Soil and water samples were collected from various regions of SIPCOT and nearby Vanappadi Lake, Ranipet, Tamilnadu, India. Based on their colony morphology and their stability during subculturing, 72 bacteria were isolated, of which 14 isolates were actinomycetes. Preliminary selection was carried out to exploit the ability of the microorganisms to utilize sodium cyanate as nitrogen source. Those organisms that were able to utilize cyanate were subjected to secondary screening viz., utilization of sodium cyanide as the nitrogen source. The oxygenolytic cleavage of cyanide is dependent on cyanide monooxygenase which obligately requires pterin cofactor for its activity. Based on this, the organisms capable of utilizing sodium cyanide were tested for the presence of pterin. Thin layer chromatography (TLC) of the cell extracts using n-butanol: 5 N glacial acetic acid (4:1) revealed that 10 out of 12 organisms that were able to utilize cyanide had the pterin-related blue fluorescent compound in the cell extract. The cell extracts of these 10 organisms were subjected to high performance thin layer chromatography (HPTLC) for further confirmation using a pterin standard. Based on the incubation period, cell biomass yield, peak height and area, strain VPW3 was selected and was identified as Bacillus subtilis. The Rf value of the cell extract was 0.73 which was consistent with the 0.74 Rf value of the pterin standard when scanned at 254 nm. The compound was extracted and purified by preparative High Performance Liquid Chromatography (HPLC). Characterization of the compound was performed by ultraviolet spectrum, fluorescence spectrum, Electrospray Ionization-Mass Spectrometry (ESI-MS), and Nuclear Magnetic Resonance spectroscopy (NMR). The compound is proposed to be 6-propionyl pterin (2-amino-6-propionyl-3H-pteridin-4-one).

In this context the present study is aimed at purifying a pteridine derivative and studying its apoptotic effect on a cancer cell line. 6-propionyl pterin have been purified from a native strain of Bacillus subtilis and administered via intra peritoneal injection to DLA induced mice. During the course of the experiment reduction in angiogenesis was visualized. Earlier report (Kim et.al., 2011) clearly suggested that sepiapterin, a precursor of tetrahydrobiopterin inhibits VEGF-A induced HUVEC proliferation and adhesion through down-regulation of VEGFR-2 downstream signaling pathways, independently of NO synthesis and this supported the present finding. Based on our preconceived view that administration of 6-pp enhances the activity of iNOS, thereby enhanced production of nitric oxide (NO), an attempt was made to check the pathway of NO induced apoptosis. The expression of iNOS, p53, caspase 3, and mdm2 gene were checked by RT PCR. But only mdm2 gene expression alone was upregulated. p53 downregulation might be due to Mdm2 which block its transcriptional activity, favours its nuclear export and stimulates its degradation (Chene, 2003). Caspase 3 activation does not participate in Mdm2 induced apoptosis as determined by the protein levels or poly (ADP-ribose) polymerase fragmentation (Dilla et al., 2000). The report of Wolf et al., 1999 strongly suggests that caspase 3 is the primary activator of apoptotic DNA fragmentation. This is inconsistent with our present finding wherein there was absence of DNA fragmentation in apoptotic cells and also expression of caspase 3 was absent. Thus overall mechanism may be initiated by expression of Mdm2 which inturn inhibited p53 expression. Mdm2 might have down regulated Bcl2 followed by a cascade of Caspase 2 and Cyt-c expression. Cyt-c activation might have regulated Caspase -7 and ROCK protein. ROCK is responsible for cell shrinking and membrane blebbing and finally leading to apoptosis. Caspase 3 activation does not participate in Mdm2 induced apoptosis and hence DNA fragmentation is not found. This study was done to evaluate the efficacy of the compound as anticancer agent and whether it induces apoptosis or not. Mechanism of action of drug was predicted from the results obtained. To derive the exact mechanism further study on the signaling proteins has to be performed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B250. Citation Format: S D. Nisshanthini, R. Sukirtha, S. Achiraman, M. Palaniswamy, Angayarkanni Jayaraman. Evaluation of anticancer efficacy of 6-propionylpterin characterized from native isolate Bacillus subtilis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B250.

Aim: Taq DNA polymerase from Thermus aquaticus is a key enzyme in the field of molecular biology that has been mostly used in polymerase chain reaction (PCR). Our aim is to produce standard grade Taq DNA polymerase free from censorious impurities like DNase, DNA and other contaminating proteins. Place and Duration of Study: The experiments were performed at the Molecular Diagnostic Division of Bhat Biotech India (P) Ltd., Bangalore from February 2017 to January 2018. Methodology: The recombinant Taq DNA polymerase clone was confirmed by PCR and DNA sequencing followed by BLAST analysis. The recombinant protein was in soluble form and was expressed by E. coli DH5α strain. The enzyme was extracted using the boil-lysis method and followed by purification with ion exchange chromatography and silica column chromatography to remove the contaminating protein, DNase and DNA.  The yield of the protein was also calculated. Results: In our laboratory, high-quality Taq DNA polymerase was purified using ion exchange chromatography columns and silica column, with a resulting yield of about 45-50 mg/L and the activity was found to be 1.5 U/µL. Conclusion: The use of a silica column to remove the residual DNA is a remarkable step in obtaining an unequalled quality of Taq DNA polymerase.

The presence of topoisomerase II inhibition activities in the intracellular extract of Streptomyces flavoviridis was investigated. One active compound inhibiting relaxation activity of topoisomerase II was determined to be a protein. This active principle was purified to homogeneity by gel filtration followed by ion exchange chromatography. The apparent molecular mass was 42 kDa as determined by SDS-PAGE. MALDI TOF peptide mass fingerprinting analysis confirmed this topoisomerase II inhibitor, as glucose-inhibited division protein A (GidA) by MOWSE score of 72. The effects of purified GidA protein on DNA relaxation and decatenation by topoisomerase II were investigated. It inhibited topoisomerase II activity and acted as a topoisomerase poison that significantly stabilized the covalent DNA-topoisomerase II reaction intermediate "cleavable complex", as observed with etoposide. Collectively, these findings indicate that GidA is a potent inhibitor of topoisomerase II enzyme, which can be exploited for rational drug design in human carcinomas.

Background & objectives: Polymerase chain reaction (PCR) has wide acceptance for rapid identification of pathogens and also for diagnosis of infectious conditions. However, because of economic and expertise constraints, a majority of small or peripheral laboratories do not use PCR. The objective of the present study was to develop a dry-reagent PCR assay as an alternative to conventional PCR to assess its applicability in routine laboratory practice using malB gene for identification of Escherichia coli as a model. Methods: A total of 184 isolates were selected for the study comprising clinical isolates of E. coli and non- E. coli including Shigella sp. and a few other control strains. The DNA was isolated from all the isolates. The isolated DNA as well as the overnight grown bacterial cultures were subjected to both conventional wet PCR and dry-reagent PCR. Results: The genomic DNA isolated from E. coli showed amplification of malB gene in both conventional wet and dry-reagent PCR and the band was observed at 491 bp. In dry-reagent PCR, the overnight grown E. coli cells also showed positive result. The non- E. coli strains other than Shigella sp. showed negative in both conventional wet and dry-reagent PCR. Shigella sp. showed positive in both conventional wet and dry-reagent PCR. Interpretation & conclusions: Considering the elimination of genomic DNA isolation step, and similar results with the conventional wet PCR, dry-reagent PCR may be a good alternative for the conventional wet PCR.