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The double-disk synergy test (DDST) using Mueller-Hinton agar and antibiotic disks with centrally positioned disks of amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam and, at a center-to-center distance of 25–30 mm, 2–4 disks with 10 various β-lactam antibiotics per one plate was performed in 58 clinical isolates of Stenotrophomonas maltophilia to determine the effectivity of 3 β-lactamase inhibitors. When tested with clavulanate as the central β-lactamase inhibitor synergic action on tested strains was the most frequent with aztreonam (81.0 % of strains), cefoperazone (63.8 %), and cefepime (60.3 %). With sulbactam the synergic action, i.e. DDST positivity, was high in the case of cefoperazone (15.5 %), ampicillin, aztreonam and piperacillin (8.6 % each); with tazobactam it was the most frequent with aztreonam (53.4 %), cefoperazone (44.8 %) and cefepime (37.9 %). No synergy was demonstrated after application of meropenem regardless of the kind of β-lactamase inhibitor used. In 58 strains of S. maltophilia, 55 different profiles of DDST positivity were found. The results confirm that clavulanate is the most effective inhibitor of S. maltophilia β-lactamases. The utilization of DDST (performed in the recommended way) for the typization of strains Stenotrophomonas species and for the estimation of potential effectiveness combinations of β-lactams with β-lactamase inhibitors for the therapy of stenotrophomonade infections was suggested. [ABSTRACT FROM AUTHOR]

Isolates of Stenotrophomonas maltophilia species display the feature “temperature-dependent susceptibility” (TDS) to antibiotics. Both 30TDS strains (at least 4 times lower value of minimum inhibitory concentration (MIC) of an antibiotic at 30 than at 37°C) and 37TDS strains (at least 4 times lower value of MIC at 37 than at 30°C) were described. Changes in the distribution of saturated and unsaturated fatty acids (FA) at 30 and 37°C were considered as one of possible causes of the TDS phenomenon. Gas chromatography was used to determine the distribution of individual FA in five 37TDS strains of S. maltophilia (Group I); in five strains with MIC values unaffected by the cultivation temperature (Group II) and in six 30TDS (four strains) or 30/37TDS (two strains) isolates (Group III). At identical temperatures, no statistically significant differences in the distribution of major FA ( iso-15:0, anteiso-15:0, 16:0 and 16:1) were registered between individual groups. Statistically significant ( p<0.05) differences between groups were found in minor FA only ( iso-16:0, iso-17:0 and iso-17:1). Distribution changes of cellular FA at 30 and 37°C can be considered to play only a minor role in the formation of the TDS phenomenon. [ABSTRACT FROM AUTHOR]

1. Biotransformation pathways of the cyclin-dependent kinase inhibitor 6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine (bohemine) by mouse liver microsomes in vitro were investigated. 2. Metabolite profiles of [8-[sup 3]H]-labelled bohemine were established by TLC/[sup 3]H-autoradiography and enzymatic and MS analyses were used to elucidate the chemical structures of the metabolites. The structures of the main primary metabolites were confirmed by synthesis of authentic compounds. 3. A schema of the primary NADPH-dependent pathways has been proposed involving N[sup 2]- and N9-dealkylation, N[sup 6]-debenzylation, aromatic hydroxylation, and C2 side chain oxidation of bohemine. Three of the primary metabolites detected, 6-(benzylamino)-2-(3-hydroxypropylamino)purine (M4), 6-amino-2-(3-hydroxypropylamino)-9-isopropylpurine (M5) and 6-(4-hydroxybenzylamino)-2-(3-hydroxypropylamino)-9-isopropylpurine (M6), all retaining their parent primary hydroxyl group, were subsequently shown to be converted, by a liver cytosolic NAD[sup +]-dependent system, into their corresponding carboxylic acids. M6 was subject to microsomal glycosidations requiring UDP-sugar donors. NADPH-dependent conversion of M6 into M5 by microsomes was also demonstrated. 4. Cytochrome P450 (CYP) enzymes-selective inhibitors were used to identify CYPs involved in bohemine biotransformation. The findings suggested that CYP2a and CYP3a substantially contributed to the NADPH-dependent bohemine transformation in vitro. 5. The findings will facilitate experiments designed to dissect enzymatic systems catalysing clearance of C2,C6,N9-trisubstituted purine compounds from mammalian tissues. [ABSTRACT FROM AUTHOR]

