[HOMOCYSTEINE-INDUCED MEMBRANE CURRENTS, CALCIUM RESPONSES AND CHANGES OF MITOCHONDRIAL POTENTIAL IN RAT CORTICAL NEURONS].

Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential.

[The effect of modulators of SK channels on simple spike firing frequency in the discharge of the cerebellar Purkinje cells in laboratory mice].

The effect of CyPPA, a positive modulator of small conductance calcium-activated potassium channels of type 3 and 2 (SK3/SK2), and of NS309, an activator of intermediate and small conductance calcium-activated potassium channels (IK/SK), on the activity of cerebellar Purkinje cells was studied in 2-month-old male mice. The use of 1 mM of CyPPA has led to a decrease of simple spike firing frequency in the discharge of Purkinje cells by 25%, on average, during 1 h after application. At the same time, application of 100 µM of NS309 has promoted a decrease in simple spike firing frequency by 47 %, on average, during 1 h after the beginning of the action. The obtained results confirm the hypothesis that SK channels participate in regulation of simple spike firing frequency in the discharge of Purkinje cells and are responsible for restriction of signal frequency. The effect of NS309 on simple spike firing frequency was more pronounced; therefore, the IK/SK channels may be suggested to play the cardinal role in regulation of spike activity of Purkinje cells. Since increasing simple spike frequency in the discharge of Purkinje cells is observed at many disturbances of motor activity, in particular, at spinocerebellar ataxia, it can be suggested that the studied compounds or substances of similar action are of interest as potential medicinal agents.

[Age-related changes in activity of cerebellum Purkinje cells, shape of the complex spike, and locomotion of wistar rats under effect of ethanol].

The work deals with study of peculiarities of effect of ethanol upon the Purkinje cell activity, shape of the complex spike, and locomotion of rats at different stages of ontogenesis, such as the stage of the morphofunstional maturation of the cerebellar cortex, the mature stage, and in the process of aging. The experiments were carried out on three age groups of Wistar rats: rat pups (2 weeks), adult rats (3-6 months), and senile animals (22-26 months). The administration of ethanol has been established to produce an increase in frequency of simple spikes, a decrease in frequency of complex spikes, a shortening of duration of depression of simple spikes, a decrease in the total duration of the complex spike, the number and frequency of its impulses as well as reduction of the motor activity of animals of all age groups. The change of the majority of the studied parameters occurred by the common temporal scheme. The earliest responding were the rat pups, later--the adult rats, and the last--the animals of the senior group. The stronger effect of ethanol was observed in adult rats. Their differences of all studied parameters, as compared with rat pups and senile animals, were characterized on the whole by the longer period of time and by the higher percent of changes relative to the initial values. Analysis of the obtained results has shown that the most pronounced changes in parameters of the cerebellum Purkinje cell activity and of the complex spike shape corresponded to the more significant decrease in the locomotion level, i. e., were recorded in adult rats. Thus, the work has demonstrated different sensitivity to administration of ethanol in the Wistar rats at different stages of ontogenetic development.

[Effect of harmaline of the complex spike waveform and depression time in cerebellar Purkinje cell discharge in rat postnatal ontogenesis].

Functional relationship between waveform of complex spike (CS) and depression time of simple spike (SS) in discharge of cerebellar Purkinje cells was studied after their activation with afferent climbing fiber at different terms of postnatal ontogenesis in norm and after treatment with harmaline. The experiments were carried out on three age groups of Wistar rats: rat pups (2 weeks), the adult (4-6 months), and the old animals (22-26 months). It was established that the CS duration in norm was approximately equal in rat pups, adult, and old animals, whereas it markedly decreased from the young to the old animals during the SS depression in the Purkinje cell discharge. Frequency of small action potential (sAP) and their number in the Purkinje cell discharge were approximately equal in young rat pups and adult animals, while in old animals these parameters were higher, on average, by 30%. After administration of harmaline, all CS parameters in rat pups and old animals increased in parallel with the depression time elongation. In adult rats, harmaline did not produce statistically significant changes of the mean values of CS parameters, but an increase of the simple spike depression time was observed. The obtained results allow concluding that the CS waveform and the simple spike depression time in norm are functionally coupled and change with age. The effect of harmaline on the CS waveforms as well as on interrelation of the CS duration and the SS depression time in the Purkinje cell discharge was more pronounced at the early and the late stages of Wistar rat postnatal ontogenesis.

[Effect of harmaline on firing pattern of rat cerebellar Purkinje cells in ontogenesis].

