putative natively unfolded protein, exhibits enzymatic activities.

Process Development for Functionalization of Cotton with Silver Nanoparticles Synthesized by Bio-based Approaches

Cotton is a widely used raw material for textiles but drawbacks regarding their poor mechanical properties often limit their applications as functional materials. The present investigation involved process development for one step coating of cotton with silver nanoparticles (SNP) synthesized using Azadirachta indica and Citrus limon extract to develop functional textiles. Addition of starch to functional textiles led to efficient binding of nanoparticles to fabric and led to drastic decrease in release of silver from fabricated textiles after ten washing cycles enhancing their environment friendliness. Differential scanning calorimetry, scanning electron microscopy, FT-IR analysis and mechanical studies demonstrated efficient binding of nanoparticles to fabric through bio-based processes. The functionalized textiles developed by the bio-based methods showed significant antibacterial activity against E. coli and S. aureus (with 99% microbial reduction). Present work offers a simple procedure for coating SNP using bio-based approaches with promising applications in specialized functions.

Antibacterial Applications of Silver Nanoparticles Synthesized by Aqueous Extract of Azadirachta Indica (Neem) Leaves

Silver nanoparticles are known to have bactericidal effects. A new generation of dressings incorporating antimicrobial agents like silver nanoparticles is being formulated to reduce or prevent infections. The particles can be incorporated in materials and cloth rendering them sterile. Recently, it was found that aqueous silver ions can be reduced by aqueous extract of plant parts to generate extremely stable silver nanoparticles in water. Apart from being environmentally friendly process, use of Neem leaves extract might add synergistic antibacterial effect of Neem leaves to the biosynthesized nanoparticles. With this hypothesis the biosynthetic production of silver nanoparticles by aqueous extract of Neem leaves and its bactericidal effect in cotton cloth against E. Coli were studied in this work. Silver nanoparticles were synthesized by short term (1 day) and long term (21 days) interaction of Neem extract (20% w/v) and 0.01 M AgNO3 solution in 1:4 mixing ratio. The synthesized particles were characterized by UV visible spectroscopy, transmission electron microscopy, and incorporated into cotton disks by (i) centrifuging the disks with liquid broth containing nanoparticles, (ii) in-situ coating process during synthesis, and (iii) coating with dried and purified nanoparticles. The antibacterial property of the nanoparticles coated cotton disks was studied by disk diffusion method. The effect of consecutive washing of the coated disks with distilled water on antibacterial property was also investigated. This work demonstrates the possible use of biologically synthesized silver nanoparticles by its incorporation in cloths leading them to sterilization.

Gossypol mediated DNA degradation

Gossypol, a polyphenolic compound isolated from cotton plant was found to degrade pBR322 DNA Image in a reaction which required the presence of a metal ion, a reducing agent (2-mercaptoethanol) and oxygen as revealed after agarose gel electrophoresis. Fe3+ and Co2+ showed maximum degradation whereas addition of Ca2+ and Mg2+ prevented the gossypol mediated DNA damage. Gossypol caused degradation of rat liver DNA incubated Image even in the absence of added metal ions and 2-mercaptoethanol. Incubation of intact rat liver nuclei with gossypol reveled DNA degradation and nuclei isolated from rats treated with gossypol Image showed higher succestibility to DNA fragmentation when incubated with gossypol Image than control nuclei. EcoRl and AIuI digestion of DNA isolated from gossypol treated rats gave clear cut evidence for DNA degradation. These observations indicate that gossypol is genotoxic and considereable care has to be exercised in its use. SDS, sodium dodecayl sulphate; TE buffer, Tris-HCL-EDTA buffer.

Bilirubin binding to polypeptides and chiral amines. Induced circular dichroism and fluorescence studies

Induced Cotton effects have been observed in the visible region on interaction of bilirubin with chiral mono- and diamines and poly-l-lysine. At alkaline pH distinct CD spectra are observed for bilirubin bound to the α-helical and β-sheet conformation of poly-l-lysine, which differ from that observed for the pigment bound to human serum albumin. The CD pattern observed on binding to N-acetyl-Lys-N1-methylamide in CH2Cl2 and dioxane is different from that observed in the presence of l-Ala-NH-(CH2)6-NH-l-Ala in dioxane. The latter case resembles the spectrum observed in the presence of human serum albumin. Binding to the helical polypeptide melittin and the antiparallel β-sheet peptide, gramicidin S, in aqueous solutions results in opposite signs of the bilirubin CD bands. The quenching of tryptophan fluorescence in melittin, in aqueous solution and enhancement of bilirubin fluorescence in dioxane on binding to gramicidin S have been used to monitor pigment-peptide interactions. The results suggest the utility of bilirubin as a conformational probe.

Aerodynamic characteristics of pretensioned elastic membrane rectangular sailwings

Presented in the paper are the details of a method for obtaining aerodynamic characteristics of pretensioned elastic membrane rectangular sailwings. This is a nonlinear problem governed by the membrane equation for the inflated sail and the lifting surface theory integral equation for aerodynamic loads on the sail. Assuming an admissible mode shape for the inflated elastic sail, an iterative procedure based on a doublet lattice method is employed to determine the inflated configuration as well as various aerodynamic characteristics. Application of the method is made to a typical nylon-cotton sailwing of AR = 6.0 and results are presented graphically to show the effect of various parameters. The results are found to tend to plane wing values when the pretensions are large in magnitude.

