Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. the gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239 bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).     

Solution structure of the covalent sterigmatocystin-DNA adduct.

Sterigmatocystin and aflatoxin are potent mutagens that contaminate foodstuffs stored under conditions that permit fungal growth. These food mycotoxins can be metabolically activated to their epoxides, which subsequently form covalent adducts with DNA and can eventually induce tumor development. We have generated the sterigmatocystin-d(A1-A2-T3-G4-C5-A6-T7-T8) covalent adduct (two sterigmatocystins per duplex) by reacting sterigmatocystin-1,2-epoxide with the self-complementary d(A-A-T-G-C-A-T-T) duplex and determined its solution structure by the combined application of two-dimensional NMR experiments and molecular dynamics calculations. The self-complementary duplex retains its 2-fold symmetry following covalent adduct formation of sterigmatocystin at the N7 position of G4 residues on each strand of the duplex. The H8 proton of [ST]G4 exchanges rapidly with water and resonates at 9.58 ppm due to the presence of the positive charge on the guanine ring following adduct formation. We have assigned the exchangeable and nonexchangeable proton resonances of sterigmatocystin and the duplex in the covalent adduct and identified the intermolecular proton-proton NOEs that define the orientation and mode of binding of the mutagen to duplex DNA. The analysis was aided by intermolecular NOEs between the sterigmatocystin protons with both the major groove and minor groove protons of the DNA. The molecular dynamics calculations were aided by 180 intramolecular nucleic acid constraints, 16 intramolecular sterigmatocystin constraints, and 56 intermolecular distance constraints between sterigmatocystin and the nucleic acid protons in the adduct. The sterigmatocystin chromophore intercalates between the [ST]G4.C5 and T3.A6 base pairs and stacks predominantly over the modified guanine ring in the adduct duplex. The overall conformation of the DNA remains right-handed on adduct formation with unwinding of the helix, as well as widening of the minor groove. Parallel NMR studies on the sterigmatocystin-d(A1-A2-A3-G4-C5-T6-T7-T8) covalent adduct (two sterigmatocystins per duplex) provide supportive evidence that the mutagen covalently adducts the N7 position of G4 and its chromophore intercalates to the 5' side of the guanine and stacks over it. The present NMR-molecular dynamics studies that define a detailed structure for the sterigmatocystin-DNA adduct support key structural conclusions proposed previously on the basis of a qualitative analysis of NMR parameters for the adduct formed by the related food mutagen aflatoxin B1 and DNA [Gopalakrishnan, S., Harris, T. M., & Stone, M. P. (1990) Biochemistry 29, 10438-10448].     

Specific high affinity binding of the calcium channel antagonist, [3H]nitrendipine, to rat liver microsomes

Some key properties of the binding of [3H]nitrendipine, an analogue of the 1,4-dihydropyridine, nifedipine, to a plasma membrane enriched microsomal fraction from the rat liver are described. Specific binding was saturable, linear with protein concentration, and reversible. The apparent equilibrium dissociation constant, KD, was 4.20 +/- 0.22 nM and the maximum density of binding, Bmax, was 3.02 +/- 0.17 pmol/mg of protein determined from Scatchard analysis of binding at 10 degrees C. Inhibition of binding was specific for dihydropyridines with competitive inhibition being noted with nifedipine and 4-chloronifedipine, as well as BAY K-8644, a calcium channel agonist. A biphasic displacement curve was recorded for methoxy verapamil (D-600), and a triphasic competition curve with lanthanum (La3+), and diltiazem demonstrated competitive kinetics. The high affinity binding site for nitrendipine in the liver, although having some similar properties to those sites described in skeletal muscle, would appear to be distinctive with respect to its unique sensitivity to D-600 and diltiazem. We speculate that this binding site may represent a Ca2+ channel responsible for regulating Ca2+ influx and hepatic glycogenolysis.     

Affinity purification of hexosaminidases

Hexosaminidases A and B were purified by affinity chromatography from normal gastric mucosa, after preliminary separation of isozymes by anion exchange chromatography. Heparin and mannosamine were coupled to Sepharose 4B and used as affinity matrices and the purified enzymes were found to be homogeneous when analysed by polyacrylamide slab gel electrophoresis. This combination of 2 novel affinity chromatographic procedures is superior to existing methods in that a final yield of over 70% could be achieved. Also, the number of steps required to obtain homogeneity are less in contrast to the conventional methods used previously.     

