Regulatory Effects of 5-Day Oral and Intraperitoneal Administration of a Thienopyrimidine Derivative on the Thyroid Status in Rats.

In experiments on rats, we studied the effect of 5-day intraperitoneal (15 mg/kg/day) and oral (40 mg/kg/day) administration of compound TPY3m, a stimulator of the production of thyroid hormones by the thyroid gland developed by us, on the blood levels of thyroxine, triiodothyronine, and thyroid-stimulating hormone and on morphology of the thyroid gland. With both routes of administration, TPY3m caused a sustained moderate elevation of thyroid hormones, mainly thyroxine, with little effect on the level of thyroid-stimulating hormone. TPY3m did not reduce the stimulating effect of thyroliberin on the levels of thyroid hormones and had no damaging effect on the thyroid gland. During long-term administration, compound TPY3m stimulates the production of thyroid hormones without weakening the activity of the thyroid axis. Thus, TPY3m is a prototype of drugs for correcting thyroid hormone deficiency.

Comparative Study of the Restoring Effect of Metformin, Gonadotropin, and Allosteric Agonist of Luteinizing Hormone Receptor on Spermatogenesis in Male Rats with Streptozotocin-Induced Type 2 Diabetes Mellitus.

We compared the effectiveness of human chorionic gonadotropin (hCG; 5 days, 20 IU/rat/day), allosteric luteinizing hormone receptor agonist TP04 (5 days, 20 mg/kg/day), and metformin (28 days, 120 mg/kg/day) in restoring spermatogenesis in male rats with type 2 diabetes mellitus. hCG and TP04 increased the levels of testosterone and expression of the steroidogenic protein StAR, the number of spermatogenic cells, thickness of the seminal epithelium, and the number and motility of mature sperm that were reduced in diabetic rats, though they did not reduce the number of defective spermatozoa. Metformin had a weak effect on steroidogenesis, but was not inferior to luteinizing hormone receptor agonist by its restorative effect on spermatogenesis and also reduced the number of defective forms of spermatozoa. Thus, the spermatogenesis-restoring effect of metformin and luteinizing hormone receptor agonist in type 2 diabetes mellitus are comparable, despite different mechanisms of action.

The Effect of Low-Molecular-Weight Allosteric Agonist of Luteinizing Hormone Receptor on Functional State of the Testes in Aging and Diabetic Rats.

Human chorionic gonadotropin that is widely used for improving spermatogenesis. The effect of chorionic gonadotropin is mediated through luteinizing hormone receptor. Treatment with gonadotropin is associated with undesirable effects due to hyperactivation of testosterone production and luteinizing hormone receptor desensitization. A promising alternative could be low-molecular-weight agonists of luteinizing hormone receptors, but their effects on spermatogenesis have not been investigated. Here we analyzed the effect of a thieno[2,3-d]pyrimidines (TP), 4-((3-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno [2,3-d]pyrimidine-4-yl) phenyl)carbamoyl)pyridine 1-oxide (TP22), an allosteric agonist of luteinizing hormone receptors, on the seminiferous tubules and spermatogenic cells in 4- and 18-month-old male rats and in animals with diabetes mellitus. TP22 and gonadotropin were administered in daily doses of 15 mg/kg and 20 U/rat for 5 days. Blood testosterone level, morphology of the seminiferous tubules, and the number of germ cells in them were estimated. Being comparable by the efficiency to gonadotropin, TP22 increased the testosterone level in all the studied groups of rats and restored epithelium thickness in the seminiferous tubules and the number of spermatogonia and pachytenic spermatocytes that are reduced in aging and diabetes, but, unlike gonadotropin, did not suppress the expression of luteinizing hormone receptor. The efficacy of TP22 as a stimulator of testicular spermatogenesis has been demonstrated both under normal conditions and in age-related and diabetes-associated reproductive dysfunctions.

[The effect of high-dose metformin on the metabolic parameters and functional state of the liver of agouti-mice with the melanocortin obesity.]