In this work, responses of rat Purkinje cells to intraperitoneal administration of the hallucinogenic alkaloid harmaline (0.15 mg/kg) were studied in the course of ontogenesis. The experiments were carried out on Wistar rats of three age groups: rat pups (13-18 days), adult animals (2-7 months), and aged rats (25-36 months). In Purkinje cell firings, two types of electric reactions were revealed; they were similar in all age group of the animals. In cells with the 1st type of reactions, in response to the harmaline administration there was recorded a significant increase of frequency of complex spikes, accompanied by disappearance of simple spikes. In the activity of Purkinje cells of the 2nd type, the complex spike frequency also increased; however, the firing simple spikes were preserved, although with a decrease of their frequency as compared with norm. Essential changes of activity of the cerebellar Purkinje cells were found in the rat pups and aged animals in comparison with adult rats, which agrees well with immaturity of various cerebellar structures in the first case and with involutionary changes in the second case.

Downregulation of Purkinje Cell Activity by Modulators of Small Conductance Calcium-Activated Potassium Channels In Rat Cerebellum.

Small-conductance calcium-activated potassium channels (SK channels) are widely expressed in CNS tissues. Their functions, however, have not been well studied. Participation of SK channels in Purkinje cell (PC) pacemaker activity has been studied predominantly in vitro. Here we studied for the first time the effects of SK channel activation by NS309 or CyPPA on the PC simple spike frequency in vivo in adult (3 - 6 months) and aged (22 - 28 months) rats using extracellular microelectrode recordings. Both pharmacological agents caused a statistically significant decrease in the PC simple spike frequency. The maximum value of the decrease in the simple spike frequency did not depend on age, whereas a statistically significant inhibition of the spike frequency was achieved faster in aged animals than in adult ones. In experiments on cultured neurons PCs were identified by the expression of calbindin as the PC-specific marker. Registration of transmembrane currents in cerebellar neurons revealed the direct action of NS309 and CyPPA on the SK channels of PC consisted in the enhancement of outward potassium currents and action potential after-hyperpolarization. Thus, SK channel activators can compensate for age-related changes of the autorhythmic functions of the cerebellum.

Basement membrane zone remodeling during appendageal development in human fetal skin. The absence of type VII collagen is associated with gelatinase-A (MMP2) activity.

Epithelial cell adhesion, migration, and differentiation are controlled by interactions at the basement membrane zone (BMZ). Type VII collagen is the major collagenous component of anchoring fibrils that are essential for the attachment of the epidermis to the dermis. Gelatinase A (MMP-2) is believed to be necessary for the degradation of type VII collagen. In this study we have examined the in vivo distribution of type VII collagen and gelatinase A (Gel A) in the developing human epidermis and its appendages. At 13-15 wk of gestation a marked decrease in type VII collagen immunoreactivity was seen in the BMZ surrounding invading appendageal buds; however, type VII collagen mRNA was strongly expressed in the budding epidermal keratinocytes adjacent to the BMZ. At these stages, Gel A-positive mesenchymal-like cells were found scattered throughout the stroma with numerous Gel A-containing cells in direct contact with the developing appendageal buds. In situ zymography was used to show Gel A-activity in vivo. Gel A-mediated lysis was present at the interface between the appendageal buds and the underlying BMZ. By 20-25 wk of gestational age, immunostaining for type VII collagen protein was absent from the BMZ surrounding the distal portion of invading appendageal epithelial cords of both hair follicles and sweat glands. In contrast, type VII collagen mRNA was present in the basal keratinocytes adjacent to the BMZ surrounding the distal portion of these invading appendageal epithelial cords. At these stages Gel A-positive cells were present in the stroma directly adjacent to the distal portion of developing appendageal cords that lacked type VII collagen. In situ zymography showed zones of Gel A-mediated stromal lysis at the distal portion of developing appendageal cords. Interestingly, no differences were seen in the distribution of type IV collagen in the BMZ of both budding and resting fetal epidermis. These observations suggest that the absence of type VII collagen protein correlates directly with the presence of Gel A-activity at the BMZ. Gel A appears to play a major role in appendageal development and contributes to remodeling of the BMZ during fetal skin morphogenesis.

Matrilysin (PUMP) correlates with dermal invasion during appendageal development and cutaneous neoplasia.