Plant-based n-3 polyunsaturated fatty acids (PUFA) possess a prospective antiatherogenic potential. Currant oil from Ribes nigrum L. is one of the few plant oils containing PUFAn-3 (15.3 mol%) in addition to PUFAn-6 (60.5 mol%). This study was aimed at comparing the effects of currant oil with those of lard fat, rich in saturated (43.8 mol%) and monounsaturated (47.0 mol%) fatty acids, on antioxidant parameters, the lipoprotein profile and liver lipids in rats fed on 1 % (w/w) cholesterol diets containing either 10 % of currant oil (COD) or lard fat (LFD). After 3 weeks of feeding, the COD induced a significant decrease in blood glutathione (GSH) and an increase in Cu(2+) induced oxidizability of serum lipids, but did not affect liver GSH and t-butyl hydroperoxide-induced lipoperoxidation of liver microsomes. Although the COD did not cause accumulation of liver triacylglycerols as LFD, the lipoprotein profile (VLDL, LDL, HDL) was not significantly improved after COD. The consumption of PUFAn-3 was reflected in LDL as an increase in eicosapentaenoic and docosahexaenoic acid. These results suggest that currant oil affects positively the lipid metabolism in the liver, above all it does not cause the development of a fatty liver. However, adverse effects of currant oil on the antioxidant status in the blood still remain of concern.

In vitro subcellular and cellular systems have important and irreplaceable roles in the metabolic investigations that precede the development of new potential drugs. Of these model systems, tissue slices are probably the nearest to in vivo conditions. From the experimental and complexity points of view, perfused organs lie midway between tissue slices and whole organism. Preparation and working with liver slices is quick and easy, and, excess material can be cryopreserved and stored untill the next experiment. Slices can be prepared from a wide variety of organs and it is possible to co-incubate them. Another important feature is the possibility of interspecies comparison of slices. Different experiments can be run both in the short-term as well as long-term incubations. Each in vitro system has an important place for example, in the development of new medicaments. It is therefore important to compare and supplement experimental results from different in vitro systems when extrapolating to in vivo situations is done.

Tissue slices represent a useful in vitro method for metabolic, pharmacologic and toxicologic studies. To their major advantage belong preserved cell to cell interactions, architecture and complexity of the tissue. They enable to reduce the number of laboratory animals, that are necessary for research purposes. Tissue slices are more similar to in vivo situation than other in vitro models such as microsomes, cell suspensions or cell cultures. Slices can be cultured up to 72 hours or even more. Preparation of the slices is rapid and easy. Slices can be prepared from any organ. There is a possibility of cross-species comparison, co-culturing of slices derived from different organs. Excess of the material can be cryopreserved for later studies.

Synthetic cyclin-dependent kinase inhibitors have recently been referred to as effective antiproliferative agents. This study was conducted to characterize clearance of a 3H-labeled, trisubstituted purine-type inhibitor, 8-[3H]bohemine [6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine], in mice. Radioactivity profiles were analyzed by liquid scintillation counting and by thin layer chromatography followed by autoradiography. Metabolite structures were elucidated by mass spectrometry, NMR, and enzymatic analyses. Bohemine was rapidly and completely metabolized in vivo and disappeared from circulation during the first 60 min following intravenous administration. The metabolites were partly eliminated by the hepatobiliary tract and partly by renal excretion. The terminal hydroxyl group located at the C2 side chain of bohemine made the compound susceptible to main metabolic attacks, i.e., distinct types of conjugation reactions with glycosyl donors as well as an oxidative reaction. Other pathways were of relatively minor significance. Bohemine O-beta-D-glucoside was the most abundant metabolite to be excreted. The enzymatic mechanism responsible for bohemine glucosidation in vitro required the presence of a UDP-glucoside donor. Additional glycosidation products were observed after inclusion of UDP-glucuronide, UDP-xylose, UDP-galactose, or UDP-N-acetylglucosamine into microsomal incubates. Glycosidations occurred faster in the kidney incubates than in hepatic ones. The second principal bohemine metabolite was a carboxylic acid, 6-benzylamino-2-(2-carboxyethylamino)-9-isopropylpurine. A cytosolic, 4-methylpyrazole-sensitive alcohol dehydrogenase class I was shown to mediate oxidation of the terminal hydroxyl group of bohemine into this acid, which was the only metabolite found in the blood in significant amounts. However, it displayed only weak cyclin-dependent kinase-1-inhibitory activity (IC(50) > 100 microM) when compared with that of bohemine (IC(50) approximately 1 microM).

