[Identification of "side population" associated with cancer stem cells by flow cytometry with violet laser].

The possibility of identification of cancer stem cells "side population" in solid tumors by using the flow cytometer equipped with 405 nm violet laser was investigated. Ovarian cancer (Skov-3) and colon cancer (Colo 320) cell lines formed the "side population" after vital staining with Hoechst 33342. Analysis of cells isolated from tumor tissue of malignant melanoma and colorectal cancer, also revealed "side population" that was a result of the fluorescent dye exclusion. The percentage of melanoma cells included in the "side population" was the same as that of cells co-expressing cancer stem cells markers--CD34 and CD44. In contrast, the colon cancer "side population" was significantly smaller than the minor populations of colon cancer cells identified by either CD133 expression or exclusion of Rhodamine 123.

[Neurogenesis in the central nervous system and prospects of regenerative neurology].

Discovery of neural stem cells (NSC) providing homeostatic adaptive and injury induced neural regeneration in the CNS of adult mammals, including Homo sapiens, is the most prominent accomplishment over the recent period of neurobiology research. NSC are concentrated in two neurogenic zones - side walls of lateral ventricle (subventricular zone) and hippocampal dentate gyrus (subgranular zone). In addition, new neurons may develop from other undifferentiated cells scattered throughout various CNS regions. Neurogenesis in adult mammals is an intensive process that leads to renewal of interneuron populations in such brain regions as olfactory bulbs and hippocampus by 5 and more percent per month. Advances in regenerative neurobiology may serve the foundation for the development of totally new technologies of treatment brain and spinal cord, as well as retina and optic nerve injuries and diseases based on the stimulation of reparative neurogenesis, design of conditions permissive for regeneration of nervous and glial cells and growth of nervous fibers, and on blocking factors inhibiting those two former processes.

[An experimental model of focal brain ischemia in rats based on the endovascular microsurgery].

An endovascular model of focal brain ischemia in rats with controlled duration of reperfusion of 1h has been studied. The reliability of experimental model for localization and volume of ischemic lesion (basal ganglia, hippocampus, parietal-temporal regions; the volume of lesion focus on MRI T2-weighted image (T2-WI) in the first day after brain ischemia 146.4 +/- 44.7 mm3) has been worked out. We conducted the clinical monitoring including assessment of neurological deficit, behavioral tests and performed MRI of the brain on 1st, 5th and 10-17th days after the occlusion of middle cerebral artery (MCA), including regimes of diffusion-weighted image (DWI) and T2-WI, and carried out a histological study of the brain. The regress of neurological deficit was seen on the 14th day after surgery. Behavioral tests revealed the reduction of movement activity of animals in the case group 7 days after the MCA occlusion. Compared to the first day after surgery, the decrease of volume lesion focus on DWI and T2-WI was observed on the 5th day (p < 0.05) as well, with the following decrease to the 10-14th days. The histological picture in the lesion foci corresponded to the brain infarction on the 5th and 14th days after surgery.

[New conceptions in complex management of spine trauma patients including reconstructive microsurgery and succeeding rehabilitation].

This work is been based upon the experience of performing 100 reconstructive microsurgical operations with transplantation of combined vascular-neural autografts, in 24 cases--with introduction cellular material into zone of transplantation. The complex approach aimed to creating optimal conditions for the functional regeneration of the spinal cord (SC), including neurosurgical methods restoring anatomical integrity of the organ and also multicomponent and staging rehabilitation of patients has been developed. On the base of complex approach during a postoperative period there has been put synchronous multifactorial action, consisting of functional multichannel electrical pacing of muscles simultaneously participating in tumble. The patient concentrates all his attention on the trial to tumble with paralized extremities by himself. At that the series and the site of application of these electrical impulses forms the algorithm of this tumble. Synchronism of produced afferent and efferent impulses creates new possibilities in activating of regenerating processes in the damaged area of the spinal cord. As a result of treatment within the bounds of the developed program the authors get clinically and electrophysiologically proved certain improvement that they can not only predict but guarantee patients of this group. Presented here results of the complex treatment of patients with trauma of the spinal cord within the framework of research international program confidently testify advantages of the new approach to solving of this problem. For the first time in history of medicine there is created close cooperation and regular continuity between molecular biology, neurosurgery and rehabilitation. This scientific alliance permits in proper time to keep a close watch, quickly correct and improve every stage of treatment. The effect of implement of innovations is been summarized at the each next stage of proposed complex treatment and eventually significantly elevates an ultimate result of medical rehabilitation of invalids with trauma of the spinal cord.

[Comparative transcriptomic and proteomic analysis of the fetal and adult human liver].

