Examination of substrate and product inhibition kinetics on the production of ethanol by suspended and immobilized cell reactors

Kinetic expressions for the fermentative production of relatively high concentrations [12% (w/v)] of ethanol have been examined. Several expressions which account for both substrate and product inhibition have been formulated, and have been applied to suspended cell and immobilized cell reactors. Experimental data have been used to validate the kinetic expressions used, and the impact of combined inhibition on optimal reactor configuration has been assessed. The process implications of combined substrate and product inhibition for suspended and immobilized cell systems have been discussed.     

Biological activity of aspartic proteinase inhibitors related to pepstatin

We have synthesized eight tripeptide analogs of pepstatin in which both the side-chain and stereochemistry of the novel amino acid statine have been altered. They have been compared to pepstatin for inhibition of pepsin and cathepsin D activity, inhibition of autolysis at pH 4, and inhibition of protein degradation in cultured cells. Effective inhibition of aspartic proteinase activity appears to require the novel amino acid to have a bulky hydrophobic side-chain and the S-configuration at both chiral centers. However, the Cbz-Val-Val-(3S4S)-statine peptide was more effective than pepstatin in cultured cells, and inhibition was also achieved, and in some cases enhanced relative to pepstatin, by its stereoisomers and by tripeptides containing valyl and alanyl analogs of statine.     

直接遗忘效应中认知抑制机制研究新进展

随着认知抑制研究的兴起,作为测量认知抑制能力的主要方法,直接遗忘效应得到了不断深入的研究。本文简要介绍了直接遗忘效应的研究范式,并就直接遗忘的内在机制是"选择性复述"的作用,还是"认知抑制"的作用展开讨论。在此基础上还就个体抑制能力的发展进程及其应用价值的研究进展做了简要介绍,并指出了已有研究存在的一些问题。     

Some physicochemical properties of modified trypsin]

Physico-chemical properties of trypsin covalently bound with human serum albumin by glutaric aldehyde have been studied. The modification of the enzyme practically caused no changes in the pH optimum of trypsin. The inhibition of modified trypsin by inhibitors from soy beans and human blood serum has been also studied. The apparent inhibition constants have been calculated. The modification has been shown to result in a deceleration of autolytic degradation. The autolysis rate constants have been calculated at 50 degrees C.     

Kinetics of substrate reaction during irreversible modification of enzyme activity for enzymes involving two substrates

Kinetic equations for the substrate reaction during simultaneous irreversible inhibition of enzyme activity for enzymes involving two substrates have been derived. It has been shown that the method proposed previously (Tsou, Acta Biochim, Biophys. Sinica 5, 398-417, 1965) for the determination of the apparent inhibition rate constants in the cases of single substrate enzymes can also be used in the present situation. Moreover, the criteria proposed to distinguish between different substrate competition types and to detect the formation of a reversible enzyme-inhibitor complex prior to the irreversible inhibition step also apply. Methods for the estimation of the microscopic rate constants have been proposed and it has been shown that irreversible inhibition kinetics can be used to distinguish between different mechanisms for substrate binding sequences.     

The action of inhibitors (histidine and AMP) on the ATP phosphoribosyltransferase of E. coli.

The inhibitors histidine and AMP cause the enzyme ATP phosphoribosyltransferase of E. coli to associate into a hexamer from its initial dimeric form. The behaviour of these inhibitors has been studied by three different methods. I) Equilibrium dialysis studies have shown that one mole of dimeric enzyme (67,000 g) binds one mole of histidine. II) By kinetic inhibition of the reaction studied at 21, 25 and 38 degrees C the enthalpy changes in the process of histidine and of AMP inhibition have been deduced. The inhibition has also been studied in function of enzyme concentration and temperature. The inhibition appears to be slightly negatively cooperative for histidine and positively cooperative for AMP. In neither case is it possible to obtain 100% maximal inhibition. III) By microcalorimetric analysis the values obtained for the enthalpies of histidine and of AMP interaction with the enzyme are similar.     

Reversal of Ethionine-Induced Inhibition of Elongation of Avena Coleoptiles by Purines, Adenosine, and Glutamic Acid

Ethionine-induced inhibition of elongation of Avena coleoptile segments has been measured in water and in indol-3yl-acetic acid. In the presence of 10 mM L-ethionine the inhibition amounts to about 70 %. It has been shown previously that the addition of adenosine triphosphate effectively counteracts this inhibition; optimal ATP concentrations are between 0.25 and 0.5 mM. Adenine, adenine sulfate, adenosine, guanine, S-adenosylmethionine, and glutamic acid have now been shown to act similarly to ATP in reversing the ethionine-induced inhibition of elongation.     