Matrix-degrading metalloproteinases play a major role in tissue remodeling. Recent studies have shown that enzymes of this class are constitutively expressed primarily by stromal cells and not by epithelium. Here we present immunohistochemical evidence that matrilysin is localized within epidermal cells in developing skin and in tumor cells of cutaneous malignancies. The expression of matrilysin protein in developing fetal skin (6-15 weeks) is localized primarily to the germinative basal cell layer of fetal epidermis and early appendageal buds. The buds continue to express matrilysin during mesenchymal invasion. As development progresses (15-19 weeks) matrilysin is concentrated only in cells at the distal portion of the invading follicular and sweat gland appendageal cords. In adult skin, matrilysin was localized specifically to the outer root sheath of the hair follicles and the secretory cells of the eccrine glands but was absent in the epidermis. Nodulocystic, keratotic, adenoid basal cell carcinomas (BCCs) did not express matrilysin. In contrast, in the more aggressive morpheaform (infiltrative) BCCs and recurrent BCCs, matrilysin was localized at the tumor-stromal interface. In squamous cell carcinomas matrilysin was present in tumor cells at the stromal interface surrounding the tumor nests. The demonstration of matrilysin protein in germinal basal cells during fetal skin development and its presence in tumor cells at the stromal junction suggests that this enzyme may contribute to the proteolytic activity associated with cell-extracellular matrix interactions during appendageal development and tumor invasion.

Matrix metalloproteinases in blood vessel development in human fetal skin and in cutaneous tumors.

In vitro angiogenesis models suggest that new blood vessel formation requires the induction and secretion by endothelial cells of matrix metalloproteinases. These enzymes assist in the controlled proteolytic degradation of the surrounding extracellular matrix during blood vessel formation. The results of in vitro studies cannot be extrapolated directly to the process of in vivo angiogenesis because the type of matrix employed and the repertoire of enzymes secreted by cells in vivo differ dramatically from in vivo conditions. To investigate the in vivo role of matrix metalloproteinases in blood vessel development, we looked for the presence of these proteinases in endothelial cells involved in fetal angiogenesis and in neovascularization of certain invasive skin tumors using immunofluorescent staining. In fetal tissue, interstitial collagenase was present in both early microvessels developing from undifferentiated mesoderm and in microvessels involved in elongation and sprout formation from preexisting blood vessels. In aggressive skin tumors, i.e., morpheaform and recurrent basal cell carcinomas and squamous cell carcinomas, there was a marked increase in the number of collagenase-containing blood vessels, often extending into the tumor nests. Immunofluorescent staining failed to detect stromelysin, matrilysin, or gelatinase A and B (72- and 92-kDa type IV collagenases, respectively) in fetal or tumor blood vessels. These findings are consistent with the hypothesis that proteolytic degradation of the extracellular matrix is required for the formation of new blood vessels. Interstitial collagenase appears to play an important role in this process.

Localization of 92-kDa type IV collagenase in human skin tumors: comparison with normal human fetal and adult skin.

To investigate the role of secreted metalloproteinases in the behavior of skin tumors we have studied immunoreactivity for 92-kDa type IV collagenase (92T4Cl) in benign tumors of sweat glands, basal cell carcinomas (BCC), baso-squamous cell carcinomas (BSCC), and squamous cell carcinomas (SCC). In all tumors, the enzyme was found in stromal cells, but not in tumor epithelium. 92T4Cl-positive cells contained the common leukocyte antigen HLe-1(CD45) and the polymorphonuclear leukocyte-specific antigen PMN-8C7. Only a few 92T4Cl-positive cells expressed either macrophage-specific Leu-M5 or eosinophil-specific cationic protein antigens. In benign sweat gland tumors, and in the majority of nodulocystic and adenoid BCCs, 92T4Cl-positive cells were relatively rare and no extracellular deposition of the enzyme was found. In the more aggressive tumors examined, SCCs, BSCC, recurrent, infiltrative, and morpheaform BCCs, 92T4Cl-positive cells were very abundant. In addition, a significant quantity of extracellular enzyme was deposited both within the extracellular matrix adjacent to the tumor nests and in their basement membrane zone. In normal adult skin only a few scattered 92T4Cl-containing cells were found in the dermis whereas in fetal skin, groups of 92T4Cl-positive, HLe-1-negative cells were present in the upper dermis. These observations suggest that in cutaneous tumors, extensive infiltration of 92T4Cl containing polymorphonuclear leukocytes and the extracellular deposition of the enzyme in the basement membrane zone are signs of more aggressive tumor behavior.

