Preparation of Site-Specific Isotopically Labelled Zervamicins,the Antibiotic Peptaibols Produced by Emericellopsis Salmosynnemata

A simple procedure for the preparation of the specifically labelled peptide antibiotic zervamicins IC, IIA and IIB has been developed. The zervamicin molecules are labelled with stable isotopes by culturing the Emericellopsis salmosynnemata on a well-defined synthetic medium containing the highly isotopically enriched amino acid. To obtain the peptide with the specifically and highly enriched amino acid residue, precautions have been taken to prevent any de novo biosynthesis of the particular amino acid from unlabelled precursors. The enrichment of the labelled peptide is determined by mass spectrometric analysis. Following this method we have incorporated [2′,4′, 5′,6′,7′-²H₅]-L -Trp-1, [1′-¹⁵N]-L -Trp-1 and [2′, 3′,4′,5′,6′-²H₅]-L - Phl-16 into zervamicins IC, IIA and IIB on the preparative scale and without scrambling of the label. Thus, using the procedures described, isotopically labelled zervamicins can be prepared, allowing them to be studied by solid- state NMR.     

Involvement of kappa-opioid receptors in mechanisms of aggressive and submissive types of behavior in male mice

Effects of the kappa-opioid receptor agonist U-50.488H (0.0, 0.6, 1.25, 2.5 mg/kg. s.c., 30 min) on behavior of the winner with repeated experience of victories and the losers with repeated experience of social defeat in 20 daily agonistic confrontations as well as the control mice were investigated in the tests estimating exploratory activity (open-field) and communication (partition test). Different effects of drug on behaviors of animals with different social story were shown in both tests. In the losers, all doses of U-50.488H had anxiolytic effect, increasing the communication in the partition test. In the winners, the drug induced an increase of aggressive motivation. The control mice were less sensitive to the treatment. In the open-field test, U-50.488H increased the locomotor and exploratory activity in high anxious losers. Winners significantly differed in their reaction to drug treatment in most behavioral forms in comparison with the controls and losers. It was concluded that kappa-opioid receptors are specifically involved into mechanisms of formation of aggressive or submissive types of behaviors under positive or negative social experience.     

Effects of caffeine on social behavior, exploration and locomotor activity: interactions with ethanol

The effects of caffeine and its interaction with ethanol were examined in a test of social behavior and a holeboard test of exploration and locomotion. Male mice were injected i.p. with 15, 30 or 60 mg/kg caffeine alone or in combination with 2 g/kg ethanol. The animals were then put in pairs into a familiar arena, or examined individually in the holeboard. Only the highest dose of caffeine (60 mg/kg) had a significant effect on the time spent in social interaction and motor activity in the social behavior test: both measures were reduced. The duration and frequency of avoidance-irritability behavior was dose-dependently increased by caffeine. In the holeboard, caffeine caused a dose-dependent increase in locomotor activity. 30 mg/kg caffeine reversed the ethanol-induced reduction of time spent in social interaction, and 60 mg/kg caffeine antagonized the ethanol-induced increase in locomotor activity in both the social behavior and holeboard tests. Caffeine's effects on ethanol-induced behavioral changes are compared with those of other drugs.     

An EEG analysis of drug effects after mild head injury in mice.

An electroencephalographic (EEG) and behavioral model of head injury in unanesthetized, free moving mice has been used to test the effects of TRH and GM1. In our experimental conditions a mechanical head injury capable of inducing loss of righting reflex for 2 to 60 sec, also induces a consistent decrease of the total power of the spectrum of EEG and a decrease of the power of fast beta band (20-40 Hz) for at least 120 min. TRH, injected after trauma in dose of 10 mg/kg, caused improvement of EEG total power of the spectrum. GM1 in high (30 mg/kg) but not in low dose (5 mg/kg) caused more rapid restoration of both the total power and fast as well as slow beta band power. These results suggest that GM1 has favorable effects on post-concussive neurophysiological symptoms in head injured animals.     

Hemodynamic effects of procainamide and quinidine and the influence of beta-blockade before and after experimental myocardial infarction.