Design, synthesis and RNase A inhibition activity of catechin and epicatechin and nucleobase chimeric molecules

Several novel catechin/epicatechin and nucleobase chimeric molecules 1-6 have been synthesized via azide-alkyne click chemistry. The structures of these hybrids have been confirmed by NMR and mass spectroscopic data. The synthesized molecules were tested for their RNase A inhibition activities. Gel-based assays showed inhibition in micromolar concentrations. The extent of inhibition was found to be dependent upon the nature of base as well as the configuration at C-3 position of catechin.     

Synthesis and biological evaluation of some phosphate triester derivatives of the anti-viral drug AraA.

A number of novel phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared by a rapid 2-step procedure, not necessitating prior sugar protection. Spectroscopic and lipophilicity data have been collected on these compounds, and they have been assayed with a range of hydrolytic enzymes. The compounds have been found to be highly resistant to hydrolysis at physiological pH, enzymatic or otherwise. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of these compounds, in the range 3-300 microM. Moreover, the degree of inhibition showed a close correlation to chemical structure; in particular, there was a direct relationship between inhibition of thymidine incorporation and lipophilicity. These results suggest cellular penetration by the phosphate triesters and intracellular hydrolysis, by an unspecified mechanism, to the free nucleotide or nucleoside.     

Kinetic and mechanism of interaction of L-cysteine with alkaline phosphatase from calf intestines

The effect of L-cysteine on activity of hydrophobic forms of calf intestine alkaline phosphatase was investigated. Apparent inhibition constants for mixed type inhibition have been determined. The kinetic results allow supposing that the mechanism of equilibrium establishment between the inhibitor and enzyme involves the initial rapid formation of intermediate complex and a subsequent slower step leading to its stabilization in the substrate binding site. The microscopic rate constants for slow step of interaction of L-cysteine with alkaline phosphatase have been calculated. Effect of pH on apparent inhibition constants and kinetic parameters for enzymatic reaction in the presence of L-cysteine was analysed.     

Synthesis of nitrated indenoisoquinolines as topoisomerase I inhibitors

Indenoisoquinolines and dihydroindenoisoquinolines have been synthesized possessing a nitro-substituted isoquinoline ring in an effort to explore the effects of electron-withdrawing substituents on biological activity. The in vitro anticancer activities of these molecules have been tested in the National Cancer Institute's screen of 55 cell lines. The compounds have also been tested for topoisomerase I (top1) inhibition. The results indicate that these substances are a potent class of top1 inhibitors with sub-micromolar cytotoxicity mean graph midpoints (MGM) and top1 inhibition equal to camptothecin.     

The inhibition of proteolytic enzyme activity by humic acids]

The inhibitory effect of peat humic acids on the hydrolysis of N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-leucine methyl ester by alpha-chymotrypsin and subtilism has been studied. Samples of humic acids with M(W) approximately 18,000 have been used in experiments. The results of kinetic studies indicates the mixed type of inhibition of proteinase activity by humic acids. The meanings of inhibition constants under the action of humic acids on alpha-chymotrypsin and subtilism have been calculated.     

Studies of reversible inhibition,irreversible inhibition,and activation of alkaline phosphatase by capillary electrophoresis

Reversible inhibition, irreversible inhibition, and activation of calf intestinal alkaline phosphatase (EC 3.1.3.1) have been studied by capillary electrophoresis. The capillary electrophoretic enzyme-inhibitor assays were based on electrophoretic mixing of inhibitor and enzyme zones in a substrate-filled capillary. Enzyme inhibition was indicated by a decrease in product formation detected in the capillary by laser-induced fluorescence. Reversible enzyme inhibitors could be quantified by Michaelis-Menten treatment of the electrophoretic data. Reversible, competitive inhibition of alkaline phosphatase by sodium vanadate and sodium arsenate has been examined, and reversible, noncompetitive inhibition by theophylline has been studied. The K(i) values determined for these reversible inhibitors using capillary electrophoresis are within the range of values reported in the literature for the same enzyme-inhibitor combinations. Irreversible inhibition of alkaline phosphatase by EDTA at concentrations of 1.0mM and above has been observed. Activation of alkaline phosphatase has also been observed for EDTA at concentrations from 20 to 400 microM.     

Action mechanisms of probiotics on Candida spp. and candidiasis prevention: an update

Due to the high incidence of fungal infections caused by Candida species and their increasing resistance to antimicrobial treatments, alternative therapies such as probiotics have been studied. It has been show that several species of the genus Lactobacillus have anti-Candida activity, probably by direct inhibition, through competition for adhesion sites or production of secondary metabolites, and by indirect inhibition, through stimulation of the immune system of their host. However, the mechanisms of inhibition of these probiotics on Candida species have not yet been fully elucidated since this effect is related to more than one inhibition pathway. In the literature, several in vitro and in vivo studies have been developed seeking to elucidate the probiotics mechanisms of action. These studies have been focused on C. albicans inhibition assays, including analysis of antimicrobial activity, adherence capacity, biofilms formation, filamentation and interference on virulence genes, as well as assays of experimental candidiasis in invertebrate and vertebrate models. In this context, the purpose of this review was to gather different studies focused on the action mechanism of probiotic strains on Candida sp. and to discuss their impact on the candidiasis prevention.     

