Biomimetic fabrication of mineralized composite films of nanosilver loaded native fibrillar collagen and chitosan.

Silver nanoparticles loaded fibrillar collagen-chitosan matrix (CC) was prepared by biomimetic approach by blending silver nanoparticles (tAgNPs), collagen fibril and chitosan hydrogel followed by cross-linking and biomineralization. Electron micrograph showed that the surface of the composites exhibited native fibrillar morphology of collagen and their cross-section revealed layer-like arrangement of native fibrillar collagen. The mineralized composites exhibited surface mineralization of calcium phosphates incorporated with magnesium. FT-IR ATR analysis revealed the uniform blending of collagen and chitosan without any chemical interaction between them. XRD analysis showed incorporation of silver nanoparticles and lamellar structure of collagen and chitosan. The mechanical property of the dry composite film showed increase in tensile strength with the addition of chitosan and raised to 4.6 fold in M-CC4 composite. The incorporation of chitosan in M-CC3 led to 2.2 fold increase in mineralization as confirmed by the TGA analysis. Contact angle analysis revealed the hydrophilic nature of the composite. Hemolysis analysis of the composites verified the hemocompatible nature of composites with hemolysis < 5%. MTT assay for the composites was carried by seeding MG-63 cells and indicated cell viability > 80%. Antibacterial activity analysis showed the percent growth inhibition of about 27% and 37% for S. aureus and E. coli respectively. The prepared composite would possess silver nanoparticles loaded collagen fibril in the native state and the formed biomineral will be similar to the bone mineral. Hence the fabricated composite -could be used as a biomaterial for bone tissue engineering applications.

Antioxidant, DNA interaction, VEGFR2 kinase, topoisomerase I and in vitro cytotoxic activities of heteroleptic copper(II) complexes of tetrazolo[1,5-a]pyrimidines and diimines.

A series of heteroleptic mononuclear copper(II) complexes of the type [Cu(L(1-3))(diimine)]ClO4 (1-6) containing three tetrazolo[1,5-a]pyrimidine core ligands, ethyl 5-methyl-7-(2-hydroxyphenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(1)), ethyl 5-methyl-7-(4-diethylamino-2-hydroxyphenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(2)) or ethyl 5-methyl-7-(2-hydroxy-4-nitrophenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(3)), and two diimine coligands, 2,2'-bipyridyl (bpy) or 1,10-phenanthroline (phen) have been synthesized and characterized by spectral methods. The geometry of complexes have been determined with the help of electronic absorption and EPR splitting patterns, which suggest four coordinated square planar geometry around copper(II) ion. The lowering of HOMO-LUMO band gap value of complex 4 implies its higher biological activity compared to other complexes. Antioxidant studies revealed that the complexes possess considerable radical scavenging potency against DPPH. The binding studies of the complexes with calf thymus DNA (CT-DNA) revealed groove mode of binding, which was further supported by docking simulation. The complexes 3 and 4 strongly inhibit the topoisomerase I, and also strongly interact with VEGFR2 kinase receptor via π-π, σ-π and hydrogen bonding interaction. Gel electrophoresis experiments demonstrated the ability of the complexes to cleave plasmid DNA in the absence of activators. In vitro cytotoxic activities of the complexes were examined on three cancerous cell lines such as human lung (A549), cervical (HeLa) and colon (HCT-15), and two normal cells such as human embryonic kidney (HEK) and peripheral blood mononuclear cells (PBMCs). The live cell and fluorescent imaging of cancer cells were observed with acridine orange/ethidium bromide staining assay. All encouraging chemical and biological findings indicate that the complex 4 is a suitable candidate for drug target.

Surface stiffening and enhanced photoluminescence of ion implanted cellulose - polyvinyl alcohol - silica composite.