The transcriptome and proteome comparative analysis of the fetal liver (9.5-10.5 weeks of gestation) and normal adult liver was developed in the present investigation. We used 44k microarrays of Agilent company and 2D-GE MALDI-TOF in transcriptome and proteome approaches, respectively. The top lists of expression genes and proteins for fetal liver obtained by these methods were compared. Transcriptome analysis confirmed proteome data only partially, but these two semi-quantitative approaches established the interdependences between expression levels of genes and proteins. The discrepancies between data obtained by two approaches were discussed. The unique feature of the fetal liver cell content is the combination of hemopoietic and nonmaturated liver cells. Fetal liver changes its hemopoietic role during embryogenesis towards the detoxicative tissue being able to metabolize different substances and produce wide serum of proteins. The status of fetal liver as the hemopoietic tissue was entirely characterized by transcriptome analysis having registered embryonic and adult hemoglobins, insufficient cytochrome-dependent detoxification system and highly developed anti-apoptotic potential.

[The role of resident and circulating stem cells in physiological and reparative regeneration].

Complete or partial restoration of the number of parenchymal cells is a vital part of the mechanism of maintaining tissue homeostasis and post-injury regeneration. It includes proliferation and differentiation of the resident stem cells, homing and differentiation of circulating stem cells, and, sometimes, transient dedifferentiation and amplification of the local parenchyma. Bone marrow serves as the principal storage site of the circulating stem cells. Several possible ways of circulating stem cells participation in tissue regeneration are discussed including trans-differentiation and fusion with resident cells. There is a theory of a common pool of circulating pluripotent stem cells present in the bone marrow and some other tissues and an alternative theory of several pools of tissue-specific circulating stem cells. The data obtained in this field of research are the starting point for development of cell-based technologies aimed at therapy of degenerative diseases and traumas.

[Enhanced control of proliferation in telomerized cells].

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

[Spinal cord regeneration in rats after thoracic segmentectomy: growth and regeneration of nerve fibers].

As it was described in a previous paper (Morphology, 2005, v.127, Iss. 2), spinal cord (SC) consolidation after thoracic segmentectomy in rats took place via connective tissue scar formation in the area of trauma, which was accelerated when SC defect was filled with by the collagen gel (Spherogel). In this paper, on the basis of semithin section analysis and transmission electron microscopic study, it was shown that regenerating nerve fibers crossed the connective tissue within the formations, which were structurally identical to peripheral nerves. In the zones of rarified SC substance, caudally to the site of trauma, myelination of growing axons was realized by glial cells without the formation of the nerve truncs. In the white matter, within the fasciculi of SC lumbar segments, multiple thin regenerating fibers were seen in the area of degenerated myelinated nerve fibers.

[Spinal cord regeneration in rats after thoracic segmentectomy: restoration of the anatomical integrity of the spinal cord].

Spinal cord (SC) segmentectomy was performed at ThX level in 28 rats. The scar formation in place of surgery and the degree of restoration of anatomical integrity were studied in control rats (group I) and in rats, in which SC defect was replaced by either Spherogel neuromatrix (group II) or Spherogel containing dispersed embryonic nerve cells (group II). While in group I rats by weeks 1-2 SC defect was filled with fibrin masses, in rats of experimental groups II and III the formation of connective tissue scar was found by the same time. By weeks 10-11 these animals demonstrated partial restoration of motion in three joints of the hindlimb. In the scar tissue and in adjacent SC zones numerous regenerating thin myelinated nerve fibers were found. They were clearly demonstrated in the zones of cranial and caudal borders along the whole distance until the appearance of typical tissue of SC. In slides from the animals of control group, thin myelinated nerve fibers were found only in close proximity to SC substance, while in the scar tissue and in the cellular bands of the intermediate zone nerve fibers were scarce.

[Subtypes of the functionally active muscarinic receptors in the heart of the frog Rana temporaria]. / Issledovanie subtipov funktsional'no aktivnykh muskarinovykh retseptorov serdtsa liagushki Rana temporaria.

Radioligand analysis of ventricular membranes from the frog Rana temporaria with muscarinic antagonist 3H-QNB (qunuclidinul benzilate) revealed homogeneous population of highly specific muscarinic receptors with high affinity (Kd = 0.502 +/- 0.020 nM and Bmax = 789 +/- 18 fmol/mg protein). Competitive binding of M1-antagonist pirenzepine revealed two binding sites on the membrane with Kd values 5.6 +/- 1.7 and 237 +/- 45 nM, and total capacity of 20.6 +/- 10.2 and 742 +/- 58 fmol/mg protein respectively, which may be attributed to M1- and M2-subtypes in accordance with Receptor Nomenclature. The inhibitory effect of pirenzepine on negative inotropic action of Ach has been studied in the intact electrically stimulated ventricle. Analysis of the data by Schild procedure showed that KB for pirenzepine is equal to 13.9 +/- 2.4 nM. The results obtained suggest that the frog ventricle contains muscarinic cholinoreceptors of M2-subtype (possibly, M4-subtype) which account for negative inotropic effect of Ach. Small population of M1-like receptors was also revealed, their functional role still remaining obscure.