Exploring the potential of 3'-O-carboxy esters of thymidine as inhibitors of ribonuclease A and angiogenin

In this study, compounds with a carboxy ester in lieu of the phosphate ester at the 3'-position have been employed to inhibit the ribonucleolytic activity of ribonuclease A (RNase A). Phosphates at the 3'-position of pyrimidine bases are well-known inhibitors of the protein. We have investigated the inhibition of RNase A by 3'-O-carboxy esters of thymidine. The compounds behave as competitive inhibitors with inhibition constants ranging from 42 to 95 microM. The mode of inhibition has also been confirmed by (1)H NMR studies of the active site histidines of RNase A. Docking studies have further substantiated the experimental results. The compounds are also found to inhibit the ribonucleolytic activity of angiogenin, a homologous protein and potent inducer of blood vessel formation.     

Inhibition of anion transport in the red blood cell membrane by anionic and non-anionic arginine-specific reagents

Arginine specific reagents are found to be powerful inhibitors of anion exchange in the red blood cell membrane. Some of these inhibitors such as cyclohexandione, phenylglyoxal and 2, 3-butandione are found to produce their inhibition by interacting covalently with band 3. In contrast to the action of these compounds, the inhibition caused by the phenylglyoxal derivative 4-hydroxy-3-nitrophenyl-glyoxal has been found to be completly reversible. In extending the studies on the mode of action of these compounds on sulfate exchange and to get some more information about their binding site, the degree of inhibition caused by different phenylglyoxal derivatives which have a similar core but differ in their substituent groups have been compared. The interaction between the binding sites of these compounds and other anion transport inhibitors have also been studied.     

Selective binding of actinomycin D and distamycin A to DNA.

The exact sites at which a number of drugs inhibit the nick translation of DNA by E.coli DNA polymerase-I have been pinpointed. In order to do this, a method has been developed for sequencing double-stranded plasmid DNA from the site of a specifically induced nick. The initial experiments have concentrated on analysis of drug inhibition of nick translation in a 200 nucleotide region near the Eco Rl origin of pBR313. Many drugs were found to inhibit nick translation in a highly sequence specific manner. For actinomycin D, significant inhibition occurred at just four sites in the nucleotide sequence under test and only one sequence (pGpCpGpCpGpGp) gave really strong inhibition. Distamycin A gave a different pattern of inhibition with particularly strong stops in just two of the many A-T rich regions in the DNA. Experiments with caffeine suggest that factors in addition to primary sequence are important in determining where major inhibition occurs.     

The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase

The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.     

Kinetics of the development of accelerated cell-cycle transit resulting from inhibition of DNA replication in the previous cycle

Shortening of the generation cycle in cells in which DNA synthesis had been temporarily inhibited in the previous generation, which has been reported several times in recent years, has been confirmed in HeLa cells. As in the previous studies, the shortening is attributable to accelerated transit of G1 resulting from the accumulation, during the inhibition, of a factor needed for initiation of DNA replication. It is shown that partial (85-96%) inhibition with any one of three inhibitors is effective when the inhibitor is added in G1 or in S, but more complete (99%) inhibition is effective only if the inhibitor is added after cells have entered S. In addition, cells begin to respond to the inhibition after a lag that increases as DNA synthesis in the early part of S is progressively inhibited with aphidicolin, indicating that competence to respond is achieved only after cells have reached a particular point in the replication of their genome.     

Voltage-independent inhibition of Ca(V)2.2 channels is delimited to a specific region of the membrane potential in rat SCG neurons

Neurotransmitters and hormones regulate Ca(V)2.2 channels through a voltage-independent pathway which is not well understood. It has been suggested that this voltage-independent inhibition is constant at all membrane voltages. However, changes in the percent of voltage-independent inhibition of Ca(V)2.2 have not been tested within a physiological voltage range. Here, we used a double-pulse protocol to isolate the voltage-independent inhibition of Ca(V)2.2 channels induced by noradrenaline in rat superior cervical ganglion neurons. To assess changes in the percent of the voltage-independent inhibition, the activation voltage of the channels was tested between -40 and +40 mV. We found that the percent of voltage-independent inhibition induced by noradrenaline changed with the activation voltage used. In addition, voltage-independent inhibition induced by oxo-M, a muscarinic agonist, exhibited the same dependence on activation voltage, which supports that this pattern is not exclusive for adrenergic activation. Our results suggested that voltage-independent inhibition of Ca(V)2.2 channels depends on the activation voltage of the channel in a physiological voltage range. This may have relevant implications in the understanding of the mechanism involved in voltage-independent inhibition.