The use of antiarrhythmie drugs in combination has been limited because of possible side effects secondary to myocardial depression in the acute myocardial infarction patient. Therefore, we investigated in intact dogs (group I) the hemodynamic interaction of propranolol plus procainamide (subgroup A) or quinidine (subgroup B) and in dogs after experimental myocardial infarction produced by coronary artery ligation (group II). Infusion of procainamide (30 mg/kg over 5 min) in animals of group IA produced a significant (P less than 0.05) decrease of 30% in mean aortic pressure, a decrease of 40% in left ventricular dp/dt and 29% in cardiac output. When procainamide was reinfuse after propranolol (1 mg/kg), its hemodynamic effects were not significantly different from those observed before propranolol in both groups IA and IIA. Infusion of quinidine (10 mg/kg over 5 min) in animals of group IB (intact dogs) also produced significant decreases of 24% in mean aortic pressure and 38% in dp/dt while cardiac output was unchanged. However, these hemodynamic changes were seen only after beta-blockade and were significantly different from those obtained before propranolol, where heart rate increased by 14%, dp/dt by 30%, and cardiac output by 35%. These changes occurred despite a similar reduction in mean aortic pressure. This drug combination produced similar response in animals after coronary artery ligation (group IIB). In conclusion, we feel that the administration of propranolol does not prevent the depressive circulatory effects of procainamide. The combined use of quinidine and propranolol also has a negative circulatory effect although not as marked as the effects observed after procainamide with propranolol.     

Total synthesis of zervamicin IIB and its deuterium-labelled analogues

For the first time the total synthesis of the peptaibol antibiotic zervamicin IIB is described. Synthesis of this peptaibol was achieved by the Fmoc/tert-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues BOP/DMAP activation was applied. The Fmoc group was removed by reaction with 0.1 M NaOH in dioxane/methanol/water (30/9/1, v/v/v). Peptide fragments were coupled by means of a new coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB and two analogues specifically deuterium-labelled at different positions of the glutamine-11 residue have been synthesized in 40% overall yield based on the isotopically labelled amino acid and with 98±2% of isotope enrichment. FAB mass spectroscopy, 600 MHz ¹H-NMR spectroscopy and high-performance liquid chromatography provided convincing evidence that the synthetic products, zervamicin IIB and its deuterium-labelled analogues, fully correspond to the naturally occurring zervamicin IIB. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Psychotropic effects of glutamic acid diethyl ester in mice

In 1 hour after intraperitoneal injection glutamic acid diethyl ester (GED) in doses 200 and 500 mg/kg decreased locomotor activity and exploratory patterns of mice in "open field" test. GED in doses 100 and 200 mg/kg diminished the immobilization period of animals in forced swimming test, that proves the reversal interaction of glutamatergic and catecholaminergic systems in CNS. Glutamate receptors antagonist--GED in doses 100-200 mg/kg disrupted passive avoidance reaction at 30 min before acquisition and retrieval, therefore glutamate receptors are involved into fixation and retrieval of memory engram.     

Evaluation of psychopharmacological effects of Clerodendrum phlomidis Linn. extract

The methanolic extract of Clerodendrum phlomidis Linn. (MECP) leaves was evaluated for its psychopharmacological activities in several experimental models using Swiss albino mice and Wistar albino rats. The MECP was found to cause significant reduction in spontaneous activity, and decreases in exploratory behavioral profiles by the Y-maze and head dip test. It also showed reduction in muscle relaxant activity by rotarod, 30° inclined screen and traction tests, as well as significantly potentiated the phenobarbitone sodium-induced sleeping time in the doses examined (200, 400 and 600 mg/kg body wt.). All the results were compared with respective controls for the evaluation of significance.     

Effect of antiepileptic drugs valproic acid, carbamazepine and ethosuccimide on exploratory behaviour in mice

Valproic acid in 100, 200 and 400 mg/kg doses produced a significant dose dependent decrease in exploratory behaviour, tested as number of head dips on the hole board. In the open field test, control mice entered less number of peripheral squares and more number of central squares on day 4 as compared to day 1 of the test. In the lower doses (100 and 200 mg/kg) valproic acid increased central square entries on day 1 with significant decrease by all doses on subsequent days indicating inhibition of exploratory behaviour. However, in peripheral square entry they followed the same pattern as control mice. Neither carbamazepine (10 and 20 mg/kg) nor ethosuccimide (100 and 200 mg/kg) affected exploratory behaviour in either the hole board or open field test.     