Tumor necrosis factor alpha (TNFalpha) was found to be significantly increased in skeletal muscles and retroperitoneal fat of obese insulin-resistant Koletsky rats as compared to control Wistar rats. This increase was accompanied by a depression of insulin receptor protein tyrosine kinase (PTK) activity. Neither the insulin-binding capacity nor insulin receptor affinity were related to this TNFalpha increase in these tissues. In the liver, no significant changes of TNFalpha content and only a lowering of insulin-binding capacity were found. It is concluded that an increased TNFalpha content in muscles and fat (but not in the liver) contributes to insulin resistance by lowering insulin receptor protein tyrosine kinase activity, while other insulin receptor characteristics (insulin-binding capacity and affinity of insulin receptors to the hormone) do not seem to be influenced by this factor.

Background: Duolip forte (ethophyllinclofibrate) is a lipid lowering drug, the effect of which was not yet adequately verified in our conditions, namely from the point of its effect on various types of hyperlipoproteinaemias., Method and Results: The therapy was applied on 45 patients with combined hyperlipidaemia, 25 men and 20 women, mean age 53.8 years (age range 41-65). About one half of patients were treated for hypertension, almost one quart of them manifested ischemic heart disease, 13% had a history of myocardial infarction and the same percentage suffered from associated diabetes type 2. Duolip forte was administered perorally in a dose of 500 mg once daily for 12-16 weeks to the patients who had complied with the prescribed diet (weight loss, improvement of lipid levels) during three-month preliminary period, but did not reach desirable parameters. After the therapy, the whole group studied manifested a significant decrease of total cholesterol (-10.8%), LDL-cholesterol (-13%), triglycerides (-40.8%) and free fatty acids (-20.6%), while the increase of HDL-cholesterol and its fractions was not significant. In hyperlipoproteinaemias of IIa type (n = 15), only a significant decrease of LDL-cholesterol appeared, in hyperlipoproteinaemias of IIb type (n = 15), a significant decrease was evidenced only in triglycerides (-33.9%). In hyperlipoproteineaemias of IV type (n = 15), a significant decrease was observed in triglycerides (-50.3%), while HDL-2 cholesterol values increased (+32.3%). A slight decrease of uric acid level and of body mass index observed in all groups was statistically not significant, as well as a slight increase of glycaemia and changes of serum transaminases. No unwanted side effects of Duolip were found., Conclusions: Ethophyllinclofibrate has proved as effective namely at hypertriglyceridemic primary hyperlipidaemias. Besides decreasing triglycerides, it increases significantly HDL-2 cholesterol. A convenient dosage, small lithogenic effect and minimum of side effects make it a drug of choice for some groups of elderly high-risk patients.

The relative content of various fatty acids in serum lipoproteins was determined in patients with type IIa (38), IIb (49) and IV (77) of hyperlipoproteinemia and compared with 52 controls. Significant changes were found in hyperlipoproteinemia associated with hypertriglyceridemia (type IV) but not in "pure" hypercholesterolemia (type IIa). In all lipoprotein fractions (VLDL, LDL, HDL) in type IV of hyperlipoproteinemia the increased oleic and linolenic acid proportions were found, while proportions of linoleic, arachidonic and docosahexaenoic acids were decreased. The saturated fatty acids (myristic, palmitic and stearic) were found increased in LDL. Linear regression analysis has shown positive correlation between the content of arachidonic and docosahexaenoic acids in HDL and LDL and the serum levels of total HDL-cholesterol, HDL2-cholesterol, HDL3-cholesterol and ApoA1, while a negative correlation between these fatty acids and serum triglycerides level appeared. These findings can be explained partly by increased content of triglycerides and free fatty acids in lipoproteins. Possible differences concerning mechanisms of accelaration of atherogenesis in various types of hyperlipidemia are discussed.

