Ligands of second generation platinum analogs decrease both platinum-induced DNA cross-linking and its ability to interact with 1-beta-D-arabinofuranosyl cytosine to potentiate cytotoxic efficacy

We evaluated the cytotoxic and DNA cross-linking (CL) ability of four second generation platinum coordination complexes (TNO-6, JM-89, JM-8 and JM-9) delivered alone or in combination with 1-beta-D-arabinofuranosyl cytosine (ara-C) to human colon cancer cells (LoVo). Cell survival varied markedly as a function of the particular substitution moiety. JM-8 and JM-9 were virtually ineffective, even at concentrations as high as 50 micrograms/ml. At that concentration cis-diamminedichloroplatinum(II) (cis-DDP) killed greater than 99.99% of the cells. JM-82 was slightly more active while TNO-6 was the only derivative with appreciably higher cytotoxic activity due to an abrogation of the shoulder region of the type C survival curve. The highest CL effect was observed for cis-DDP followed closely by TNO-6. Very little CL effects were demonstrated for the other three analogs JM-82, JM-8 and JM-9 when measured 6 h after treatment. The combination of cis-DDP and ara-C augmented 10-fold the cytotoxic activity of cis-DDP alone, an effect accompanied by an almost 2-fold increase in CL; every other analog failed to interact in a potentiating manner (either cytotoxicity, or CL at 6 h) with the antimetabolite. Thus, it appears clear that the associated moieties of the Pt coordination complex play a fundamental role in reducing the interaction of the analogs with DNA (as reflected by the decreased CL and cytotoxic effects produced by each agent alone) and in totally preventing their interaction with ara-C to yield a potentiating lethal effect.     

Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

Background  

Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS) in non-ribosomal peptide ligation.     

High Altitude Frogs (Rana kukonoris) Adopt a Diversified Bethedging Strategy in the Face of Environmental Unpredictability

Environmental unpredictability can influence strategies of maternal investment among eggs within a clutch. Models predict that breeding females should adopt a diversified bet-hedging strategy in unpredictable environments, but empirical field evidence from Asia is scarce. Here we tested this hypothesis by exploring spatial patterns in egg size along an altitudinal gradient in a frog species(Rana kukunoris) inhabiting the Tibetan Plateau. Within-clutch variability in egg size increased as the environment became variable(e.g., lower mean monthly temperature and mean monthly rainfall at higher altitudes), and populations in environments with more unpredictable rainfall produced eggs that were smaller and more variable in size. We provide support for a diversified bet-hedging strategy in high-altitude environments, which experience dynamic weather patterns and therefore are of unpredictable environmental quality. This strategy may be an adaptive response to lower environmental quality and higher unpredictable environmental variance. Such a strategy should increase the likelihood of breeding success and maximize maternal lifetime fitness by producing offspring that are adapted to current environmental conditions. We speculate that in high-altitude environments prone to physical disturbance, breeding females are unable to consistently produce the optimal egg size due to physiological constraints imposed by environmental conditions(e.g., duration of the active season, food availability). Species and populations whose breeding strategies are adapted to cope with uncertain environmental conditions by adjusting offspring size and therefore quality show a remarkable degree of ability to cope with future climatic changes.     

Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.     

Anomeric and other substrate specificity studies with myo-inositol-1-P synthase

L-myo-Inositol-1-phosphate synthase has been found to have at least a 5-fold preference for the beta-anomer of its natural substrate D-Glc-6-P. The alpha-anomer appears to be an inhibitor of the reaction and may be converted to product as well. As well as showing an enzymatic preference for the equatorial C-1 hydroxyl of D-Glc-6-P, our results suggest that it is the pyranose form of D-Glc-6-P that binds to the enzyme and that ring-opening is an enzymatic step. We have also found D-2-dGlc-6-P, D-2-F-2-dGlc-6-P, and D-Man-6-P each to be both competitive inhibitors and substrates that are converted to inositol phosphates by the synthase. D-Allose-6-P is a weak inhibitor of the enzyme, but not a substrate. D-Gal-6-P is neither substrate nor inhibitor. Thus the specificity of the synthase with respect to single position epimers of D-Glc-6-P increases in the order C1 less than C2 much less than C3 less than C4.