Reciprocal relation between reporter gene transcription and translation efficiency in fission yeast.

The fission yeast, Schizosaccharomyces pombe, is an excellent model for basic research but is not useful for commercial scale protein expression due to lack of strong expression vectors. Earlier, we showed that the lsd90 promoter elicited significantly greater GFP expression level than the adh1 and nmt1 promoters, albeit in different vector backbones. Here, we have systematically investigated the contribution of selectable markers, LEU2 and URA3m to GFP expression: while LEU2 elicited very low expression, the URA3m gene, with truncated promoter, elicited much greater GFP expression level with all promoters. Paradoxically, an inverse correlation was observed between the GFP transcription and translation efficiency. This system can be useful for understanding the factors governing recombinant gene expression and optimization of protein production.

Role of Cdc23/Mcm10 in generating the ribonucleotide imprint at the mat1 locus in fission yeast.

The developmental asymmetry of fission yeast daughter cells derives from inheriting 'older Watson' versus 'older Crick' DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific 'imprint', installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The catalytic subunit of DNA Polymerase α (Polα) has been implicated in the imprinting process. Based on its known biochemical function, Polα might install the mat1 imprint during lagging strand synthesis. The nature of the imprint is not clear: it is either a nick or a ribonucleotide insertion. Our investigations do not support a direct role of Polα in nicking through putative endonuclease domains but confirm its indirect role in installing an alkali-labile moiety as the imprint. While ruling out the role of the primase subunit of Polα holoenzyme, we find that mutations in the Polα-recruitment and putative primase homology domain in Mcm10/Cdc23 abrogate the ribonucleotide imprint formation. These results, while confirming the ribonucleotide nature of the imprint suggest the possibility of a direct role of Mcm10/Cdc23 in installing it in cooperation with Polα and Swi1.

Ablation of RNA interference and retrotransposons accompany acquisition and evolution of transposases to heterochromatin protein CENPB.

Inactivation of retrotransposons is accompanied by the emergence of centromere-binding protein-B (CENPB) in Schizosaccharomyces, as well as in metazoans. The RNA interference (RNAi)-induced transcriptional silencing (RITS) complex, comprising chromodomain protein-1 (Chp1), Tas3 (protein with unknown function), and Argonaute (Ago1), plays an important role in RNAi-mediated heterochromatinization. We find that whereas the Ago1 subunit of the RITS complex is highly conserved, Tas3 is lost and Chp1 is truncated in Schizosaccharomyces cryophilus and Schizosaccharomyces octosporus We show that truncated Chp1 loses the property of heterochromatin localization and silencing when transformed in Schizosaccharomyces pombe Furthermore, multiple copies of CENPB, related to Tc1/mariner and Tc5 transposons, occur in all Schizosaccharomyces species, as well as in humans, but with loss of transposase function (except Schizosaccharomyces japonicus). We propose that acquisition of Tc1/mariner and Tc5 elements by horizontal transfer in S. pombe (and humans) is accompanied by alteration of their function from a transposase/endonuclease to a heterochromatin protein, designed to suppress transposon expression and recombination. The resulting redundancy of RITS may have eased the selection pressure, resulting in progressive loss or truncation of tas3 and chp1 genes in S. octosporus and S. cryophilus and triggered similar evolutionary dynamics in the metazoan orthologues.

α-(-)-bisabolol reduces pro-inflammatory cytokine production and ameliorates skin inflammation.

α-(-)-bisabolol is a natural monocyclic sesquiterpene present in the essential oil has generated considerable interest in the chemical and pharmaceutical industries and currently in use in various formulations, mainly in cosmetics. This study was undertaken to evaluate its therapeutic profile against skin inflammation using in-vitro, in-vivo and in-silico assays. Lipopolysachharide (LPS) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced production of proinflammatory cytokines (TNF-α and IL-6) in macrophage cells as well as in TPA-induced skin inflammation in mice was significantly inhibited by α-(-)-bisabolol. TPA-induced ear thickness, ear weight and lipid peroxidation and histopathological damage in the ear tissue were also significantly inhibited by topical application of α-(-)-bisabolol in a dose dependent manner. In-vitro and in-vivo toxicity profiles indicate that it is safe for topical application on skin. Molecular docking study also revealed its strong binding affinity to the active site of the pro-inflammatory proteins. These findings suggested that α-(-)-bisabolol may be a useful therapeutic candidate for the treatment of skin inflammation.

Experimental Assessment of Moringa oleifera Leaf and Fruit for Its Antistress, Antioxidant, and Scavenging Potential Using In Vitro and In Vivo Assays.

We have investigated effect of Moringa oleifera leaf and fruit extracts on markers of oxidative stress, its toxicity evaluation, and correlation with antioxidant properties using in vitro and in vitro assays. The aqueous extract of leaf was able to increase the GSH and reduce MDA level in a concentration-dependent manner. The ethanolic extract of fruit showed highest phenolic content, strong reducing power and free radical scavenging capacity. The antioxidant capacity of ethanolic extract of both fruit and leaf was higher in the in vitro assay compared to aqueous extract which showed higher potential in vivo. Safety evaluation studies showed no toxicity of the extracts up to a dose of 100 mg/kg body weight. Our results support the potent antioxidant activity of aqueous and ethanolic extract of Moringa oleifera which adds one more positive attribute to its known pharmacological importance.

Antiproliferative and antioxidant activities of Juglans regia fruit extracts.

