Absence of human herpesvirus 8 genomes in coronary atherosclerosis in immunocompetent patients.

Human herpevirus 8 (HHV8) has been localized to the endothelial and spindle cells of KS, suggesting a role for HHV8 in atherosclerosis. None of the 38 coronary atherectomy specimens contained HHV8 with both sensitive nested PCR assays, making it unlikely that persistent viral infection with HHV8 plays a role in coronary atherogenesis in the general population of the United States.

HIV-1 protease specificity derived from a complex mixture of synthetic substrates.

A rapid and semiquantitative method is described for determining the relative kcat/Km for individual peptides in defined substrate mixtures. The method utilizes electrospray ionization/mass spectrometry alone to semiquantitatively determine relative peptide substrate turnover rates. Unlike previous studies, in which chromatographic separation of individual peptide species was required, this mass spectrometric-based method relies strictly on the ability to ionize and detect simultaneously all peptide species in a defined mixture. Differences in the ion intensities of the individual components before and after incubation with protease are used to semiquantitatively determine preferred substrates. This method was used to the identify preferred peptide substrates for HIV-1 protease. Optimal substrates were identified from a defined synthetic peptide substrate mixture based on Ser-Gln-Asn-Tyr-Pro-Ile-Val, where the P1' proline was substituted with 20 naturally occurring amino acids. The hydrophobic residues Leu, Ile, Val, Phe, and Tyr were preferred in addition to Pro at the P1' site. The results were corroborated by performing the more laborious HPLC/Frit-fast atom bombardment/MS analyses.

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease.

Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease represent a promising addition to the available agents used to inhibit virus replication in a therapeutic setting. HIV-1 is capable of generating phenotypic variants in the face of a variety of selective pressures. The potential to generate variants with reduced sensitivity to a protease inhibitor was examined by selecting for virus growth in cell culture in the presence of the protease inhibitor A-77003. Virus variants grew out in the presence of the inhibitor, and these variants encoded proteases with reduced sensitivity to the inhibitor. Variants were identified that encoded changes in each of the three subsites of the protease that interact with the inhibitor. HIV-1 displays significant potential for altering its interaction with this protease inhibitor, suggesting the need for multiple protease inhibitors with varying specificities.

[The role of malate in regulating the rate of mitochondrial respiration in vitro]. / Rol' malata v reguliatsii skorosti mitokhondrial'nogo dykhaniia in vitro.

Depletion of endogenous malate by preincubation of mitochondria at 30 degrees C in substrate-free media sharply decreases the rate of citrate oxidation and inhibits mitochondrial respiration in the presence of pyruvate and alpha-ketoglutarate. Addition of catalytic amounts of endogenous malate and its production via succinate oxidation promote rapid oxidation of citrate and pyruvate in the mitochondria and abolishes the lag period with alpha-ketoglutarate Malate increases the rate of membrane potential generation after addition of citrate, pyruvate or alpha-ketoglutarate to mitochondrial suspensions. Studies with controlled malate concentrations revealed that the changes in malate concentrations observed in the mitochondria in the presence of gluconeogenesis-inducing hormones may be due to the influence of these hormones on mitochondrial oxidation.

Inhibition by caltrin of calcium transport into spermatozoa, liver and heart mitochondria.

The calcium-transport inhibitor, caltrin, isolated from bovine seminal fluid inhibits calcium accumulation by bovine epididymal spermatozoa, spermatozoal mitochondria, rat liver mitochondria and beef heart mitochondria. Respiration studies demonstrate a marked stimulation of oxygen consumption by caltrin in filipin-treated spermatozoa and rat liver mitochondria. A biphasic effect of caltrin on rat liver mitochondrial respiration was noted, with stimulation at low caltrin concentrations and inhibition as the concentration of caltrin is increased. The ability of caltrin to uncouple and/or inhibit respiration in filipin-treated spermatozoa and isolated liver mitochondria indicates that inhibition of mitochondrial calcium accumulation by caltrin results from one of these mechanisms. Only a marginal effect of caltrin on respiration of intact spermatozoa was observed; indicating that the plasma membrane is impermeable to this protein. The differential effect of caltrin on respiration of intact and permeabilized spermatozoa indicates that caltrin inhibition of Ca2+ uptake into spermatozoa in vivo occurs at the level of the plasma membrane.

Rat liver gamma-butyrobetaine hydroxylase catalyzed reaction: influence of potassium, substrates, and substrate analogues on hydroxylation and decarboxylation.

