Lipolytic activity of porcine pancreas lipase on fatty acid esters of dialkylglycerols: a structural basis for the design of new substrates for the assay of pancreatic lipases activity.

For the design of new synthetic substrates for the assay of pancreatic lipases activity, acyl dialkylglycerols of variable chain length were prepared. Titrimetric assay of these substrates showed the highest lipolytic activity of porcine pancreas lipase (pPL) with butanoyl dibutylglycerol. The activity is lower but comparable to that shown by pPL towards the classical substrate tributyrin. The 4-nitrophenylcarbonate of 1,2-di-O-butylglycerol, has been prepared and proposed as synthetic substrate for a new spectrophotometric assay of pancreatic lipases.

Lipase-catalyzed protection of the hydroxy groups of the nucleosides inosine and 2'-deoxyinosine: a new chemoenzymatic synthesis of the antiviral drug 2',3'-dideoxyinosine.

The selective acylation of the hydroxy groups of the nucleosides inosine 1a and 2'-deoxyinosine 1b has been achieved in the presence of Candida antarctica and Pseudomonas sp. lipases in organic solvents; starting from the 5'-acetyl derivative of 2'-deoxyinosine, compound 5a, an efficient chemoenzymatic synthesis of the antiviral drug 2',3'-dideoxyinosine 1c has been achieved.

Pharmacological control of the mevalonate pathway: effect on arterial smooth muscle cell proliferation.

The mevalonate (MVA) pathway is involved in cell proliferation. We investigated drugs acting at different enzymatic steps on rat aorta smooth muscle cell (SMC) proliferation. Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (0.1-10 microM) dose-dependently decreased (up to 90%) SMC proliferation. This effect was prevented by 100 microM MVA, 10 microM all-trans farnesol (F-OH) and 5 microM all-trans geranylgeraniol (GG-OH), precursors of protein prenyl groups, but not by 2-cis GG-OH, precursor of dolichols, squalene and ubiquinone. The same inhibitory effect was obtained with 6-fluoromevalonate (1-50 microM), an inhibitor of MVA-pyrophosphate decarboxylase. Partial recovery of cell proliferation was possible by all-trans F-OH and all-trans GG-OH, but not MVA. Squalestatin 1 (1-25 microM), a potent squalene synthase inhibitor, blocked cholesterol synthesis and slightly inhibited (21% decrease) SMC proliferation only at the highest tested concentration. NB-598 (1-10 microM), a potent squalene epoxidase inhibitor, blocked cholesterol synthesis without affecting SMC proliferation. Finally, the benzodiazepine peptidomimetic BZA-5B (10-100 microM), a specific inhibitor of protein farnesyltransferase, time- and dose-dependently decreased SMC proliferation (up to 62%) after 9 days. This effect of BZA-5B was prevented by MVA and all-trans GG-OH, but not by all-trans F-OH. SMC proliferation was not affected by the closely related compound BZA-7B, which does not inhibit protein farnesyltransferase. Altogether, these findings focus the role of the MVA pathway in cell proliferation and call attention to the involvement of specific isoprenoid metabolites, probably through farnesylated and geranylgeranylated proteins, in the control of this cellular event.

Synthesis of buffers for generating immobilized pH gradients. II: Basic acrylamido buffers.

Six basic acrylamido buffers, for isoelectric focusing in immobilized pH gradients, are described: a strong titrant (pK greater than 12), QAE-acrylamide, and five weaker bases, namely: 2-morpholinoethyl acrylamide (pK 6.2), 3-morpholinopropyl acrylamide (pK 7.0), N,N-dimethyl aminoethyl acrylamide (pK 8.5), N,N-dimethyl aminopropyl acrylamide (pK 9.3) and N,N-diethyl aminopropyl acrylamide (pK 10.3). Their synthesis and general properties are described.

Synthesis of buffers for generating immobilized pH gradients. I: Acidic acrylamido buffers.

Five acidic acrylamido buffers for isoelectric focusing in immobilized pH gradients, are described: a strong titrant (pK 1.0) 2-acrylamido-2-methyl propane sulphonic acid and four weaker acids, namely 2-acrylamido glycolic acid (pK 3.1), N-acryloil glycine (pK 3.6), 3-acrylamido propanoic acid (pK 4.4) and 4-acrylamido butyric acid (pK 4.6). Their synthesis and general properties are described.

Focusing of pepsin in strongly acidic immobilized pH gradients.

A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25 degrees C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.

Studies on analogues of succinic semialdehyde as substrates for succinate semialdehyde dehydrogenase from rat brain.

