Synthesis and characterization of long-chain tartaric acid diamides as novel ceramide-like compounds.

Ceramides play a crucial role in the barrier function of the skin as well as in transmembrane signaling. In this study long aliphatic chain tartaric acid diamides able to replace ceramides in an in vitro model of the stratum corneum lipid matrix due to their similar physico-chemical properties were synthesized from diacetoxysuccinic anhydride in four steps. Their pro-apoptotic effect on fibroblast cells was also investigated.

CE-LIF determination of salivary cadaverine and lysine concentration ratio as an indicator of lysine decarboxylase enzyme activity.

Salivary bacteria produce the enzyme lysine decarboxylase which converts lysine to cadaverine. In the absence of appropriate oral hygiene, overgrowth of these bacteria depletes lysine. This may contribute to gingival inflammation, while cadaverine contributes to oral malodor. A selective and sensitive capillary electrophoresis method with laser-induced fluorescence detection has been developed for the determination of cadaverine and lysine in saliva, as an indicator of lysine decarboxylase enzyme activity. The diamino compounds were separated in acidic background electrolyte in their mono-labeled form after derivatization with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F). Linearity and reproducibility of the method in the range 1-50 µmol L(-1) have been demonstrated using saliva samples. The method was applied for the measurement of cadaverine and lysine in the saliva of healthy volunteers with or without proper oral hygiene. In the absence of oral hygiene, the mol fraction of cadaverine to cadaverine plus lysine in saliva increased significantly (0.65 ± 0.13 vs. 0.39 ± 0.18, P < 0.001), indicating the presence of higher amount of bacterial lysine decarboxylase, that may contribute to periodontal diseases.

[Change of the nitric oxide synthase activity after administration of neurotoxic compounds in mice]. / A nitrogen-oxid szintáz enzim aktivitásának megváltozása neurotoxinok adását követoen egérben.

The possible contribution of reactive oxygen and nitrogen species (RONS) to the development of the neurode-generation came up after the investigations with neurotoxic compounds. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH) have detrimental effect on the dopaminergic neurons. The aim of our study was to examine whether altered nitric oxide synthase (NOS) enzyme activity can be involved in the damage induced by these neurotoxins. The other goal of the study was to investigate the applicability of the measurement of the ratio of NADP+/L-citrulline formed by the enzyme to assess the coupled state of the NOS. Elevated NOS activity in mouse striatum and declined enzyme activity in mouse hippocampus have been found after administration of MPTP Decreased NOS activity in mouse striatum and hippocampus was observed after administration of METH. The ratio of the NADP+/L-citrulline produced by the enzyme provides useful information about the coupling state of the NOS. The ratio in the presence of saturating substrate concentration measured in our experiments was comparable with the data found in the literature. Dramatically increased ratio could be observed in case of decreasing substrate concentration indicating the uncoupled function of the enzyme.

[Molecular mechanisms of the neuroprotective effect of (-)-deprenyl]. / A (-)-deprenyl neuroprotektív hatásának molekuláris mechanizmusai.

(-)-Deprenyl, the irreversible inhibitor of monoamine oxidase B, has been used for decades in the therapy of Parkinson's disease. It improves parkinsonian symptoms due to its dopamine potentiating and antioxidant properties and presumedly delays disease progression. Its complex pharmacological action cannot be explained solely by its monoamine oxidase B inhibitory property. Recently, (-)-deprenyl has been demonstrated to exert antiapoptotic, neuroprotective effects on a number of in vitro and in vivo models in a dose significantly lower than required for monoamine oxidase B inhibition. (-)-Deprenyl and related propargylamines prevent apoptotic cell death by preserving the integrity of the mitochondrion that may be based on the activation of a complex transcriptional program. The changes in gene expression initiated by propargylamines incited to search for further possible target molecules that would explain more accurately the antiapoptotic effect of these compounds. The latest molecular targets include such classical metabolic enzymes, the homologues of which may participate in the regulation of gene expression as a part of transcriptional factor complexes. Some of the propargylamine targets--glyceraldehyde-3-phosphate dehydrogenase, poly(ADP-ribose) polymerase, nuclear amine oxidases--have already been demonstrated to be capable of transforming the metabolic changes in the cell to transcriptional responses. Data are accumulating about the relationship of these enzymes and propargyl compounds, but the real significance of this issue will only be established by future research.

High sensitivity analysis of nitrite and nitrate in biological samples by capillary zone electrophoresis with transient isotachophoretic sample stacking.

Tissue level of nitrate and nitrite are established indicators of altered nitric oxide metabolism under various pathological conditions. Determination of these anions in biological samples, in the presence of high chloride concentration, using capillary zone electrophoresis suffers from poor detection sensitivity. Separation conditions providing excellent resolution and submicromolar detection sensitivity of nitrate and nitrite have been developed and validated. Simple sample preparation was applied that maintains nitrite stability in tissue extracts and at the same time allows transient isotachophoresis stacking of the analytes. Nitrate and nitrite concentrations in rat brain and liver tissue samples were determined in control and lipopolysaccharide treated animals.

Application of the measurement of oxidized pyridine dinucleotides with high-performance liquid chromatography-fluorescence detection to assay the uncoupled oxidation of NADPH by neuronal nitric oxide synthase.

A rapid and sensitive high-performance liquid chromatography method has been developed for the measurement of oxidized pyridine dinucleotides (NAD+, NADP+) in biological samples following fluorescence derivatization. Under strongly alkaline conditions the pyridinium ring of the nicotinamide moiety reacts with carbonyl compounds, resulting in stable fluorescent products. Upon subsequent addition of concentrated formic acid and treatment with heat, this fluorescence is further amplified and is shifted to higher-wavelength regions. From among the ketones assayed (acetone, ethylmethyl ketone, acetophenone) the condensation product with acetophenone possesses the highest molar relative fluorescence, thus allowing the most sensitive detection in our experimental setup (limit of detection: 0.02pmol/50 microliter injected volume). The fluorescent products have been separated on a reverse-phase C-18 column using 0.1M citric acid (pH 3.2)/acetonitrile (92/8, v/v) as mobile phase. Our method is suitable for assaying NADH- and NADPH-dependent enzyme reactions by quantifying oxidized coenzyme products. As an example, the activity of neuronal nitric oxide synthase (nNOS), a NADPH-requiring enzyme, has been assessed by measuring the products NADP+ and l-citrulline at various substrate (l-arginine) concentrations. The rate of the uncoupled NADPH oxidation by nNOS can be estimated from the ratio of NADP+/l-citrulline produced.

Chiral separation of deprenyl-N-oxide isomers by capillary electrophoresis using various cyclodextrin derivatives.

Chiral separation of deprenyl-N-oxide isomers is presented using capillary electrophoresis in the presence of various cyclodextrin (CD) derivatives. This recently identified metabolite of R-(-)-deprenyl may possess desirable pharmacological activities. The effect of the cavity size and the substituents of the CD are examined on the enantiomer resolution of the compound having an asymmetric center on a heteroatom. The importance of hydrophilic or hydrogen bonding interaction, as well as the position of the interacting groups is demonstrated. Outstanding selectivity and resolution values are achieved using the chargeable carboxymethyl-beta-CD. 2-Hydroxypropyl-beta-CD is also suitable for the enantiomer separation of the analyte. Native beta-CD and carboxyethyl-beta-CD provide only poor enantioselectivity, whereas heptakis-(2,6-di-O-methyl)-beta-CD is capable of separating only the diastereomers. No chiral resolution can be observed in the presence of gamma-CD.