1H-NMR lipid profiles of human blood platelets; links with coronary artery disease.

Blood platelets are closely involved in the early development of atherosclerosis and in the events that lead to thrombosis, both of which are dominating factors in coronary artery disease (CAD). The aim of the present study was to evaluate the platelet lipid profiles of patients suffering from CAD and explore the possibility of a link between platelet lipids and CAD, using high-resolution high-field proton nuclear magnetic resonance spectroscopy as the analytical tool. The total platelet lipid profiles of healthy volunteers were compared with those of patients presenting with chest pain requiring coronary angiography. Two lipid groups changed significantly: cholesterol increased by 16.5% and total diacylglycerophospholipids decreased by 15.7%. There was also a significant decrease of the ethanolamine-containing phospholipids, by 4.7%; the extent of unsaturation of the fatty acid chains, by 0.2, and increase of the linoleate content of the fatty acid chains, by 1.9%. Our results suggest that platelet lipid abnormalities occur in patients with CAD and these changes may predate the development of overt atherosclerosis.

Effect of hydrazine upon vitamin B12-dependent methionine synthase activity and the sulphur amino acid pathway in isolated rat hepatocytes.

The effect of the industrial chemical, hydrazine (4-12 mM), on methionine synthase (EC 2.1.1.13) activity and levels of the sulphur amino acids homocysteine, cysteine, and taurine as well as GSH were investigated in vitro in isolated rat hepatocyte suspensions and monolayers in order to explain some of the adverse in vivo effects of hydrazine. None of the concentrations of hydrazine were overtly cytotoxic in hepatocyte suspensions (measured as lactate dehydrogenase [LDH] leakage) after 3 hr. However, after 24 hr in culture cells treated with 12 mM, hydrazine showed a significant increase in LDH leakage. Methionine synthase activity was reduced by hydrazine (8 and 12 mM) in suspensions (by 45 and 55%, after 3 hr) and monolayers (12 mM; 65-80% after 24 hr). This was not due to nitric oxide production and the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine, failed to protect against the hydrazine-induced loss of ATP and GSH and the reduction in urea synthesis at 24 hr. Homocysteine export was increased by 6 mM hydrazine, and total taurine content of treated cells was increased by 12 mM hydrazine. Thus, hydrazine was found to have several important and possibly deleterious effects on some parts of the sulphur amino acid pathway.

The effect of ethanol and its metabolites upon methionine synthase activity in vitro.

The association of alcoholism with macrocytic anaemia has lead to investigation of the role of cobalamin-dependent methionine synthase in mediating alcohol toxicity. Several studies have found that long-term ingestion of large quantities of ethanol causes inhibition of liver methionine synthase activity in vivo: however, ethanol has not been found to inhibit the enzyme directly. The effect of ethanol and its breakdown products, acetate and acetaldehyde, on highly purified rat liver methionine synthase was tested in vitro. Enzyme activity was not inhibited by ethanol or acetate. Acetaldehyde was found to inhibit methionine synthase activity, with an apparent IC50 of 2 mM. The reported inhibition by acetaldehyde was found to become irreversible over time. Acetaldehyde-induced inhibition of liver methionine synthase activity is thus proposed as the most likely explanation of the reported in vivo effect of ethanol upon methionine synthase.

Synthesis and in vitro cytotoxicity of lipophilic platinum(II) complexes.

A number of lipophilic platinum(II) complexes of the general structures cis-[Pt(LA)2Cl2] and [Pt(LD)Cl2] were synthesised. Long chain amines (LA) and diamines (LD), prepared from lipidic amino acids, were used as ligands. The in vitro cytotoxicity of the complexes was evaluated against four cell lines (P388, NSCLC-N6, E39, M96). cis-Dichloro-bis(2-aminohexadecanol)platinum(II) was the most active against P388, NSCLC-N6 and E39 (IC50: 11 micrograms/ml, 25 micrograms/ml, 31 micrograms/ml), while dichloro(1,3-heptadecanediamine)platinum(II) presented the highest activity against M96 (IC50: 13 micrograms/ml).

Synthesis and properties of novel lipopeptides and lipid mimetics.

Lipid mimetics, synthetic molecules that resemble natural lipids either structurally or functionally, have been developed as potential medicinal substances. They have been successfully applied in the development of drug and peptide delivery systems and for the development of inhibitors or lipid metabolizing enzymes. Phospholipase A2 is considered to be involved as the rate-limiting step in the production of lipid mediators of inflammatory responses and, as such, it has been a target for drug design. A series of lipid mimetics including lipopeptides, amides and alcohols of lipidic alpha-amino acids, have been tested by bulk and monolayer assay techniques. The findings suggested the direct interaction of the tested compounds with porcine pancreatic phospholipase A2. The inactivation of the enzyme occurred in a competitive manner. The most active compound I (2-amino-N-hexadecyl-L-hexanamide) showed an apparent IC50 of 12 microM and inhibitory power Z = 13 in the monolayer assay.

