[Subtilisin like protease secreted in Bacillus pumilus KMM 62 on different growth stages].

A protease secreted in Bacillus pumilus KMM 62 culture liquid on different growth stages was isolated using ion-exchange chromatography. On the basis of pattern of specific chromogenic substrates hydrolysis and inhibitory analysis the protease was classified as subtilisin like serine protease. The molecular weight ofprotease is 31 kDa. Proteolytic activity towards Z-Ala-Ala-Leu-pNa substrate was maximal at pH 8-8.5. The optimal temperature for proteolytic activity was observed at a temperature of 30 degrees C, and the protein was stable within the pH range of 7.5-10.0. Bacillus pumilus KMM 62 subtilisin like serine protease was shown to have thrombolytic activity.

Anxiety and predisposition to audiogenic epilepsy in rats of different genotypes.

We studied plus-maze behavior of inbred Krushinskii-Molodkina, Wistar, and black-hooded rats (originating from the Long-Evans outbred strain) differing by predisposition to audiogenic seizures. The severity of audiogenic seizures partially correlated with anxiety and negatively correlated with the total level of locomotor activity in the elevated plus-maze. The anxiety parameters in Krushinskii-Molodkina rats were evaluated after injection of anticonvulsant levetiracetam and anxiolytic afobazol. Levetiracetam and afobazol somewhat stimulated locomotor activity.

[Levetiracetam modifies the pattern of audiogenic locomotive response in Wistar and Krushinsky-Molodkina strain rats].

The novel antiepileptic drug levetiracetam (keppra, 80 mg/kg, i.p.) produces a pronounced anticonvulsant effect on all parameters of the audiogenic locomotion response in Wistar rats, leading to the prevention of acoustic seizures approximately in 50% animals. Keppra injection also led to a decrease in the intensity of single audiogenic convulsive episode, a twofold prolongation of the latency of motor reaction, and a change in the pattern of seizure reaction toward increasing number of rats with "one-wave" response. In contrast, a lack of sound resistant animals and the change from one- to two-wave audiogenic response were observed in Krushinsky-Molodkina strain rats.

[The investigation of cataleptic muscle tonus changes in rats after audiogenic seizures].

The duration and severity of cataleptic freezing after audiogenic seizures were investigated in rats of several genotypes with different predisposition to audiogenic epilepsy. These genotypes were the Krushinsky-Molodkina strain (KM--the audiogenic seizure prone strain), the Wistar and two new substrains selected from hybrids KM x Wistar for high ("4") and low ("0") audiogenic seizure susceptibility. The drug injections, which modulated the audiogenic fit severity, induced the changes in catalepsy too. It was found, that in intact animals cataleptic freezing developed after audiogenic seizures only and was more intense after severe seizures, the correlation coefficient being statistically significant. Levetiracetam (anticonvulsant drug) injections resulted in significant decrease of audiogenic fit severity and cataleptic state in Wistar and KM rats. Caffeine injections resulted in audiogenic seizures accompanied by cataleptic freezing in rats of the substrain "0", selected for audiogenic non-pronness.

Ribotyping and biotyping of Staphylococcus epidermidis isolated from hospital environment.

The Staphylococcus strains acquired from scrapings from hospital environments were identified to the species level based on their biochemical properties. From the monitored sample the Staphylococcus epidermidis strains were selected for more accurate typing and tested on their virulence factor and ribotyped. The biotyping of S. epidermidis did not show any considerable intraspecific variation of these isolates and there were no atypical reactions, with the exception of three strains (out of 33). In contrast, the results of ribotyping showed greater heterogeneity of strains and unequivocally demonstrated the relation between the ribotype and the place of sample drawing. In addition to this fact, the found ribotypes repeat in the same environment in the long-term which suggests the occurrence and persistence of the same strains of conditionally pathogenic bacteria in hospital environment. We showed that ribotyping is a suitable method for precise and reliable detection of some coagulase-negative staphylococci.

[Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62].

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.

Purification and characterization of a subtilisin-like proteinases secreted in the stationary growth phase of Bacillus amyloliquefaciens H2.

Proteinases secreted during the early and late stationary phases have been isolated from the culture liquid of Bacillus amyloliquefaciens H2 using CM-cellulose ion-exchange chromatography with subsequent FPLC on a Mono S column. Considering the character of hydrolysis of specific chromogenic substrates and the type of inhibition, these enzymes were identified as subtilisin-like proteinases. The molecular weight of both proteinases is 29 kD. The proteolytic activity of the proteinases secreted during the early and late stationary phases towards the synthetic substrate Z-Ala-Ala-Leu-pNA was maximal at pH 8.5 and 9.0, respectively. The maximal activity of both proteinases was observed at 37 degrees C, and the proteins were stable within the pH range of 7.2-9.5. The subtilisin-like proteinases from B. amyloliquefaciens were shown to catalyze synthesis of peptide bonds.

