TLC separation of carbohydrates on silica gel modified with copper (II) salts.

Impregnation of silica gel was conducted by immersion method with the help of copper (II) sulfate and ammonia complex: Cu(NH3)4(2+). The adsorbents thus obtained were used for the analysis of carbohydrates of mono-di-oligosaccharides. The influence of the pH of the impregnating solution (pH = 5 and pH = 8) on the retention of the analysed compounds was examined. The results were interpreted in view of the process of metal ions complexation by sugar molecules in water. The effects of organic modifiers: propanol-2, methyl-ethyl ketone, acetonitrile, acetone added to the water phase on the retention and selectivity of the sugars determination on the modified stationary phase were studied.

Correlation of fungostatic activity with log P and sigma parameters in the group of thiobenzanilides.

2,4-Dihydroxythiobenzanilide and its analogues were recently found to show strong biological activity. Earlier works exhibited that their biological activity depends strongly on their lipophilicity determined by means of RP-HPLC. Among others, fungostatic activity against tested yeast and mould strains appeared to be comparatively poorer modelled by chromatographic lipophilicity parameters. Very high correlation (r approximately 0.85-0.9) with these activities was obtained by means of multiparameter regression analysis where calculated log P and Hammet sigma values were involved as the independent variables. Analysed data show that activity against yeast and mould fungi is a superposition of two basic properties of thiobenzanilides: their lipophilicity and electronic properties.

Use of reversed-phase high-performance liquid chromatography in QSAR analysis of 2,4-dihydroxythiobenzanilide analogues.

Thiobenzanilides are found to show strong biological activity as antimicrobial, antimycotic, and tuberculostatic agents. In addition, they are relatively weakly toxic to higher organisms. A large set of new (N-phenyl-)-2,4-dihydroxybenzenecarbothioamide derivatives was obtained. Preliminary studies showed high microbiological action of some of them. In the process of chromatographic analysis, several different chromatographic parameters were obtained. In case of RP-HPLC, these parameters correspond to hydrophobicity of the solute. Obtained chromatographic parameters exhibited moderate correlation with calculated log P parameter. Linear dependence of bacteriostatic or fungostatic activity on lipophilicity was observed. The degree of correlation of different parameters was compared. The lipophilicity of analysed tioamides was the most important factor responsible for fungostatic and bacteriostatic activity. In comparison to methanol eluent system, chromatographic parameters obtained in acetonitrile system were better correlated with bioactivity. Conversely with the calculated log P values, the experimentally derived parameters exhibited significant higher correlation to fungostatic activity determined on dermatophytes. While in case of other tested microorganisms log P was comparably or sometimes slightly better correlated.

Purification to homogeneity and properties of UDP-GlcNAc (GalNAc) pyrophosphorylase.

The pyrophosphorylase that condenses UTP and GlcNAc-1-P was purified 9500-fold to near homogeneity from the soluble fraction of pig liver extracts. At the final stage of purification, the enzyme was quite stable and could be kept for at least 4 months in the freezer with only slight loss of activity. On native gels, the purified enzyme showed a single protein band, and this band was estimated to have a molecular mass of approximately125 kDa on Sephacryl S-300. SDS-polyacrylamide gel electrophoresis analysis of the enzyme gave three protein bands of 64, 57, and 49 kDa, but these polypeptides are all closely related based on the following. 1) All three polypeptides show strong cross-reactivity with antibody prepared against the 64-kDa band. 2) All three proteins become labeled with either the UDP-GlcNAc photoaffinity probe azido-125I-salicylate-allylamine-UDP-GlcNAc or a similar UDP-GalNAc photoaffinity probe, and either labeling was inhibited in a specific and concentration-dependent manner by unlabeled UDP-GlcNAc or UDP-GalNAc. Thus, the enzyme is probably a homodimer composed of two 64-kDa subunits. The purified enzyme had an unusual specificity in that, at higher substrate concentrations, it utilized UDP-GalNAc as a substrate as well as UDP-GlcNAc in the reverse direction and GalNAc-1-P as well as GlcNAc-1-P in the forward direction. However, the Km for the GalNAc substrates was considerably higher than that for GlcNAc derivatives. This activity for synthesizing UDP-GalNAc was not due to epimerase activity since no UDP-GalNAc could be detected when the enzyme was incubated with UDP-GlcNAc for various periods of time. The pyrophosphorylase required a divalent cation, with Mn2+ being best at 0.5-1 mM, and the pH optimum was between 8.5 and 8.9.

