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Effects of some inhibitors of serine/threonine and tyrosine protein phosphatases on the depression and spontaneous recovery of the acetylcholine-induced inward current (ACh-current) in command Helix neurons of defensive behavior at the cellular correlate of habituation were investigated. The following drugs were used: okadaic acid (reduces activity ofphosphatases PP1 and PP2A), endothall (PP2A), cyclosporine A and cypermethrin (PP2B), CCT007093 (PPM1D), dephostatin (blocks tyrosine phosphatases). All used inhibitors modify the depression flow, and endothall reduces spontaneous recovery of ACh-current also. Obtained results indicate that changes in cholinosensitivity of command neurons depend on activity of all investigated protein phosphatases. Mathematical model considers the possibility of different localizations of receptors in a neuron and regularity of transitions between them. This model makes it possible to conclude participation indicated phosphatases in mobility of membrane cholinoreceptors ensuring the ACh-current modification at the cellular correlate of habituations. Comparison of experimental and calculated curves of ACh-current change allows to conclude that the main target of protein phosphatases is the transport system of a neuron--cytoskeleton and motor proteins.

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We investigated the involvement of cytoskeleton motor proteins, myosins, in the molecular mechanism of sensitivity depression to acetylcholine in Helix command neurons of defensive behavior in a cellular analog of habituation. There were analyzed the effects of several drugs disturbing myosin function: ML-7 and MLCK-IP-18--blockers of myosin light chain kinase, blebbistatin--an inhibitor of non-muscle myosin II, Y-27632--inhibitor of kinases ROCK-I and ROCK-II (activate mainly non-muscle myosin II) on the depression of acetylcholine-induced inward current. It was found that ML-7 and MLCK-IP- 18 weakened current depression; blebbistatin and Y-27632 did not change the depression. The results of experimental inhibitory analysis and mathematical modeling of the effects of inhibitors on the number of membrane-bound cholinergic receptors allow to suggest the involvement ofmyosins (excluding non-muscle myosin II) in the transports of acetylcholine receptors (endo- and exocytosis) that are responsible for sensitivity changes in neuron somatic membrane to acetylcholine in a cellular analog of habituation.

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Inhibitor ofadenylate cyclase (SQ 22,536) and inhibitors ofserin/threonine protein kinases A (PKA -Rp-cAMPS), G (PKG - H-Arg-Lys-Arg-Ala-Arg-Lys-Glu-OH), calcium/calmodulin-dependent kinase II (CaMKII - KN-93), p38mitogen-activated (MAPK - PD 169316), and tyrosine protein kinases (genistein), including their Src-family (PP2), weaken the depression of the acetylcholine-induced inward current (ACh-current) in command Helix neurons of defensive behavior under conditions of rhythmical local acetylcholine applications to the soma in the cellular analogue of habituation. Selective inhibitor of protein kinase C (PKC - chelerythrine) does not change the depression of the ACh-current. Mathematical simulation of the influence of the inhibitors applied on a number of membrane-connected acetylcholine receptors made it possible to obtain the design curves consistent with the experimental curves of the ACh-current depression. The experimental data and the results of calculations allowed us to make the following assumptions. The reversible depression of sensitivity to ACh of command Helix neurons of defensive behavior in the cellular correlate of habituation depends on the decrease in the number of membrane-connected ACh receptors as a result of activation of several serine/threonine protein kinases: A, G, CaMKII, p38 MAPK (without the participation of PKC), and tyrosine protein kinases including the family of Src-kinases. The main targets of all protein kinases under study (excluding PKC) in command neurons are the proteins of cytoskeleton (actin microfilaments and microtubules). Phosphorylation of these proteins evokes polymerization and stabilization ofactin microfilaments, stabilization of the main microtubule protein tubulin, a change in the activity of motor proteins responsible for the speed of receptor endocytosis and exocytosis. The PKG action is indirect via the modification of actin-myosin interaction. Protein kinase A, CaMKII, and tyrosine Src-kinase phosphorylate also proteins activating receptor translocation into clathrin-coated membrane invaginations during endocytosis.

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Inhibitors of dynamin-dependent endocytosis (dynamin inhibitory peptide and dynasore) and inhibitors of tubulin (colchicine and vinblastine) decrease the depression of acetylcholine-induced inward current in command neurons of Helix defensive behavior under conditions of rhythmical local applications of acetylcholine to a neuron soma in a cellular model of habituation. Mathematical model which allows for the possibility of different localizations of receptors in a neuron and characteristics of receptor travel makes it possible to analyze the dependence of the current depression on some intracellular processes. We suggest that the cholinosensitivity depression of extrasynaptic membrane zones in command neurons of Helix defensive behavior in the cellular model of habituation under study is associated with dynamin-dependent endocytosis of cholinoreceptors with involvement of cytoskeleton microtubules.

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Thyroid lesions were histologically studied on surgical material obtained in 1992-1996 in 12 regions of Russian Federation and Republic of Belarus. Various forms of thyroid pathology were considered with reference to local ecological situation. An increase of thyroid carcinoma incidence and particularly that of papillary carcinoma was observed in regions with radiation pollution (Chelyabinsk and Bryansk regions). A high degree of correlation exists between the incidence of various forms of thyroid pathology and radiation situation. This is also true for pollution of sewage and the presence of ferrous metallurgy factories. No correlation was found with the pollution of atmosphere.

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Successful application of a double immunostaining of oxyphilic tumors with a specific antimitochondrial antibody and proliferative marker Ki-67 was made for the first time. Comparative study of proliferative activity did not show a statistically significant difference between Hurthle-cell adenomas and carcinomas. However, oxyphilic carcinomas had a statistically significant positive correlation between proliferative index and aggressive course of tumor, tendency to recurrence, high metastatic activity, tumor size, presence of necrosis and capsule penetration.

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Standardization of the morphometric studies of the thyroid is performed. The following table for a semiquantitative analysis of thyroid morphofunctional state is given: 1) epithelial components with characteristics of main cell types and their proliferative activity; 2) stromal components; 3) some pathologic changes (cyst, hemorrhages, etc.). A quantitative study of the thyroid includes: I) on the organ (macroscopic level)--thyroid volume, weight, length, width, height; II) light microscopy: point counting, stereological methods. Main parameters: 1) epithelial components (18 indices are suggested); 2) proliferative activity (7 indices: nuclear diameter and surface, mitotic activity, etc.); 3) desquamation (mean number of cells per follicle, relative volume of the desquamated epithelium); 4) colloid characteristics; 5) stromal characteristics; 6) vascular characteristics; III) electron microscopy. The first stage of the morphometric study is a description, then semiquantitative method supported by a deep morphometric analysis with the use of statistics.

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Under normal conditions the number and proportion of four main cell types of Apud cells (EC, DI, D, L) is stable. The number of Apud cells is always lower when adenomas or adenocarcinomas are present. A decrease of Apud cells number is inversely proportional to the degree of dysplasia. In parallel, increased proportion of EC-cells occurs, 20-30% as compared to normal condition. A decrease of epithelial tumor differentiation correlation with an increase of ultrastructural signs of active proliferation and Apud cells secretory activity, their degranulation and destruction, appearance of "agranular" and "mixed" endo-endocrine cells.

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