Blockade of the expression of mecamylamine-precipitated nicotine withdrawal by calcium channel antagonists.

The present study focused on the evaluation of nicotine abstinence syndrome in mice and on the influence of calcium channel blockers on the expression of the somatic signs of nicotine withdrawal. Our experimental protocol consisted of intermittent administration of nicotine, 2.5 mgkg(-1), subcutaneously (s.c.), four times daily for 7 days. In attempt to precipitate nicotine abstinence, mice were given one injection of mecamylamine (3 mgkg(-1), intraperitoneally (i.p.)), 1h after the last nicotine injection, on the test day (day 8) in the morning. Additionally, body weight changes, locomotor activity and anxiogenic responses in the elevated plus maze test were also evaluated in nicotine withdrawn mice. Our data shown that the L-type calcium channel antagonists, nimodipine, verapamil, flunarizine and diltiazem (5 and 10 mgkg(-1), i.p., each), injected before mecamylamine administration, dose-dependently attenuated the expression of nicotine withdrawal signs. Moreover, 24h after terminating nicotine treatment, we also observed additional nicotine abstinence measures, such as loss of body weight followed by a slight body weight gain, decrease of spontaneous locomotor activity and anxiogenic responses. These findings obtained using our valuable rodent model of nicotine dependence suggest the involvement of calcium-dependent mechanisms in the expression of mecamylamine-precipitated nicotine abstinence syndrome.     

Optimization of determination of aflatoxins in foods with bromine postcolumn derivatization

The method for determination of aflatoxin B1, B2, G1 i G2 in nuts, culinary spices, cereals and cereal products was described. To optimize the analytical procedure in several products, condition of proper extraction, clean-up, HPLC and detection were selected. After extraction by means of methanol and water (80+20 v/v) or (70+30 v/v), clean-up on IAC columns, HPLC on C18 columns--Nucleosil and Nova Pak with mobile phase-methanol, acetonitrile, water (20+20+60 v/v) was performed. For fluorometric detection at 362/430 nm, post-column derivatization of aflatoxin B1 and G1 with bromine was carried out. The mean recovery of the method depending on matrix and aflatoxin, was 52-102% at RSD% 0.2-8.3. LOD and LOQ, respectively were: 0.01 and 0.02 microg/kg for nuts and 0.05 and 0.1 microg/kg for culinary spices and cereal products. The concentrations of aflatoxins in 86 samples of foods from market were below the permissible maximum levels legally binding.     

Clinical and epidemiological analysis of patients with botulism hospitalized at the Department of Infectious Disease,Medical University of Lublin in 1990-2000

In the paper we presented results of clinical and epidemiological analysis of 32 patients with botulism hospitalized at the Department of Infectious Diseases, Medical University of Lublin in 1990-2000. In the studied group, the relationships between botulism incidence and sex and place of residence were not significant. The incubation period ranged from 7 hours to 5 days (average 36 hrs). The clinical manifestations of botulism were typical in all cases. In one female patient the course of disease was complicated. She developed right-sided bronchopneumonia and left-sided purulent parotitis. The type B botulinum toxin occurred more frequently than the other types and the cases without serological confirmation (Chi 2 = 6.125 p = 0.01). It was found in serum of 23 patients (in 2 cases together with the type A toxin). The type E toxin was found in serum of one patient. The presence of toxin in serum was not detected in 8 patients. In all patients trivalent (types A, B and E) equine antitoxin was administered. The dose ranged from 50 to 150 cm3. Symptomatic treatment was given in all cases. Nobody required mechanical ventilation. The duration of hospitalization ranged from 5 to 28 days (average 16.6 days). A few patients complained of long-lasting blurred vision or dry mouth.     

Anxiolytic-like effects of preferential dopamine D3 receptor agonists in an animal model

The aim of the present study was to examine a potential anxiolytic-like action of (+)-7-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (7-OH-DPAT), a preferential dopamine D(3) receptor agonist, and (N-[4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl]-2-naphthylcarboxamide (BP 897), partial dopamine D(3) receptor agonist. Diazepam was used as a reference compound. The anxiolytic-like effect of those drugs was tested in the conflict drinking test (Vogel test) in male Wistar rats. The obtained results showed that 7-OH-DPAT and BP 897 (like diazepam) induced anxiolytic-like effects in the conflict drinking test. 7-OH-DPAT (0.05 and 0.1 mg/kg), BP 897 (0.5 mg/kg) and diazepam (5 and 10 mg/kg), tested at the effective doses in an animal model, did not affect motor coordination but produced significant reduction in exploratory activity in the open field test. These data suggest that preferential dopamine D(3) agonists may play a role in the therapy of anxiety, however, further studies are necessary to elucidate the mechanism of these actions.     

Characterization of substrate binding to cytochrome P450 1A1 using molecular modeling and kinetic analyses: case of residue 382.

Key residue Val-382 in P450 1A1 has been predicted to interact with the alkoxy chain of resorufin derivatives. Therefore, we undertook a detailed analysis of substrate mobility in the active site of the P450 1A1 homology model and assessed the effect of mutations at position 382. Dynamic trajectories of 7-methoxy-, 7-ethoxy-, and 7-pentoxyresorufin indicated that 7-ethoxyresorufin would be oxidized most efficiently by the wild-type enzyme. The Val-382-->Ala mutation would increase the O-dealkylation of 7-pentoxyresorufin but decrease the oxidation of other substrates. In the case of the V382L mutant, the large bulk of Leu would block alkoxyresorufins from productive binding orientations leading to lowered activities. Binding free energy calculations for three substrates with 1A1 WT and two mutants indicated that binding constants would be similar for all enzyme-substrate combinations. Modeling predictions were tested experimentally. The plasmid containing the cDNA for human P450 1A1 modified for bacterial expression was altered to include a C-terminal PCR-generated six histidine domain to facilitate enzyme purification. The V382A and V382L mutants were constructed by site-directed mutagenesis and Escherichia coli-expressed enzymes purified using Ni-NTA affinity chromatography. The activity of the WT 1A1 was highest toward 7-ethoxyresorufin and lowest toward 7-pentoxyresorufin. Both mutants displayed a decrease in V(max) with 7-methoxy- and 7-ethoxyresorufin, whereas for the V382A mutant, V(max) with 7-pentoxyresorufin was increased. No significant changes in K(m) were observed relative to the wild-type enzyme. The experimental results are thus in good agreement with modeling predictions.     

