Analysis of the Intensity of Nitric Oxide Production in Different Parts of the Spinal Cord after Modeling Combined Cerebral and Spinal Injury.

Using the method of electron paramagnetic resonance spectroscopy, we showed that NO production decreases by 60% (p<0.05) in the region located rostral to the spinal cord injury 7 days after combined injury to the brain and spinal cord. At the same time, NO production did not change in the site of spinal cord injury and caudal to the injury. The intensity of NO production in similar parts of the spinal cord in intact animals remained unchanged.

Aromatic sulphonamides of aziridine-2-carboxylic acid derivatives as novel PDIA1 and PDIA3 inhibitors.

In this study, we report a series of newly synthesised sulphonamides of aziridine-2-carboxylic acid (Az-COOH) ester and amide analogues as potent protein disulphide isomerase (PDI, EC 5.3.4.1) inhibitors. The inhibitory activity on PDI was determined against recombinant human PDIA1 and PDIA3 proteins using an insulin reduction assay. These compounds in low micromolar to low nanomolar concentrations showed the effective in vitro inhibitory properties of PDIA1 with weaker effects on PDIA3. Complexes of 15N- and 15N,13C- uniformly labelled recombinant human PDIA1a with two PDIA1 inhibitors were produced and investigated by a protein nuclear magnetic resonance (NMR) spectroscopy. It was found that both C53 and C56 of the PDIA1 enzyme were involved in covalent binding. Finally, in a range of pharmacological studies, we demonstrated that investigated compounds displayed anti-cancer and anti-thrombotic activity. These findings demonstrate that sulphonamides of Az-COOH derivatives are promising candidates for the development of novel anti-cancer and anti-thrombotic agents.

The Effects of Repeated Administration of the Micellar Complex of Methylprednisolone on the Locomotor Activity of a Terrestrial Snails.

We studied the effects of repeated injections of methylprednisolone and its micellar complex with block-copolymer on locomotor activity of a terrestrial snail. It was shown that methylprednisolone solution injected into the hemolymph of the animal produced a direct effect on the muscle system of the animal as soon as 1 h after administration: it slowed down snail locomotion and reduced contractile activity of the foot muscles. The micellar complex of methylprednisolone with block-copolymer prevented this effect during the first 2 days of injection and negatively affected locomotion only in 2 days after injection, the decrease in locomotion in this case was not accompanied by a decrease in contractile activity of the foot muscle.

Changes in Membrane and Threshold Potentials of Command Neurons in Terrestrial Snail during Development of a Conditioned Situational Defensive Reflex.

Changes of the electrical characteristics of command neurons of defensive behavior caused by the development of a conditioned situational defensive reflex were studied experimentally under in vitro conditions on preparations of the nervous system of snails. After learning, the membrane and threshold potentials of command neurons LPa3 and RPa3 significantly decreased and excitability of the studied neurons increased.

Spermine Induces Autophagy in Plants: Possible Role of NO and Reactive Oxygen Species.

This is the first study to show that polyamine spermine, a low-molecular-weight nitrogen-containing compound, can induce autophagy in plants. This process is accompanied by an increased generation of reactive oxygen species and nitric oxide, which play a signal role and are required for triggering autophagy.

Effects of NO Synthase Blocker L-NAME on Functional State of the Neuromotor System during Traumatic Disease of the Spinal Cord.

Functional state of the neuromotor system after administration of a nonspecific NO synthase blocker L-NAME was studied on the model of experimental contusion of the spinal cord. Electron paramagnetic resonance measurements of NO production in the damaged segment of the spinal cord were performed for estimation of the dynamics of intensity of NO production during traumatic disease of the spinal cord and selection of optimal period for L-NAME administration. The status of the neuromotor system was evaluated by stimulation electromyography. Treatment with L-NAME during the acute period of traumatic injury to the spinal cord sharply reduced the intensity of evoked motor responses and more pronounced increase in excitability of peripheral motor structures. The results suggest that NO system is a factor of regulation of the stress-induced and adaptive responses of the body at the early stage of spinal cord injury.

[The role of ATP-dependent potassium channels and nitric oxide system in the neuroprotective effect of preconditioning]. / Rol' ATF-chuvstvitel'nykh kalievykh kanalov i oksida azota v zashchitnom effekte prekonditsionirovaniya mozga.