[Current theories on the nature of myoepithelial cells and their role in the morphogenesis of dyshormonal hyperplasia and breast neoplasms]. / Sovremennye predstavleniia o prirode mioépitelial'nykh kletok i uchastii ikh v morfogeneze disgormonal'nykh giperplazii i opukholei molochnykh zhelez.

An analysis of current concepts on the origin and potentials of neoplastic transformation of myoepithelial cells (MC) of the mammary glands is presented. The epithelial and smooth-muscle nature of these cells is discussed. The hypothesis of mixed (epithelial and myoepithelial) nature of mammary gland carcinoma and possibilities of neoplastic transformation of poorly-differentiated forms of MC are considered. Methods for identification of cells of the myoepithelial origin are described.

[Role of myoepithelial cells in the histogenesis of lobular breast cancer]. / O roli mioepitelial'nykh kletok v gistogeneze dol'kovogo raka molochnoi zhelezy.

Myoepithelial cells (MC) and their distribution in lobular carcinoma (LC) of woman (4 cases) and dog (3 cases) mammary gland were studied by indirect Coons' method using monospecific antiserum against smooth muscle myosin. The following types of MC distribution in the duct-lobular system were distinguished: strictly peripheral localization and close connection with a basal membrane in LC in situ; disorderly distribution among tumour cells and complete disappearance from solid complexes of the infiltrating LC. It is shown that the alteration of normal architectonics of lobular structure is followed by MC move along the basal membrane resulting in the appearance of poorly differentiated MC. These facts do not allow one to draw the conclusion on the tumour nature of poorly differentiated MC; the decrease of their differentiation might depend upon the substrate.

[Patterns of keratin protein expression in basal cell, metatypical and squamous cell carcinoma of human skin]. / Nekotorye zakonomernosti ékspressii keratinovykh belkov v bazaliomakh, metatipicheskom i ploskokletochnom rake kozhi cheloveka.

Comparative immunomorphological study of keratinous proteins was performed in 17 basaliomas, 4 metatypical (MT) and 1 squamous cell carcinomas by means of a spectrum of antibodies to the individual keratins. It is found that cells of MT carcinoma are distinguished from basalioma cells by the absence of keratins N8 and 17 and this may be used for the differential diagnosis of these two tumours. The spectrum of keratins expressed by basalioma cells coincides with that of early stages of hair follicles. Keratin N17 is locally induced in parabasal layers of the morphologically intact epidermis adjacent to the tumour.

[Pathomorphology of Kaposi's sarcoma]. / Patomorfologiia sarkomy Kaposhi.

Chaotic angiogenesis and permanent presence of spindle cells with a variable degree of nuclear atypia in the nodular aid tumourous foci represent important diagnostic criteria of Kaposi's sarcoma (KS). At early stages or, on the contrary, in the persistent lesions morphological picture is not sufficiently characteristic and the diagnosis cannot be established on the basis of histological examination only; evaluation of the clinical and morphological data and repeated biopsy are necessary in such cases. The degree of manifestation of a spindle-cell component and the degree of its anaplasia are not determining factors in the prognosis of disease.

[Histogenesis of Kaposi's sarcoma]. / O gistogeneze sarkomy Kaposhi.

The origin of Kaposi's sarcoma from the endothelium and fibroblast-like cells of a vascular wall is proven on the basis of complex pathomorphological, immunomorphological, autoradiographic and electron microscopical investigation of tumour biopsies. Depending upon the predominating cell type of origin, the tumour in its different parts, may form the structures of either different variants of angiomas or fibrosarcomas.

[Immunocytochemical study of the myoepithelial cells in fibrocystic disease and ductal carcinoma of the breast]. / Immunotsitokhimicheskoe izuchenie mioepitelial'nykh kletok pri fibrozno-kistoznoi mastopatii i protokovom rake molochnoi zhelezy.

Myoepithelial cells (MC) were identified and the pattern of their distribution in dysplasia and duct carcinoma of the mammary gland was studied using a monospecific antiserum to smooth-muscle myosin of the human uterus, by the indirect Coons' method under luminescent microscope. Samples of the operation material from 10 patients with fibrous-cystic mastopathy and 26 patients with duct carcinoma were examined. In solid (7 observations) and comedo (4 observations) carcinomas with the structure of cancer in situ, MC were found intact in the periphery. In invasive growth of these tumors as well as in scirrhus (5 observations) and adenocarcinoma (10 observations) MC disappeared from the duct structures and were absent in carcinoma proliferates. Positive immunofluorescence in the conventional location of MC in the alveolar duct system of the mammary gland allows sufficiently valid identification and study of the distribution and changes in these cells. The immunocytochemical verification of MC in dysplasia and duct carcinoma indicates the possibility of using MC as markers of duct carcinoma in situ; MC disappearance from the duct structures characterizes the beginning of the infiltrative tumor growth.