Proportions of unsaturated fatty acids of high density lipoprotein (HDL) lipids and their relationships to the HDL-cholesterol level were compared in hypo- and hyperalphalipoproteinemic subjects. Both groups did not differ in the level of serum cholesterol. However, hypoalphalipoproteinemia was associated with hypertriglyceridemia, higher HDL-triacylglycerol level, and higher proportion of HDL-18:1 (oleic acid) and lower proportions of HDL-18:2 (linoleic acid) and 20:4 (arachidonic acid) than hyperalphalipoproteinemia. The HDL-20:4 was the only fatty acid correlating (negatively) with HDL-cholesterol. However, HDL-18:1 correlated positively and HDL-18:2 negatively with HDL-triacylglycerols, lipids related to the fall of HDL-cholesterol. These results suggest 1) an antagonism of 20:4 and 18:2 as structural components of HDL lipids in relation to the HDL-cholesterol level, and 2) an association of replacement of HDL 18:2 by 18:1 with the disorder of plasma triacylglycerol metabolism.

The supposed antistress effect of vitamins-alpha-tocopherol, pyridoxine and dexpanthenol (pantothenic acid precursor)--was followed on the model of nociceptive stress in laboratory rats. The decrease of the stress enhancement of nonesterified fatty acids (NEFA), estimated in the brain cortex, hypothalamus and the brain stem, was taken for the indicator of the antistress effect. Nonesterified fatty acids were determined with the help of gas chromatography following the separation performed by thin layer chromatographic method. Five-day application of alpha-tocopherol acetate (per os, 300 mg.kg-1) led to a decrease of the stress enhancement of arachidonic acid level in the brain stem.

In 29 samples of recurrent nasoethmoidal polyps removed from 11 patients, the relative fatty acids composition was established by means of gas chromatography. Four samples of nasal mucosa of patients without polyps were subjected to the same procedure. A significant increase in the content of linoleic acid (18:2, n-6) as well as in the index "linoleic acid/oleic acid" was found. Proportion of arachidonic acid was high both in polyps and in control samples of nasal mucosa. An orientation examination of fatty acids in individual fractions of lipids, compared to serum values, showed that the main source of arachidonic acid appears to be phospholipids.

There was studied the effect of ketoconazole on the growth, the amount of ergosterol and the relative distribution of fatty acids in the sensitive strain MG-155 of M. gypseum and its two ketoconazole-resistant mutants UV-1 and UV-2 (induction by UV radiation) in the exponential stage growth. After three-day cultivation in the medium with ketoconazole (0.64 microgram.ml-1) there appeared 40% inhibition of growth in MG-155, 10% inhibition in UV-2 and only 4% inhibition UV-1. The amount of ergosterol decreased in MG-155, in both mutants the amount of ergosterol increased by the effect of ketoconazole (by more than 50%). The main saturate fatty acids are palmitic acid (10% and stearic acid (4%). The major fraction of unsaturate fatty acids is formed by linoleic acid (50%) and by oleic acid (4%). Ketoconazole does not affect the quality of fatty acids spectre, it changes only the size of the relative distribution of individual fatty acids. In this point of view, the mutant UV-2, and namely the mutant UV-1 (the higher degree of resistance) differ from the initial sensitive strain MG-155. Ketoconazole provokes in mutants a higher reduction of stearic acid fraction, of saturate fatty acids C greater than 18 and of triunsaturate fatty acids and it causes only minimum (4% for UV-1) decrease of oleic acid. The results of cultivations with postponed application of ketoconazole to the medium (on the 2nd the 1st day before the mycelium harvest) show in a decisive way that effect of ketoconazole is fully developed only on condition that ketoconazole is added to the medium simultaneously with the inoculum. There are discussed several conclusions with regard to the findings in the identical strains in stationary stage of growth.