Synthesis of 5-IASA-UDP-GlcNAc and its use for the photoaffinity labeling of a novel UDP-GlcNAc pyrophosphorylase.

Photoreactivable 5-[3-(p-iodoazidosalicylamide)allyl]-UDP-GlcNAc (5-IASA-UDP-GlcNAc) was synthesized by a four-step procedure and used for photoaffinity labeling of UDP-GlcNAc-dependent enzymes. Upon iodination with 125I, the compound was successfully applied to probe a purified UDP-GlcNAc pyrophosphorylase from pig liver. The enzyme was photoinactivated by the probe in the concentration-dependent manner, and was protected by UDP-GlcNAc and, to a lesser extent, by UTP and UDP-GlcCOOH.

2,4-Dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone activate the glutathione S-conjugate transport ATPase of human erythrocyte membranes.

2,4-Dinitrophenol (DNPOH) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), two classical uncouplers of mitochondrial oxidative phosphorylation, were found to stimulate human erythrocyte membrane vesicle ATPase activity. Both compounds competed with S-(2,4-dinitrophenyl)glutathione (DNPSG) for activation of the glutathione S-conjugate transport ATPase. Stimulation of the ATPase by DNPOH or FCCP occurred with Vmax values 4-6 times greater than that with DNPSG. The K0.5 for DNPOH (195 microM) was similar to that of DNPSG (196 microM), while that for FCCP (4.3 microM) was 40 times lower. Vanadate inhibits both the DNPOH- and FCCP-stimulated ATPase activities, as previously reported for the glutathione S-conjugate ATPase. The stimulation of erythrocyte vesicle ATPase activities by these classical uncoupling agents does not result from increased proton conductance across the vesicle membrane: monensin, gramicidin and nystatin, all of which increase proton conductance, but by different mechanisms, do not stimulate erythrocyte vesicle ATPase activity. Verapamil, a known P-glycoprotein ATPase activator also does not stimulate human erythrocyte membrane ATPase activity. These results show that relatively small, monoanionic lipophilic compounds such as DNPOH and FCCP can activate the glutathione S-conjugate transport ATPase. The higher Vmax values for activation by these agents than by DNPSG make possible a more sensitive assay of this transport ATPase activity. The results raise the question of whether these substances and other small anionic, lipophilic compounds are also transported by this system.

Pharmacokinetics of tryptamide following intravenous administration in rats.

Plasma and tissue levels of N-3-pyridoyl-tryptamine (tryptamide) vs. time were determined in rats following iv administration of various doses: 6.25, 12.5, 25.0 and 37.5 mg/kg. The course of changes was described by the two-compartment open model. The distribution rate constant (alpha, min-1), the elimination rate constant (beta, min-1), the body clearance (Cltot, ml/min) and its fraction absorbed (f,%) values changed depending on the dose and were respectively: 0.107-0.266, 0.0068-0.0126, 5.39-11.59 and 31.77-66.70.

[Testing of some dibenzoazepines and dibenzocycloheptadiene derivatives with high pressure thin layer chromatography]. / Badania niektórych pochodnych dibenzoazepiny i dibenzocykloheptadienu metoda chromatografii cienkowarstwoweji wysokosprawnej cieczowej.

An influence of several adsorbents and the composition of the eluent mixtures on the separation of dibenzoazepine and dibenzocycloheptadiene derivatives was examined by thin-layer chromatography. The best system (RP-18 and methanol-buffer solution pH 8.5 19:1) was employed for the separation of amitryptiline and doxepine by HPLC. Both compounds were determined in ng amounts in pharmaceutical preparations and in blood by addition and in patients blood. Doxepine was used as internal standard for the determination of amitryptiline.

Pharmacokinetics of tryptamide following oral and intraperitoneal administration in rats.

Tryptamide, either suspended in 1% methylcellulose or dissolved in polyethyleneglycol 400-ethanol-physiological salt solution (4:1:5) was administered po or ip at a dose 25 mg/kg to rats, and blood level of this drug was determined by HPLC method. The obtained results were used to construct the semilogarithmic plots of tryptamide concentration vs. time and pharmacokinetic parameters of tryptamide were calculated. We found that tryptamide undergoes fast absorption (t 1/2 = 4.92-17.5 min) and elimination (t 1/2 = 55.72-74.52 min), can reach C(max) values (11-13 microg/cm3) after 30 min, and gives AUC values in the range of 21.40-27.30 (microg x h/cm3).