AIM: To study a role of ATP-dependent potassium channels (K+ATP) in the neuroprotective effect of ischemic (IP) and pharmacological (PP) preconditioning and evaluate the dynamics of blood nitric oxide (NO) metabolites in cerebral ischemia. MATERIAL AND METHODS: A model of ischemic stroke induced by the electrocoagulation of a middle cerebral artery (MCA) branch was used in male rats (n=86). Glibenclamide, a selective inhibitor of ATP-sensitive K+ channels, and diazoxide, a potassium channel activator, were used. IP and PP were performed 24 h before MCA occlusion. Blood concentrations of NO, NO3- and NO2-were measured 5, 24 and 72 h after occlusion. RESULTS: IP decreased a lesion area by 37% (p<0/05) and the preliminary introduction ofglibenclamide levelled the effect of IP. A protective effect of PP was similar to that of IP. A decrease in oxygenated R-conformers of Hb-NO and a reverse increase in non-oxygenated T-conformers as well as NO3- и NO2-were noted 5h after MCA occlusion. In the first 24 h after MCA occlusion, contents of NO3- and NO2- returned to normal values. There were changes in the concentrations of Hb-NO complexes as well, with the predominance of R-conformers and minimal contents of T-conformers. Moreover, the correlations between K+ATP channel blockade and the decrease in serum NO3- and NO2 were found (p<0/03). CONCLUSION: The neuroprotective effect of preconditioning is caused by the activation of K+ATP channels. An analysis of NO metabolite concentrations in the blood of rats with IP suggests that Hb-NO complexes belonging to R-conformers deposit and carry NO in tissues releasing NO accumulated via R→T transfer in conditions of ischemia.

Effects of NO Donors and Inhibitors of NO Synthase and Guanylate Cyclase on the Acquisition of a Conditioned Defense Food Aversion Response in Edible Snails.

Experiments on edible snails revealed that NO synthase blockade with a nonspecific neuronal NO synthase inhibitor L-NAME before defense food aversion conditioning is followed by a decrease in the rate of learning. Exogenous NO donors, sodium nitroprusside and dinitrosyl iron complex, were shown to improve learning. Chronic administration of a specific soluble guanylate cyclase inhibitor ODQ also increased the rate of learning. Our results indicate that NO has a modulatory effect on the formation of a conditioned response in this type of learning.

Sex- and age-related morphological characteristics of European elk (Alces alces) pelves.

There is no data in the literature concerning the frequency of birthing difficulties in the elk cow. This study aims to describe the pelves of elk cows and adult bulls. The measurements of an elk pelvis depend on living conditions rather than age. The dimensions of primiparous pelves are more homogeneous than those of pluriparous cows because heifers have to reach a certain weight prior to successful conception. The elk pelvis has a rather slim shape; the cavum pelvis is deep, oval lengthwise in the median plane, and narrows in the caudal direction. Primiparous and pluriparous cows were significantly different regarding the following measurements: the lengths of the diameter conjugata, the diameter transversa and the caudal transverse and the area of the aperturae pelvis. The pelvic cavity of bulls is longer and narrower in comparison with that of cows, and the areas of the pelvic apertures are smaller. A rapid delivery of the foetus is facilitated by the following factors: a uniform diameter and an appropriate angle of inclination of the bony birth canal, relative mobility of the articulatio sacroiliaca, a concave pelvic surface of the os sacrum, a slow ossification of the symphysis pelvina and the degeneration of the tuberculum pubicum dorsale. In order to differentiate between elks pelves by sex and age, the measurements of the aperturae pelvis and their ratios, the surface area of the symphysis pelvina and the pecten ossis pubis, and the position of the eminentiae iliopubicae and os interischiadicum should be recorded and analysed.

[Influence of hypokinezia of varying duration on the nitric oxide productions dynamics in rat's heart, spinal cord and liver].

By electron paramagnetic resonance method analyzed the production of nitric oxide (NO) in atrias and ventricles of the rat's heart, spinal cord and liver of rats after the contained in conditions of hypokinesia during 30, 60 and 90 days. It was discovered that the regime of hypokinesia leads to increase NO production in all the investigated tissues, and the largest increases were observed in 30-day hypokinesia.

[Aqueous and salt solutions of quinine of low concentrations: self-organization, physicochemical properties and actions on the electrical characteristics of neurons].