Dihydrofolate reductase from soybean seedlings: A fluorescence and circular dichroism study

The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of −181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes.

Evaluation of anti-inflammatory potential of Chloranthus erectus (Buch.-Ham.) Verd. leaf extract in rats

Aim of the study: Chloranthus erectus (Buch.-Ham.) Verdcourt (Chloranthaceae) is a shrub native to tropical and temperate zone of Eastern Himalaya of India and South-East Asia and have traditionally been used as a folklore medicine to treat localised swelling, joint pain, skin inflammation, fever and bodyache. In this study, an attempthas been made to demonstrate the anti-inflammatory activity of methanol extract obtained from Chloranthus erectus leaves (MECEL) in acute, sub-acute and chronic mouse models. Materials and methods: Inflammation in the hind paw of Wistar albino rat was induced by carrageenan, histamine and serotonin, and tissue granuloma pouch was induced by cotton pellet method. Antiinflammatory drug-phenylbutazone was used as standard drug for comparison. Results: In acute carrageenan-induced rat hind paw edema, oral administration of MECEL at 200 mg/kg produced significant inhibition of edema by 38.34 % (p<0.01) while the histamine- and serotonin-induced sub-acute model, the inhibition of paw edema reached 52.54 % (p < 0.001) and 25.5 % (p < 0.01), respectively. in a 7-day study, MECEL at 20 and 50 mg/kg produced significant suppression of cotton pellet-induced tissue granuloma formation in rats. Conclusions: This preliminary study revealed that the methanol extract of Chloranthus erectus exhibited significant anti-inflammatory activity in the tested models, and may provide the scientific rationale for its popular folk medicine as anti-inflammatory agent.

Experimental study on shrinkage behaviour and prediction of shrinkage magnitudes of residual soils

The present study examines the shrinkage behaviour of residually derived black cotton (BC) soil and red soil compacted specimens that were subjected to air-drying from the swollen state. The soil specimens were compacted at varying dry density and moisture contents to simulate varied field conditions. The void ratio and moisture content of the swollen specimens were monitored during the drying process and relationship between them is analyzed. Shrinkage is represented as reduction in void ratio with decrease in water content of soil specimens. It is found to occur in three distinct stages. Total shrinkage magnitude depends on the type of clay mineral present. Variation in compaction conditions effect marginally total shrinkage magnitudes of BC soil specimens but have relatively more effect on red soil specimens. A linear relation is obtained between total shrinkage magnitude and volumetric water content of soil specimens in swollen state and can be used to predict the shrinkage magnitude of soils.

Mineralogical changes and geotechnical properties of an expansive soil interacted with caustic solution

The type and amount of clay mineral plays an important role in the behaviour of fine-grained soils. Clay minerals are the primary source and moisture is often the external agent of swelling in soils. Also soils may exhibit increased/reduced swelling due to interaction with chemicals. Alkalis used in industrial operations are one such example. Concentrations of alkali and mineral type are the key factors in such interactions. The present paper reports the changes in the properties of an expansive Black Cotton soil containing a mixed layer mineral, rectorite upon interaction with high concentration caustic solutions. X-ray diffraction studies have shown that the rectorite present in the soil undergoes changes with increase in the concentration of alkali. Saponite gets transformed to nantronite. Small amount of kaolinitic mineral present in the soil also reacts with alkali producing some changes in its mineralogy. Many hydroxides are produced. Differential thermal analysis studies have been supportive of these changes. Consequent of these changes, the soil-specific surface increases, changes its Atterberg limits and free swell volume increases. The results have been supported by the characteristics and behaviour of samples contaminated in the field with alkali from an alumina extraction plant.

Optical, Field-Emission, and Antimicrobial Properties of ZnO Nanostructured Films Deposited at Room Temperature by Activated Reactive Evaporation

ZnO nanostructures were deposited on flexible polymer sheet and cotton fabrics at room temperature by activated reactive evaporation. Room-temperature photoluminescence spectrum of ZnO nanostructured film exhibited a week intrinsic UV emission and a strong broad yellow-orange visible emission. TEM and HRTEM studies show that the grown nanostructures are crystalline in nature and their growth direction was indentified to be along [002]. ZnO nanostructures grown on the copper-coated flexible polymer sheets exhibited stable field-emissio characteristics with a threshold voltage of 2.74 V/mu m (250 mu A) and a very large field enhancement factor (beta) of 23,213. Cotton fabric coated with ZnO nanostructures show an excellent antimicrobial activity against Staphylococcus aureus bacteria (Gram positive), and similar to 73% reduction in the bacterial population is achieved compared to uncoated fabrics after 4 h in viability. Using a shadow mask technique, we also selectively deposited the nanostructures at room temperature on polymer substrates.