In recent years, the effectiveness of high-dose metformin (MF) to treat the endocrine and oncological diseases has been shown. However, the use of high-dose MF may be associated with the lactic acidosis and the liver dysfunctions. The aim of the work was to study the effect of long-term (10 days) oral administration of a relatively high dose of MF (600 mg/kg per day) into yellow C57Bl/6J (Ay/a) Agouti line mice with the melanocortin type obesity on the liver function, which was evaluated by the morphology of hepatocytes and the severity of steatosis, the expression of the inflammatory and apoptotic factors of and the activity of aminotransferases, as well as on the plasma lactate level in the animals. In Agouti line mice, MF (600 mg/kg per day) caused a decrease in the body and fat weight, led to the reduced hyperglycemia, hyperinsulinemia and hyperleptinemia, and restored the sensitivity to glucose and insulin. At the same time, in the liver of Agouti line mice treated with MF, the small-drop and large-drop fatty degeneration and the hydropic degeneration were attenuated, and the expression of pro-inflammatory IL-1ß and pro-apoptotic Bax protein and the Bax/Bcl-2 ratio did not differ from the control C57Bl/6J (a/a) mice. In the blood of Agouti line mice treated with MF, the activity of alanine aminotransferase was normalized, and the lactate levels was increased, but to a moderate degree. It was concluded that the high-dose MF did not induce the lactic acidosis in Agouti line mice, and at the same time it restored the liver functions impaired in the melanocortin obesity. This allows us to consider the use of the high doses of MF as one of the possible ways to treat obesity and metabolic disorders that are associated with the hepatic steatosis.

Metabolic parameters and functional state of hypothalamic signaling systems in AY/a mice with genetic predisposition to obesity and the effect of metformin.

The metabolic parameters and functional state of hypothalamic systems in mice with the Yellow mutation in the Agouti locus and with obesity of the melanocortin type and the effect of metformin (MF) treatment (9 days, 200 mg/kg/day) were studied. The MF treatment led to decreased body weight and to normalization of glucose tolerance in mice. In the hypothalamus, MF restored the decreased activity of Akt kinase, the main component of leptin pathway, and normalized the increased expression of subtype 1B serotonin receptor. The obtained data suggest the efficiency of MF to treat obesity of the melanocortin type.

Localization of the dopamine receptors of types 1 and 2 on the bodies of POMC-expressing neurons of the arcuate nucleus of the hypothalamus in mice and rats.

Using immunofluorescent techniques, we have demonstrated the presence of two main types of dopamine receptors, D1 and D2, on the bodies of neurons of the arcuate nucleus of the hypothalamus expressing the precursor of peptides of the melanocortin family proopiomelanocortin in C57Bl/6J mice and Wistar rats. These data show close functional relationship between the dopamine and melanocortin systems of the brain and involvement of dopamine in the control of synthesis and secretion of peptides of the melanocortin family.

[Analysis of development of morphofunctional interconnections between CART- and AGRP-ergic neurons with dopaminergic structures in ontogenesis of mammals].

There are obtained data indicating that morphogenesis and, probably, formation of structurally functional interaction of the CART- and dopaminergic neurons occur as early as during embryonic development. Meanwhile development of the AGRergic system and formation of its structural and functional communications are observed in the course of postnatal development of rat. Analysis of literature and re- sults of our own investigation show that differentiation of dopaminergic neurons in hypothalamus and midbrain occurs on the background of development of CARTergic neurons and in the absence of AGRP. Taking into account our data on activating action of the CART-peptide and inhibitory effect of AGRP on dopaminergic brain neurons, we believe that the leading component at formation of functional interactions of these systems is the later maturing AGRPergic system.

[Morphfunctional interaction of cart-peptide and dopaminergic brain neurons].

