Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

Loss of the SWI/SNF ATPase subunits BRM and BRG1 drives lung cancer development.

Inactivation of Brg1 and Brm accelerated lung tumor development, shortened tumor latency, and caused a loss of differentiation. Tumors with Brg1 and/or Brm loss recapitulated the evolution of human lung cancer as observed by the development of local tumor invasion as well as distal tumor metastasis, thereby making this model useful in lung cancer studies. Brg1 loss contributed to metastasis in part by driving E-cadherin loss and Vimentin up-regulation. By changing more than 6% of the murine genome with the down-regulation of tumor suppressors, DNA repair, differentiation and cell adhesion genes, and the concomitant up-regulation of oncogenes, angiogenesis, metastasis and antiapoptosis genes, caused by the dual loss of Brg1/Brm further accelerated tumor development. Additionally, this Brg1/Brm-driven change in gene expression resulted in a nearly two-fold increase in tumorigenicity in Brg1/Brm knockout mice compared with wild type mice. Most importantly, Brg1/Brm-driven lung cancer development histologically and clinically reflects human lung cancer development thereby making this GEMM model potentially useful.

BRG1 and BRM loss selectively impacts RB and P53, respectively: BRG1 and BRM have differential functions in vivo.

The SWI/SNF complex is an important regulator of gene expression that functions by interacting with a diverse array of cellular proteins. The catalytic subunits of SWI/SNF, BRG1 and BRM, are frequently lost alone or concomitantly in a range of different cancer types. This loss abrogates SWI/SNF complex function as well as the functions of proteins that are required for SWI/SNF function, such as RB1 and TP53. Yet while both proteins are known to be dependent on SWI/SNF, we found that BRG1, but not BRM, is functionally linked to RB1, such that loss of BRG1 can directly or indirectly inactivate the RB1 pathway. This newly discovered dependence of RB1 on BRG1 is important because it explains why BRG1 loss can blunt the growth-inhibitory effect of tyrosine kinase inhibitors (TKIs). We also observed that selection for Trp53 mutations occurred in Brm-positive tumors but did not occur in Brm-negative tumors. Hence, these data indicate that, during cancer development, Trp53 is functionally dependent on Brm but not Brg1. Our findings show for the first time the key differences in Brm- and Brg1-specific SWI/SNF complexes and help explain why concomitant loss of Brg1 and Brm frequently occurs in cancer, as well as how their loss impacts cancer development.

Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency.

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent K(m) and V(max) values of Bafibrinase towards fibrin were determined as 0.24 µM and 2.8 µmol/min, respectively. It showed a K(m) value of 0.139 mM towards a chromogenic substrate for plasmin (D-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,ß-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.

Biodegradable and biocompatible epoxidized vegetable oil modified thermostable poly(vinyl chloride): thermal and performance characteristics post biodegradation with Pseudomonas aeruginosa and Achromobacter sp.

The increased production of municipal solid waste by the disposal of plastic materials heightens the urgency to develop biodegradable materials for daily use. In vitro-biodegradation study on poly(vinyl chloride) (PVC) plasticized by epoxidized Mesua ferrea L. seed oil at three different weight percentages (PVC/ENO ratio of 75/25, 50/50 and 25/75) was conducted by using Pseudomonas aeruginosa and Achromobacter sp. bacteria. The test bacterial species were able to grow on the polymer matrix by using it as a source of energy; however the pristine PVC did not support the microbial growth. The PVC/ENO material of 25/75 ratio showed the highest percent (%) of biodegradation compared to other tested systems. The bacterial count and the dry biomass post 180 days of inoculation in 25/75 plasticized PVC suggested bacterial growth at the expense of degradation of the system. The tensile strength of 25/75 PVC/ENO system, post 180 days of inoculation by Pseudomonas aeruginosa and Achromobacter sp. decreased by about 53% and 43% respectively. Further, surface erosion phenomenon and structural change of the matrix after bacterial growth, as studied by FTIR and SEM analysis of PVC/ENO of 25/75 ratio exhibited noticeable deterioration post 180 days of inoculation.

Characterization and application of a detergent-stable alkaline α-amylase from Bacillus subtilis strain AS-S01a.