Self-organization, the physicochemical properties of aqueous and salt solutions of quinine and the effects of salt quinine solutions in a wide range of concentrations (1 x 10(-22) - 1 x 10(-3) M) on the electrical characteristics of the edible snail's identified neurons were studied. Similar non-monotonic concentration dependencies of physicochemical properties of aqueous and salt quinine solutions at low concentrations are obtained. This allows of predicting the occurrence of biological effects at low concentrations of quinine solutions. Intrinsic (within 5% of the interval) changes in membrane potential, the amplitude and duration of the neuron action potential under the influence of quinine salt solutions at concentrations of quinine of 1 x 10(-20), 1 x 10(-18), 1 x 10(-10) M are found. For these concentrations the extreme values of specific conductivity and pH are shown.

[Electron paramagnetic resonance study of nitric oxide production in rat heart during hypokinesia].

By the method of EPR spectroscopy we studied the intensity of nitric oxide (NO) production after modeling of hypokinesia in rats (limitation of moving activity) through analyses of the amount of NO-containing paramagnetic complexes in tissues of heart and liver. Experimental animals were kept during 60 day protracted hypokinesia. NO amount was assessed by the intensity of EPR spectra of complex (DEDTC)2-Fe(2+)-NO. It was established that after 60 day hypokinesia the 2-3 fold increase of NO quantity occured in heart and liver tissues. The application of a nonspecific inhibitor of NO-synthases, L-NAME, in suspended rats led to a decreased NO quantity up to the value more lower than in control. The obtained results show that the main contribution to the increase in the intensity of NO production during hypokinesia belongs to the fermentative pathway of NO production and the formation of NO by nitrite-reductase activity is not enhanced during hypokinesia.

Nitric oxide level in the rat tissues increases after 30-day hypokinesia: studies by electron paramagnetic resonance (EPR) spectroscopy.

Studies by EPR spectroscopy showed that 30-day exposure of rats to augmenting hypokinesia led to a 3-fold increase in nitric oxide (NO) production in the heart and 2-fold in the liver. These results indicated that long-term hypokinesia stimulated NO synthesis.

A novel combined method of osteosynthesis in treatment of tibial fractures: a comparative study on sheep with application of rod-through-plate fixator and bone plating.

The study compares the efficiency of a new bone fixator combining periostal and intramedullary osteosynthesis to bone plating in treatment of tibial fractures in sheep. Experimental osteotomies were performed in the middle third of the left tibia. Animals were divided into two groups: in one group (four animals) combined osteosynthesis (rod-through-plate fixator, RTP fixator) was applied, and in the other group (three animals) bone plating was used. The experiments lasted for 10 weeks during which fracture union was followed by radiography, and the healing process was studied by blood serum markers reflecting bone turnover and by histological and immunohistochemical investigations. In the RTP fixator group, animals started to load body weight on the operated limbs the next day after the surgery, while in the bone plating group, this happened only on the seventh day. In the RTP fixator group, consolidation of fractures was also faster, as demonstrated by radiographical, histological, and immunohistochemical investigations and in part by blood serum markers for bone formation. It can be concluded that application of RTP fixation is more efficient than plate fixation in the treatment of experimental osteotomies of long bones in sheep.

[Changes of the neuronal membrane excitability as cellular mechanisms of learning and memory].

In the presented review given literature and results of own studies of dynamics of electrical characteristics of neurons, which change are included in processes both an elaboration of learning, and retention of the long-term memory. Literary datas and our results allow to conclusion, that long-term retention of behavioural reactions during learning is accompanied not only by changing efficiency of synaptic transmission, as well as increasing of excitability of command neurons of the defensive reflex. This means, that in the process of learning are involved long-term changes of the characteristics a membrane of certain elements of neuronal network, dependent from the metabolism of the cells. see text). Thou phenomena possible mark as cellular (electrophysiological) correlates of long-term plastic modifications of the behaviour. The analyses of having results demonstrates an important role of membrane characteristics of neurons (their excitability) and parameters an synaptic transmission not only in initial stage of learning, as well as in long-term modifications of the behaviour (long-term memory).

[The effect of serotonin precursor 5-hydroxytryptophan and neurotoxic analogue 5,7-dihydroxytryptamine on defensive conditioning in a snail].

This study is devoted to investigation of the influence of precursor of serotonin 5-hydroxytryptophane (5-HTP) and neurotoxic analogue 5,7-dihydroxytryptamine (5,7-DHT) on defensive conditioning and electrical characteristics of command neurones of defensive behaviour after learning. Snails injected with 5-HTP learned faster as compared to control group injected with physiological solution. After the 5,7-DHT injection, snails failed to form the conditioned reflex. Injection of 5-HTP after the preliminary injection of neurotoxin 5,7-DHT restored the capability of snails for learning. Injections of 5-HTP prevented the effect of 5,7-DHT at the behavioural level, but not at the level of electrical characteristics of the command neurones.

