Genistein induces the metastasis suppressor kangai-1 which mediates its anti-invasive effects in TRAMP cancer cells

Previous studies demonstrated a direct correlation with loss of kangai-1 (KAI1), a metastasis suppressor, and poor prognosis in human prostate and other cancers. In this study, we have characterized the age-dependent downregulation of KAI1 in the TRAMP model which was reversed when mice were fed a genistein-enriched diet. We demonstrated here that doses of genistein (5 and 10 microM)--achievable by supplement intake--significantly induced the expression of KAI1, both at the mRNA and protein levels (up to 2.5-fold), and decreased the invasiveness of TRAMP-C2 cells >2.0-fold. We have pinpointed KAI1 as the invasion suppressor, since its knockdown by siRNA restored the invasive potential of genistein-treated TRAMP-C2 cells to control levels. This work provides the first evidence that genistein treatment may counteract KAI1 downregulation, which is observed in many cancer types and therefore, could be used in anti-metastatic therapies.     

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.     

Immunomodulatory effects of seabuckthorn (Hippophae rhamnoides L.) against chromium (VI) induced immunosuppression

The present study reports the immunomodulatory effects of seabuckthorn (Hippophae rhamnoides L.) leaf extract on cellular and humoral immune response by studying delayed-type hypersensitivity response, IL-2, IL-4 and γ-IFN levels and antibody titres in chromium-induced immunosuppressed animals. Oral feeding of chromium (30 mg/kg bw) significantly inhibited antibody production and S-RBC induced delayed-type hypersensitivity response. Administration of leaf extract (100 mg/kg bw) along with chromium significantly inhibited chromium-induced immunosuppression. To understand the immunomodulatory mechanism of leaf extract, in vitro studies were carried out using rat lymphocytes. Addition of chromium resulted in a significant decrease in lymphocyte size and increased ROS generation. The leaf extract of seabuckthorn significantly inhibited chromium-induced reactive oxygen species (ROS) generation and maintained the cell size identical to that of control cells. Chromium treatment markedly inhibited the mitochondrial transmembrane potential by larger lymphocytes in particular, while the leaf extract restored the same significantly. Chromium also inhibited significantly concanavalin A (ConA) induced IL-2, IL-4 and γ-IFN production in rat lymphocytes. The leaf extract (100 μg/ml) alone stimulated IL-2 and γ-IFN production even in the absence of ConA and also inhibited chromium-induced decline in IL-2 and γ-IFN production but it did not change IL-4 production. These observations suggest that the leaf extract of seabuckthorn has significant immunomodulatory activity and specifically activates the cell-mediated immune response. (Mol Cell Biochem 278: 101–109, 2005)     

Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.     

Free radical scavenging, anti-glycation and tyrosinase inhibition properties of a polysaccharide fraction isolated from the rind from Punica granatum

The present investigation deals with the isolation of a polysaccharide fraction from Pomegranate (PFP), which was found to inhibit 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonate] ABTS(+) radical activities by 69% and 88%, respectively with 4 microg/ml concentration. The activity of PFP for free radical scavenging was also evaluated by electron spin resonance (ESR) Spectrophotometer and DPPH dot blot test. Anti-glycation ability of PFP was tested using BSA, which inhibited the formation of advanced glycation end-products (AGEs) by 28% and also inhibited the formation of fructosamine in the BSA/Glucose system. The inhibition of mushroom tyrosinase by 43% at 10 microg/ml concentration of PFP strongly suggested its efficacy as a possible skin whitener.     

Optimization of extraction and purification of glucoamylase produced by Aspergillus awamori in solid-state fermentation

Various parameters such as solvent selection, concentration, soaking time, and temperature were tested in a single bioreactor in order to determine optimum extraction conditions of glucoamylase, when produced simultaneously with protease by Aspergillus awamari nakazawa MTCC 6652. Optimum conditions were achieved in a 10% glycerol solution soaked for 2 h at 40°C, followed by concentration of extracted glucoamylase (9,157 U/gds) by acetone precipitation (1:2, v/v), which yielded 51.9% recovery. Ion exchange chromatography and gel filtration showed specific activities of 270.5 and 337.5 U/mg, respectively, while SDS-PAGE and zymogram analysis of glucoamylase indicated the presence of three starch-hydrolyzing isoforms with molecular weights of approximately 109.6, 87.1, and 59.4 kDa, respectively     

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH₄)₂SO₄ fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k _cat/K _m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.     

Stereoselective enzymatic hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol diacetate ester in a biphasic system

Summary A key chiral intermediate lactol(3)[3aS (3a,4,7,7a)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one was prepared for the total synthesis of a new thromboxane antagonist. The stereoselective hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (1) to the corresponding chiral monoacetate ester (2) was carried out with lipases, among which Amano P-30 lipase from Pseudomonas sp. was most effective since it gave the desired enantiomer of monoacetate ester. A yield of 75 mol% and optical purity of >99% was obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/l of the substrate. Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (five cycles) without loss of enzyme activity, productivity or optical purity. The reaction process was scaled-up to 80 1 (400 g substrate) and monoacetate (2) was isolated in 80 mol% yield with 99.3% optical purity as determined by chiral HPLC and nuclear magnetic resonance (NMR) analysis. A gas chromatography of 99.5% and specific rotation, []_D of -7.6° was obtained. The chiral monoacetate ester (2) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give lactol (3).     

Cloning and DNA sequence of plasmid determinant iss, coding for increased serum survival and surface exclusion, which has homology with lambda DNA

Summary Escherichia coli K12 cells carrying a cloned 1.4 kb HindIII fragment from plasmid ColV2-K94, showed increased survival in guinea pig serum. The recombinant plasmid also conferred group II surface exclusion, i.e. the cells were reduced in recipient ability towards the incoming plasmid R538drd in conjugation experiments. Southern blotting suggested homology with bacteriophage lambda DNA and to the insertion element IS2. Determination of the DNA sequence of the fragment demonstrated the presence of a truncated IS2 (165 bp), separated by 250 bp from a 900 bp stretch of homology with lambda DNA, beginning within the Rz gene and continuing in the rightward direction on the lambda map. A 97 amino acid open reading frame (ORF) adjacent to Rz and on the opposite strand, remained intact in iss, with several amino acid changes. The ORF in iss is preceded by sequences resembling prokaryotic ribosome binding sites and promoters.