The methyl ester of succinic semialdehyde (SSA) was examined as a substrate for succinate semialdehyde dehydrogenase (SSADH) from rat brain. It was found that the ester can be oxidized by the enzyme. Values of Km for SSA-Me were higher than for those for SSA, and for this substrate the enzyme showed a substrate-dependent inhibition. This finding suggests that the carboxylate group of SSA is not essential in the process of inhibition of SSADH by the substrate. Cyclopropyl analogues of SSA, cis- and trans-1-formyl-cyclopropan-2-carboxylic acids, were also individually tested as substrates of SSADH. Only the trans isomer was found to be oxidized to the corresponding dicarboxylic acid; it inhibited the enzyme in the same range of concentrations as SSA. The above data suggest that, as for gamma-aminobutyric acid, SSA is present in an unfolded, transoid conformation at the active site of SSADH.

New routes to a-ring substituted estrogens.

Estrogens can be regiospecifically functionalized at the position 2 by a few electrophilic reagents. Reactions of estradiol 3-methyl ether-17 beta-acetate with Hg(OAc)2 and BF3 . Et2O/Ac2O allows the preparation of 2-chloromercurio- and 2-organoboron-derivatives respectively. Both the above compounds are useful starting material for easy preparations of 2-catecholestrogens and 2-chloromercurio-derivative can be transformed in other functionalized estrogens. New procedures for the preparation of 2,4-dinitroestrone and 2-nitroestrone are also described.

Synthesis of C-2 and C-4 deuterium-labeled estradiol-17 beta.

Estradiol-17 beta labeled with deuterium in the positions 2 or 4 can be prepared from 2-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol 3-methyl ether 17-acetate or 4-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol, respectively, in refluxing CH3COO(2)H/(2)H2O. The same reaction performed on 4-acetoxymercurio-1,3,5(10)-estratriene-3,17 beta-diol afforded 2,4-dideuterio-estradiol-17 beta in good yields.

Effects of 6- and 7-hydroxy metabolites of 3 beta,17 beta-dihydroxy-5 alpha-androstane on gonadotrophin and prolactin secretion and on sex accessories weight of male rats.

It has recently been shown that 3 beta,17 beta-dihydroxy-5 alpha-androstane (3 beta-diol), a known testosterone metabolite, may be further hydroxylated in position 6 and 7. Because of the possible involvement of 3 beta-diol in the control of gonadotrophin secretion, this work was aimed at investigating the effects of 3 beta,6 alpha,17 beta-trihydroxy-5 alpha-androstane (6 alpha-triol), 3 beta,7 alpha,17 beta-trihydroxy-5 alpha-androstane (7 alpha-triol), 3 beta,6 beta,17 beta-trihydroxy-5 alpha-androstane (6 beta-triol) and 3 beta,7 beta,17 beta-trihydroxy-5 alpha-androstane (7 beta-triol) on the secretion of LH, FSH and prolactin in long term castrated male rats. The four triols, 3 beta-diol and 3 alpha,17 beta-dihydroxy-5 alpha-androstane (3 alpha-diol), used for comparison, were given in a single subcutaneous dose of 2 mg/rat. Animals were killed 2, 5, 8 and 24 h after injection. None of the six steroids produces any significant effect on serum levels of FSH and prolactin at the 4 time intervals considered. On the contrary, LH levels are significantly reduced 2, 5 and 8 h after the injection of 7 beta-triol. This effect appears rapidly but is short lasting, since it disappears in the 24 h blood sample, 3 alpha-Diol also inhibits LH secretion but at later time intervals (8 and 24 h). None of the other steroids has any significant effect on serum LH levels. The four triols administered at the daily dose of 2 and 4 mg/rat for 6 days are totally ineffective in increasing the weight of the ventral prostate and of the seminal vesicles of prepuberal castrated male rats.

Reversed-phase high-performance liquid chromatography analysis of apigenin and its glucosides in flowers of Matricaria chamomilla and chamomile extracts.

Quantitative estimation of the flavones apigenin, apigenin-7-glucoside and apigenin-7-acetylglucoside in ligulate florets of Matricaria chamomilla was performed by HPLC using a reverse-phase column and eluting with acetonitrile/water, acetic acid system. These flavonoids were detected at 335 nm. Apigenin and its glucosides were not found in the tubular florets. The method was also applied to the estimation of these flavonoids in chamomile extracts.

Bioavailability of plafibride in healthy volunteers.

The pharmacokinetics of N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) in rats, and its intestinal absorption, plasmatic biotransformation, urinary elimination and fecal excretion were previously studied. 1200 mg of plafibride orally were administered to 5 healthy volunteers and then the presence of the drug and its metabolites was studied in the serum. The presence of unchanged plafibride and clofibric acid was detected, whereas that of 2-(p-chlorophenoxy) isobutyrylurea was not. At the same time the platelet antiaggregating activity of plafibride was investigated in the 5 volunteers. In this study plafibride presented a good and long-lasting platelet antiaggregating activity.