Interaction of rat liver gamma-butyrobetaine hydroxylase (EC 1.14.11.1) with various ligands was studied by following the decarboxylation of alpha-ketoglutarate, formation of L-carnitine, or both. Potassium ion stimulates rat liver gamma-butyrobetaine hydroxylase catalyzed L-carnitine synthesis and alpha-ketoglutarate decarboxylation by 630% and 240%, respectively, and optimizes the coupling efficiency of these two activities. Affinities for alpha-ketoglutarate and gamma-butyrobetaine are increased in the presence of potassium. gamma-Butyrobetaine hydroxylase catalyzed decarboxylation of alpha-ketoglutarate was dependent on the presence of gamma-butyrobetaine, L-carnitine, or D-carnitine in the reaction and exhibited Km(app) values of 29, 52, and 470 microM, respectively. gamma-Butyrobetaine saturation of the enzyme indicated a substrate inhibition pattern in both the assays. Omission of potassium decreased the apparent maximum velocity of decarboxylation supported by all three compounds by a similar percent. beta-Bromo-alpha-ketoglutarate supported gamma-butyrobetaine hydroxylation, although less effectively than alpha-ketoglutarate. The rat liver enzyme was rapidly inactivated by 1 mM beta-bromo-alpha-ketoglutarate at pH 7.0. This inactivation reaction did not show a rate saturation with increasing concentrations of beta-bromo-alpha-ketoglutarate. None of the substrates or cofactors, including alpha-ketoglutarate, protected the enzyme against this inactivation. Unlike beta-bromo-alpha-ketoglutarate, beta-mercapto-alpha-ketoglutarate did not replace alpha-ketoglutarate as a cosubstrate. Both beta-mercapto-alpha-ketoglutarate and beta-glutathione-alpha-ketoglutarate were noncompetitive inhibitors with respect to alpha-ketoglutarate.

The antibiotic W341C, its ion transport properties and inhibitory effects on mitochondrial substrate oxidation.

We have examined the ion transport properties and the inhibition of rat liver mitochondrial substrate oxidation by the antibiotic W341C. W341C was able to transport 22Na+ and 42K+ across a bulk carbon tetrachloride layer. A preference was shown for K+ transport. With equal molar antibiotic concentrations, W341C transported 42K+ at a greater rate than the K+-selective ionophore nigericin, but transported 22Na+ at a lesser rate than the Na+-selective ionophore monensin. Like nigericin, W341C was able to deplete mitochondrial K+, but not Mg2+ nor Ca2+. The inhibition of mitochondrial substrate oxidation by W341C paralleled the patterns obtained with nigericin. These data indicate that W341C is a K+-selective ionophore that inhibits mitochondrial substrate oxidation by a mechanism analogous to that of nigericin.

gamma-Butyrobetaine hydroxylase and the protective role of glutathione peroxidase.

The selenoenzyme glutathione peroxidase in the presence of GSH effectively replaced catalase in the in vitro assay for gamma-butyrobetaine hydroxylase. Quantitatively, glutathione peroxidase was an order of magnitude more efficient than catalase, with maximal activity at less than 0.1 microM glutathione peroxidase in a standard reaction. Glutathione peroxidase prevented the loss of gamma-butyrobetaine hydroxylase during preliminary incubation with ferrous ions but without other substrates as well as in the course of the reaction. Regardless of whether glutathione peroxidase or catalase was present in the assay, the ascorbate concentrations needed to achieve half-maximal rates were similar (about 1 mM). Phosphate stimulated the rate of L-carnitine synthesis. Ferrous ion saturation indicated a pronounced effect of phosphate on the maximal velocity of the enzyme-catalyzed reaction, but its mechanism of action remains to be elucidated. Based on the subcellular distribution of gamma-butyrobetaine hydroxylase, catalase, and glutathione peroxidase, the role of glutathione peroxidase assumes importance. However, initial studies indicated that the assayable activity of liver gamma-butyrobetaine hydroxylase and L-carnitine concentrations in liver, blood plasma, and muscle were not significantly altered in selenium-deficient rats.

The role of malate in hormone-induced enhancement of mitochondrial respiration.

Shortly after the injection of glucagon, epinephrine, norepinephrine, vasopressin, or angiotensin II into fasted rats, mitochondria isolated from their livers contained elevated concentrations of malate and oxidized citrate, alpha-ketoglutarate, and, in some cases, succinate more rapidly than mitochondria from fasted, control rats. The administration of tryptophan, lactate, or ethanol and refeeding of rats fasted 24 h result in similar elevations of mitochondrial malate concentration and oxidation of added substrates. Treatments that resulted in elevated mitochondrial malate resulted also in increased uptake of added citrate, alpha-ketoglutarate, pyruvate, and, in some cases, succinate. It is postulated that the well-documented effect of gluconeogenic hormones on mitochondrial oxidation of carboxylic substrates may be mediated by malate which not only yields oxalacetate to support the tricarboxylic acid cycle but also facilitates the transport of added substrates, and which is regenerated in the tricarboxylic acid cycle.