In Wistar rats, after 6 h of sleep deprivation and subsequent 2 h postdeprivation sleep, we found significant changes in optical density of CART-peptide in neurons of hypothalamic nucleus accumbens and nucleus arcuatus as well as in processes coming into substantia nigra from nucleus accumbens. The obtained data revealed unidirectional changes of optical density of tyrosine hydroxylase: a decrease after sleep deprivation (p < 0.05) and, on the contrary, an increase on the background of postdeprivation sleep (p < 0.05). Confocal laser microscopy showed morphological connections of CART and dopaminergic neurons and possible colocalization of these both substances in the same neuron at the postdeprivation sleep. In experiments in vitro, after 1 h of incubation of survived brain slices from the substantia nigra area in the medium with CART-peptide there was revealed a rise of optical density of tyrosine hydroxylase in the substantia nigra compact part by 55 % (p < 0.05). The obtained data indicate an activating effect of CART-peptide on brain dopaminergic neurons and its as a modulator of their functional activity.

[Biodistribution of the recombinant heat shock protein rhHsp70 in intracranial C6 glioma models in Wistar rats and subcutaneos B16/F10 melanoma in C57BL/6 mice].

For the first time, the biodistribution of recombinant heat shock protein in rhHsp70 rats with grafted intracranial C6 glioma was evaluated. It was assessed using the fluorescent antibody accumulation chaperone rhHsp70 conjugated with fluorochrome Alexa Fluor 555 in tumor cells by intratumoral or intravenous administration. Assessment of the distribution and accumulation of labeled protein was carried out on the model of subcutaneous B16/F10 melanoma in C57BL/6 mice with the use of single-photon emission computer tomography. After 60 minutes after intravenous administration rhHsp70-I123 (20 MBq, 5 mg chaperone) accumulation of the drug mainly in the liver and tumor tissue was showed. The coefficient of the differential accumulation of the labeled protein KDN(tumor/background) was 3.14. It was turned out that comparing the level of fixation of rhHsp70-I123 in the liver and the tumor KDN(tumor/ liver) = 0.76. After 24 hours from the time of injection of rhHsp70-I123 it was observed increase the level of fixation of the labeled protein in the liver and melanoma: KDN(tumor/background) = 3.43; KDN(tumor/liver = 0.78.

[Participation of Agouti related peptide in machanisms of wakefulness-sleep cycle regulation].

Agouti-related protein (AGRP) is expresses in hypothalamic neurons in human and animals. Immunohistochemical study in rats Wistar rats demonstrates significant changes AGRP optical density in the neurons of arcuate hypothalamic nucleus as well as in processes in the hypothalamus and nucleus accumbens after the 6 hours of sleep deprivation (increase) and after 2 hours of post-deprivative sleep (decrease). Comparison of these results with earlier obtained shows the opposite trend changes in AGRP optical density and speed limiting enzyme of dopamine synthesis-tyrosine hydroxylase in the hypothalamus and in striatonigral system. The increase of AGRP was accompanied by a decrease of tyrosine hydroxylase and the decrease of AGRP, apposite, it increases. The obtained data demonstrate the role ofAGRP as a modulator of the functional activity of the dopaminergic brain neurons. The problem of the relationship of various functions of organism (food behavior, sleep, stress) is discusses by their participation in the regulation of the same neurotransmitter systems.

[Effect of retinol on interaction of the protein period1, oxytocin, and GABA at the prenatal period of formation of the circadian clock-mechanism in rats].

Clock-gene proteins are expressed in mammals in neurons of the hypothalamus suprachiasmatic nucleus and of other CNS structures, in muscles, viscercal organs, and vessels, and form circadian rhythms of many functions. Little is known about the factors of formation of the circadian mechanism at the prenatal period. In rats the E20 stage is characterized by a high level of oxytocin and selective expression of the first protein of the clock-genes PER1. The foal of the present study was to check the suggestion about the positive feedback between PER1 and oxytocin at the prenatal period as well as to elucidate a possible role of PER1 in regulation of interactions between oxytocin and GABA at the period of formation of the cerebral circadian mechanism of clock-genes. With aid of western-blotting, we analyzed the nuclear and cytoplasmic fractions from anterior hypothalamus homogenate in pregnant females and embryos of rats (E20). The retinol metabolites through their nuclear receptor RORalpha are known to be bound to promoters of genes of oxytocin and per 1. Next day after administration of retinol to the females, a rise of PER1 content was noted in their cytoplasm, whereas in their embryos the PER1 content was elevated in the nucleus. In the embryo cytoplasm there was a significant rise of production of oxytocin receptors and a decrease of the level of enzymes of GABA synthesis (glutamate decarboxylases 67 and 65). The results indicate the oxytocin- and retinol-dependent increase of the PER1 expression and the subsequent change of ratio of efficiency of oxytocin and GABA at the prenatal stage of formation of the circadian clock-mechanism of the rat embryo anterior hypothalamus.