A strain AS-S01a, capable of producing high-titer alkaline α-amylase, was isolated from a soil sample of Assam, India and was taxonomically identified as Bacillus subtilis strain AS-S01a. Optimized α-amylase yield by response surface method (RSM) was obtained as 799.0 U with a specific activity of 201.0 U/mg in a process control bioreactor. A 21.0 kDa alkaline α-amylase purified from this strain showed optimum activity at 55°C and pH 9.0, and it produced high molecular weight oligosaccharides including small amount of glucose from starch as the end product. The K(m) and V(max) values for this enzyme towards starch were determined as 1.9 mg/ml and 198.21 µmol/min/mg, respectively. The purified α-amylase retained its activity in presence of oxidant, surfactants, EDTA and various commercial laundry detergents, thus advocating its suitability for various industrial applications.

A statistical approach for the enhanced production of alkaline protease showing fibrinolytic activity from a newly isolated Gram-negative Bacillus sp. strain AS-S20-I.

An alkaline-fibrinolytic protease-producing bacterial strain (AS-S20-I) isolated from a soil sample in Assam was a Gram-negative rod and grown at temperatures ranging from 25 to 55 °C, and pH 6.5 to 11.0. Taxonomic identification of isolated strain by polyphasic approach (phenotypic characterization, chemotaxonomic properties, and ribotyping data of the strain) suggested that it belongs to the genus Bacillus, for which the name Bacillus sp. strain AS-S20-I (MTCC 8961) was proposed. The initial screening by using Plackett-Burman's design demonstrated that among the tested factors, casein, ammonium sulphate and pH of the medium significantly (p < 0.05) enhanced the protease (fibrinolytic enzyme) yield in submerged fermentation. Further optimization of fibrinolytic protease production by Bacillus. sp. strain AS-S20-I in SmF by applying RSM was achieved as 749.0 × 10(3)UL(-1) in the presence of 3.0% (w/v) casein and 0.12% (w/v) ammonium sulphate at pH 10.9 and 45 °C. This was a 4.0-fold increase in yield compared with that obtained before applying the Plackett-Burman and RSM experimental design. The protease preparation preferentially degraded the fibrin (specific activity 2408.0 ± 70.0 U mg(-1); mean ± S.D.) suggesting that its future application in pharmaceutical industry as thrombolytic and anticancer drugs is highly promising.

Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II.

An alkaline-protease-producing bacterial strain (AS-S24-II) isolated from a soil sample in Assam is a Gram-stain-positive, catalase-positive, endospore-forming rod and grows at temperatures ranging from 30 degrees C to 60 degrees C and salinity ranging from 0% to 7% (w/v) NaCl. Phenotypic characterisation, chemotaxonomic properties, presence of Paenibacillus-specific signature sequences, and ribotyping data suggested that the strain AS-S24-II represents a novel species of the genus Paenibacillus, for which the name Paenibacillus tezpurensis sp. nov. (MTCC 8959) is proposed. Phylogenetic analysis revealed that P. lentimorbus strain DNG-14 and P. lentimorbus strain DNG-16 represent the closest phylogenetic neighbour of this novel strain. Alkaline protease production (598 x 10(3) U l(-1)) by P. tezpurensis sp. nov. in SmF was optimised by response surface method. A laundry-detergent-stable, Ca(2+)-independent, 43-kDa molecular weight alkaline serine protease from this strain was purified with a 1.7-fold increase in specific activity. The purified protease displayed optimum activity at pH 9.5 and 45-50 degrees C temperature range and exhibited a significant stability and compatibility with surfactants and most of the tested commercial laundry detergents at room temperature. Further, the protease improved the wash performance of detergents, thus demonstrating its feasibility for inclusion in laundry detergent formulations.

Ecological significance and some biotechnological application of an organic solvent stable alkaline serine protease from Bacillus subtilis strain DM-04.

An organic solvent stable, alkaline serine protease (Bsubap-I) with molecular mass of 33.1 kDa, purified from Bacillus subtilis DM-04 showed optimum activity at temperature and pH range of 37-45 degrees C and 10.0-10.5, respectively. The enzyme activity of Bsubap-I was significantly enhanced in presence of Fe(2+). The thermal resistance and stability and of Bsubap-I in presence of surfactants, detergents, and organic solvents, and its dehairing activity supported its candidature for application in laundry detergent formulations, ultrafiltration membrane cleaning, peptide synthesis and in leather industry. The broad substrate specificity and differential antibacterial property of Bsubap-I suggested the natural ecological role of this enzyme for the producing bacterium.