[Neuronal and neurochemical mechanisms underlying the effect of a novel antiepileptic drug levetiracetam].

Published data on the mechanisms involved in the action of levetiracetam (LVT, 2S-(oxo-1-pyrrolidinyl)butanoic acid amide), a widely used agent for the adjunctive therapy of partial epilepsy of different genesis, have been analyzed. A unique profile of the anticonvulsant action of LVT was already revealed at the screening stage: this agent was not effective in the traditional tests, but it demonstrated a pronounced protective effect in kindling models and in the experiments on mice sensitive to the audiogenic convulsions. Now it is commonly accepted that the LVT mode of action is different from that of the other known antiepileptic drugs. LVT was shown to have specific binding sites in the brain. It is believed that the synaptic vesicle protein SV2A is the binding site for LVT, and the main mechanism of its action involves the modulation of this protein functions, which probably accounts for the unique profile of the antiepileptic activity of LVT. The results of investigations of the neurochemical mechanisms of LVT action suggest that proteins involved in the vesicular exocytosis, in particular SV2, can be promising targets in the search for new effective agents for the treatment of CNS diseases.

[Anticonvulsant effects of levetiracetam on audiogenic epileptiform siezures in Krushinsky-Molodkina rats].

The new antiepileptic drug levetiracetam (LVT) at doses of 40 and 80 mg/kg exhibits a pronounced anticonvulsant effect, influencing all the parameters of audiogenic epileptiform seizures (AES) in Krushinsky-Molodkina (KM) rats. The latent period of the motor reaction was increased 3-15 times compared to control, and the intensity of single convulsive episodes was significantly decreased. Changes were also detected in the profile of the first stage of motor activity, and the "two-wave" reaction was observed in 50-80% of animals. The mechanism of LVT anticonvulsant action in KM rats is probably associated with an increase in the inhibitory and a decrease in the excitatory processes in the CNS.

Effect of single and repeated administration of high-molecular-weight heparin in low doses on brain content of neurotransmitters in Wistar rats.

We demonstrated high sensitivity of the brain neurotransmitter system to high-molecular-weight heparin in Wistar rats. Single and repeated administration of heparin modulated the content of monoamines and neurotransmitter amino acids in the hippocampus, striatum, and cerebral cortex. Antistress activity of high-molecular-weight heparin and its positive effects on memory under experimental conditions suggest that this preparation in low doses can be used in the therapy of neurological and age-related diseases.

Role of nitric oxide and lipid peroxidation in pathophysiological mechanisms of audiogenic seizures in GEP Rats and DBA/2 mice.

We evaluated the role of nitric oxide and lipid peroxidation in the pathophysiological mechanisms of seizures in genetically epilepsy prone (GEP) rats and DBA/2 mice with genetically determined audiogenic epilepsy. In rats and mice acoustic stimulation led to locomotor activation followed by clonic-tonic seizures. The contents of nitric oxide and lipid peroxidation products at the peak of seizures markedly surpassed the control level.

Levels of neurotransmitter amino acids in the cerebrospinal fluid of patients with acute ischemic insult.

The dynamics of excitatory (glutamate, aspartate) and inhibitory (GABA, glycine) neurotransmitter amino acid contents in the cerebrospinal fluid were studied in 110 patients with hemispheric ischemic insult. These studies revealed significant increases in the levels of glutamate and aspartate in the first six hours of illness, and the level and duration of these changes correlated with the severity of the insult. Peak GABA and glycine levels were seen at the end of the first day after strokes, reflecting the delayed activation of the mechanisms of protective inhibition. The insufficiency of GABAergic mediation in strokes located in the hemispheres to a significant extent mirrored the severity of clinical features and the potential of restorative processes. Early significant biochemical criteria were identified for objective assessment of the severity of brain ischemia, and these had prognostic value for the course and outcome of strokes. The most unfavorable prognostic signs were the presence of low (or undetectable) GABA levels in the first days after insult and progressive increases in aspartate levels to the third day on the background of sharp reductions in glutamate levels (after initial elevation on the first day).

Effect of cyclic GABA derivative TZ-146 on the content of neurotransmitters in rat brain stem.

The effect of cyclic GABA derivative TZ-146 on the content of catecholamines, serotonin, their metabolites, and neurotransmitter amino acids in rat hypothalamus and brain stem was studied by high-performance liquid chromatography with electrochemical detection. Opposite changes in the content of homovanillic acid, aspartate, glutamate, and glycine in the examined structures were accompanied by inhibition of dopamine metabolism. Possible participation of cerebral glutamatergic system in the effect of TZ-146 is discussed.