The structural and aggregation properties of the synthetic C-terminal half (104-mer) polypeptide from HIV p24gag resemble those of full-length protein.

The aggregation and structural properties of the synthetic C-terminal half [Ala330, Ala350(270-373; 104-mer)] polypeptide from HIV-1 p24gag were studied. In concentrated solutions the synthetic polypeptide aggregated to tetramers which, upon dilution, gave a mixture of monomeric and dimeric and dimeric species. These results correlated well with the in vitro aggregation properties of recombinant p24. The tetrameric form of the synthetic polypeptide had a pI which differed by about four units from that of the mixture of monomeric and dimeric species. CD studies indicated that the latter contained, in aqueous solutions, a compact molecule lacking, however, a defined tertiary structure. Addition of MeOH to aqueous solutions of both tetramer and monomer/dimer mixture induced a more defined structure, which was assigned to that of an alpha + beta protein in agreement with secondary structure predictions. A model of the dimeric form of the 104-mer, which takes into account the results presented here and those from a study on the specificity of a set of anti-104-mer MoAbs, is presented. Finally, the results indicated that the structure of the 104-mer in its dimeric form is similar to that adopted by the same sequence when part of full-length p24.

The inactivation of methionine synthase in isolated rat hepatocytes by sodium nitroprusside.

Methionine synthase, the enzyme that catalyses the transfer of a methyl group from 5-methyl tetrahydrofolate to homocysteine via the cofactor methylcobalamin, is one of the two established mammalian enzymes that utilise a biologically active vitamin B-12 derivative. Through its substrates, products and downstream metabolites, methionine synthase is directly involved in the sulphur amino acid pathways, polyamine biosynthesis, biological methylations and one-carbon-unit transfers. Rat liver methionine synthase was shown to be inactivated by the nitric oxide donor sodium nitroprusside. The inactivation occurred during the treatment of isolated rat hepatocytes in a time-dependent and dose-dependent manner with an apparent IC50 value of 170 microM. Highly purified rat liver methionine synthase was inactivated in a partially irreversible manner with an apparent IC50 value of 10 microM. The inactivation has been attributed to nitric oxide released by sodium nitroprusside. Since biomolecules possessing transition state metals are targets for nitric oxide, the possibility of a nitric oxide-cobalamin interaction could explain the observed inactivation. Nitric oxide is directly involved in different aspects of liver metabolic functions both under physiological and pathological conditions like sepsis and inflammation. The nitric-oxide-induced inactivation of methionine synthase could offer a rational explanation for the cellular and cytotoxic effects of this highly reactive molecule.

Synthesis of lipidic amino acid and dipeptide inhibitors of human platelet phospholipase A2.

Phospholipases A2 (PLA2s) catalyze the hydrolysis of glycerophospholipids at the sn-2 position, and their inhibition is considered a rational approach for the prevention and treatment of inflammation. A number of amides and esters of alpha-amino acids with saturated linear side chains, esters of alpha-amino alcohols and derivatives of lipidic peptides were prepared and tested against secreted humal platelet phospholipase A2. Among the compounds tested the amides of free amino acids with long-chain amines presented the highest in vitro inhibitory activity.

Application of proton NMR spectroscopy to measurement of whole-body RNA degradation rates: effects of surgical stress in human patients.

The urinary catabolites, N2,N2-dimethylguanosine (DMG), pseudouridine (PSU) and 7-methylguanine (m7-Gua) are formed from post-transcriptional methylation of RNA bases and are not reincorporated into RNA upon its degradation. Their quantitative urinary excretion may be used to determine rates of whole body degradation of individual RNA species since DMG occurs exclusively in tRNA, PSU occurs in rRNA and tRNA and m7-Gua occurs in all RNA species. Conventional HPLC analysis has several drawbacks since pre-analytical steps may involve selective losses and, under certain conditions, other urinary analytes may co-elute. In the present paper, we report analysis of these compounds by high-field 1H-nuclear magnetic resonance (1H-NMR) spectroscopy. Urinary concentrations of these metabolites were found to be in agreement with previously published HPLC and ELISA determinations. However, NMR analysis required minimal sample preparation (other than lyophilisation and reconstitution) and was capable of the simultaneous determination of other relevant analytes such as creatinine. This technique was therefore applied to urine samples from patients who had undergone surgical stress and insulin-like growth factor-1 (IGF-I) therapy. Surgical stress increased the excretion of DMG and m7-Gua. Degradation rates for tRNA and mRNA were also higher in surgically stressed subjects when compared with controls but degradation rates of rRNA decreased by approx. 30%. However, injection of IGF-I (40 micrograms/kg s.c.) had no significant effect on the excretion of these nucleosides. These data indicated that IGF-I therapy has no marked effects on RNA turnover following trauma. We suggest that this technique can be applied to study of RNA metabolism in any surgical or medical condition. Furthermore, since only 0.6 ml of urine is required, studies in neonates seem to be feasible.