There was studied the spectre of fatty acids and its changes induced by ketoconazole in the strain M. gypseum and its two mutants (UV-1, UV-2). The main saturate fatty acid in MG-155 and in both mutants is palmitic acid, the main unsaturate fatty acid is linoleic acid. In comparison with MG-155, UV-1 and UV-2 manifest a lower amount of stearic and oleic acids and a higher amount of stearic acid) in MG-155 and UV-1, an increase of saturate fatty acids is observed in UV-2. The unsaturate fatty acids affected by ketoconazole increase in MG-155 and UV-1, they decrease in UV-2 (the changes are most marked in linoleic acid). Oleic acid, as the main representant of monounsaturate fatty acids, decreases in MG-155 and in both mutants. There is discussed the relationship of these changes in fatty acids distribution to the eventual damage of membrane function, to the growth characteristics and to the metabolism of ergosterol.

In the brain tissue there exists a highly active "pool" of nonesterified fatty acids (NEFA). The greatest part of NEFA comes from the fraction of nerve endings. It was evidenced that the model convulsive activity provoked by Bemegrid increased NEFA in the brain cortex, hypothalamus and brain stem. Cinnarizine (StugeronR) markedly decreases the convulsive increase of NEFA. The greatest inhibitory effect was observed in the brain cortex. Cinnarizine has an objective stabilizing and anticonvulsive effect on the brain tissue.

The levels of NEFA (oleic acid, palmitic acid, stearic acid, arachidonic acid) were studied in the brain cortex, hypothalamus and the brain stem of rat brain. Inhibition of phospholipase A2 by Delagil (Mepacrinium dichloratum CsL 3) decreased statistically significantly the values of arachidonic aci (-5.5%) in the brain cortex and the values of stearic acid (-11.2%) in the hypothalamua. The cyclooxygenase inhibition by aspirin led to a significant increase of palmitic acid (+12.7%), oleic acid (+18.7%) and arachidonic acid (+14.3%) in the hypothalamus. Total NEFA increased by 11.2%. A significant increase of palmitic acid (+26.9%), arachidonic acid (+33.9%) and of total NEFA (+22.9%) was evidenced in the brain stem. Delagil administered in combination with acetylsalicylic acid suppressed the NEFA increase observed after the application of acetylsalicylic acid alone. A statistically significant decrease was found in stearic acid (-12.6%) in the hypothalamus and in all studied NEFA in the brain stem (palmitic acid, -14.2% stearic acid -11.7%, oleic acid -3.8%, arachidonic acid -16.6% and total NEFA -11.8%).

An acute 30 min exposure to toluene vapours (0.32 mmol/l of the air) provoked in the rat brain a decrease of total nonesterified fatty acids (arachidonic acid -21.7%, oleic acid -18.3%, stearic acid -7.9% in the brain cortex and arachidonic acid -6.8% in the hypothalamus). The results are in harmony with the hypothesis of the NEFA decreasing effect of the inhibitory substances in the brain.

There was studied the effect of repeated administration of delta-9-THC (10 mg/kg per os for two days) on the level of nonesterified fatty acids (NEFA) in the brain structures of laboratory rats. Palmitic acid, stearic acid, oleic acid and arachidonic acid were estimated with the help of gas chromatography after preliminary separation by TLC. After administration of delta-9-THC, the levels of NEFA decreased in the brain cortex, the brain stem and most in the hypothalamus. The NEFA decrease after delta-9-THC administration is in harmony with the functional inhibition of CNS, provoked by this substance.

A method is proposed for the determination of trimecaine (diethylglycylmesidide) and its de-ethylated metabolites (monoethylglycylmesidide and glycylmesidide) in blood plasma by capillary isotachophoresis. The deproteinated plasma is extracted into chloroform after alkalinization and the total solids in the organic layer are dissolved in acidified 25% 2-propanol. Subsequent isotachophoretic analysis is performed in an operational system consisting of potassium acetate buffer (pH 4.75) as the leading and beta-alanine as the terminating electrolyte. The order of the zones corresponds to the molecular weights of the separated compounds. The recovery of all substances of interest is 55% and the limit of determination is 0.05 micrograms of each substance in 1 ml of plasma.

The administration of physostigmine (0.3 mg/kg s.c.) compensated toluene intoxication in rats together with NEFA return toward the normal values. This interaction of toluene and physostigmine supports the hypothesis of NEFA functioning as moderators of the cerebral activity.