Novel Cellulose (Cel) reinforced polyvinyl alcohol (PVA)-Silica (Si) composite which has good stability and in vitro degradation was prepared by lyophilization technique and implanted using N(3+) ions of energy 24keV in the fluences of 1×10(15), 5×10(15) and 1×10(16)ions/cm(2). SEM analysis revealed the formation of microstructures, and improved the surface roughness on ion implantation. In addition to these structural changes, the implantation significantly modified the luminescent, thermal and mechanical properties of the samples. The elastic modulus of the implanted samples has increased by about 50 times compared to the pristine which confirms that the stiffness of the sample surface has increased remarkably on ion implantation. The photoluminescence of the native cellulose has improved greatly due to defect site, dangling bonds and hydrogen passivation. Electric conductivity of the ion implanted samples was improved by about 25%. Hence, low energy ion implantation tunes the mechanical property, surface roughness and further induces the formation of nano structures. MG63 cells seeded onto the scaffolds reveals that with the increase in implantation fluence, the cell attachment, viability and proliferation have improved greatly compared to pristine. The enhancement of cell growth of about 59% was observed in the implanted samples compared to pristine. These properties will enable the scaffolds to be ideal for bone tissue engineering and imaging applications.

Anti-leukemic, anti-lung, and anti-breast cancer potential of the microbial polyketide 2, 4-diacetylphloroglucinol (DAPG) and its interaction with the metastatic proteins than the antiapoptotic Bcl-2 proteins.

The microbial polyketide, 2, 4-diacetylphloroglucinol (DAPG), exhibited a broad-spectrum of anti-leukemic, anti-lung, and anti-breast cancer properties. The aim of the present investigation was to study the interactive potentials of DAPG with the metastatic proteins such as MMP-2, MMP-9, and NF-κB and antiapoptotic Bcl-2 family proteins such as Bcl-2, Bcl-w, and Bcl-xL through in silico interaction and in vitro studies. The in silico modeling predicted high interactions of DAPG with the metastatic proteins, especially MMP-2, MMP-9, and NF-κB with the glide score of -7.028, -6.304, and -5.231, respectively. Similarly, the DAPG had weak interactions with the antiapoptotic Bcl-2, Bcl-w, and Bcl-xL with the glide score of -4.505, -3.839, and -4.003, respectively. The interaction studies further revealed the inhibition of MMP-2, MMP-9, and NF-κB activities with the low IC50 concentration of 5.82 ± 1.6, 6.74 ± 1.2, and 10.7 ± 1.5 µM respectively, in the presence of DAPG. Similarly, DAPG inhibited the Bcl-2, Bcl-xL and Bcl-w activities with the high IC50 concentration of 29.8 ± 1.9, 85.9 ± 2.7, and 97.4 ± 1.5 µM, respectively. These results correlate with the relatively high IC50 concentration of 16.3 ± 1.76, 7.67 ± 0.78, and 10.7 ± 0.96 µM in the Bcl-2-overexpressing HL-60, K562 and Raji leukemic cells than the metastatic A549 and MDA MB-231 cancer cells with the low IC50 concentration of 0.06 ± 0.02 and 0.08 ± 0.01 µM, respectively, compared to the healthy, human embryonic kidney (HEK-293) cells with the high IC50 concentration of 54.7 ± 1.43 µM. In summary, the affinity of DAPG with proteins are in the order of MMP-2 > MMP-9 > NF-κB > Bcl-2 > Bcl-xL > Bcl-w. Results presented in this study confirmed the high interaction of DAPG with the metastatic proteins than the antiapoptotic Bcl-2 family proteins.

Anticancer potential of pyrrole (1, 2, a) pyrazine 1, 4, dione, hexahydro 3-(2-methyl propyl) (PPDHMP) extracted from a new marine bacterium, Staphylococcus sp. strain MB30.