Crotoxin acceptor protein isolated from Torpedo electric organ: binding properties to crotoxin by surface plasmon resonance.

Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A(2) (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a crotoxin column. SDS-PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (K(d)(app)) of 3.4 nM was calculated for the interaction of pure CAPT and crotoxin from the dissociation rate constant (k(off)=1.2 x 10(-2)s(-1)) and the association rate constant (k(on)=3.5 x 10(6)M(-1)s(-1)).     

Frequency intake and energy value of breakfast consumed by school youth

The aim of study was evaluation of frequency intake and energy value of breakfast consumed by high school youth. The group of 126 pupils aged 16-18 years was investigated. Frequency intake was estimated using questionnaire and energy value by 24 hours recall method. The irregularity in breakfast intake as well as changing energy value of them has been shown.     

Mechanism of synergistic action following co-treatment with pramipexole and fluoxetine or sertraline in the forced swimming test in rats

The aim of the present study was to examine the effect of combined treatment of male Wistar rats with pramipexole and fluoxetine or sertraline in the forced swimming test. The obtained results showed that co-treatment with pramipexole (0.1 mg/kg) and fluoxetine (10 mg/kg) or sertraline (5 mg/kg) (in doses inactive per se) exhibited antidepressant-like activity in the forced swimming test. Sulpiride (a dopamine D(2/3) receptor antagonist) and WAY 100635 (a 5-HT(1A) receptor antagonist), either being ineffective in the forced swimming test, inhibited the antidepressant-like effect induced by co-administration of pramipexole and fluoxetine or sertraline. However, SCH 23390 (a dopamine D(1) receptor antagonist) only partly did not alter the effect of pramipexole given jointly with antidepressant drugs; on the other hand, S 33084 (a dopamine D(3) receptor antagonist) only partly decreased (in a statistically insignificant manner) that effect. Moreover, progesterone and BD 1047 (a sigma(1) receptor antagonist) counteracted the antidepressant-like effect induced by co-administration of pramipexole and sertraline (but not pramipexole and fluoxetine). In that test, active behavior did not reflect the increases in general activity, since combined administration of pramipexole and fluoxetine or sertraline failed to enhance the locomotor activity of rats. None of the tested drugs (SCH 23390, sulpiride, S 33084, WAY 100635, BD 1047 and progesterone) - alone or in combination with pramipexole and fluoxetine or sertraline - changed locomotor activity. The results described in the present paper indicate that co-administration of pramipexole and fluoxetine or sertraline may induce a more pronounced antidepressive activity than does treatment with pramipexole alone, and that in addition to other mechanisms, dopamine D(2/3) and 5-HT(1A) receptors may contribute to the antidepressant-like activity of pramipexole and fluoxetine or sertraline in the forced swimming test in rats. Moreover, sigma(1) receptors may constitute one of the possible mechanisms by which co-administration of pramipexole and sertraline induces antidepressant-like activity in that test.     

Respiratory hypersensitivity to trimellitic anhydride in Brown Norway rats: a comparison of endpoints

A rat bioassay has been developed to provide an objective approach for the identification and classification of respiratory allergy using trimellitic anhydride (TMA), which is a known respiratory tract irritant and asthmagen. Particular emphasis was placed on the study of route-of-induction-dependent effects and their progression upon inhalation challenge with TMA (approximately 23 mg m(-3) for a duration of 30 min), which included analysis of specific and non-specific airway hyperreactivity and pulmonary inflammation initiated and sustained by immunological processes. Refinement of the bioassay focused on procedures to probe changes occurring upon challenge with TMA or methacholine aerosols using physiological, biochemical and immunological procedures. Following challenge with TMA, the rats sensitized to TMA showed marked changes in peak inspiratory and expiratory air flows and respiratory minute volume. In these animals, a sustained pulmonary inflammation occurred, characterized by specific endpoints determined in bronchoalveolar lavage (lactate dehydrogenase, protein, nitrite, eosinophil peroxidase, myeloperoxidase). When compared with the naive controls, lung weights were increased significantly, as were the weights of lung-associated lymph nodes following inhalation induction and auricular lymph nodes following topical induction. The extent of changes observed was equal or more pronounced in animals sensitized epicutaneously (day 0:150 microl vehicle/50% TMA on each flank, day 7; booster administration to the skin of the dorsum of both ears using half the concentration and volume used on day 0) when compared with rats sensitized by 5 x 3 h day(-1) inhalation exposures (low dose: 25 mg TMA m(-3), high dose: 120 mg TMA m(-3)). In summary, the findings support the conclusion that the Brown Norway rat model is suitable for identifying TMA as an agent that causes both an immediate-type change of breathing patterns and a delayed-type sustained pulmonary inflammatory response. However, it remains unresolved whether the marked effects observed in the topically sensitized rats are more related to a route-of-induction or dose-dependent phenomenon.