Purification and characterization of ß-galactosidase from newly isolated Aspergillus terreus (KUBCF1306) and evaluating its efficacy on breast cancer cell line (MCF-7).

ß-galactosidases (EC 3.2.1.23) are able to catalyze two different types of reactions, namely hydrolysis and transgalactosylation. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of ß-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from microbial sources including bacteria, yeasts, and filamentous fungi. The enzyme was purified from the crude enzyme using ammonium sulfate precipitation, dialysis, ion exchange chromatography using DEAE cellulose, fast protein liquid chromatography and high performance liquid chromatography. The enzyme was purified with 10.78 -fold with specific activity of 62 U/mg of protein and yield of 28.26%. Molecular weight of ß -galactosidase as estimated by using SDS-PAGE was 42 kDa. Kinetic parameters Km and Vmax for purified enzyme were 0.48 and 0.96 respectively. Further the characterization and kinetic studies of purified enzyme were carried out. The optimum pH and temperature for maximum ß-galactosidase activity were found to be 6, 40 °C, respectively. The present study is aimed to purification, characterization and in vitro efficacy assessment in breast cancer cell line. The ß-galactosidase isolated from Aspergillus terreus was found to be effective in the proliferation of MCF-7 breast cancer cells in vitro. The present study is aimed to purification and characterization of enzyme to assess in vitro efficacy of ß-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Quinazoline derivative from indigenous isolate, Nocardiopsis alba inhibits human telomerase enzyme.

AIM: Aim of this study was isolation and screening of various secondary metabolites produced by indigenous isolates of soil Actinomycetes for human telomerase inhibitory activity. METHODS AND RESULTS: Extracellular extract from culture suspension of various soil Actinomycetes species were tested for telomerase inhibitory activity. The organism which produced telomerase inhibitor was identified by 16S rRNA gene sequencing. The active fraction was purified by HPLC and analysed by GC-MS to identify the compound. In GC-MS analysis, the active principle was identified as 3-[4'-(2″-chlorophenyl)-2'-thiazolyl]-2,4-dioxo-1,2,3,4-tetrahydro quinazoline. The G-quadruplex stabilizing ability of the compound was checked by molecular docking and simulation experiments with G-quadruplex model (PDB ID-1L1H). The selective binding ability of the compound with G-quadruplex over Dickerson-Drew dodecamer DNA structures showed that the compound possess high selectivity towards G-quadruplex. CONCLUSIONS: Quinazoline derivative isolated from an indigenous strain of Nocardiopsis alba inhibited telomerase. Molecular docking and simulation studies predicted that this compound is a strong stabilizer of G-quadruplex conformation. It also showed a preferable binding to G-quadruplex DNA over normal DNA duplex. SIGNIFICANCE AND IMPACT OF THE STUDY: This particular compound can be suggested as a suitable compound for developing a future anticancer drug. The selectivity towards G-quadruplex over normal DNA duplex gives a clue that it is likely to show lower cytotoxicity in normal cells.

Spectral characterization of a pteridine derivative from cyanide-utilizing bacterium Bacillus subtilis - JN989651.

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).

Purification of GidA protein, a novel topoisomerase II inhibitor produced by Streptomyces flavoviridis.

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.

A potent mitogenic lectin from the mycelia of a phytopathogenic fungus, Rhizoctonia bataticola, with complex sugar specificity and cytotoxic effect on human ovarian cancer cells.

A lectin with strong mitogenic activity towards human peripheral blood mononuclear cells (PBMCs) and cytotoxic effect on human ovarian cancer cells has been purified from the mycelium of a phytopathogenic fungus, Rhizoctonia bataticola, using ion exchange chromatography and affinity chromatography on asialofetuin-Sepharose. The lectin, termed RBL, is a tetramer of 11-kDa subunits and has unique amino acid sequence at its blocked N-terminus. The purified RBL was blood group nonspecific and its hemagglutination activity was inhibited by mucin (porcine stomach), fetuin (fetal calf serum) and asialofetuin. Glycan array analysis revealed high affinity binding of RBL towards N-glycans and also the glycoproteins containing complex N-glycan chains. Interestingly, the lectin showed high affinity for glycans which are part of ovarian cancer marker CA125, a high molecular weight mucin containing high mannose and complex bisecting type N-linked glycans as well core 1 and 2 type O-glycans. RBL bound to human PBMCs eliciting strong mitogenic response, which could be blocked by mucin, fetuin and asialofetuin demonstrating the carbohydrate-mediated interaction with the cells. Analysis of the kinetics of binding of RBL to PBMCs revealed a delayed mitogenic response indicating a different signaling pathway compared to phytohemagglutinin-L. RBL had a significant cytotoxic effect on human ovarian cancer cell line, PA-1.

In Vitro Anti-plasmodial activity of Trigonella foenum-graecum L.

Developing countries, where malaria is one of the most prevalent diseases, still rely on traditional medicine as a source for the treatment of this disease. For the present study, Trigonella foenum-graecum L. (fenugreek) were collected from Coimbatore, Tamilnadu, India. The test plant has been used in India by traditional healers for the treatment of fever as well as other diseases. The active principle was extracted out in different solvent systems to assess the anti-plasmodial potential, with an aim that they can further be utilized to formulate drugs. In vitro anti-plasmodial assay of the extracted fractions of fenugreek leaves was carried out using laboratory adapted chloroquine sensitive and resistant Plasmodium falciparum isolates. Schizont maturation inhibition assay was adopted to analyze the potential of the extracts. Ethanol extract (50%) seemed to possess profound anti-plasmodial activity with IC(50) value of 8.75 ± 0.35 µg ml(-1) and 10.25 ± 0.35 µg ml(-1) against chloroquine sensitive and resistant P. falciparum isolates, respectively. Among the investigated six fractions of the plant extracts, two were found to have significant anti-plasmodial activity with IC(50) values <10 µg ml(-1), namely ethanol and butanol extracts. Two extracts chloroform and ethyl acetate showed moderate activity with IC(50) values ranging from 10 to 20 µg ml(-1), and the other two extracts, hexane and water appeared to be inactive with IC(50) values >85 µg ml(-1). In addition, preliminary phytochemical screening of the various extracts indicated the presence of alkaloids, saponin, tannin like phenolic compounds, flavonoids and steroids.