Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

1,3-丙二醇发酵中甘油代谢及产物抑制作用

对微生物法甘油转化成1,3-丙二醇过程中代谢规律及代谢控制的相关酶作以简述,并着重分析了丁酸梭菌发酵生产中乙酸、丁酸等副产物对细胞的抑制作用,对未来的研究发展方向进行了展望。     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.     

Green synthesis,biological evaluation,molecular docking studies and 3D-QSAR analysis of novel phenylalanine linked quinazoline-4(3H)-one-sulphonamide hybrid entities distorting the malarial reductase activity in folate pathway

A modified Grimmel's method for N-heterocyclization of phenylalanine linked sulphonamide side arm at position-2 was optimized leading to 2,3-disustituted-4-quinazolin-(3H)-ones. Further, [Bmim][BF₄]-H2O (IL) was used as green solvent as well as catalyst for the synthesis of twenty two hybrid quinazolinone motifs (4a-4v) by N-heterocyclization reaction using microwave irradiation technique. The in vitro screening of the hybrid entities against the malarial species Plasmodium falciparum yielded five potent molecules 4l, 4n, 4r, 4t & 4u owing comparable antimalarial activity to the reference drugs. In continuation, an in silico study was carried out to obtain a pharmacophoric model and quantitative structure activity relationship. We also built a 3D-QSAR model to procure more information that could be applied to design new molecules with more potent Pf-DHFR inhibitory activity. The designed pharmacophore was recognized to be more potent for the selected molecules, exhibiting five pharmacophoric features. The active scaffolds were further evaluated for enzyme inhibition efficacy against alleged receptor Pf-DHFR computationally and in vitro, proving their candidature as lead dihydrofolate reductase inhibitors as well as the selectivity of the test candidates was ascertained by toxicity study against vero cells. The perception of good oral bioavailability was also proved by study of pharmacokinetic properties.     

Classification of the mode of inhibition of high-affinity choline uptake using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode

A nonradiochemical in vitro assay using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode has been developed to evaluate the inhibition of high-affinity choline transport in synaptosomes. Quantitative analysis of high-affinity choline transporter rates as a function of inhibitor and substrate concentrations allowed determination of the mode of inhibition for the quaternary ammonium-catechol-based inhibitors 3-[(trimethylammonio)methyl]catechol, N,N-dimethylepinephrine, and 6-hydroxy-N,N-dimethylepinephrine. The results are compared to the well-characterized inhibitor of choline transport, hemicholinium-3.     

Phosphate inhibition of secondary metabolism in Streptomyces hygroscopicus and its reversal by cyclic AMP

Inorganic phosphate inhibited the biosynthesis of the macrolide antibiotic turimycin in different strains of Streptomyces hygroscopicus. In the wild type strain a depression was observed with increasing phosphate concentrations. A total inhibition was found at 0.1 M phosphate. In a high producing mutant a minimum of turimycin production occured when the phosphate concentration was between 5 mM and 10 mM. Above this concentration the antibiotic synthesis increased again but the production period shifted to a later period of cultivation. Addition of inorganic phosphate resulted in an initial increase of intracellular cyclic AMP content. But a second elevation characterizing the normal level of cyclic AMP throughout the growth phase was prevented by phosphate. Exogenous cyclic AMP as well as positive effectors of the adenylyl cyclase system were able to overcome the phosphate suppression. Cyclic AMP abolished the reduction of protein synthesis following phosphate addition and caused the reappearance of a protein band which may be responsible for the turimycin biosynthesis.     

Effects of alloxan on the islets of Langerhans inhibition of leucine metabolism and insulin secretion

The action of alloxan on the metabolism of the islets of Langerhans was studied in vitro. Isolated mouse islets were exposed to the drug at 4°C to prevent its decomposition. Islet uptake of leucine was subsequently estimated at 37°C, and was found not to be affected by the drug. However, islet leucine oxidation was strongly inhibited by the preceding alloxan exposure. The islets were protected against this inhibition by an incubation at a high glucose concentration prior to alloxan exposure. In contrast, a high concentration of leucine failed to provide full protection of either islet leucine oxidation or islet glucose oxidation. Furthermore, it was shown that alloxan impeded islet insulin response to both leucine and glucose. In addition, the potentiation of insulin release by theophylline was abolished after alloxan treatment of the islets. The results reinforce the hypothesis that the B-cytotoxicity of alloxan reflects an interaction with intracellular sites involved in the oxidative metabolism of the B-cell, and that these sites may be protected against the action of the drug by some metabolite of glucose.     

Correlating the fractional inhibition of caspase-3 in NT2 cells with apoptotic markers using an active-caspase-3 enzyme-linked immunosorbent assay

A rapid and quantitative method for measuring the activity and fractional inhibition of enzymes within their natural cellular environment remains an unmet need in drug discovery. We describe the use of a nonradioactive quantitative enzyme-linked immunosorbent assay (ELISA) for measuring intracellular caspase activity that is amenable to robotic automation. The ELISA specifically detects active-caspase-3 and was used to correlate the in-cell activity of caspase-3 with the progress of caspase-3-mediated events under varying concentrations of caspase-3 inhibitors in NT2 cells. We examined the cleavage of endogenous substrates (poly(ADP-ribose)polymerase and alphaII-spectrin), the extent of DNA fragmentation, and the autocatalytic removal of the caspase-3 prodomain as markers of caspase-3 activity. To impart inhibition of the downstream markers, a greater level of caspase-3 inhibition was required. Although the functional markers were found not to accurately predict intracellular caspase-3 activity, we found that the inhibition of intracellular caspase-3 was highly correlated (R(2) = 0.96) to the inhibition of DNA fragmentation. Also, by comparing the potency of the different inhibitors against the intracellular enzyme versus the purified enzyme, the effects of inhibitor functional groups on whole-cell activity were addressed.