Antioxidant Effects of Nerolidol in Mice Hippocampus After Open Field Test

The aim of this study was to evaluate the neuroprotective effects of nerolidol in mice hippocampus against oxidative stress in neuronal cells compared to ascorbic acid (positive control) as well as evaluated the nerolidol sedative effects by open field test compared to diazepam (positive control). Thirty minutes prior to behavioral observation on open field test, mice were intraperitoneally treated with vehicle, nerolidol (25, 50 and 75 mg/kg), diazepam (1 mg/kg) or ascorbic acid (250 mg/kg). To clarify the action mechanism of of nerolidol on oxidative stress in animals subjected to the open field test, Western blot analysis of Mn-superoxide dismutase and catalase in mice hippocampus were performed. In nerolidol group, there was a significant decrease in lipid peroxidation and nitrite levels when compared to negative control (vehicle). However, a significant increase was observed in superoxide dismutase and catalase activities in this group when compared to the other groups. Vehicle, diazepam, ascorbic acid and nerolidol groups did not affected Mn-superoxide dismutase, catalase mRNA or protein levels. Our findings strongly support the hypothesis that oxidative stress occurs in hippocampus. Nerolidol showed sedative effects in animals subjected to the open field test. Oxidative process plays a crucial role on neuronal pathological consequence, and implies that antioxidant effects could be achieved using this sesquiterpene.     

Clycerolphosphate oxidase and succinate dehydrogenase activities in IIA and IIB fibres of mouse and rabbit tibialis anterior muscles

Summary Quantitative microphotometric measurements of two mitochondrial flavoproteins, glycerolphosphate oxidase (GP-OX) and succinate dehydrogenase (SDH), were performed on serial sections of mouse and rabbit tibialis anterior (TA) muscles in order to study the distribution of these two enzymes and their activity ratios in IIA and IIB fibres. The measurements showed a large scatter of the two enzyme activities in these two myosin-based fibre types. In rabbit TA, IIA and IIB fibres have similar GP-OX activities, whereas generally IIA fibres have higher SDH activities than IIB fibres. An inverse distribution of the two enzymes exists in mouse muscle. Generally, IIA fibres of mouse TA display low SDH and IIB fibres high SDH activities. The mean activity of GP-OX is slightly higher in IIA than in IIB fibres of mouse TA. Since measurements of both enzymes were taken in the same fibres, the ratio of their activities in each fibre could be evaluated. The SDH/ GP-OX activity ratios vary significantly between the two fibre populations both in rabbit and in mouse. The ratio is high in IIA and low in IIB fibres of rabbit TA, whereas it is low in IIA and high in IIB fibres of mouse TA.     

Glycerolphosphate oxidase and succinate dehydrogenase activities in IIA and IIB fibres of mouse and rabbit tibialis anterior muscles

Quantitative microphotometric measurements of two mitochondrial flavoproteins, glycerolphosphate oxidase (GP-OX) and succinate dehydrogenase (SDH), were performed on serial sections of mouse and rabbit tibialis anterior (TA) muscles in order to study the distribution of these two enzymes and their activity ratios in IIA and IIB fibres. The measurements showed a large scatter of the two enzyme activities in these two myosin-based fibre types. In rabbit TA, IIA and IIB fibres have similar GP-OX activities, whereas generally IIA fibres have higher SDH activities than IIB fibres. An inverse distribution of the two enzymes exists in mouse muscle. Generally, IIA fibres of mouse TA display low SDH and IIB fibres high SDH activities. The mean activity of GP-OX is slightly higher in IIA than in IIB fibres of mouse TA. Since measurements of both enzymes were taken in the same fibres, the ratio of their activities in each fibre could be evaluated. The SDH/GP-OX activity ratios vary significantly between the two fibre populations both in rabbit and in mouse. The ratio is high in IIA and low in IIB fibres of rabbit TA, whereas it is low in IIA and high in IIB fibres of mouse TA.     

Neuroprotective effects of tanshinones in transient focal cerebral ischemia in mice.

Tanshinones are the major lipid soluble pharmacological constituents of Danshen, the dried roots of Salvia miltiorrhiza Bunge (Labiatae), a well known traditional Chinese medicine used for the treatment of cerebrovascular diseases including stroke. Potential neuroprotective effects of tanshinones IIA (TsIIA) and IIB (TsIIB) were examined in adult mice subjected to transient focal cerebral ischemia caused by middle cerebral artery occlusion (MCAo). Our results revealed that TsIIA (16 mg/kg) readily penetrated the blood brain barrier reaching a peak concentration of 0.41 nmol/g brain wet weight 60 minutes after intraperitoneal injection and decreased slowly over several hours. Twenty-four hours after middle cerebral artery occlusion, brain infarct volume was reduced by 30% and 37% following treatment with TsIIA and TsIIB, respectively. The reduction in brain infarct volume was accompanied by a significant decrease in the observed neurological deficit. Tanshinones or other structurally related compounds may have potential for further development as neuroprotective drugs.     