CONTEXT: Cancer chemopreventive action of walnut [Juglans regia L. (Juglandaceae)] has been explored. OBJECTIVE: This study evaluated antiproliferative and antioxidant activities of walnut. MATERIALS AND METHODS: Various fractions of walnut extract have been screened for antiproliferative activity against human cancer cell lines using the MTT assay. All these fractions have also been evaluated for total phenolic content, antioxidant activity, and reducing power capacity. RESULTS AND DISCUSSION: Chloroform and ethyl acetate fractions exhibited a high level of antiproliferation against HepG-2, liver cancer cell line (IC(50) = 9 and 15 µg/mL, respectively). CONCLUSION: Exhibiting high phenolic content, antioxidant activity, and potent antiproliferative activity, walnut may act as a cancer chemopreventive agent.

Metabolic pathway reconstruction of eugenol to vanillin bioconversion in Aspergillus niger.

Identification of missing genes or proteins participating in the metabolic pathways as enzymes are of great interest. One such class of pathway is involved in the eugenol to vanillin bioconversion. Our goal is to develop an integral approach for identifying the topology of a reference or known pathway in other organism. We successfully identify the missing enzymes and then reconstruct the vanillin biosynthetic pathway in Aspergillus niger. The procedure combines enzyme sequence similarity searched through BLAST homology search and orthologs detection through COG & KEGG databases. Conservation of protein domains and motifs was searched through CDD, PFAM & PROSITE databases. Predictions regarding how proteins act in pathway were validated experimentally and also compared with reported data. The bioconversion of vanillin was screened on UV-TLC plates and later confirmed through GC and GC-MS techniques. We applied a procedure for identifying missing enzymes on the basis of conserved functional motifs and later reconstruct the metabolic pathway in target organism. Using the vanillin biosynthetic pathway of Pseudomonas fluorescens as a case study, we indicate how this approach can be used to reconstruct the reference pathway in A. niger and later results were experimentally validated through chromatography and spectroscopy techniques.

Design and synthesis of C-ring lactone- and lactam-based podophyllotoxin analogues as anticancer agents.

A series of novel podophyllotoxin (PDT) analogues was synthesized in which the lactone moiety was shifted to C ring. Some of the derivatives were also synthesized with modified A ring. Analogues 23 and 25 exhibited potent in vitro cytotoxicity against colon cancer (CaCO(2)) cell line. p-Demethylated E-ring analogues exhibited better potency than the corresponding methylated analogues. These analogues showed toxicity comparable to PDT against human erythrocytes albeit at much higher concentrations (100 microg/ml) than their cytotoxicity values.

Antioxidant potential of the root of Vetiveria zizanioides (L.) Nash.

Vetiveria zizanioides, an aromatic plant commonly known as vetiver has been used for various ailments. The essential oil of vetiver root has been shown to possess antioxidant activity. However, antioxidant potential of spent root extract has not been reported. Hence, in the present study, ferric reducing, free radical scavenging and antioxidant activity of two genotypes namely KS1 and gulabi of V. zizanioides L. Nash root were investigated using in vitro assays - the ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), total phenolic content (TPC), total antioxidant capacity (TAC) and reducing power (RP). KS1 genotype showed higher FRAP values, DPPH inhibition, TPC and RP potential compared to gulabi and the antioxidant activity increased with the concentration of the extract (10-1000 microg/mL). A significant protective effect of cv KS1 (100 microg/mL) extract was also observed in reduced glutathione (GSH) and malondialdehyde (MDA) concentrations of erythrocytes subjected to oxidative stress by tert-butyl hydroperoxide (t-BHP) and hydrogen peroxide (H202). The cv KS1 showed better antioxidant activity, compared to cv gulabi indicating the possibility of exploring the presence of different phytoconstituents in the two varieties.

Antifungal activity of Glycyrrhiza glabra extracts and its active constituent glabridin.

Glabridin, an active constituent of Glycyrrhiza glabra roots, was found to be active against both yeast and filamentous fungi. Glabridin also showed resistance modifying activity against drug resistant mutants of Candida albicans at a minimum inhibitory concentration of 31.25-250 microg/mL. Although the compound was reported earlier to be active against Candida albicans, but this is the first report of its activity against drug resistant mutants.

A promising anticancer and antimalarial component from the leaves of Bidens pilosa.

Bidens pilosa is used in folk medicine for various applications due to the presence of polyacetylenes, flavonoids, terpenoids, phenylpropanoids and others. Bioactivity-guided fractionation of different extracts of B. pilosa leaf showed potential in vitro anticancer and antimalarial activity and led to the identification of a potential marker compound, phenyl-1,3,5-heptatriyne. Erythrocyte osmotic fragility experiments revealed the various extracts as well as the marker component's toxicity profiles on normal blood cells.

Gallic acid-based indanone derivatives as anticancer agents.

Gallic acid-based indanone derivatives have been synthesised. Some of the indanones showed very good anticancer activity in MTT assay. Compounds 10, 11, 12 and 14 possessed potent anticancer activity against various human cancer cell lines. The most potent indanone (10, IC(50)=2.2 microM), against MCF-7, that is, hormone-dependent breast cancer cell line, showed no toxicity to human erythrocytes even at higher concentrations (100 microg/ml, 258 microM). While, indanones 11, 12 and 14 showed toxicities to erythrocytes at higher concentrations.