Phylogenetic characterization of archaea in saltpan sediments.

A study was undertaken to investigate the presence of archaeal diversity in saltpan sediments of Goa, India by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA) from saltpan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. 10 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonuclease Msp 1, which was most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain archaea including both crenarchaeota and euryarcheaota. None of the retrieved crenarchaeota sequences could be grouped with previously cultured crenarchaeota however; two sequences were related with haloarchaea. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of low archaeal population but showed yet unclassified species, may specially adapted to the salt pan sediment of Goa.

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L.

BACKGROUND: Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide. RESULTS: Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 microg/ml for yeasts, 125 to 500 microg/ml for Aspergillus species, and 7.81 to 62.5 microg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 x MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 x MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 x to 8 x MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol. CONCLUSIONS: The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.

Functional cloning and predictive structural modeling of a novel esterase from Bacillus subtilis strain, RRL 1789.

We have recently reported the purification and characterization of a novel esterase from the Bacillus subtilis strain. In the present study we report the genomic DNA cloning and predictive structural modeling of this novel esterase. Tributyrin- and Rhodamine B-based functional screen of a Bacillus subtilis genomic library led to the identification of a potential lipolytic gene. DNA sequence analysis of the cloned gene showed that it encodes a protein of 489 amino acid residues. Sequence homology search and multiple sequence alignment showed that the protein was highly homologous to known esterases. Secondary structure-driven multiple sequence alignment with the homologous esterase of known three-dimensional structures was performed and a 3D structure model of this enzyme was constructed. Based on the topological organization of the secondary structures, this protein belongs to the alpha/beta hydrolase superfamily. Moreover, the presence of serine in the context of amino acid sequence G/A-X-S-X-G (with X an arbitrary amino acid residue) in the protein indicates that it belong to the class of serine hydrolases of this superfamily.

Immune modulation and apoptosis induction: Two sides of antitumoural activity of a standardised herbal formulation of Withania somnifera.

Deregulated apoptosis and suppressed tumour reactive immunity render tumour cells to grow amok in the host body. Traditionally used botanicals may offer potential anticancer chemo-immunotherapeutic leads. We report in this study a chemically standardised herbal formulation (WSF) of Withania somnifera possessing anticancer and Th1 immune up-regulatory activities. WSF produced cytotoxicity in a panel of human cancer cell lines in vitro. The molecular mechanism of cell cytotoxicity, IC(50) 48h approximately 20mug/ml, was investigated in HL-60, where it induced apoptosis by activating both intrinsic and extrinsic signalling pathways. It induced early generation of reactive nitrogen and oxygen species (RNOS), thus producing oxidative stress mediated mitochondrial membrane potential (MMP) loss leading to the release of cytochrome c, the translocation of Bax to mitochondria and apoptosis-inducing factor to the nuclei. These events paralleled the activation of caspase-9, -3 and PARP cleavage. WSF also activated caspase-8 through enhanced expression of TNF-R1 and DR-4, suggesting also the involvement of extrinsic pathway of apoptosis. WSF at 150mg/kg, i.p., inhibited >50% tumour growth in the mouse tumour models. In tumour-bearing mice, WSF inhibited the expression of pStat-3, with a selective stimulation of Th1 immunity as evidenced by enhanced secretion of IFN-gamma and IL-2. In parallel, it enhanced the proliferation of CD4(+)/CD8(+) and NK cells along with an increased expression of CD40/CD40L/CD80. In addition, WSF also enhanced T cell activation in camptothecin treated tumour-bearing mice. WSF being safe when given orally up to 1500mg/kg to rats for 6 months may be found useful in the management of malignancy by targeting at multiple pathways.

Anticancer activity of an essential oil from Cymbopogon flexuosus.

