Surface Plasmon Resonance Investigations of Bioselective Element Based on the Recombinant Protein A for Immunoglobulin Detection.

The developed surface plasmon resonance (SPR) biosensor based on the recombinant Staphylococcal protein A with an additional cysteine residue (SPA-Cys) used as a biorecognition component showed a good selectivity and sensitivity for the immunoglobulin detection. The developed biosensor with SPA-Cys-based bioselective element can also be used as a first step of immunosensor creation. The successful immobilization of SPA-Cys on the nanolayer gold sensor surface of the SPR spectrometer was performed. The efficiency of blocking nonspecific sorption sites on the sensor surface with milk proteins, gelatin, BSA, and HSA was studied, and a rather high efficiency of using gelatin was confirmed. The SPR biosensor selectively interacted with IgG and did not interact with the control proteins. The linear dependence of the sensor response on the IgG concentration in the range from 2 to 10 µg/ml was shown. Using the calibration curve, the IgG concentration was measured in the model samples. The determined concentrations are in good agreement (r 2 = 0.97) with the given concentration of IgG.

[Polyclonal antibodies against human cell-surface antigen CD34].

An efficient and inexpensive laboratory approach for the generation and the purification of polyclonal antibodies to human antigen CD34 was developed. It was shown that cloned refolded and purified from Escherichia coli recombinant extracellular fragment of CD34 antigen retained immunogenic determinants of cell-surface expressed CD34. Immunization of mice with unglycosylated truncated recombinant protein elicit polyclonal antibodies specific for the native human antigen CD34. The antibodies generated are applicable for phenotyping of CD34+ cells using immunocytochemistry and flow cytometry assays.

[Repeated exposure in hypergravity: morphology of locus coeruleus, hypothalamic paraventricular nucleus and vagal nerve dorsal nucleus in rats]. / Povtornoe vozdeistvie gipergravitatsii: morfologiia sinego piatna, paraventrikuliarnogo iadra gipotalamusa i dorsal'nogo iadra vagusa golovnogo mozga krys.

As compared to analogous single rotation at 2 g and in contrast to 5-d single and repeated exposures to Coriolis accelerations, repeated 5-day hypergravity (2 g generated by centrifuge rotation) gave rise to structural alterations in rat's neurons of locus coeruleus, vasopressinergic neurons of the lateral magnocellular subnucleus paraventricular nucleus and nervi vagi dorsal nucleus suggesting involvement of these structures of brain in the mechanism of facilitation of adaptation to repeated long-term hypergravity. Results of the study point to the ability of mammals to remember changes in gravity. Findings of the study may help develop an algorithm of intermittent exposure to artificial gravity aboard space vehicle.

[The participation of the nontraditional neuromediator nitric oxide in the mechanisms of adaptation to extreme conditions]. / Uchastie netraditsionnogo neiromediatora okisi azota v mekhanizmakh adaptatsii k ékstremal'nym usloviiam.

The investigation was performed on the medial (MMS) and lateral (LMS) magnocellular subdivisions of the hypothalamic paraventricular nuclei (HPN). The histochemical activity NO synthesizing enzyme nitric oxide synthase or NOS whose histochemical marker is NADPH-diaphorase (NADPH-D), immunocytochemical content of oxytocin (OXY), vasopressin (VP) and nucleoli sizes (squares) were studied in the mature male rats under experimental reconstruction of the both micro- and macrogravity, which are factors of the gravity field changes acting to the body during the space flight. Two experimental effects were used: B--tail suspending (imitation of the microgravity effects), C--centrifugation at 2 G (imitation of the macrogravity effects). The effect durations were designed as a time period when body is mostly affected by (1 day) and adapted (15 days) to the stress. There were 6 animal groups. 1--B(15 days), 2--B(15 days) succeeded by C(1 day), 3--B(15 days) succeeded by C(15 days), 4--C(1 day), 5--C(15 days), 6--intact animals. The histochemically and immuno-cytochemically stained neurons developing the high, moderate and small reaction intensity were counted in serial HPN sections under the light microscope and the results obtained were transformed to percent neuron contents. The nucleoli squares were examined by using the TV analyser. The histochemical staining intensity of NADPH-D in MMS is enhanced in the animals of the groups 1-4; the number of NADPH-D staining neurons with high enzyme activity was increased in 8-14 times. In the animals of group 5 the NADPH-D activity did not differ from the intact animals. The number of MMS neurons with high OXY immunoreactivities was increased up to 1.5-1.7 times in groups 1-5 if compared to those of intact controls. VP-positive neurons of LMS developed the similar increase in number of the high staining neurons in experimental animals as well as OXY-positive neurons of MMS. The nucleoli enlargement was observed in MMS (in 1.3-1.5 times) of groups 1-5 (insignificantly in group 5) and in the most magnocellular neurons LMS (in 1.5-1.7 times) of group 2-5 except group 1 where nucleoli were insignificantly decreased. The nucleoli sizes of group 4 were more than group 5. So the hypothalamo-neurohypophyseal system was activated in the animals subjected of the earthly correlates of micro- and macrogravity. The data obtained suggest involvement both the nonconventional neurotransmitter NO and stress-related peptides OXY and VP in the mechanisms subserving adaptation to the extreme factors by what a human has to be faced with during the space flight.

[Structural and neurochemical characteristics of sex-dependent neurons of the brain transplanted to the anterior chamber of the eye]. / Strukturnye i neirokhimicheskie osobennosti sekszavisimykh neironov mozga, transplantirovannykh v peredniuiu kameru glaza.

Histological, electron microscopic and histoautoradiographic methods were used to study structural and neurochemical features of the development of neurons of the hypothalamic paraventricular nuclei (PVN) and the medullary nucleus of the solitary tract (NST), which had been grafted from 20-day-old rat fetuses into the anterior eye chamber of ovariectomized female rats not exposed (control group) and exposed (experimental group) to prolonged estrogen action (during 4 weeks). The exposure to estrogen stimulated the growth, cell differentiation, RNA transcription and synaptogenesis in the grafted PVN and NST neurons. The data obtained seem to provide additional evidence that PVN and NST neurons belong to sex-dependent brain neurons.

[Sexual dimorphism in the response of the large-cell neurons of the rat hypothalamic paraventricular nuclei to water-salt loading and water deprivation]. / Polovoi dimorfizm v reaktsii krupnokletochnykh neironov paraventrikuliarnykh iader gipotalamusa krys na vodno-solevuiu nagruzku i obezvozhivanie.

The responses of large-cell populations of the hypothalamic paraventricular nuclei (PVN) to salt loading and water deprivation in adult male and female rats were studied using morphometric methods at the level of light microscopy. Sexual dimorphism, especially pronounced during functional stress, has been observed in the responses of all large-cell PVN populations. The males proved to be more sensitive.

[Testosterone metabolism in discrete areas of the brain in rat fetuses]. / Metabolizm testosterona v diskretnykh oblastiakh mozga plodov krys.

Experiments in vitro with further thin-layer chromatography of steroids were staged to investigate changing of 3H-testosterone into 3H-estradiol-17 beta and 3H-5 alpha-reduced metabolites in a fraction of 1000 g of homogenates of the medial preoptic area (MPA), the paraventricular nuclei (PVN), the dorsal vagal complex (DVC) of the lower cerebral trunk, the rostral and caudal parts of the amygdaloid complex (ACr and ACc) of the brain of 21-day male and female rat fetuses. The activity of aromatase (AA) and 5 alpha-reductase (AR) was detected in all investigated areas. AA was maximum in PVN of females and minimum in ACc of males and females. The production of 5 alpha-dehydrotestosterone prevailed among 5 alpha-reduced products in PVN, DVC and ACr, 5 alpha- and rostane-3 alpha and 17 beta-diol--in MPA and ACr, AR in ACr was much higher than in ACc. AR in DVC of females was slightly higher than in males. Sex differences in the other cerebral areas were undetectable. The results of the investigation suggest that the above cerebral structures are dependent on sex hormones.

[Reaction of specific cell populations of hypothalamic paraventricular nuclei of the rat to carbohydrate loading and fasting]. / Reaktsiia otdel'nykh kletochnykh populiatsii paraventrikuliarnykh iader gipotalamusa krys na uglevodnuiu nagruzku i golodanie.

The structural correlates of the cell responses in subnuclei of the hypothalamic paraventricular nuclei (PVN) in fasting and carbohydrate loaded adult male rats were studied using light microscopic morphometric methods. The data obtained revealed difference in responsiveness of the subnuclei studied to alternative shifts in the carbohydrate homeostasis: histophysiological features in five of ten cell-divisions, distinguished in the rat RVN, demonstrated their dependence on impaired carbohydrate homeostasis.