[Interaction of tetrahydroisoquinolines and 3-aminotetralines with opioid mu-receptors]. / Vzaimodeistvie tetragidroizokhinolinov i 3-aminotetralinov s opioidnymi mu-retseptorami.

The interaction of the tetrahydroisoquinoline (THIQ) and 3-aminotetraline (3-AT derivatives with opioid mu-receptors has been studied. It is shown that THIQ and 3-AT derivatives bind to a site on the mu-receptor which these compounds are likely to share with "classical" opiates, whose structure also includes the 3-AT group. The binding site for nonpeptide substances is in a strong allosteric interaction with the binding site for enkephalins. Some biological effects of THIQ and 3-AT derivatives can be explained in terms of their interaction with opioid receptors. One may speculate that the evolution of the endogenous opioid receptor ligands proceeded from simple 3-AT derivatives towards morphinans and, probably, benzomorphans.

[Alpha-tocopherol induced activation of the endogenous opioid system]. / alfa-tokoferol-indutsirovannaia aktivatsiia éndogennoi opioidnoi sistemy.

In an attempt to clarify the nature of analgesic effect of alpha-tocopherol, the influence of alpha-tocopherol on the levels of endogenic opioids was studied in the experiments in vitro. Main changes of the level of adenohypophyseal (tissue) beta-endorphins and in the incubation media were evaluated as well as the involvement of the endogenous opioid system in the analgesic effect of alpha-tocopherol.

[Sigma receptors of the loach embryo regulate ornithine decarboxylase activity]. / Sigma-retseptory zarodyshei v'iuna reguliruiut aktivnost' ornitindekarboksilazy.

When loach embryos (Miagurnus fossilis) were incubated with opioid substance, sigma-receptors ligand SKF 10.047, activity of the key enzyme of polyamine synthesis - ornithine decarboxylase (ODC), increased. A correlation between teratogenic and ODC-stimulated action of SKF 10.047 was stage-specific and depend on opioid concentration. Possible mechanism of embryonic cell aggregation under SKF 10.047 action and polyamines role in that process is discussed.

[Receptor characteristics of the duodenal mucosa in peptic ulcer: a new approach to the individual selection of the therapy]. / Retseptornaia kharakteristika duodenal'noi slizistoi pri iazvennoi bolezni: novyi podkhod k individualizirovannomu vyboru terapii.

The radioligand-receptor method was used to assay the M-cholinereceptors and opioid binding centers in biopsy specimens of the duodenal mucosa from 40 patients with duodenal ulcer as compared with the results of subsequent therapy by means of gastrocepin (20 patients) and dalargin (20 patients). Analysis of the isothermal curve of 3H-QNB-binding with duodenal mucosa membranes within Skatchard's coordinates showed the presence of 2 subtypes of muscarinic receptors blocked (M1) and not blocked (M2) by gastrocepin. The majority of the patients in both groups demonstrated a significant decrease of the dissociation constant as compared to the control (20 patients). In patients with high receptor sensitivity to gastrocepin and dalargin, ulcer cicatrization was noted during the treatment in all the cases. Meanwhile the treatment proved little effective in patients with appreciably decreased receptor sensitivity to the drugs under consideration. The data obtained indicate the possibility of predicting the results of duodenal ulcer treatment by means of the radioreceptor analysis, providing the basis for the individualized treatment choice.

[Specific binding of a synthetic antioxidant from a series of spatially screened phenols, H3-phenozan, with isolated rat enterocytes]. / Spetsificheskoe sviazyvanie sinteticheskogo antioksidanta iz riada prostranstvenno zatrudnennykh fenolov 3H-fenozana s izolirovannymi énterotsitami krys.

Specific binding of gamma-(4 hydroxy-3,5-ditret. butyl-phenol) propionate (phenozan), a synthetic water-soluble screened phenol with antioxidant activity, with isolated rat enterocytes has been demonstrated. The Scatchard analysis of binding isotherms revealed two types of 3H-phenozan binding sites. It is concluded that the effect of synthetic screened phenol antioxidants on cellular activity is the result of two mechanisms of action--alteration of physicochemical properties of membrane lipids caused by the inhibitors of free-radical reactions and specific binding of these compounds to cell plasma membranes.

[Effect of low doses of the hydra peptide morphogen on protein biosynthesis in the intact and regenerating rat liver]. / Vliianie nizkikh doz peptidnogo morfogena gidry na biosintez belka v intaktnoi i regeneriruiushchei pecheni krys.

The dependence of ornithine decarboxylase (ODC) activity on hydra peptide morphogen doses has been established. The parameters of protein synthesis were determined in normal and regenerating rat liver, using the peptide in a dose of 20 micrograms/kg body weight, initiating maximum enzyme activity. It was established that intraperitoneal injection of the peptide in partially hepatectomized animals stimulated ODS activity in dose-dependent manner and was dome-shaped. The peptide injection in intact animals does not affect the intensity of 3H-leucine inclusion into the liver protein and the protein content in rat liver. However, the peptide injection in partially hepatectomized animals increased the level of 3H-leucine inclusion into the protein of regenerating liver and stimulated protein accumulation in this type of tissue.

[Development of magnocellular neurosecretory neurons in embryonic hypothalamic tissue transplanted into the cavity of the 3d cerebral ventricle of adult rats]. / Razvitie magnotselliuliarnykh neirosekretornykh neironov émbrional'noi tkani gipotalamusa, peresazhennoi v polost' III zheludochka mozga vzroslykh krys.

Magnocellular neurosecretory cell (MSC) ontogenesis was comparatively assessed in normal rat hypothalamus and in fetal hypothalamic tissue transplanted into the third ventricle of the adult rat brain. 3H-thymidine uptake has shown that the time of origin of NSC in grafted tissue and supraoptic nucleus of normal rat fetuses was similar. NSC pericarions in grafts were well developed and contained neurosecretory material (NSM). Nuclear and nucleolar volumes did not differ from those in adult animals. Water deprivation was followed by a significant increase in nuclear and nucleolar volumes and NSM content in pericarions of grafted tissue, which suggests that raised synthetic activity was not accompanied by an increased NSM release. It is concluded that proliferation and specific differentiation of NSC precursor cells are invariably determined by genetic factors. The origin of neurosecretory nuclei and their specific connections is considerably influenced by conditions of tissue surroundings.

[Endogenous opioid system in the realization of the analgesic effect of alpha-tocopherol in reference to algomenorrhea]. / Endogennaia opioidnaia sistema v realizatsii anal'geticheskogo éffekta al'fa-tokoferola na primere al'gomenorei.

Beta-endorphin-like immunoreactivity was studied in 7 patients with algomenorrhea during pain attack and 15 minutes after alpha-tocopherol administration with a therapeutic aim (till the analgetic effect was reached). There was an increase in beta-endorphin-like immunoreactivity after alpha-tocopherol administration. Naloxone administration to 9 patients with algomenorrhea of various etiology resumed the pain. The effect of alpha-tocopherol application for pain relief depended on the pathogenesis of algomenorrhea. At the same time naloxone administration failed to resume the pain in patients, in whom alpha-tocopherol had a strong analgetic effect. It is assumed that the endogenous opioid system participates in alpha-tocopherol effect on pain relief in patients with algomenorrhea.

[Specific binding of (+)-3H-SKF 10047 by mouse splenocytes]. / Spetsificheskoe sviazyvanie (+)-3H-SKF 10047 splenotsitami myshi.

Stereoselective, saturable, reversible and temperature-dependent binding of ligand of sigma opioid receptors (+)-3H-SKF 10047 to mouse splenocytes has been demonstrated. The estimated Kd value of this interaction (20-100 nM) was similar to that of (+)-3H-SKF 10047-sigma receptor complex. The binding capacity was 100 molecules per cell. A comparatively low affinity, non-stereoselective and temperature-independent binding of (+)-3H-SKF 10047 to mouse splenocytes has also been found.

[Opioid activity of peptides and wound healing of the skin]. / Opioidnaia aktivnost' peptidov i zazhivlenie ran kozhi.

The binding of dalargin, its four analogues and FK-33824, DADLE, met-enkephalin and morphine to peripheral mu- and delta-receptors and to brain receptors has been investigated in comparison with their influence on skin wound healing in rats. It has been shown that only substances with opiate activity, including morphine, stimulated wound healing. No correlation between wound healing effect of peptides and their binding to a definite receptor has been found. Naloxone inhibited wound healing and suppressed opiate peptide-mediated healing process. It is suggested that endogenous opiate peptides are involved in the maintenance of structural homeostasis.