The essential oil from a lemon grass variety of Cymbopogon flexuosus was studied for its in vitro cytotoxicity against twelve human cancer cell lines. The in vivo anticancer activity of the oil was also studied using both solid and ascitic Ehrlich and Sarcoma-180 tumor models in mice. In addition, the morphological changes in tumor cells were studied to ascertain the mechanism of cell death. The in vitro cytotoxicity studies showed dose-dependent effects against various human cancer cell lines. The IC(50) values of oil ranged from 4.2 to 79 microg/ml depending upon the cell line. In 502713 (colon) and IMR-32 (neuroblastoma) cell lines, the oil showed highest cytotoxicity with IC(50) value of 4.2 and 4.7 microg/ml, respectively. Intra-peritoneal administration of the oil significantly inhibited both ascitic and solid forms of Ehrlich and Sarcoma-180 tumors in a dose-dependent manner. The tumor growth inhibition at 200 mg/kg (i.p.) of the oil observed with both ascitic and solid tumor forms of Ehrlich Ascites carcinoma was 97.34 and 57.83 respectively. In case of Sarcoma-180, the growth inhibition at similar dose of oil was 94.07 and 36.97% in ascitic and solid forms respectively. Morphological studies of the oil treated HL-60 cells revealed loss of surface projections, chromatin condensation and apoptosis. The mitochondria showed apparent loss of cristae in the cells undergoing apoptosis. The morphological studies of Sarcoma-180 solid tumor cells from animals treated with the oil revealed condensation and fragmentation of nuclei typical of apoptosis. Morphological studies of ascites cells from animals treated with the oil too revealed the changes typical of apoptosis. Our results indicate that the oil has a promising anticancer activity and causes loss in tumor cell viability by activating the apoptotic process as identified by electron microscopy.

Arthrobacter sp. lipase immobilization for preparation of enantiopure masked beta-amino alcohols.

Recent reports on immobilization of lipase from Arthrobacter sp. (ABL, MTCC 5125; IIIM isolate) on insoluble polymers have shown altered properties including stability and enantioselectivity. Present work demonstrates a facile method for the preparation of enantiopure beta-amino alcohols by modulation of ABL enzyme properties via immobilization on insoluble as well as soluble supports using entrapment/covalent binding techniques. Efficacies of immobilized ABL on insoluble supports prepared from tetraethylorthosilicate/aminopropyltriethoxy silane and soluble supports derived from copolymerization of N-vinyl pyrrolidone-allylglycidyl ether (ANP type)/N-vinyl pyrrolidone-glycidyl methacrylate (GNP type) for kinetic resolution of masked beta-amino alcohols have been studied vis-à-vis free ABL enzyme/wet cell biomass. The immobilized lipase on different insoluble/soluble supports has shown 21-110 mg/g protein binding and 30-700 U/g activity for hydrolyzing tributyrin substrate. The findings have shown a significant enhancement in enantioselectivity (ee 99%) vis-à-vis wet cell biomass providing ee 70-90% for resolution of beta-amino alcohols.

Induction of apoptosis by a synergistic lignan composition from Cedrus deodara in human cancer cells.

AP9-cd, a synergistic lignan mixture from Cedrus deodara (Pinaceae) consisting of (-)-wikstromal, (-)-matairesinol and dibenzyl butyrolactol, depicted cytotoxic effects against numerous human cancer cell lines reported previously. The aim of this study was to investigate the mechanism of cell death in human cancer cells. The viability, morphological and ultrastructural changes in Molt-4 cells were investigated. Using the trypan blue exclusion assay, we demonstrated that AP9-cd significantly reduced the viability of Molt-4 cells in a time- and dose-dependent manner. Apoptotic assays using light microscopy revealed that this agent induced Molt-4 cell apoptosis at varied concentrations. The treatment causes a loss in cell viability by activating the apoptotic process as identified by light and electron microscopy. The morphological changes of intracellular organelles in Molt-4 cells treated with 30 microg/ml of AP9-cd revealed the disruption of mitochondrial cristae. Other features included the vacuolization, chromatin condensation and formation of micronuclei. Surface ultrastructural studies of four different tumor cell lines (Molt-4, HL-60, PC-3 and A-549) treated with AP9-cd depicted loss of surface projections, condensation and formation of apoptotic bodies. AP9-cd treatment to transgenic fruit fly, Drosophila, carrying human adenomatous polpyposis coli (hAPC) gene enhanced eye phenotypes and therefore may inhibit Wnt/Wg pathway which is important in the aetiology of a number of human cancers.

Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors.

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.

Medicinal plants and cancer chemoprevention.

Cancer is the second leading cause of death worldwide. Although great advancements have been made in the treatment and control of cancer progression, significant deficiencies and room for improvement remain. A number of undesired side effects sometimes occur during chemotherapy. Natural therapies, such as the use of plant-derived products in cancer treatment, may reduce adverse side effects. Currently, a few plant products are being used to treat cancer. However, a myriad of many plant products exist that have shown very promising anti-cancer properties in vitro, but have yet to be evaluated in humans. Further study is required to determine the efficacy of these plant products in treating cancers in humans. This review will focus on the various plant-derived chemical compounds that have, in recent years, shown promise as anticancer agents and will outline their potential mechanism of action.

Citral derived amides as potent bacterial NorA efflux pump inhibitors.

Monoterpene citral and citronellal have been used as starting material for the preparation of 5,9-dimethyl-deca-2,4,8-trienoic acid amides and 9-formyl-5-methyl-deca-2,4,8-trienoic acid amides. The amides on bioevaluation as efflux pump inhibitors (EPIs) against Staphylococcus aureus 1199 and NorA overexpressing S. aureus 1199B bacteria resulted in the identification of several of these as potent EPIs. Many of these amides have been shown to possess potency higher or equivalent to known EPIs such as reserpine, verapamil, carsonic acid, and piperine. In this communication, we report a convenient synthesis of alkenyl amides, their bioevaluation and identification as efflux pump inhibitors against S. aureus.

Phylogenetic analyses of archaeal ribosomal DNA sequences from salt pan sediment of Mumbai, India.

The archaeal diversity in salt pan sediment from Mumbai, India, was investigated by 16S rDNA-dependent molecular phylogeny. Small-subunit rRNA (16S rDNA) from salt pan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. Thirty-two unique phylotypes were obtained by PCR-based RFLP of 16S rRNA genes using endonucleases Hae111 and Msp1, which were most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain Archaea including both Crenarchaeota and Euryarcheaota. However, none of the retrieved Crenarchaeota sequences could be grouped with previously cultured Crenarchaeota. Of all the Euryarcheaota sequences, three sequences were related to Haloarchaea, two were related to cultured Methanosarcina sp., and the remaining sequences were affiliated with uncultured Methanosarcina sp., Methanogenium sp., and Methanolobus sp. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of a highly diverse archaeal population and may represent novel sequences and organisms specially adapted to the salt pan sediment of Mumbai. These findings are of fundamental value for understanding the complexity of salt pan ecosystems.

Design, synthesis, and biological testing of 4beta-[(4-substituted)-1,2,3-triazol-1-yl]podophyllotoxin analogues as antitumor agents.

A series of 4beta-[(4-substituted)-1,2,3-triazol-1-yl]podophyllotoxin analogues have been synthesized with high regio-selectivity by employing copper(I)-catalyzed 1,3-dipolar cycloaddition of 1-O-propargyl monosaccharides with C4beta-azido podophyllotoxin and C4beta-azido-4'-O-demethyl podophyllotoxin. All the compounds were evaluated for their anticancer activity against a panel of six human cancer cell lines. Among these, 4'-O-demethyl podophyllotoxin congeners are showing promising anticancer activity mainly against HCT-15 (colon) and DU-145 (prostate) cells.

An essential oil and its major constituent isointermedeol induce apoptosis by increased expression of mitochondrial cytochrome c and apical death receptors in human leukaemia HL-60 cells.