Marine bacterium, strain MB30 isolated from the deep sea sediment of Bay of Bengal, India, exhibited antimicrobial activity against human pathogenic bacteria. Based on the 16S rRNA sequence homology and subsequent phylogenetic tree analysis, the strain MB30 was identified as Staphylococcus sp. The bioactive metabolite produced by the strain MB30 was purified through silica gel column chromatography and preparative HPLC. Purified metabolite was further characterized by FT-IR, LC-MS and NMR analyses. On the basis of spectroscopic data, the metabolite was identified as pyrrole (1, 2, a) pyrazine 1, 4, dione, hexahydro 3-(2-methyl propyl) (PPDHMP). The PPDHMP exhibited in vitro anticancer potential against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner with the IC50 concentration of 19.94 ± 1.23 and 16.73 ± 1.78 µg ml(-1) respectively. The acridine orange (AO)/ethidium bromide (EB) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining of the IC50 concentration of PPDHMP-treated cancer cells exhibited an array of morphological changes such as nuclear condensation, cell shrinkage and formation of apoptotic bodies. The PPDHMP-treated cancer cells induced the progressive accumulation of fragmented DNA in a time-dependent manner. Based on the flow cytometric analysis, it has become evident that the compound was also effective in arresting the cell cycle at G1 phase. Further, the Western blotting analysis confirmed the down-regulation of cyclin-D1, cyclin dependent kinase (CDK-2), anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL), activation of caspase-9 and 3 with the cleavage of PARP. The PPDHMP-treated cancer cells also showed the inhibition of migration and invasive capacity of cancer cells. In the present investigation, for the first time, we have reported the extraction, purification and characterization of an anticancer metabolite, PPDHMP from a new marine bacterium, Staphylococcus sp. strain MB30.

Self-assembly of oxamidato bridged ester functionalised dirhenium metallastirrups: synthesis, characterisation and cytotoxicity studies.

A new set of ester functionalised Re(i)-based oxamidato bridged neutral dinuclear metallacycles were synthesised by self-assembly of four components from three building blocks in a facile one-pot reaction via an orthogonal bonding approach. Oxidative addition of oxamide ligands (H2L = N,N'-diphenyloxamide, and N,N'-dibenzyloxamide) to rhenium carbonyl (Re2(CO)10) in the presence of semi-rigid and flexible ditopic pyridyl ligands (L' = o-phenylene diisonicotinate (pdi), ethane diyl di-4-pyridine carboxylate (etdp) and 1,4-butane diyl di-4-pyridine carboxylate (budp)) having ester functionality afforded neutral dirhenium metallacycles of the general formula [(CO)3Re(µ-L)(µ-L')Re(CO)3] (1-5) under solvothermal reaction conditions. The metallacyclic compounds were characterised using elemental analyses, IR, UV-vis and NMR spectroscopic techniques. Structural analyses of 2-5 by single crystal X-ray diffraction methods revealed a stirrup like molecular framework in which two fac-Re(CO)3 units are bridged together by dissymmetrical NO∩ON bis-chelation of oxamide ligands (as a pedestal of stirrups) and further connected by a flexible ditopic tecton (as an arched anchor of stirrups) in an orthogonal fashion. The cytotoxicity activities of dirhenium metallacycles 1-5 were studied in vitro against three different cancer cell lines and normal cells.

Isolation and characterization of a novel thermostable ß-glucosidase from Bacillus subtilis SU40.

A new bacterial strain SU40 with ability to produce thermostable ß-glucosidase was isolated from soil located at Puducherry (India) and identified as Bacillus subtilis. The conditions like temperature, pH, carbon and nitrogen sources were optimized conditions for the maximum production of ß-glucosidase. The enzyme was purified using ammonium sulphate precipitation and BioSep and HPLC chromatography. Purified enzyme was found to be a monomeric protein with an apparent molecular mass of 38.3 kDa. The enzyme was stable over a wide range of pH and temperatures and exhibited maximal activity at 75°C and pH 12.0. The enzyme was found to retain approximately 60% of maximal activity for 1 h at 70°C and hydrolyzed a wide range of aryl-ß-D-glucosides and ß-linked oligosaccharides with highest activity towards cellobiose and p-nitrophenyl-ß-D-glucopyranoside. The Km and Vmax values of the enzyme were 0.21 µM and 9.38 µmoles mg(-1) min(-1), respectively. The enzyme demonstrated stability in the presence of 0.5% surfactants and was completely inhibited after addition of EDTA and Hg2+ ions. Synergistic action of ß-glucosidase from B. subtilis SU40 with endoglucanase from Paenibacillus barcinonensis MG7 completely degraded the rice straw.

