l-asparaginase induces intrinsic mitochondrial-mediated apoptosis in human gastric adenocarcinoma cells and impedes tumor progression.

l-asparagine essentially regulates growth and proliferation of cancer cells. l-asparaginase is an anti-cancer enzyme that deprives the cancer cells of l-asparagine. The purpose of this study was to explore the mechanism of a novel l-asparaginase from Pseudomonas fluorescens on l-asparagine deprivation mediated anti-proliferation, apoptosis in human gastric adenocarcinoma cells and to evaluate inhibition of angiogenesis. We observed that, the presence of extracellular l-asparagine was essential for the growth of AGS cells. l-asparagine deprivation by l-asparaginase induced metabolic stress, cytotoxicity and apoptosis by G0 phase cell-cycle arrest, modulated the mitochondrial membrane integrity, accelerated caspase-3 activation and instigated DNA damage. The RT-PCR analysis of pro-apoptosis genes: bak1, bax, bbc3, bik, pmaip1, bnip3l, apaf1, casp3, casp7 and casp9 were significantly higher (P < 0.05), while anti-apoptotic markers xiap, bid, mcl1, and death receptor genes tnf and tradd were significantly down-regulated (P < 0.05). Additionally, higher protein expressions of p53, caspase-3 and TEM analysis showing modulations in mitochondria confirmed intrinsic apoptosis pathway. The enzyme impeded tumor progression through inhibition of cell migration and vascular remodelling of endothelial cells. Our findings suggests that the action of l-asparaginase alters mitochondrial membrane permeability and auxiliary activates intrinsic apoptosis. Therefore, this mechanistic approach might be considered as a targeted enzymotherapy against gastric adenocarcinoma.

Expression and characterization of recombinant l-asparaginase from Pseudomonas fluorescens.

l-asparaginase (E.C. 3.5.1.1), an anti-cancer drug has been used in the treatment of acute lymphoblastic leukemia. A novel source of l-asparaginase from Pseudomonas fluorescens was addressed in the present studies. The ANS gene in Pseudomonas fluorescens MTCC 8127 which produces l-asparaginase was cloned and expressed in E. coli BL21 (DE3). The expressed recombinant protein (PfAns) which was purified, showed l-asparaginase activity. The enzyme was further characterized. The pH and temperature optima were found to be 7.5 and 37 °C. The recombinant enzyme was stable to temperature and pH. The enzyme was homotetramer with the molecular weight of the monomer being 35 kDa and a whole protein molecular weight of 140 kDa. The purified l-asparaginase had a specific activity of 26 U/mg with a Km and Vmax of 0.050 M and 4.032 mol-1min. The enzyme exhibited a high affinity towards l-asparagine and showed a very minimal activity with glutamine as a substrate. The enzyme activity was inhibited by PMSF, suggesting the presence of serine at the active site. The presence of Mg2+ enhanced PfAns activity by 49%, and SDS strongly inhibited the enzyme activity. The in vitro half-life of the recombinant enzyme was ∼40 h. The enzyme demonstrated deglycosylation activity which could exhibit an additional barrier for proliferating cancer cells.

Purification and characterization of carbon-phosphorus bond-cleavage enzyme from glyphosate degrading Pseudomonas putida T5.

An inducible, carbon-phosphorus bond-cleavage enzyme was purified from cells of Pseudomonas putida T5 grown on N-phosphonomethyl glycine. The native enzyme had a molecular mass of approximately 70 kD and upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), yielded a homogeneous protein band with an apparent molecular mass of about 70 kD. Activity of purified enzyme was increased by 627-fold compared to the crude extract and showed pH and temperature optima of approximately 7 and 30°C, respectively. The purified enzyme had an apparent Km and Vmax of 3.7 mM and 6.8 mM/min, respectively, for its sole substrate N-phosphonomethyl glycine. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), indicating the presence of serine at the active site. The enzyme was not inhibited by SDS, suggesting the absence of disulfide linkage in the enzyme. The enzyme was found to be inhibited by most of the metals studied except Mg(2+). Detergents studied also inhibited glyphosate acting as a carbon-phosphorus bond-cleavage enzyme. Thus initial characterization of the purified enzyme suggested that it could be used as a potential candidate for glyphosate bioremediation.

Prodigiosin and its potential applications.

Since a decade, there has been a strong consumer demand for more natural products. This has augmented inclination towards substitution of synthetic colorants with natural pigments. Natural pigments not only have the capacity to increase the marketability of products, they also demonstrate valuable biological activities as antioxidants and anticancer agents. There is a long history of exploitation of natural products produced by bacteria as sources of pharmaceutically important, bioactive compounds. Among natural pigments, pigments from microbial sources are potentially suitable alternatives to synthetic pigments. The red pigment prodigiosin (PG) has unusual properties, which have long been documented. The red-pigmented prodiginines are bioactive secondary metabolites produced by both Gram-negative and Gram-positive bacteria. Prodigiosins are characterized by a common pyrrolyl pyrromethene skeleton, and the biological role of these pigments in the producer organisms remains unclear. Bacterial prodigiosins and their synthetic derivatives are effective proapoptotic agents against various cancer cell lines, with multiple cellular targets including multi-drug resistant cells with little or no toxicity towards normal cell lines. However, research into the biology of pigment production will stimulate interest in the bioengineering of strains to synthesize useful prodiginine derivatives. This review article highlights the characteristics and potential applications of prodigiosin pigment from Serratia as prodigiosins are real potential therapeutic drugs.

Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

Immunoassay for lead ions: analysis of spiked food samples.

A rapid, simple, and reliable competitive immunoassay was developed for measurement of lead ions in spiked food samples. Avian antibodies were produced against Pb(II). Since lead ions are too small to elicit an immune response, the metal was coupled to protein carrier Bovine Serum Albumin (BSA) using a bifunctional chelator 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N',N'-tetraacetic acid (ITCBE). Poultry birds (layers) were immunized with this Pb(II)-ITCBE-BSA immunoconjugate and avian antibodies (IgY) were isolated from egg yolk. This avian antibody (IgY) produced recognized Pb(II)-ITCBE complexes as capture reagent. The assay depended on a competitive binding reaction between the antibody and Pb(II). Antibody reaction was optimized for different concentrations of antigen and antibody dilutions. Cross reactivity with other metals were below 1% in competitive ELISA. The detection range and the detection limit were 0.02-1000 mg · kg⁻¹ and 0.2 mg · kg⁻¹, respectively. Spike recovery studies in different food samples showed that the avian antibodies could recognize Pb(II) in food samples without much matrix effect.

Chemical modification of L-asparaginase from Cladosporium sp. for improved activity and thermal stability.

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (kd), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.

Detection of isoprothiolane in food, soil, and water samples by immunosorbent assay using avian antibodies.

A simple competitive immunoassay was developed for the measurement of isoprothiolane in rice, soil, and water samples. It employed the avian antibodies (IgY) that recognized isoprothiolane as a capture reagent and isoprothiolane-alkaline phosphatase conjugate as an enzyme label. The assay depended on the competitive binding between the anti-isoprothiolane antibody and isoprothiolane derived from rice, soil, and water samples for binding sites with immobilized isoprothiolane-ovalbumin (OVA) conjugate. The concentration of isoprothiolane in the rice, soil, and water samples was quantified by the ability of the pesticide present in the samples to inhibit the binding of the enzyme conjugate to the antibody and subsequently the color formation in the assay. The assay was specific to isoprothiolane with a limit of detection of 2 ng/mL. Mean analytical recovery of isoprothiolane in different rice matrices was 87.20%-98.02%, for soil samples recovery was 74.24%-111.20%, and water samples recovery was 35.2%-95.73%. The precision of the assay was satisfactory. The assay compared favorably with gas chromatography (GC) in its ability to accurately measure isoprothiolane in the different rice, soil, and water samples.

Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp.

L-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 µmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, L-asparaginase has greater affinity towards L-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of L-glutamine. L-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, ß-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca(2+), Co(2+), Cu(2+), Mg(2+), Na(+), K(+) and Zn(2+) significantly affected enzyme activity whereas presence of Fe(3+), Pb(2+) and KI stimulated the activity. Detergents studied also enhanced L-asparaginase activity. In-vitro half-life of purified L-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.

Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods.

AIMS: The study describes the development of simple and rapid DNA extraction method in combination with loop-mediated isothermal amplification (LAMP) to detect enterotoxigenic Staphylococcus aureus in food samples. METHODS AND RESULTS: In this study, isolation of genomic DNA of enterotoxigenic Staph. aureus from spiked milk, milk burfi, khoa, sugarcane juice and boiled rice was carried out by boiling the isolated sample pellets for 10 min with 1% Triton X-100. The isolated DNA was evaluated by polymerase chain reaction (PCR) and LAMP method. The LAMP was found to be 100 times more sensitive than PCR. The LAMP assay was very specific for Staph. aureus, and the presence of other contaminating bacterial DNAs and food matrix did not interfere or inhibit the LAMP assay. CONCLUSIONS: The template DNA extraction method developed in this study for food samples is simple, rapid and cost-effective. LAMP was found to be less sensitive to matrix effect of food, compared to PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for direct detection of Staph. aureus without any enrichment in contaminated food samples and hence finds its application in food safety analysis, in permutation with LAMP.

In silico evaluation of naturally isolated triterpene glycosides (TG) from Gymnema sylvestre towards diabetic treatment.

Diabetes is a metabolic disorder which is characterised by high levels of blood glucose. Most of the oral drugs available today for the treatment of diabetes are associated with various side-effects. Herbal medicines are considered relatively safer alternatives and Gymnema sylvestre (GS) is one such known traditional medicinal plant widely used for the treatment of diabetes. In our previous work, we isolated active triterpene glycosides (TG) from Gymnema sylvestre (GS) and screened for yeast α-glucosidase inhibitory activity in vitro. The present study aims to use in silico techniques to understand and predict the inhibitory role of the isolated triterpene glycosides (TG); Gymnemic acid I, IV, VII and gymnemagenin against disaccharidase enzymes. enzyme kinetic analysis using Lineweaver-Burk plot indicated that TG competitively inhibited yeast α-glucosidase at IC50 concentration with Ki 0.0028 µM. TG also exhibited significant inhibitory activity against mammalian sucrase and maltase respectively, compared to control. PRACTICAL APPLICATIONS: The molecular docking simulation reveals that TG is capable of docking well with crystallographic structures of the selected enzyme targets. Inhibition of α-glucosidases could delay the absorption of glucose in the blood during post-meal digestion. Thus the current study highlights the dietary intervention of TG towards the selected enzyme targets, thus making TG a potential nutraceutical candidate towards management of blood glucose.

Aptamer as capture agent in enzyme-linked apta-sorbent assay (ELASA) for ultrasensitive detection of Aflatoxin B1.

Aflatoxin B1 (AFB1), is one of the most toxic mycotoxins found to contaminate various food commodities like cereals, dried fruits, tree nuts, spices and crude vegetable oils. In spite of considerable progress in analytical techniques, there is still a need to develop rapid and highly sensitive detection platforms for AFB1. In this study, AFB1 specific aptamer was used as a capture molecule to develop an enzyme-linked apta-sorbent assay (ELASA) for ultrasensitive detection of AFB1. Under optimized conditions, the assay had a linear detection range from 1 µg to 1 pg with a limit of detection (LOD) of 1 pg/mL in buffer. Conventional ELISA with AFB1 hapten as the capture agent (LOD = 10 pg/mL) was also carried out to compare the results with the present method. Recovery studies in food samples like dried red chillies, groundnut and pepper using both the methods was found to be in the range of 88.49-106.4% at 10 ng/mL and 87.4% to 95.8% at 5 ng/mL for ELASA and 76.56-127.68% at 10 ng/mL and 82-101.2% at 5 ng/mL for ELISA. Higher detection (10 fold) and better recovery using ELASA suggest that the method could offer an early, ultrasensitive, high-throughput, qualitative and semi-quantitative detection of AFB1 in contaminated food samples.

In Vitro Antidiabetic Effects of Isolated Triterpene Glycoside Fraction from Gymnema sylvestre.

A triterpene glycoside (TG) fraction isolated and purified from ethanolic extract of Gymnema sylvestre (EEGS) was investigated for blood glucose control benefit using in vitro methods. The HPLC purified active fraction TG was characterized using FTIR, LC-MS, and NMR. The purified fraction (TG) exhibited effective inhibition of yeast α-glucosidase, sucrase, maltase, and pancreatic α-amylase with IC50 values 3.16 ± 0.05 µg/mL, 74.07 ± 0.51, 5.69 ± 0.02, and 1.17 ± 0.24 µg/mL, respectively, compared to control. TG was characterized to be a mixture of triterpene glycosides: gymnemic acids I, IV, and VII and gymnemagenin. In vitro studies were performed using mouse pancreatic ß-cell lines (MIN6). TG did not exhibit any toxic effects on ß-cell viability and showed protection against H2O2 induced ROS generation. There was up to 1.34-fold increase in glucose stimulated insulin secretion (p<0.05) in a dose-dependent manner relative to standard antidiabetic drug glibenclamide. Also, there was further one-fold enhancement in the expression of GLUT2 compared to commercial standard DAG (deacylgymnemic acid). Thus, the present study highlights the effective isolation and therapeutic potential of TG, making it a functional food ingredient and a safe nutraceutical candidate for management of diabetes.

Aerobic degradation of technical hexachlorocyclohexane by a defined microbial consortium.

Organochlorine pesticides including hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) are largely used in developing countries like India for public health and agricultural purposes. Even though the agricultural use of technical mixture (tech-HCH) is banned, countries like India are still using gamma-HCH for economic purposes. Thus, in addition to already contaminated sites, new sites are being contaminated with gamma-HCH and its stereoisomers. In the environment, these isomers have a half-life of 8-10 years. In our laboratory, we developed a microbial consortium capable of degrading tech-HCH. Conditions such as induction, inoculum level, concentration of the substrate, pH of degradation and interaction between isomers were optimized for tech-HCH degradation. Up to 25 ppm tech-HCH was degraded at an inoculum level of 100 microg protein/mL, pH 7.5 at ambient temperature (26-28 degrees C). The degradation of HCH-isomers was in the order of gamma>alpha>beta>delta. The rate of degradation was also determined.

Prodigiosin inhibits motility and activates bacterial cell death revealing molecular biomarkers of programmed cell death.

The antimicrobial activity of prodigiosin from Serratia nematodiphila darsh1, a bacterial pigment was tested against few food borne bacterial pathogens Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The mode of action of prodigiosin was studied. Prodigiosin induced bactericidal activity indicating a stereotypical set of biochemical and morphological feature of Programmed cell death (PCD). PCD involves DNA fragmentation, generation of ROS, and expression of a protein with caspase-like substrate specificity in bacterial cells. Prodigiosin was observed to be internalized into bacterial cells and was localized predominantly in the membrane and the nuclear fraction, thus, facilitating intracellular trafficking and then binding of prodigiosin to the bacterial DNA. Corresponding to an increasing concentration of prodigiosin, the level of certain proteases were observed to increase in bacteria studied, thus initiating the onset of PCD. Prodigiosin at a sub-inhibitory concentration inhibits motility of pathogens. Our observations indicated that prodigiosin could be a promising antibacterial agent and could be used in the prevention of bacterial infections.

Cloning, expression and characterization of L-asparaginase from Pseudomonas fluorescens for large scale production in E. coli BL21.

L-Asparaginase (E.C. 3.5.1.1) is used as an anti-neoplastic drug in the treatment of acute lymphoblastic leukemia. L-Asparaginase from Pseudomonas fluorescens was cloned and overexpressed in E. coli BL21. The Enzyme was found to be a Fusion protein-asparaginase complex which was given a lysozyme treatment and sonication, and then was purified in a Sepharose 6B column. The enzymatic properties of the recombinant enzyme were studied and the kinetic parameters were determined with kilometre of 109.99 mM and V max of 2.88 µM/min. Recombinant enzyme showed pH optima at 6.3 and temperature optima at 34 °C. Asp gene was successfully cloned into E. coli BL21 which produced high level of asparaginase intracellularly with 85.25 % recovery of enzyme with a specific activity of 0.94 IU/mg protein. The enzyme was a tetramer with molecular weight of approximately 141 kDa.

Isolation and acclimation of a microbial consortium for improved aerobic degradation of alpha-hexachlorocyclohexane.

A microbial consortium that can utilize alpha-hexachlorocyclohexane (alpha-HCH) as a sole source of carbon and energy was isolated from soil and sewage through a novel technique involving an initial enrichment in a glass column reactor followed by a shake flask enrichment. This consortium took 14 days to completely mineralize 5 and 10 microg mL(-)(1) alpha-HCH in mineral salts medium in shake flasks. The degradative ability of this consortium improved very markedly on acclimation by successive and repeated passages through media containing increasing concentrations of alpha-HCH. The acclimated consortium could degrade 100 microg mL(-)(1) of alpha-HCH within 72 h at a degradation rate of 58 microg mL(-)(1) day(-)(1) with concomitant release of stoichiometric amounts of chloride. Accumulation of any intermediary metabolites was not detected in the culture broth as tested by TLC and GC, implying complete mineralization of the substrate. The acclimated consortium contained eight bacterial strains and a fungus. The individual strains and the different permutations and combinations of them, however, were able to utilize only 10 microg mL(-)(1) of alpha-HCH. Mesophilic temperatures (20-30 degrees C) and near-neutral pH (6.0-8.0) were most favorable for alpha-HCH degradation. Among the auxiliary carbon sources tested, ethanol, benzoate, and glucose (at higher concentrations) retarded the degradation of alpha-HCH, whereas the addition of cellulose, sawdust, and low concentrations of glucose (<200 microg mL(-)(1)) and acetone enhanced the rate of degradation.

Bioremediation of HCH-contaminated soil: elimination of inhibitory effects of the insecticide on radish and green gram seed germination.

The effects of technical grade hexachlorocyclohexane (tech-HCH) on the germination of different seeds were tested. Two types of seeds, radish and green gram showed marked reduction in germination percentage and seeding vigour index. The abnormalities and reduction in germination increased with increasing concentration of tech-HCH. At 100 microg HCH level the germination of radish and green gram seeds was inhibited almost completely on moist filter paper and soil. Protease and amylase activities were reduced in seeds grown in soil spiked with tech-HCH. Bioremediation of HCH-spiked soils with a HCH-degrading microbial consortium helped in eliminating the toxic effects of tech-HCH towards seed germination. The degradation of 25 microg tech-HCH g(-1) soil was complete by 120 h. The seed germination and the activities of the assayed enzymes, amylase and protease, were same as before or better in bioremediated soils.

Gold nanoparticles based dipstick immunoassay for the rapid detection of dichlorodiphenyltrichloroethane: an organochlorine pesticide.

Gold nanoparticles (GNPs) based dipstick competitive immunoassay was developed to detect organochlorine pesticide such as DDT at nanogram level (ppb). GNPs of definite size were synthesized and conjugated to anti-DDT antibodies (IgY), which served as the detecting reagent. DDA-BSA conjugate (antigen) was immobilized on to nitro cellulose (NC) membrane containing strip. GNPs conjugated anti-DDT antibodies were treated with different concentrations of free DDT ranging from 0.7 ng mL(-1) to 1000 ng mL(-1) to form an immunocomplex. This immunocomplex solution was further reacted with DDA-BSA conjugate immobilized NC membrane containing strips by dipping the strip in the immunocomplex solution. The free GNPs conjugated anti-DDT antibodies present in the immunocomplex solution were targeted for competitive binding with immobilized DDA-BSA on NC membrane containing strip. Depending on the concentration of free DDT in the sample the binding of GNPs conjugated anti-DDT antibodies to the immobilized DDA-BSA varied and was detected by the development of red color (due to gold nanoparticles) in the detection zone of NC membrane containing strips. The intensity of color development was inversely proportional to the DDT concentration with maximum intensity at zero DDT concentration. The lowest detection limit of DDT was determined to be 27 ng mL(-1) with the optimized conditions. The dipstick technique based on GNPs is suitable for the detection of several toxins in food and environmental samples and can be applied for rapid on-site testing of pesticides.