An essential oil from a lemon grass variety of Cymbopogon flexuosus (CFO) and its major chemical constituent sesquiterpene isointermedeol (ISO) were investigated for their ability to induce apoptosis in human leukaemia HL-60 cells because dysregulation of apoptosis is the hallmark of cancer cells. CFO and ISO inhibited cell proliferation with 48 h IC50 of approximately 30 and 20 microg/ml, respectively. Both induced concentration dependent strong and early apoptosis as measured by various end-points, e.g. annexinV binding, DNA laddering, apoptotic bodies formation and an increase in hypo diploid sub-G0 DNA content during the early 6h period of study. This could be because of early surge in ROS formation with concurrent loss of mitochondrial membrane potential observed. Both CFO and ISO activated apical death receptors TNFR1, DR4 and caspase-8 activity. Simultaneously, both increased the expression of mitochondrial cytochrome c protein with its concomitant release to cytosol leading to caspase-9 activation, suggesting thereby the involvement of both the intrinsic and extrinsic pathways of apoptosis. Further, Bax translocation, and decrease in nuclear NF-kappaB expression predict multi-target effects of the essential oil and ISO while both appeared to follow similar signaling apoptosis pathways. The easy and abundant availability of the oil combined with its suggested mechanism of cytotoxicity make CFO highly useful in the development of anti-cancer therapeutics.

Development of a rapid normal-phase LC-positive ion APCI-MS method for simultaneous detection and quantitation of cholesterol, androst-4-ene-3,1 7-dione, and androsta-1,4-diene-3,17-dione.

This paper describes the development of a normal-phase liquid chromatograph-UV-diode array detection-positive ion atmospheric pressure chemical ionization-mass spectrometry method for the simultaneous identification and quantitation of cholesterol, androst-4-ene-3,17-dione (AD), and androsta-1,4-diene-3,17-dione (ADD) in fermentation broths. The compounds detected under positive ion atmospheric pressure chemical ionization on a mass spectrometer by selected ion monitoring are separated by normal-phase high-performance liquid chromatography. [M+H]+ ions are taken into consideration for quantitation of AD and ADD, and [M-H2O+H]+ ions are considered for quantitation of cholesterol. The compounds are analyzed on a Si60 silica (5 microm, 125 x 4-mm i.d.) Merck column using a 2:3 isocratic mixture of isopropyl alcohol and hexane. The calibration curves resulting from the reference compounds in the concentration range of 100-5000 pg on column exhibit a good linear correlation (r2 > or = 0.996). The method is validated by analyzing six replicates of broth samples fortified with three compounds, namely, cholesterol, AD, and ADD, at 0.050 and 0.5 microg/g levels. The mean recoveries for the fortifications range from 90% to 98% with relative standard deviations in the range of 3.36% to 9.78%. The method is developed to study the qualitative as well as quantitative conversion of cholesterol to AD and ADD by a microorganism identified as Nocardia sp. These studies helped the investigation of the reaction kinetics, which showed that the molar biotransformation of cholesterol into AD and ADD was 21%, even when the reaction was prolonged for 96 h.

Cytotoxic and apoptotic activities of novel amino analogues of boswellic acids.

4-Amino analogues prepared from beta-boswellic acid and 11-keto-beta-boswellic acid, wherein the carboxyl group in ursane nucleus was replaced by an amino function via Curtius reaction, displayed improved cytotoxicity than the parent molecules. The same molecules also exhibited apoptotic activity by inducing DNA fragmentation.

Immunomodulatory effect of bergenin and norbergenin against adjuvant-induced arthritis--a flow cytometric study.

Bergenin (1), a C-glycoside of 4-O-methyl gallic acid, isolated from rhizomes of Bergenia stracheyi (Saxifragaceae) and its O-demethylated derivative norbergenin (2), prepared from 1, are reported to show anti-arthritic activity through possible modulation of Th1/Th2 cytokine balance. Flow cytometric study showed that the oral administration of 1 and 2 at doses of 5, 10, 20, 40 and 80 mg/kg per oral dose inhibit the production of proinflammatory Th1 cytokines (IL-2, IFN-gamma and TNF-alpha) while as potentiate anti-inflammatory Th2 cytokines (IL-4 and IL-5) in the peripheral blood of adjuvant-induced arthritic balb/c mice. This shows the potential Th1/Th2 cytokine balancing activity of 1 and 2 which is strongly correlated with their anti-arthritic activity. At similar dose levels, the effect of 2 was found to be more than that of 1. The oral LD(0) for 1 and 2 was more than 2000 mg/kg body weight of the mice.

A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up regulation of Th1-dominant polarization in BALB/c mice.