[Chaperone therapy in the rat model of intracranial glioblastoma].

Molecular chaperons can effectively activate innate and adaptive anti-tumor immune response. In the model of intracranial glioma C6 in Wistar rats we assessed immunomodulatory activity of recombinant protein Hsp70 in case of local, intratumoral injection. Single intratumoral infusion of chaperone had led to dramatic delay in tumor volume growth (on MRI of rat brain), which was accompanied by increase in survival rates. Incubation of rat spleenocytes with C6 cells elevated the levels of INF-gamma, that shows an immunologically specific T-cell response. With immunohistochemical assay we observed a marked infiltration of the tumor by T-lymphocytes and NK-cells. Thus, purified Hsp70 can efficiently induce innate and adaptive anti-tumor response and could be used as adjuvant in treatment of malignant brain tumors of central nervous system.

[Participation of vertebrate forebrain activating systems in organization of the wakefulness--sleep cycle].

The article considers mechanisms of diencephalic-telencephlic interactions in regulation of the wakefulness-sleep cycle in various classes of vertebrates. In such interactions a special role is played by the dopaminergic systems that perform neurosecretory function at the level of diencephalon and neurotransmitter function at the level of telencephalon. Concepts of A. I. Karamyan and A. L. Polenov about the stage pattern of development of CNS and neurosecretory systems are presented as well as the interconnection of dopaminergic and glutamatergic neurotransmitter system in the mammalian neostriatum in the wakefulness-sleep cycle is considered. Comparison of dynamics of expression of the dopamine metabotropic receptors and of the glutamate ionotropic receptors in neostriatum showed unidirectional changes of D1 and AMPA on the background of the 6-h sleep deprivation as well as of D2 and NMDA on the background of postdeprivative sleep. The corticofugal direction of glutamate impulsation and its relatively fast actions allow admitting its triggering action on generation of the sleep-inducing processes in the underlying brain parts.

[Immunohistochemical investigation of Bcl-2 and p53 levels in rat hypothalamus after sleep deprivation].

On Wistar rats in view of electrophysiological parameters after sleep deprivation (SD; awake by gentle handling method) and the subsequent postdeprivative sleep (PDS) immunohistochemical investigation of Bcl-2 and p53 peptides optical density levels in neurons of paraventricular (PVN), supraoptic (SON) and median (MnPN) hypothalamus nuclei was carried out. The Bcl-2 was increased in all nuclei both after SD and PDS. The level of p53 was increased in PVN and SON after SD and PDS, but in MnPN only on PDS. Any morphological attributes of apoptosis in the nuclei was not revealed. Obtained data testify an active role of p53 and Bcl-2 peptides in regulation of neuronal activity in hypothalamus at change of a cycle wakefulness-sleep.

Immunohistochemical analysis of active caspase-3 expression in structures of neonatal brain.

The hippocampal fields of neonatal rats differ by the level of active caspase-3: dentate gyrus >CA3>CA1>CA2. In the dentate gyrus it was 70% of its maximum value in the cortex, while in CA2 it corresponded to the minimum level in the brain stem. Taking into account the role of caspase-3 in apoptosis, these differences can indicate different intensity of programmed cell death in different fields of the forming hippocampus.

The dopaminergic nigrostriatal system in sleep deprivation in cats.