Compartive behavioral effects of dibutyryl cyclic AMP and prostaglandin E1 in mice.

Three behavioral tests, spontaneous locomotor activity (SLMA), exploratory behavior (EB) and rotarod performance (RP), a measure of neuromuscular coordination, were used to stuey the interaction of PGE1 (1 mg/kg i.p., 10 min. pretreatment) with DBcAMP (25 mg/kg i.p., 25 min. pretreatment) in mice. A dose-response relationship of PGE1 (0.01-5.0 mg/kg) to SLMA was determined, with a significant decrease in SLMA produced by a dose of 0.1 mg/kg. decreases in SLMA were produced by PGE1 (79%), DBcAMP (41%) and DBcAMP-PGE1 combination (71%). Similar decreases in EB were observed. Although no significant difference between controls and DBcAMP was observed in RP, 52% of mice tested were RP failures following PGE1 and a 100% failure rate was induced by the combination. Mice were treated with a second injection of DBcAMP or PGE1 or the combination 24 hr following the first injection. Behavioral activity of these mice was observed 25 min (DBcAMP) or 10 min (PGE1) after the second dose was administered. A second injection of DBcAMP failed to decrease SLMA and EB from controls; moreover, SLMA began to return towards control levels as early as 2 hr between injections. The second injection of PGE1 or DBcAMP+PGE1 produced the same behavior as that produced by the first injection. On the basis of these results, the relationship of cyclic nucleotides and PGs to behavioral activity is discussed.     

Self-aggregation properties of spin-labeled zervamicin IIA as studied by PELDOR spectroscopy

In this article, the pulsed double electron-electron resonance in electron spin-echo (PELDOR) technique is applied to study the self-aggregation of spin-labeled zervamicin IIA, a hexadecapeptide antibiotic of fungal origin, which is known to form ion channels in a phospholipid double layer. Measurements of the ion channel forming properties and the antibiotic activity of the analog indicate that replacement of the C-terminal phenylalaninol by the amino-2,2,6,6-tetramethylpiperidinyloxy (TEMPO) residue does not influence the biophysical and biological properties. The dipole-dipole interaction between the spin labels of the fully biologically active peptide analog was studied in frozen (77 K) glassy solutions in different ratios of toluene-methanol. The spin-labeled zervamicin IIA molecules were shown to form aggregates. An average distance between the spin labels in the aggregates was estimated to be in the range of 25-35 A (depending on the solvent composition), indicating that the amphiphilic helical peptide molecules are oriented in an antiparallel fashion. Increasing of methanol content in the solution results in a loosening of the aggregate structure. It was shown that the fraction of aggregated zervamicin IIA molecules is less than 44-67% depending on the solvent composition. The general usefulness of the method to obtain structural long-range information in a range of several tens of angstroms is demonstrated by comparison with the peptide cluster of trichogin GA IV.     

The participation of CCK-8-ergic mechanisms in the regulation of emotional behavior in rodents

Effects of CCK-8 receptor agonists caerulein and pentagastrin and CCK-8 receptor antagonist proglumide on exploratory and locomotor activity of mice and rats were studied. Systemic administration of caerulein (500 ng/kg 1 mcg/kg) decreased significantly the exploratory activity of mice in elevated plus-maze. This anxiogenic-like action of caerulein was attenuated by acute pretreatment with proglumide (1 and 15 mg/kg) but not with diazepam (up to 0.75 mg/kg). Proglumide slightly increased the exploratory activity of rats in plus-maze; on the other hand, caerulein and pentagastrin potently decreased the measures of exploration in this test. Caerulein (10-100 mcg/kg) and proglumide (1 and 15 mg/kg) inhibited 3H-pentagastrin binding in mice brain in in vivo experiments. The data obtained indicate that CCK-8-ergic mechanisms in brain play an important role in the generation of anxiety states in rodents.     

Possible GABAergic Modulation in the Protective Effect of Zolpidem in Acute Hypoxic Stress-induced Behavior Alterations and Oxidative Damage

Hypoxia is an environmental stressor that is known to elicit alterations in both the autonomic nervous system and endocrine functions. The free radical or oxidative stress theory holds that oxidative reactions are mainly underlying neurodegenerative disorders. In fact among complex metabolic reactions occurring during hypoxia, many could be related to the formation of oxygen derived free radicals, causing a wide spectrum of cell damage. In present study, we investigated possible involvement of GABAergic mechanism in the protective effect of zolpidem against acute hypoxia-induced behavioral modification and biochemical alterations in mice. Mice were subjected to acute hypoxic stress for a period of 2 h. Acute hypoxic stress for 2 h caused significant impairment in locomotor activity, anxiety-like behavior, and antinocioceptive effect in mice. Biochemical analysis revealed a significant increased malondialdehyde, nitrite concentrations and depleted reduced glutathione and catalase levels. Pretreatment with zolpidem (5 and 10 mg/kg, i.p.) significantly improved locomotor activity, anti-anxiety effect, reduced tail flick latency and attenuated oxidative damage (reduced malondialdehyde, nitrite concentration, and restoration of reduced glutathione and catalase levels) as compared to stressed control (hypoxia) (P < 0.05). Besides, protective effect of zolpidem (5 mg/kg) was blocked significantly by picrotoxin (1.0 mg/kg) or flumazenil (2 mg/kg) and potentiated by muscimol (0.05 mg/kg) in hypoxic animals (P < 0.05). These effects were significant as compared to zolpidem (5 mg/kg) per se (P < 0.05). Present study suggest that the possible involvement of GABAergic modulation in the protective effect of zolpidem against hypoxic stress.     

Characterization of two poly(A) polymerases from cultured hamster fibroblasts.

The enzymatic and physiochemical properties of poly(A) polymerases IIA and IIB from cultured hamster fibroblasts were investigated. The enzymes show an absolute requirement for Mn2+ as the divalent ion. Although Mg2+ alone is inactive, maximum activity is observed in the presence of both Mn2+ and Mg2+. An optimal pH of approx. 8 is found for polymerases IIA and IIB. The enzymes, however, differ somewhat in the pH curves as well as in the Mn2+ and Mg2+ concentration curves. Poly(A) polymerases IIA and IIB have an isoelectric point of about 6 and a sedimentation coefficient of 3.5--4 S. The molecular weights, obtained by gel filtration chromatography, are 145 000 and 155 000 for enzymes IIA and IIB, respectively. Poly(A) polymerases IIA and IIB can utilize a variety of natural and synthetic RNAs as well as DNA as primers. Poly(A) polymerase IIA is saturated at much lower concentrations of primer than enzyme IIB. On the other hand, the chain length of the product synthesized by polymerase IIA is independent of the primer concentration, whereas, with polymerase IIB, the length of the product decreases when the concentration of RNA is increased.     

Behavioral effects of acute and chronic triazolam treatments in albino rats

Previous behavioral studies on triazolam (TZ), which are small in number, could only speculate about tolerance to the anxiolytic effect of TZ, as the experiments did not cover sufficient time (of 4 to 7 days) for tolerance to develop. Therefore longer time for chronic TZ administration is used. We investigated the effects of TZ on motor activity and exploratory behavior using plus maze and open field. Three experiments were conducted. In the first, five groups of rats were acutely treated with different doses of TZ (0.25 mg/kg-4.0 mg/kg). In the second set of experiments, rats were treated chronically with a single daily dose of TZ (started with 0.25 mg/kg and increased by time to 1.0 mg/kg) for 5 weeks (representing clinical use). In the third, rats were treated chronically with three daily doses of TZ (started with 0.25 mg/kg and increased by time to 0.5 mg/kg) for 20 days (mimicking drug abuse). Acute TZ administration produced dose dependent anxiolytic effects and a decrease in motor activity with higher doses. Chronically treated rats, either once daily or three times daily doses, showed tolerance to both anxiolytic and sedative effects of TZ. It may be concluded that tolerance to the anxiolytic and sedative effects of TZ would develop after chronic administration either with clinical use or its abuse.     

Effect of GABA receptor agonist phenibut on behavior and respiration of rabbits in the negative emotional situation

The influence of systemic injection of GABA-receptor agonist--phenibut (40 mg/kg, s/c) on open field behavior, behavioral reactivity and changes in respiratory parameters after exposure of negative emotional stimuli was studied in three rabbit groups differentiated by locomotor activity in open field (active, passive and medium-active animals). The injection of phenibut results in decrease of rabbits horizontal locomotor activity and some components of research behavior in open field and also decrease of behavioral reactivity on emotional stimuli. At the same time the probability of both an active orienting exploratory or defensive reactions and passive reactions (freezing) were decreased. The influence of phenibut depended on typological features of rabbits: the most potent effect occurred upon behavior of active rabbits, less on passive animals and practically none on medium-active rabbits. The phenibut injection results in increase of duration of inhalation during exposure to emotional stimuli, whereas it decreased in normal.