The effects of graded doses of a chemically standardized aqueous alcoholic (1:1) root extract (AGB) of Withania somnifera on the immune system of SRBC immunized BALB/c mice were investigated. Mice were administrated AGB orally for 15 days. AGB stimulated cell mediated immunity, IgM and IgG titers reaching peak value with 30 mg/kg b.wt. Flow cytometric analysis of lymphocyte surface markers of T cells (CD3(+), CD4(+) and CD8(+)) and B cells (CD19(+)) indicated prominent enhancement in proliferation and differentiation of lymphocytes. The extract selectively, induced type 1 immunity because it guided enhanced expression of T helper cells (Th)1 cytokines interferon (IFN)-gamma and interleukin (IL)-2 while Th2 cytokine IL-4 observed a moderate decline. Confirmation of Th1 polarization was obtained from augmented levels of IgG2a over IgG1 in the blood sera of AGB treated groups. Withanolide-A, a major constituent of AGB appeared responsible for Th1 skewing effect of the extract as it significantly increased the levels of Th1 cytokines, decreased moderately IL-4 and significantly restored the selective dexamethasone inhibition of Th1 cytokines in mouse splenocytes cultures in vitro. In addition, AGB also strongly activated macrophage functions ex vivo and in vitro indicated by enhanced secretion of nitrite, IL-12 and TNF-alpha. In contrast IL-10 remained unchanged again suggesting that AGB critically influenced Th1 profile of the cytokines. The studies suggested that AGB supports predominantly Th1 immunity with increase in macrophage functions. The standardized root extract of no toxicological consequences might therefore, find useful applications against the intracellular pathogens and in the management of immune suppressed diseases.

Arthrobacter sp. lipase immobilization for improvement in stability and enantioselectivity.

Arthrobacter sp. lipase (ABL, MTCC no. 5125) is being recognized as an efficient enzyme for the resolution of drugs and their intermediates. The immobilization of ABL on various matrices for its enantioselectivity, stability, and reusability has been studied. Immobilization by covalent bonding on sepharose and silica afforded a maximum of 380 and 40 IU/g activity, respectively, whereas sol-gel entrapment provided a maximum of 150 IU/g activity in dry powder. The immobilized enzyme displayed excellent stability in the pH range of 4-10 and even at higher temperature, i.e., 50-60 degrees C, compared to free enzyme, which is unstable under extreme conditions. The resolution of racemic auxiliaries like 1-phenyl ethanol and an intermediate of antidepressant drug fluoxetine, i.e., ethyl 3-hydroxy-3-phenylpropanoate alkyl acylates, provided exclusively R-(+) products ( approximately 99% ee, E=646 and 473), compared to cell free extract/whole cells which gave a product with approximately 96% ee (E=106 and 150). The repeated use (ten times) of covalently immobilized and entrapped ABL resulted in no loss in activity, thus demonstrating its prospects for commercial applications.

Molecular cloning of carboxylesterase gene and biochemical characterization of encoded protein from Bacillus subtilis (RRL BB1).

An isolated strain of Bacillus subtilis identified by 16S rDNA sequence analysis produces an enantioselective ester hydrolase. Whole cells of B. subtilis (RRL BB1) and enzyme derived from it was capable of enantioselective hydrolysis of several racemates including drug intermediates with moderate to high enantioselectivity as already reported by us. In this communication, we describe cloning of the gene encoding the enantioselective esterase designated as estBB1. The primary structure of the enzyme determined from the nucleotide sequence indicated that esterase estBB1 has Mw approximately 52kDa and pI approximately 5.2 and belongs to the family of type B carboxylesterases with 50-60% similarity at amino acid level. Alignment studies of sequences of the estBB1 and Pnb esterase 56C8 from B. subtilis showed that estBB1 has an alpha/beta hydrolase fold with catalytic triad formed by Ser190, Glu305 and His394 at active site and Ser190 is located in the conserved motif -G-X-S-X-G-.

A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme.

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase (BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is approximately 52 kDa monomer, maximally activity at 37 degrees C and pH 8.0 and fairly stable up to 55 degrees C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by approximately 20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Gly- revealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis.