Diagnosis and follow-up of cystinuria: use of proton magnetic resonance spectroscopy.

Proton Nuclear Magnetic Resonance (NMR) Spectroscopy of urine (as well as of other biological fluids) is a very powerful technique enabling multi-component analysis useful in both diagnosis and follow-up of a wide range of inherited metabolic diseases. Among these pathologies, cystinuria is characterised by accumulation in urine of four dibasic amino acids, namely lysine, arginine, ornithine and cystine; the last one, being only slightly water soluble, generates urolithiasis. The mentioned aminoacids can be detected in the urine NMR spectrum of cystinuric patients, the most abundant being the lysine (5 mM and over are often detected), whose typical signals become very high; arginine and ornithine are also usually detectable, although pathologic concentrations are lower (usually below 2mM).

Molecular modeling study of the differential ligand-receptor interaction at the mu, delta and kappa opioid receptors.

3D models of the opioid receptors mu, delta and kappa were constructed using BUNDLE, an in-house program to build de novo models of G-protein coupled receptors at the atomic level. Once the three opioid receptors were constructed and before any energy refinement, models were assessed for their compatibility with the results available from point-site mutations carried out on these receptors. In a subsequent step, three selective antagonists to each of three receptors (naltrindole, naltrexone and nor-binaltorphamine) were docked onto each of the three receptors and subsequently energy minimized.

Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase.

Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different gamma(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro.

New insights into the conformational requirements of B2 bradykinin antagonism.

The conformational profiles of a selected group of a new series of small linear and cyclic penta- and hexapeptides, inspired on the C-terminal segment of second-generation bradykinin (BK) antagonists, were independently computed in order to assess the chemical and geometrical requirements necessary for BK antagonism. Specifically, four cyclic peptides: cyclo-(Gly-Thi-D-Tic-Oic-Arg), cyclo-(Gly-Ala-D-Tic-Oic-Arg), cyclo-(Abu-Ala-Ser-D-Tic-Oic-Arg), cyclo-(Abu-D-Phe-Ala-D-Tic-Oic-Arg), and a linear peptide: Thi-Ser-D-Tic-Oic-Arg were selected for the present study. The first three BK analogs are capable to antagonize kinin-induced rabbit jugular vein and rabbit aorta smooth muscle contraction, while last two show no detectable affinity for the BK B2 receptor.

BUNDLE: a program for building the transmembrane domains of G-protein-coupled receptors.

The only information available at present about the structural features of G-protein-coupled receptors (GPCRs) comes from low resolution electron density maps of rhodopsin obtained from electron microscopy studies on 2D crystals. Despite their low resolution, maps can be used to extract information about transmembrane helix relative positions and their tilt. This information, together with a reliable algorithm to assess the residues involved in each of the membrane spanning regions, can be used to construct a 3D model of the transmembrane domains of rhodopsin at atomic resolution.

Conformational analysis of the highly potent bradykinin antagonist Hoe-140 by means of two different computational methods.

The AMBER 4.0 force field was used to perform the characterization of the conformational profile of the highly potent bradykinin antagonist Hoe-140 (D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-D-++ +Tic7-Oic8-Arg9). The structural features of the peptide were assessed using two different computational methods, both capable to provide a good sampling of the low-energy conformations of the molecule.

Conformational study of vasoactive intestinal peptide by computational methods.

The conformational profile of vasoactive intestinal peptide (VIP) was characterized using computational methods. The strategy devised included a close examination of the conformational profile of the first 11 residues fragment followed by a study that considered the compatibility of the different conformations found with a continuation of the polypeptide chain in a alpha-helical conformation. Accordingly, a detailed analysis of the conformational preferences of the N-terminal fragment of VIP(1-11) was carried out within the framework of the molecular mechanics approach, using simulated annealing in an iterative fashion as the sampling technique.

Diagnosis and follow-up of inborn errors of amino acid metabolism: Use of proton magnetic resonance of biological fluids.

Proton magnetic resonance spectra of biological fluids such as urine, plasma and cerebro-spinal fluid can be used for multi-component analysis of highly concentrated species, thus providing information about the general metabolism of the patient. Hydrogen containing analytes in concentration higher than 10µM are indeed often detectable in biological fluid in 15 minutes by means of an unexpensive 200 MHz spectrometer essentially without sample manipulation. Amino acids, keton bodies, organic acids and other metabolites can be easily estimated by this approach; consequently this technique represents a powerful tool particularly in the diagnosis of inborn errors of amino acid metabolism, when improving the prognosis often depends on a very early diagnosis and on an effective method for monitoring the effects of therapy.

Diet treatment of branched chain ketoaciduria studied by proton magnetic resonance spectroscopy.

A novel nuclear magnetic resonance method is proposed for the diagnosis and follow-up of patients affected by branched chain ketoaciduria. The method allows quantitation of the branched chain amino acids (BCAA's) such as leucine, isoleucine and valine and of related keto- and hydroxy acids by means of a single spectrum. The method implies short time of analysis, as opposed to the very long time required by the techniques currently in use (amino acid analyzer combined with gaschromatography/mass spectrometry of keto- and hydroxyacids), it is easy and suitable for adjustements of the dietary treatment even on a daily basis.