Phenazine-1-carboxamide (PCN) from Pseudomonas sp. strain PUP6 selectively induced apoptosis in lung (A549) and breast (MDA MB-231) cancer cells by inhibition of antiapoptotic Bcl-2 family proteins.

Phenazine-1-carboxamide (PCN), a naturally occurring simple phenazine derivative isolated from Pseudomonas sp. strain PUP6, exhibited selective cytotoxic activity against lung (A549) and breast (MDA-MB-231) cancer cell lines in differential and dose-dependent manner compared to normal peripheral blood mononuclear cells. PCN-treated cancer cells showed the induction of apoptosis as evidenced by the release of low level of LDH, morphological characteristics, production of reactive oxygen species, loss of mitochondrial membrane potential (ΔΨm) and induction of caspase-3. At molecular level, PCN instigates apoptosis by mitochondrial intrinsic apoptotic pathway via the overexpression of p53, Bax, cytochrome C release and activation of caspase-3 with the inhibition of oncogenic anti-apoptotic proteins such as PARP and Bcl-2 family proteins (Bcl-2, Bcl-w and Bcl-xL). The in silico docking studies of PCN targeted against the anti-apoptotic members of Bcl-2 family proteins revealed the interaction of PCN with the BH3 domain, which might lead to the induction of apoptosis due to the inhibition of antiapoptotic proteins. Due to its innate inhibition potential of antiapoptotic Bcl-2 family proteins, PCN may be used as potent anticancer agent against both lung and breast cancer.

Stochastic sampled-data control for synchronization of complex dynamical networks with control packet loss and additive time-varying delays.

This study examines the exponential synchronization of complex dynamical networks with control packet loss and additive time-varying delays. Additionally, sampled-data controller with time-varying sampling period is considered and is assumed to switch between m different values in a random way with given probability. Then, a novel Lyapunov-Krasovskii functional (LKF) with triple integral terms is constructed and by using Jensen's inequality and reciprocally convex approach, sufficient conditions under which the dynamical network is exponentially mean-square stable are derived. When applying Jensen's inequality to partition double integral terms in the derivation of linear matrix inequality (LMI) conditions, a new kind of linear combination of positive functions weighted by the inverses of squared convex parameters appears. In order to handle such a combination, an effective method is introduced by extending the lower bound lemma. To design the sampled-data controller, the synchronization error system is represented as a switched system. Based on the derived LMI conditions and average dwell-time method, sufficient conditions for the synchronization of switched error system are derived in terms of LMIs. Finally, numerical example is employed to show the effectiveness of the proposed methods.

5-Methyl phenazine-1-carboxylic acid: a novel bioactive metabolite by a rhizosphere soil bacterium that exhibits potent antimicrobial and anticancer activities.

A new rhizosphere soil bacterium that exhibits antimicrobial potential against human pathogens was isolated. On the basis of 16S ribosomal RNA nucleotide sequence homology and subsequent phylogenetic tree analysis, the strain PUW5 was identified as Pseudomonas putida. A bioactive metabolite was extracted and purified using silica gel column chromatography and preparative HPLC. Characterization of metabolite was done by employing Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and mass spectroscopy (MS). On the basis of spectroscopic data, the metabolite was structurally elucidated as 5-methyl phenazine-1-carboxylic acid betaine (MPCAB). The MPCAB exhibits selective cytotoxicity towards lung (A549) and breast (MDA MB-231) cancer cell lines in dose-dependent manner with IC50 value of 488.7±2.52 nM and 458.6±2.48 nM respectively. The MPCAB exhibited inhibition of cell viability, DNA synthesis, induced G1 cell cycle arrest and apoptosis in cancer cells. The docking and interaction studies confirmed the binding potential of MPCAB with Bcl-2 than Bcl-xL and Bcl-w proteins. These results strongly suggest that the MPCAB induces apoptosis in A549 and MDA MB-231 cancer cells through mitochondrial intrinsic pathway via activation of caspase-3 and down regulation of Bcl-2 protein.

Silver ion impregnated composite biomaterial optimally prepared using zeta potential measurements.