Production of hydrogen peroxide and nitric oxide following introduction of nitrate and nitrite into wheat leaf apoplast.

Infiltration of wheat (Triticum aestivum L.) seedling leaves with excess of nitrate, nitrite, or the NO donor sodium nitroprusside leads to increase both in content of hydroperoxide and activity of peroxidase and decrease in superoxide dismutase (SOD) activity in the leaf apoplast. Polymorphism of extracellular peroxidases and the presence of Cu/Zn-SOD have been shown in apoplast. Using an ESR assay, a considerable increase in the level of NO following infiltration of leaf tissues with nitrite has been demonstrated. These data suggest development of both oxidative and nitrosative stresses in leaves exposed to high levels of nitrate or nitrite. A possible interplay of NO and reactive oxygen species in plant cells is discussed.

Production of recombinant anthrax toxin receptor (ATR/CMG2) fused with human Fc in planta.

Mass vaccination against anthrax with existing vaccines is costly and unsafe due to potential side effects. For post-infection treatment, passive immunotherapy measures are currently available, most based on anthrax protective antigen (PA)-specific therapeutic antibodies. Efficient against wild-type strains, these treatment(s) might fail to protect against infections caused by genetically engineered Bacillus anthracis strains. A recent discovery revealed that the von Willebrand factor A (VWA) domain of human capillary morphogenesis protein 2 (CMG2) is an exceptionally effective anthrax toxin receptor (ATR) proficient in helping to resolve this issue. Here we describe in planta production of chimeric recombinant protein (immunoadhesin) comprised of functional ATR domain fused with the human immunoglobulin Fc fragment (pATR-Fc). The fusion design allowed us to obtain pATR-Fc in plant green tissues in a soluble form making it fairly easy to purify by Protein-A chromatography. Standardized pATR-Fc preparations (purity>90%) were shown to efficiently bind anthrax PA as demonstrated by ELISA and Western blot analysis. Recombinant pATR-Fc was also shown to protect J774A1 macrophage cells against the anthrax toxin. This study confirmed that plant-derived pATR-Fc antibody-like protein is a prospective candidate for anthrax immunotherapy.

Calorimetric low temperature detectors for low-energetic heavy ions and their application in accelerator mass spectrometry.

The energy-sensitive detection of heavy ions with calorimetric low temperature detectors was investigated in the energy range of E=0.1-1 MeV/amu, commonly used for accelerator mass spectrometry (AMS). The detectors used consist of sapphire absorbers and superconducting aluminum transition edge thermometers operated at T approximately 1.5 K. They were irradiated with various ion beams (13C, 197Au, 238U) provided by the VERA tandem accelerator in Vienna, Austria. The relative energy resolution obtained was DeltaE/E=(5-9) x 10(-3), even for the heaviest ions such as 238U. In addition, no evidence for a pulse height defect was observed. This performance allowed for the first time to apply a calorimetric low temperature detector in an AMS experiment. The aim was to precisely determine the isotope ratio of 236U/238U for several samples of natural uranium, 236U being known as a sensitive monitor for neutron fluxes. Replacing a conventionally used detection system at VERA by the calorimetric detector enabled to substantially reduce background from neighboring isotopes and to increase the detection efficiency. Due to the high sensitivity achieved, a value of 236U/238U=6.1 x 10(-12) could be obtained, representing the smallest 236U/238U ratio measured at the time. In addition, we contributed to establishing an improved material standard of 236U/238U, which can be used as a reference for future AMS measurements.

Electrophysiological studies of the effects of chronic administration of caffeine on the formation of a conditioned defensive reflex in the common snail.

Chronic administration of caffeine to common snails increased the rate of formation of a conditioned defensive reflex. When daily caffeine injections were given immediately after the training procedure, the conditioned defensive reflex was acquired more quickly than when caffeine injections were given before the training procedure started. Chronic caffeine administration to both trained and untrained snails led to depolarization changes in the membrane potential and reductions in the threshold potential of defensive behavior command neurons in common snails. Comparative studies showed that addition of caffeine to the solution bathing the mollusk nervous system resulted in decreases in the threshold potential of command neurons in both intact and trained snails; there was, however, no change in the membrane resting potential.