A new class of potent, slowly reversible dehydropeptidase inhibitors

Dehydrodipeptide analogs whose scissile carboxamide has been replaced with a PO(OH)CH2 group have been found to be potent inhibitors of the zinc protease dehydrodipeptidase 1 (DHP-1, renal dipeptidase, EC 3.4.13.11). The best of these inhibitors, compound 25 (Ki = 0.52 nM), is two hundred times more potent than cilastatin 2 which is used clinically as a component of the broad-spectrum antibiotic combination Primaxin. Compound 25 is a tight binding inhibitor exhibiting slow binding kinetics with a remarkably slow off rate from DHP-1 (half life greater than 8 hours). The kinetics of its binding are consistent with a simple on-off mechanism whereas the less active D-enantiomer 26 appears to bind in an initial loose complex with the enzyme which slowly rearranges to a tighter complex (Ki = 83 nM).     

A standardized two-sex stable population

The “problem of the sexes” has been one of trying to reconcile inconsistent male and female demographic rates. The present paper deals with that question in the context of a two-sex stable population. A “rectangular” population, with equal numbers of persons in each age-sex group, is introduced as a standard, and a standardization relationship expressed in Eq. (3) relates changes in rectangular population rates to changes in age-sex composition. The standardization relationship is shown to satisfy a number of desirable properties and produce a realistic two-sex model. The standardization approach is then applied to data from Sweden 1973 and the United States 1963, and the results and their implications are discussed. In particular, it is seen that the intrinsic growth rate in a two-sex stable population is not necessarily bounded by the growth rate of the associated male and female one-sex stable populations.     

Statistical analysis of growth limitations in Paracoccus denitrificans: an experiment with a completely randomized two-way factorial design with replications

Cultivation of microorganisms under growth limitation is a widely used technique in microbiology. The relevant investigations, though seemingly based on straightforward experiments, have generated conflicting results, e.g., concerning molar growth yields. The purpose of this paper is to show that discrepancies in the literature could be solved if more attention were paid to methodology, especially statistics. New experimental results, concerning growth limitations in Paracoccus denitrificans, will exemplify this. They include the following items. Two limitations, one of them being succinate limitation, were established in the present study. Molar growth yields on succinate were 41 g·mol^-1 for succinate limitation (95% confidence limits were 38 and 44) and 32 g·mol^-1 for the other limitation (95% confidence limits were 29 and 35). The latter result is compatible with sulphate limitation, but the present experimental design does not really permit this conclusion.     

Differential regulation of angiotensin II-induced expression of connective tissue growth factor by protein kinase C isoforms in the myocardium

Protein kinase C (PKC) and angiotensin II (AngII) can regulate cardiac function in pathological conditions such as in diabetes or ischemic heart disease. We have reported that expression of connective tissue growth factor (CTGF) is increased in the myocardium of diabetic mice. Now we showed that the increase in CTGF expression in cardiac tissues of streptozotocin-induced diabetic rats was reversed by captopril and islet cell transplantation. Infusion of AngII in rats increased CTGF mRNA expression by 15-fold, which was completely inhibited by co-infusion with AT1 receptor antagonist, candesartan. Similarly, incubation of cultured cardiomyocytes with AngII increased CTGF mRNA expression by 2-fold, which was blocked by candesartan and a general PKC inhibitor, GF109203X. The role of PKC isoform-dependent action was further studied using adenoviral vector-mediated gene transfer of dominant negative (dn) PKC or wild type PKC isoforms. Expression of dnPKCalpha, -epsilon, and -zeta isoforms suppressed AngII-induced CTGF expression in cardiomyocytes. In contrast, expression of dominant negative PKCdelta significantly increased AngII-induced CTGF expression, whereas expression of wild type PKCdelta inhibited this induction. This inhibitory effect was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of PKCdelta (deltaTg mice). Thus, AngII can regulate CTGF expression in cardiomyocytes through a PKC activation-mediated pathway in an isoform-selective manner both in physiological and diabetic states and may contribute to the development of cardiac fibrosis in diabetic cardiomyopathy.     

Thrombin generation and inactivation in the presence of antithrombin III and heparin

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of multiple hydrogen-bonding groups in specific alcohol binding sites in proteins: insights from structural studies of LUSH

It is now generally accepted that many of the physiological effects of alcohol consumption are a direct result of binding to specific sites in neuronal proteins such as ion channels or other components of neuronal signaling cascades. Binding to these targets generally occurs in water-filled pockets and leads to alterations in protein structure and dynamics. However, the precise interactions required to confer alcohol sensitivity to a particular protein remain undefined.Using information from the previously solved crystal structures of the Drosophila melanogaster protein LUSH in complexes with short-chain alcohols, we have designed and tested the effects of specific amino acid substitutions on alcohol binding. The effects of these substitutions, specifically S52A, T57S, and T57A, were examined using a combination of molecular dynamics, X-ray crystallography, fluorescence spectroscopy, and thermal unfolding. These studies reveal that the binding of ethanol is highly sensitive to small changes in the composition of the alcohol binding site. We find that T57 is the most critical residue for binding alcohols; the T57A substitution completely abolishes binding, while the T57S substitution differentially affects ethanol binding compared to longer-chain alcohols. The additional requirement for a potential hydrogen-bond acceptor at position 52 suggests that both the presence of multiple hydrogen-bonding groups and the identity of the hydrogen-bonding residues are critical for defining an ethanol binding site. These results provide new insights into the detailed chemistry of alcohol's interactions with proteins.