The dynamics of changes in electrophysiological measures of the sleep-waking cycle were analyzed in Wistar rats after 6 h of sleep deprivation by gentle waking and subsequent 9-h post-deprivation sleep. A delayed sleep "overshoot" reaction was observed 2.5-3 h after sleep deprivation, as a moderate increase in the proportions of slow-wave and fast-wave sleep in the sleep-waking cycle. Immunohistochemical studies were performed in relation to changes in the sleep-waking cycle, with the aim of identifying changes in the quantities of immunoreactive dopamine D1 and D2 receptor material and tyrosine hydroxylase, the key enzyme in dopamine synthesis in the nigrostriatal system. In conditions of sleep deprivation, the caudate nucleus showed increases in the quantities of dopamine D1 and D2 receptor material, while there was a simultaneous decrease in the amount of immunoreactive material in the substantia nigra. Post-deprivation sleep was accompanied by decreases in the quantities of immunoreactive D1 receptor material and increases in D2 receptor material in the caudate nucleus, with an increase in the quantity of immunoreactive tyrosine hydroxylase in the substantia nigra. These data provide evidence of the active role of the dopaminergic nigrostriatal system which, along with other CNS transmitter systems, supports telencephalic-diencephalic interactions, in the sleep-waking cycle.

[Diencephalo-telencephalic changes of tyrosine hydroxylase in rats and common frogs (Rana temporaria) after sleep deprivation].

Based on sleep deprivation-produced changes of electrographic parameters of the wakefulness--sleep cycle (WSC) in rats and common frogs, dynamics of activity of tyrosine hydroxylase, the key enzyme of dopamine synthesis, was studied immunohistochemically in substantia nigra and nigrostriatal pathway in rats and in striatum, paraventricular organ, and extrahypothalamic pathways in frogs. There are revealed changes in dynamics of tyrosine hydroxylase in rats and in common frogs after the 6-h sleep deprivation and after 2 h of postdeprivation sleep. This allows determining the degree of participation of corticostriatal neuroregulatory and hypothalamo-pituitary neurosecretory systems and their role in regulation of WSC. Possible evolutionary peculiarities of morphofunctional differences in homoiothermal and poikilothermal animals are discussed.

[FUNCTIONAL INTERACTION BETWEEN THE DOPAMINE AND MELANOCORTIN SYSTEMS IN THE BRAIN].

The brain melanocortin system plays a key role in the regulation of energy metabolism and feeding behavior and is involved in memory formation, and its abnormalities lead to metabolic disorders and cognitive deficit. The brain dopamine system controls the motor activity, the functions of the nervous, endocrine and cardiovascular systems, and is also involved in the regulation of peripheral metabolism and feeding behavior and in the formation of effect of reward and reinforcement. The fact that a large number of the important physiological functions are controlled by the melanocortin and dopamine systems indicates a close functional relationship between them in different areas of the brain. The basis of this relationship is colocalization of the components of melanocortin and dopamine signaling systems in neurons and interaction between them, which ensures a cross-talk between melanocortin and dopamine pathways in the CNS. The review includes a comprehensive analysis of the current state of the problem concerning the interaction between the melanocortin and dopamine systems of the brain at both the structural and functional levels.

p-Azidophenyl phosphate is a photoactivatable phosphorylating reagent and p-benzoquinone monoimine precursor.

p-Azidophenyl phosphate (I) has been exposed to ultraviolet light (lambda=313 nm) in aqueous solution with or without Lys. Analysis of the photoproducts by means of UV-VIS, IR, (1)H, (13)C and (31)P NMR spectroscopy has revealed that under irradiation of I inorganic phosphate (P(i)) is released, and p-benzoquinone monoimine (II) and p-benzoquinone (III) have appeared. The electrophilic nature of the intermediate results in a high tendency to react with lysine molecules, whereas the reaction with water is less favourable when I is irradiated in the presence of Lys. The product formed in this case is a phosphoramidate whose structure has been tentatively supported by (31)P NMR spectroscopy. These results imply that a p-azidophenyl phosphate is a highly potent aryl nitrene-precursor, which can be transformed easily into p-benzoquinone monoimine and is able to phosphorylate nucleophilic groups of amino acids. This finding is of great importance for the discussions about the chemical nature of protein photomodification products when p-azidophenyl phosphate derivatives are used as modifying reagents.