[Levels of neurotransmitter amino acids in the cerebrospinal fluid of patients with acute ischemic stroke]. / Soderzhanie neirotransmitternykh aminokislot v spinnomozgovoi zhidkosti bol'nykh ostrym ishemicheskim insul'tom.

The dynamics of the levels of both excitatory (aspartate, glutamate) and inhibitory (GABA, glycine) neurotransmitter amino acids was estimated in cerebrospinal fluid of 110 patients with hemispheric ischemic stroke. A significant increase of the contents of glutamate and aspartate was found beginning with the first 6 hours of the disease onset. The degree and duration of such elevation correlated with severity of the stroke. Maximal GABA and glycine levels were registered to the end of the 1st day of the stroke, that reflected delayed triggering of the protective inhibitory mechanisms. It was established that insufficiency of GABA-mediation in hemispherical location of the stroke was much responsible for both the severity of its clinical manifestations and potential of the restorative processes. Early significant biochemical criteria for objective assessment of the severity of cerebral ischemia as well as of the stroke course and outcome were defined. The most unfavourable prognostic signs were low GABA concentration (or impossibility of its evaluation) during the first days of the stroke, progredient elevation of the aspartate level until the 3d day of the disease and the severe fall of glutamate concentration (in spite of its initial increase on the 1st day).

[The function of the peripheral adrenergic link under the action of neuroleptics in immobilization stress]. / Sostoianie perifericheskogo adrenergicheskogo zvena pri deistvii neiroleptikov v usloviiakh immobilizatsionnogo stressa.

The morphofunctional state of different links of the sympathoadrenal system under the action of neuroleptics haloperidol and sulpiride was studied on the model of immobilization stress in rats. The drugs were shown to possess the effect of the pharmacological correction of hormone content in the adrenals and the level of the neuromediator activity of the adrenergic nerves at different stages of immobilization stress. The data obtained indicate the anti-stress action of the studied neuroleptics. The effect of sulpiride is noted both in the early (the stage of anxiety) and late (the stage of exhaustion) periods of immobilization. The action of haloperidol is mainly limited to the stage of anxiety.

[Effect of the N-terminal fragment of substance P1-4 on the somatic manifestations of the stress reaction and on the catecholamine content of the adrenals in rats]. / Vliianie N-terminal'nogo fragmenta substantsii P1-4 na somaticheskie proiavleniia stressovoi reaktsii i soderzhanie katekholaminov v nadpochechnikakh krys.

The antistress affect of the substance P1-4 N-terminal fragment (ARG-Pro-Lys-Pro, 100 mkg/kg, i.p.) has been studied on the model of immobilization stress in rats. It was ascertained that the preparation of protective effect is revealed to the greatest extent on the exhaustion stage (48 h of immobilization), which served to prevent the lymphoid organs mass reduction and ulcer development and also accounted for greater adrenaline and noradrenaline content preservation in tissues and chromaffin cells of adrenal glands in stressed animals.

[Role of the peripheral catecholaminergic systems in the antistress action of neuropeptides]. / Rol' perifericheskikh katekholaminergicheskikh sistem v antistressovom deistvii neiropeptidov.

The mediator activity of the peripheral catecholaminergic systems (the adrenergic nerves of dura mater and the concentrations of noradrenaline and adrenaline in the adrenals of rats) during dynamic and immobilization stress was investigated with the help of fluorescent microscopy and spectrofluorometry. Neuropeptides--dalargin and another enkephalin analog--were injected intraperitoneally, 150 mg/kg. A visible antistress action of these neuropeptides has been demonstrated, it was more marked after treatment with dalargin. The role of peripheral catecholaminergic system in the mechanism of stress-protective effects of neuropeptides is discussed.

[Morphometric ultrastructural study of the adreno- and noradrenocytes of the rat adrenal during immobilization stress of varying duration]. / Morfometricheskoe ul'trastrukturnoe izuchenie adreno- i noradrenotsitov nadpochechnika krysy v usloviiakh immobilizatsionnogo stressa razlichnoi dlitel'nosti.

The ultrastructural organization of secretory granules, containing adrenaline (A) or noradrenaline (NA) was studied in chromaffin cells of the rat adrenal gland after 3-, 24- and 48-hour immobilization stress. Using cytochemical electron microscopic Tranzer's method and the method of morphometry, the number of normal dense cores, "empty" and "semiempty" vesicles was calculated. It was shown that the total content of vesicles and the ratio of investigated types of both adrenaline- and noradrenaline-accumulating granules were markedly changed during stress. The degree of such stress-induced reorganization depended on the stage of stress reaction and involved first A-cells and then NA-cells.