A physico-chemical approach to the study of the binding interaction between S-adenosyl-L-methionine and polyanions: binding constants and nature of the interaction with sodium poly(styrene sulfonate).

The interaction between S-adenosyl-L-methionine (AdoMet) and sodium poly(styrene sulfonate) NaPSS) was studied by means of ultrafiltration and ultraviolet absorption spectroscopy at several pH values and sodium sulfate concentrations. The results obtained are interpreted mainly in terms of electrostatic interactions and permit the evaluation of the binding constants under different experimental conditions. Furthermore, ultraviolet absorption spectroscopy data show a specific short-range interaction between the aromatic electronic system of AdoMet and the NaPSS aromatic ring.

S-adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus. Purification and characterization of two isoforms.

Two isoforms of methionine adenosyltransferase (S-adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87 degrees C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl-Sepharose column as a major step, results in 330-fold and 150-fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B.

Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium.

The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87 degrees C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis.

Purification and characterization of 5'-deoxy-5'-methylthioadenosine phosphorylase from human placenta.

5'-Methylthioadenosine phosphorylase has been purified to homogeneity (30,000-fold) from human full-term placenta by a procedure involving covalent chromatography on organomercurial-agarose as the major step. The specific activity of the homogeneous enzyme is 10.2 mumol of 5'-methylthioadenosine cleaved per min per mg of protein, and the overall yield is about 20%.

Escherichia coli S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action.

S-Adenosylhomocysteine/5'-methylthioadenosine nucleosidase (EC 3.2.2.

Studies on the metabolic effects of methylthioformycin.

5'-Methylthioformycin, a structural analog of 5'-methylthioadenosine in which the N-C glycosidic bond is substituted by a C-C bond, has been synthesized by a newly developed procedure. Membrane permeability of the molecule has been compared to that of methylthioadenosine in intact human erythrocytes and Friend erythroleukemia cells. The formycinyl compound is taken up with a rate significantly lower than that of 5'-methylthioadenosine and is not metabolized by the cells.

Inhibition of dimethyl sulfoxide-induced differentiation of Friend erythroleukemic cells by 5'-methylthioadenosine.

5'-Methylthioadenosine is a sulfur-containing nucleoside derived from the metabolism of polyamines which is known to exert an antiproliferative effect on several cell systems in vitro, including the Friend leukemia cell system. We have investigated the role of 5'-methylthioadenosine on the dimethyl sulfoxide-induced differentiation of this system. At a concentration of 400 microM, the drug strongly inhibited (80%) the induced differentiation of Friend cells, and this effect was already observable at a concentration as low as 10 microM (36% inhibition), as evidenced by the benzidine staining procedure and by the dot-blot hybridization of globin mRNA with a human beta-globin probe.

Transport and metabolism of 5'-methylthioadenosine in human erythrocytes.

The transport and metabolism of 5'-deoxy-5'-S-methylthioadenosine have been studied in intact human erythrocytes. The sulfur nucleoside is rapidly accumulated into red cells and the extent of uptake largely exceeds the theoretical equilibrium between inner and outer compartment owing to its conversion into a non-permeable compound, namely 5-methylthioribose 1-phosphate. To characterize the nucleoside transport, phosphate-depleted erythrocytes, in which the methylthioadenosine metabolism is negligible, have been employed.

Studies on 5'-methylthioadenosine uptake by human erythrocytes.

The transport of 5'-methylthioadenosine across plasma membrane of human erythrocytes occurs by a carrier-mediated mechanism displaying a Km of congruent to 3 mM and a Vmax of congruent to 600 pmol/10(6) cells/min. Phosphate-depleted erythrocytes were employed to distinguish between 5'-methylthioadenosine transport and metabolic trapping. In phosphate medium, where 5'-methylthioadenosine phosphorylase is operative, the uptake of radioactivity increases over the theoretical calculated equilibrium owing principally to conversion of the molecular into 5-methylthioribose-1-phosphate.

[Possible regulatory role of 5'-methylthioadenosine on enzymatic methyl esterification of membrane protein].

In the present study the effect of 5'methylthioadenosine (MTA), a natural metabolite of S-adenosylmethionine (AdoMet), on the enzymatic methyl esterification of intact erythrocyte membrane proteins has been investigated. The thioether significantly affects the methylation process :50% inhibition being observable at 100 microM MTA. This inhibition is due to the action of MTA on the enzyme protein methylase II.

[A new method of measuring 5'-methylthioadenosine with high pressure liquid chromatography].

A rapid, sensitive and specific high performance liquid chromatographic method for the estimation of 5'-methylthioadenosine in biological samples has been developed. A double-step chromatographic on Dowex-50, H+ form, and Affi-Gel 601 were required before the chromatographic separation of sample on Partisil 10 SCX. The yield is quantitative and the procedure is simple and highly reproducible.