[Types of myoepithelial cell proliferation in dyshormonal breast dysplasias and benign breast tumors]. / Tipy proliferatsii mioépitelial'nykh kletok pri disgormonal'nykh displaziiakh i dobrokachestvennykh opukholiakh molochnoi zhelezy.

Myoepithelial cells (MC) were identified and types and forms of their hyperplasia in dysplasias and bening mammary gland tumors in dog and man were studied by indirect Coons' method using highly purified monospecific antiserum to smooth muscle myosin and by performing alkaline phosphatase test. Operation material from 75 patients and 12 dogs was studied by immunohistochemical method and from 26 persons and 12 dogs by histochemical method. Comparative analysis of immunohistochemical and histochemical identification of MC revealed differences in the results of staining in 7 out of 38 observations due to negative test for alkaline phosphatase in the presence of fluorescence. A high degree of coincidence of positive tests in immunohistochemical and histochemical methods of the study suggests that the test for alkaline phosphatase is a sufficiently reliable marker of MC. The principal similarity of types and forms of MC hyperplasia in canine and human mammary gland tissue indicates that dogs may be used as an adequate model for the study of various diseases of this organ. In addition to the known centripetal and centrifugal types, a uniformly concentric and smooth-muscle proliferations of MC were distinguished in parallel immunohistochemical and histochemical studies on variants of MC proliferation.

[Immunomorphological identification of myoepithelial cells in mixed tumors of the mammary gland in dogs]. / Immunomorfologicheskaia identifikatsiia mioépitelial'nykh kletok v smeshannykh opukholiakh molochnoi zhelezy u sobak.

Myoepithelial cells (MC) in mixed tumors of the mammary gland in dogs were identified with the Coons indirect method with the aid of monospecific antiserum to smooth muscle myosin. In 2 of 5 observations, anaplastic carcinoma and adenocarcinomas were detected in the presence of a mixed tumor. Immunochemical study of the myoepithelium demonstrated varying fluorescence intensity and ununiform pattern of MC distribution in tumor tissue of the mammary gland. MC were detected in lobular structures in the form of islet accumulations or diffuse vegetations from cells with a poorly fluorescent rim of the cytoplasm. In the ducts with papillary epimyoepithelial proliferations, MC occupied the peripheral position, showing bright fluorescence of the cytoplasm and hypertrophied processes. The fluorescence intensity decreased as MC displaced towards the stroma or lumen of the ducts. The changeability of specific staining of the myoepithelium may attest to different levels of cell differentiation. The presence of immature forms of MC in mixed tumors is likely to be due to the modulatory character of cell differentiation and is determined by the totality of factors that apparently play the triggerring part in derepression of the genes of specific synthesis of smooth muscle proteins in undifferentiated cells of the epithelium of the terminal ducts and alveoli.

Interstitial collagenase and the ED-B oncofetal domain of fibronectin are markers of angiogenesis in human skin tumors.

Collagenase-1 (C1) is the predominant matrix metalloproteinase present in newly formed microvessels and serves as a marker of neovascularization. The expression of the oncofetal fragment of fibronectin (Fn-f) was found to be increased during angiogenesis. In the present study, we investigated the relationship between the expression of collagenase-1 and the oncofetal fragment of fibronectin in newly formed microvessels as markers of tumor angiogenesis. In aggressive skin tumors (i.e., morpheaform and recurrent basal cell carcinomas) and squamous cell carcinomas, neovascularization was associated with a marked increase in the number of C1-positive and Fn-f-positive microvessels. At the beginning of elongation, microvessels begin to produce C1 but lose their ability to express type IV collagen and FVIII-related antigen. Later, this endothelium produces both Fn-f and C1. As maturation of microvessels occurs, C1-containing endothelium fails to express Fn-f but begins to produce a type IV collagen-containing basement membrane and FVIII-related antigen. These studies show that there is a selective expression of both Fn-f and collagenase by immature endothelial cells. C1 production begins at early stages of blood vessel formation and continues throughout angiogenesis. In contrast, Fn-f expression is limited to later stages of vasculogenesis, indicating that these proteins are reliable markers of angiogenesis.