Expression pattern of synaptic vesicle protein 2 (SV2) isoforms in patients with temporal lobe epilepsy and hippocampal sclerosis.

AIMS: Synaptic vesicle proteins 2 (SV2) are neuronal vesicles membrane glycoproteins that appear as important targets in the treatment of partial and generalized epilepsies. Therefore, we analysed the expression of SV2 isoforms in the hippocampus of patients with temporal lobe epilepsy (TLE). METHODS: SV2A, SV2B and SV2C immunostaining and QuantiGene branched DNA assay were performed on biopsies from 31 consecutive TLE patients with mesial temporal sclerosis (MTS) and compared with 10 autopsy controls. SV2 expression was further compared with Timm's staining, and synaptophysin, Zinc transporter 3 (ZnT3), dynorphin, vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter (VGAT) expression. RESULTS: In TLE patients, SV2A and SV2B expression was decreased in areas of synaptic loss. SV2C, which is weakly expressed or absent in the hippocampus of controls, was overexpressed in 10/11 cases with classical MTS1A and mossy fibre sprouting but not in cases with other types of MTS. SV2C staining was located in the inner molecular layer of the dentate gyrus and colocalized with dynorphin, ZnT3 and VGLUT1, suggesting selective expression in presynaptic glutamatergic Zn(2+) -rich terminals of abnormal sprouting fibres. SV2 expression patterns correlated with histological subtypes of MTS, but not with clinical features or therapeutic regimens in this patient cohort. CONCLUSION: In classical MTS1A, the expression of SV2 isoforms is altered with a marked decrease of SV2A and SV2B paralleling synaptic loss and a selective increase of SV2C in sprouting mossy fibres. These findings suggest a different physiology of sprouting synapses and the possibility to target them with SV2C-specific strategies.

Mechanism of inactivation of S-adenosyl-L-homocysteine hydrolase by 5'-deoxy-5'-S-allenylthioadenosine and 5'-deoxy-5'-S-propynylthioadenosine.

5'-Deoxy-5'-S-allenylthioadenosine 1 and 5'-deoxy-5'-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II "mechanism-based" inhibitors.

Inactivation of S-adenosyl-L-homocysteine hydrolase with fluorinated analogs of 2'- and 3'-deoxy-5'-methylthioadenosine.

Fluorinated analogs of 2'- and 3'-deoxy-5'-methylthioadenosine 1-4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.

A new series of mechanism-based inhibitors of S-adenosyl-L-homocysteine hydrolase from beef liver.

Nucleosides I, II, III caused irreversible inactivation of AdoHcy hydrolase. A mechanism of inactivation is proposed.

The mechanism of inactivation of S-adenosylhomocysteine hydrolase by fluorinated analogs of 5'-methylthioadenosine.

5'-Deoxy-5'-difluoromethylthioadenosine (DFMTA) 1a and 5'-deoxy-5'-trifluoromethyl-thioadenosine (TFMTA) 1b are inhibitors of beef liver S-adenosyl-L-homocysteine hydrolase. DFMTA and TFMTA are time-dependent and irreversible inhibitors of the enzyme. Both 1a and 1b are oxidized by E-NAD+ to produce E-NADH and fluoride anion is formed in the inactivation reaction (2.2 mol of fluoride/mole of enzyme subunit and 3.1 fluoride/mole of enzyme subunit from DFMTA and TFMTA respectively). Using [8-3H]-1a or [8-3H]-1b no trace of labelled adenosine was detected during the inactivation reaction but adenine was formed. The mechanism of inhibition of S-adenosyl-L-homocysteine hydrolase by these two fluorinated nucleosides is discussed.

Pharmacodynamic activity of lipoprotein lipase and hepatic lipase, and pharmacokinetic parameters measured in normolipidaemic subjects receiving ciprofibrate (100 or 200 mg/day) or micronised fenofibrate (200 mg/day) therapy for 23 days.

The activities of lipoprotein lipase (LPL) and hepatic lipase (HL) were investigated after 23 days of ciprofibrate (100 mg or 200 mg) therapy or fenofibrate (200 mg) therapy. In a double-blind, double-placebo, cross-over study, three groups of six healthy volunteers received either 100 mg ciprofibrate/day followed by 200 mg fenofibrate 'high bioavailability' (HB)/day, or vice versa (group A), 200 mg ciprofibrate HB/day followed by 200 mg fenofibrate HB/day, or vice versa (group B), or 100 mg ciprofibrate/day followed by 200 mg ciprofibrate/day, or vice versa (group C). Fasting plasma lipid levels and safety parameters were evaluated before and after treatment. One hundred milligrams ciprofibrate/day therapy was found to be approximately as effective as 200 mg fenofibrate HB/day therapy in altering the lipid profile. The highest activation of LPL was obtained after treatment with 200 mg ciprofibrate/day. A modest, but statistically significant, increase in HL activity was found after 100 or 200 mg ciprofibrate treatment. Investigation of the pharmacokinetics of ciprofibrate and fenofibric acid revealed a shorter time to reach peak plasma levels, but a longer elimination half life for the ciprofibrate preparations in comparison with fenofibrate. A dose of 200 mg ciprofibrate/day is more effective than 100 mg ciprofibrate/day at increasing LPL and HL activity; however, 200 mg ciprofibrate/day is also associated with a potential detrimental change in safety parameters. Two hundred milligrams fenofibrate HB/day therapy may represent an alternative therapy to 100 mg ciprofibrate/day for hyperlipidaemic patients.

Fast determination of low-level cytochrome P-450 1A1 activity by high-performance liquid chromatography with fluorescence or visible absorbance detection.

A method to determine the activity of the cytochrome P-450 1A1 enzyme, by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity using high-performance liquid chromatography (HPLC) with fluorescence or with visible absorbance detection of resorufin, is described. The lowest quantifiable activity (0.2 pmol/mg min) is obtained by incubation of 0.3 mg of human duodenal microsomal proteins using HPLC fluorescence detection. Using HPLC with visible absorbance detection, sensivity was ten times lower. However, the equipment for this last method is available in most laboratories. The use of both HPLC assays allows determination of the low EROD activity level in samples of small size, such as two or three human duodenal biopsies obtained by routine endoscopy. These methods will be a useful tool to study the role of drug intestinal metabolism by cytochrome P-450 1A1.