Stimulation in vitro of vitamin B12-dependent methionine synthase by polyamines.

Vitamin B12-dependent methionine synthase is an important enzyme for sulphur amino acid, folate polyamine metabolism, S-adenosylmethionine metabolism and also in the methylation pathway of DNA, RNA, proteins and lipids. Consequently, studies aiming at exploring the control and regulation of methionine synthase are of particular interest. Here we report the modulation of enzyme activity in vitro by polyamines. Although putrescine, cadaverine, spermine and spermidine all stimulated enzyme activity, the last two were the most potent, causing increases in enzyme activity up to 400%. The EC50 for spermine was determined as 8 microM and for spermidine 40 microM. The physiological concentration for spermine has been reported to be 15-19 microM. Spermine was found to increase both the Km and the V(max) with respect to methyltetrahydrofolate for the enzyme. These data support the hypothesis that spermine and spermidine are feedback regulators of methionine synthase both in vivo and in vitro and are consistent with the polyamines' regulating cell signalling pathways.

The solution structure of the immunodominant and cell receptor binding regions of foot-and-mouth disease virus serotype A, variant A.

Abstract: The solution structure of a 20 amino acid long peptide corresponding to the region 141-160 of the envelope protein Vp1 from foot-and-mouth disease virus (FMDV) serotype A, variant A, has been determined by a combination of NMR experiments and computer calculations. The peptide contains both the immunodominant epitope as well as the sequence (RGD) used by the virus to bind the cell receptor in the initial stages of infection. These two sites have been shown to partially overlap. One hundred and thirty-five NMR distance constraints were used to obtain a set of 11 structures by distance geometry, minimization and molecular dynamics simulations. These structures were divided into two homogeneous families based upon backbone superimposition. The first and most populated family was characterized by a backbone RMS of 1.5 +/- 0.4 A, the second by a backbone RMS of 0.8 +/- 0.2 A. The two families had similar structural features and differed mainly in the backbone angles of G149. In the larger of the two families these angles favoured the formation of a loop comprising residues 147 to 152 and stabilized by a H-bond between NH of D147 and the CO of A152. In the second family, where this bond was absent, the peptide adopted in this region the shape of an irregular helix. The C-terminal half of the peptide (152-159) was similar in both families and largely helical. Similar structural features were also found within the VRGDS sequence (144-148) which was assigned to a beta-turn type IV. The features of the two families of structures were found to be different from those of the recently published X-ray structure of the antigenic loop of a chemically modified form of FMDV. Proposals accounting for these differences are provided which take into account the dual activity of the 141-160 sequence (i.e. antibody binding and cell invasion through receptor binding).

The solution conformational features of two highly homologous antigenic peptides of foot-and-mouth disease virus serotype A, variant A and USA, correlate with their serological properties.

The solution structure of a peptide corresponding to the VP1 region 141-160 of foot-and-mouth disease virus (FMDV) serotype A variant USA has been studied by NMR and computer calculations and compared with the results from a study on a highly homologous peptide deriving from serotype A, variant A. The two peptides differ in their serological behavior and contain the immunodominant epitope of the virus which partly overlaps with its receptor binding region. Distance constraints, derived both from 2D and 3D homonuclear NMR and 2D-heteronuclear NMR experiments, were combined with DG calculations to yield 50 structures. After refinement through EM and restrained molecular dynamics simulations the selected structures shared several general features. In particular the 151-158 region was a helix in all cases while a large loop similar to that found in peptide A but comprising less residues and stabilized by an H-bond between the side chains of D147 and S150 was found in the majority of structures. A further loop, common to all structures, was identified around the RGD sequence (145-147). This was different from that found in the corresponding region of peptide A as were the conformations of the individual residues within the RGDX sequence. The different structural features shown by the two peptides were rationalized in terms of the S148 (peptide A) to F148 (peptide USA) mutation. The second mutation, that at position 153 (L in A, P in USA) did not appear to affect the structure of the peptide significantly although the different dimensions of the loop in the central region and the type of H-bond stabilizing it could be potentially ascribed to this second mutation. All criteria used pointed to different structural features for the two peptides consistent with their serological behaviour.