Biodegradable, antimicrobial composite of various silver ion concentrations was synthesized using zeta potential and isoelectric point measurements, for a controlled release of silver ions, and in addition to assess the effect of protein adsorption with the increase of the silver ion concentration. The interaction between hydroxyapatite (HAp) and silver incorporated hydroxyapatite (AgHAp) with gelatin was increased by optimally adjusting the zeta potential and isoelectric point of the ceramic (HAp and AgHAp), and bio-polymer individually. The electrostatic interactions between the ceramic and biopolymer were confirmed, through shifts in N-H stretching, decrease in the swelling ratio, and increase in the degradation temperature observed by the derivative thermo-gravimetric analysis (DTG). These results substantiate that, the zeta potential is a novel tool to increase the ceramic-biopolymer interaction. Increasing electrostatic interaction between the biopolymer and ceramic, decreases the release of silver ions in the simulated body fluid, due to the controlled degradation of the biopolymer. The isoelectric point decreases with the increase of the silver ion concentration, which evidenced the change in the net surface charge. With the increase of the silver ion concentration, the protein adsorption decreases due to an increase in hydrophilic character of the composite. This study examines the minimum concentration of silver ion essential for maximum protein adsorption, antimicrobial and hemocompatibility. This study provides a novel route to control the release of silver ions by enhancing the ceramic-polymer interaction and estimate the silver ion concentration suitable for protein adsorption. The prepared composite is nontoxic, degradable, and antimicrobial, with the controlled release of silver ions in the simulated body fluid.

Voxel based parallel post processor for void nucleation and growth analysis of atomistic simulations of material fracture.

Molecular dynamics (MD) simulations are used in the study of void nucleation and growth in crystals that are subjected to tensile deformation. These simulations are run for typically several hundred thousand time steps depending on the problem. We output the atom positions at a required frequency for post processing to determine the void nucleation, growth and coalescence due to tensile deformation. The simulation volume is broken up into voxels of size equal to the unit cell size of crystal. In this paper, we present the algorithm to identify the empty unit cells (voids), their connections (void size) and dynamic changes (growth and coalescence of voids) for MD simulations of large atomic systems (multi-million atoms). We discuss the parallel algorithms that were implemented and discuss their relative applicability in terms of their speedup and scalability. We also present the results on scalability of our algorithm when it is incorporated into MD software LAMMPS.

Freeze dried cross linking free biodegradable composites with microstructures for tissue engineering and drug delivery application.

Biodegradable laminated polymer composites of agarose (A), gelatin (G) and hydroxyapatite (HAp) having 3D network of interconnected pores (1-500 µm) were fabricated without using cross linking agents. The incorporation of HAp to A, G and AG had considerable influence on the swelling behaviour, drug release and haemolytic activity. A-HAp scaffolds demonstrated interconnected porosity with extended drug release. G-HAp scaffolds possessed enhanced mechanical property. AG-HAp scaffolds exhibited extended drug delivery, haemocompatibility and efficacy against Gram positive bacteria compared with G-HAp. Hence, AG-HAp composites could be used us a scaffold for tissue engineering and drug delivery system. This method provides non toxic, versatile and cost effective biodegradable scaffolds which could be used for various biomedical applications.

Characterization of chryseobacterium aquaticum strain PUPC1 producing a novel antifungal protease from rice rhizosphere soil.

Strain PUPC1 produces an antifungal protease as well as plant growth promoting enzymes such as 1- aminocyclopropane-1-carboxylate (ACC) deaminase and phosphatase. Morphological, cultural, and physiological characteristics as well as 16S rRNA gene-sequence-based phylogenetic analysis confirmed the taxonomic affiliation of PUPC1 as Chryseobacterium aquaticum. The optimum growth of PUPC1 was observed at pH 6.0 and 30 degrees , and maximum protease production was observed in medium B amended with 1% tryptone, 0.5% sucrose, and 0.005% MnCl2. The protease was purified by ammonium sulfate precipitation, Sephadex G-75 gel filtration chromatography, and electroelution from preparative SDS-PAGE. The protease had a molecular mass of 18.5 kDa. The optimum pH and temperature stability of the protease were pH 5.0- 10.0 and temperature 40-70 degrees . Chryseobacterium aquaticum PUPC1 and its protease showed a broad-spectrum antifungal activity against phytopathogenic fungi. Strain PUPC1 also exhibited plant growth promoting traits. The objective of the present investigation was to isolate a strain for agricultural application for plant growth promotion and biocontrol of fungal diseases.