Biochemical characterisation of grain mould resistant and susceptible genotypes and PGPR-induced resistance in the host to Curvularia lunata and Fusarium proliferatum

Resistance to biotic stresses in plants is either due to the presence of preformed biochemical compounds or induced in response to external stimulus. In this study, 13 grain mould resistant and seven susceptible lines of sorghum were analysed for biochemical defence mechanism. The levels of total phenols and phenylalanine ammonia lyase were almost same in the resistant and susceptible genotypes. However, two additional isoforms of peroxidase were found in the three of the 13 resistant genotypes. The isoform peroxidase corresponding to the R _f value of 0.25 was found in the resistant genotypes IS 13969, ICSB 377 and IS 8219-1, and two genotypes IS 13969 and ICSB 377 had an additional isoform corresponding to the R _f value of 0.32. The results indicated the genotype specific association of peroxidases with grain mould resistance in sorghum. Nine bacterial strains (Bacillus pumilus SB 21, Bacillus megaterium HiB 9, Bacillus subtilis BCB 19, Pseudomonas plecoglossicida SRI 156, Brevibacterium antiquum SRI 158, B. pumilus INR 7, P. fluorescens UOM SAR 80, P. fluorescens UOM SAR 14, B. pumilus SE 34) were tested to induce systemic resistance in sorghum cultivars 296B and Bulk Y against the highly pathogenic grain mould pathogens Curvularia lunata and Fusarium proliferatum, respectively. The bacterial isolates were effective in inducing resistance in sorghum. Among the strains tested, SRI 158 was found highly effective in reducing grain mould severity in both the genotypes.     

Contraceptive potential of an antiprogestin ZK 98.734: reversal of ZK 98.734--induced blockade of folliculogenesis with FSH and LH and their differential effects in bonnet monkeys.

Studies were undertaken in adult bonnet monkeys to investigate whether treatment with an antiprogestin ZK 98.734 at weekly intervals, starting from day one of menstrual cycle, could arrest ovulation and also to determine if ZK 98.734 induced blockade of ovulation could be reversed with gonadotropins. Adult animals have ovulatory menstrual cycles of normal duration were treated at weekly intervals with ZK 98.734 (25 mg/dose, sc, oil base) for 10 consecutive weeks and its effects on serum levels of estradiol, bioactive LH and progesterone, and endometrial histology were investigated. Following treatment with the antiprogestin they were treated with hMG or hFSH alone. Ovulation was blocked during treatment period in all the animals (n = 14). Typical follicular phase rise in estradiol levels was inhibited, mid cycle surge in the levels of bioactive LH was abolished and serum progesterone levels remained below 1 ng/ml throughout the treatment period. However, prolonged treatment had no significant effect on the basal levels of estradiol which were around 50 pg/ml. ZK 98.734 also had no significant effect on cortisol levels. In animals (n = 4) followed for recovery after the last dose, the treatment cycle length was increased to 117.8 + 6.8 days. In three animals the treatment cycles were anovulatory, whereas in one delayed ovulation with luteal insufficiency was observed. The endometrium had become atrophic. Treatment with hMG (Pergonal: 35 I.U. hLH and 35 I.U. hFSH) or hFSH (Metrodin, 35 I.U.) for 7 consecutive days initiated folliculogenesis and the animals ovulated either spontaneously or after a single im injection of hCG (100 I.U.) on day 8 in ZK 98.734 treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective effect of vanillic acid against benzo(a)pyrene induced lung cancer in Swiss albino mice

Vanillic acid (VA) is found in high concentrations in various plants and used as traditional medicine for various diseases. The aim of the existing study is to illustrate the protective effects of VA against benzo(a)pyrene (B(a)P)‐induced lung cancer in Swiss albino mice. B(a)P (50 mg/kg b.wt.) was given orally to induce lung cancer in mice. The body weight, tumor incidence, carcinoembryonic antigen (CEA), neuron‐specific enolase (NSE), and enzymatic/nonenzymatic antioxidants (superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and glutathione) were estimated. Further histochemical investigation through hematoxylin and eosin staining was also carried out. B(a)P administered groups showed increased levels of serum pathological markers CEA, NSE along with reduced final body weight as well as decreased tissue enzymatic and nonenzymatic antioxidants activities, whereas VA treatment (200mg/kg/b.wt) along with B(a)P showed significantly reverted the above changes, which proves as prominent anticancer effects in experimentally induced lung cancer. Overall, these results suggest that VA has an efficient preventive action against B(a)P‐induced lung cancer, and this is attributed to its free‐radical scavenging antioxidant activities.