Characterization of a new antifungal lipid transfer protein from wheat.

Lipid transfer proteins (LTPs) are members of the family of pathogenesis-related proteins (PR-14) that are believed to be involved in plant defense responses. In this study, a novel gene Ltp 3F1 encoding an antifungal protein from wheat (Sumai 3) was subcloned, overexpressed in Escherichia coli BL-21 (DE3) and enriched using ammonium sulfate fractionation followed by gel permeation chromatography. Molecular phylogeny analyses of wheat Ltp 3F1 gene showed a strong identity to other plant LTPs. Predicted three-dimensional structural model showed the presence of 6 alpha-helices and 9 loop turns. The active site catalytic residues Gly30, Pro50, Ala52 and Cys55 may be suggested for catalyzing the reaction involved in lipid binding. SDS-PAGE analysis confirmed the production of recombinant fusion protein. The LTP fusion protein exhibited a broad-spectrum antifungal activity against Alternaria sp., Rhizoctonia solani, Curvularia lunata, Bipolaris oryzae, Cylindrocladium scoparium, Botrytis cinerea and Sarocladium oryzae. Gene cassette with cyanamide hydratase (cah) marker and Ltp 3F1 gene was constructed for genetic transformation in tobacco. Efficient regeneration was achieved in selective media amended with cyanamide. Transgenic plants with normal phenotype were obtained. Results of PCR and Southern, Northern and Western hybridization analyses confirmed the integration and expression of genes in transgenic plants. Experiments with detached leaves from transgenic tobacco expressing Ltp 3F1 gene showed fungal resistance. Due to the innate potential of broad-spectrum antifungal activity, wheat Ltp 3F1 gene can be used to enhance resistance against fungi in crop plants.

Functional characterization of antagonistic fluorescent pseudomonads associated with rhizospheric soil of rice (Oryza sativa L.).

Antagonistic fluorescent pseudomonads isolated from rhizospheric soil of rice were characterized by 16S rRNA amplicon and fatty acid methyl ester (FAME) analyses. Antagonistic isolates were grown in the fermentation media, and production of antibiotics was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Production of fungal cell-wall-degrading enzymes such as protease, cellulase, pectinase, and chitinase was determined. Dendrogram based on the major and differentiating fatty acids resulted into 5 clusters, viz., cluster I (P. pseudoalcaligenes group), cluster II (P. plecoglossicida group), cluster III (P. fluorescens group), cluster IV (P. aeruginosa group), and cluster V (P. putida group). Characteristic presence of high relative proportions of cyclopropane (17:0 CYCLO w7c) was observed in antagonistic bacteria. Data revealed biodiversity among antagonistic fluorescent pseudomonads associated with the rice rhizosphere. Results presented in this study will help to identify the antagonistic isolates and to determine their mechanisms that mediate antagonism against fungal pathogens of rice.

Heterologous expression of new antifungal chitinase from wheat.

Chitinases (EC 3.2.1.14) have been grouped into seven classes (class I-VII) on the basis of their structural properties. Chitinases expressed during plant-microbe interaction are involved in defense responses of host plant against pathogens. In the present investigation, chitinase gene from wheat has been subcloned and overexpressed in Escherichia coli BL-21 (DE3). Molecular phylogeny analyses of wheat chitinase indicated that it belongs to an acidic form of class VII chitinase (glycosyl hydrolase family 19) and shows 77% identity with other wheat chitinase of class IV and low level identity to other plant chitinases. The three-dimensional structural model of wheat chitinase showed the presence of 10 alpha-helices, 3 beta-strands, 21 loop turns and the presence of 6 cysteine residues that are responsible for the formation of 3 disulphide bridges. The active site residues (Glu94 and Glu103) may be suggested for its antifungal activity. Expression of chitinase (33 kDa) was confirmed by SDS-PAGE and Western hybridization analyses. The yield of purified chitinase was 20 mg/L with chitinase activity of 1.9 U/mg. Purified chitinase exerted a broad-spectrum antifungal activity against Colletotrichum falcatum (red rot of sugarcane) Pestalotia theae (leaf spot of tea), Rhizoctonia solani (sheath blight of rice), Sarocladium oryzae (sheath rot of rice) Alternaria sp. (grain discoloration of rice) and Fusarium sp. (scab of rye). Due to its innate antifungal potential wheat chitinase can be used to enhance fungal-resistance in crop plants.

Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli.

Plant chitinases are pathogenesis-related proteins, which are believed to be involved in plant defense responses to pathogen infection. In this study, chitinase gene from barley was cloned and overexpressed in Escherichia coli. Chitinase (35 kDa) was isolated and purified. Since the protein was produced as insoluble inclusion bodies, the protein was solubilized and refolded. Purified chitinase exerted broad-spectrum antifungal activity against Botrytis cinerea (blight of tobacco), Pestalotia theae (leaf spot of tea), Bipolaris oryzae (brown spot of rice), Alternaria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and Rhizoctonia solani (sheath blight of rice). Due to the potential of broad-spectrum antifungal activity barley chitinase gene can be used to enhance fungal-resistance in crop plants such as rice, tobacco, tea and clover.

Isolation and characterization of a novel banana rhizosphere bacterium as fungal antagonist and microbial adjuvant in micropropagation of banana.

AIM: Isolation and characterization of a bacterial isolate (strain FP10) from banana rhizosphere with innate potential as fungal antagonist and microbial adjuvant in micropropagation of banana. METHODS AND RESULTS: Bacterium FP10 was isolated from the banana rhizosphere and identified as Pseudomonas aeruginosa based on phenotypic, biochemical traits and sequence homology of partial 622-bp fragment of 16S ribosomal DNA (rDNA) amplicon, with the ribosomal database sequences. Strain FP10 displayed antibiosis towards fungi causing wilt and root necrosis diseases of banana. Production of plant growth hormone, indole-3-acetic acid (IAA), siderophores and phosphate-solubilizing enzyme in FP10 was determined. Strain FP10 tested negative for hydrogen cyanide, cellulase and pectinase, the deleterious traits for plant growth. Screening of antibiotic genes was carried out by polymerase chain reaction using gene-specific primers. Amplification of a 745-bp DNA fragment confirmed the presence of phlD, which is a key gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG) in FP10. The antibiotic produced by FP10 was confirmed as DAPG using thin layer chromatography, high performance liquid chromatography and Fourier transform infrared and tested for fungal antibiosis towards banana pathogens. Procedures for encapsulation of banana shoot tips with FP10 are described. CONCLUSIONS: Strain FP10 exhibited broad-spectrum antibiosis towards banana fungi causing wilt and root necrosis. DAPG by FP10 induced bulb formation and lysis of fungal mycelia. Encapsulation of banana shoot tips with FP10 induced higher frequency of germination (plantlet development) than nontreated controls on Murashige and Skoog basal medium. Treatment of banana plants with FP10 enhanced plant height and reduced the vascular discolouration as a result of Fusarium oxysporum f. sp. cubense FOC. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the innate potential of fungal antibiosis by DAPG antibiotic and production of siderophore, plant-growth-promoting IAA and phosphatase, the strain FP10 can be used as biofertilizer as well as a biocontrol agent.

Identification and characterization of IS1112 and IS1113 insertion element sequences in Xanthomonas oryzae pv. oryzae.

Two new insertion sequences (IS1112 and IS1113) were identified in the genome of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice. Three copies of IS1112 were trapped, one containing 1052-bp and the other two with 1055-bp. They all have 25-bp imperfect inverted repeats with a 3-bp duplication at the site of insertion. They contain an open reading frame (ORF) of 317 and 318 amino acid residues, respectively. IS1113 is 1306-bp, contains 25-bp imperfect terminal inverted repeats, and is flanked by a 9-bp direct repeat at the site of insertion. It contains an ORF of 395 amino acid residues.