A common system of sparsely-branched projection (reticular) NADPH-diaphorase neurons in formations of densely-branched cells in the human forebrain.

Morphometric studies of human forebrain formations composed of densely branched cells - the entorhinal cortex, the basolateral amygdala, the nucleus accumbens, the striatum, and the dorsal thalamus - were performed using nine parameters, with statistical analysis of the resulting data; measurements addressed the major projection-type densely branched and sparsely branched reticular neurons (scattered reticular and marginal reticular cells of the dorsal thalamus) stained by the Golgi method and with NADPH-diaphorase. Scattered reticular cells in the various formations showed no differences in any of the nine measures, while there were significant differences (in 5-7 measures, apart from one comparison, where there were differences in two measures) in their major projection-type densely branched cells. Scattered reticular and main projection-type densely branched neurons in each formation differed in terms of 7-9 measures. In endbrain formations, scattered reticular neurons contained NADPH-diaphorase; in the dorsal thalamus, only intermediate marginal reticular neurons were NADPH-diaphorase-positive. Thus, these human formations contained a common system of ancient integrative NADPH-diaphorase-containing reticular cells. Our results, along with published data, show these to be projection-type cells with projections to layers V and VI of the neocortex, which suggests that they have modulatory influences on its descending systems.

[Common system of sparsely branched projection (reticular) NADPH-diaphorase neurons in the human forebrain formations built from densely branched cells].

Cell morphometry with statistical analysis (using 9 parameters) of densely branched projection and sparsely branched reticular neurons was performed in the human forebrain formations built from densely branched projection neurons (the entorhinal cortex, striatum, nucleus accumbens basolateral amygdala, and dorsal thalamus). The reticular neurons included scattered reticular neurons and marginal reticular neurons of the dorsal thalamus. Golgi method and staining for NADPH-diaphorase were used. The scattered reticular neurons of different formations under study did not differ in any of the 9 parameters, whereas they significantly differed from the main projection neurons in 5 to 7 parameters (except one comparison with the difference in 2 parameters). Within the same formation, the scattered reticular and main projection densely branched neurons differed in 7 to 9 parameters. The endbrain scattered reticular neurons expressed NADPH-diaphorase, while in the dorsal thalamus only the medium marginal reticular neurons were NADPH-diaphorase-positive. Thus, a common system of ancient integrative reticular neurons expressing NADPH-diaphorase exists in the examined human forebrain formations. The evidence obtained by us and the literature data point to the projection nature of the scattered reticular neurons (to the V and VI neocortical layers), which suggests their modulatory influence on descending neocortical pathways.

Mechanisms of cycloheximide-induced apoptosis in liver cells.

Cycloheximide in sublethal doses caused apoptosis in liver cells in vivo, inducing c-myc, c-fos, c-jun and p53 genes and accumulation of sphingosine, a toxic product of the sphingomyelin cycle. These data support the hypothesis that continuous synthesis of labile protective proteins is required to restrain apoptosis in liver; sphingosine might be important in mediating cycloheximide-induced apoptosis as an endogenous modulator of protein kinase C activity.

[Effect of embryonal nerve tissue transplantation on regeneration of dorsal roots of the rat spinal nerves]. / Regeneratsiia zadnikh koreshkov spinnomozgovykh nervov krysy v usloviiakh transplantatsii émbrional'noi nervnoi tkani.

Using cobalt salts axonal ionophoresis posttraumatic regeneration of TXII dorsal roots nerve fibres in the zone of hemisection in conditions of 14 wks embryo spinal cord transplantation into the zone of trauma of spinal cord. Regro Invasion of dorsal roots nerve fibres into recipients posterior cords and Lissawers tract through the transitional zone "spinal cord--dorsal roots" was observed on posttransplantation d 14-120. It was show that afferent axons predominantly spread in substantia alba and substantia grisea caudal to the level of spinal cord transection with only individual fibres invading rostrad through the neuronal plate. In the transplants neurons were encountered up to d 120 of the observation although transplant neuropil was limited from recipient tissue brain by a glial and connective tissue scar. The influence of embryonal nervous tissue transplantation on intraspinal regeneration of dorsal roots afferents was discussed.

[The role of sphingomyelin cycle products in development of apoptosis mediated by Fas receptors and tumor necrosis factor alpha]. / Rol' produktov sfingomielinovogo tsikla v razvitii apoptoza, indutsirovannogo cherez retseptory Fas, i faktora nekrosa opukholi al'fa.

Programmed cell death or apoptosis is a cell suicide developing according to a specific program, in which the sphingomyelin cycle products, ceramide and sphingosine, play the central role. The present review provides published data and the authors' results suggesting that the content of ceramide and sphingosine in the cell is controlled by the tumor necrosis factor alpha and activators of Fas receptor. The results of many experiments confirmed that the sphingomyelin cycle products induce cells death by the apoptotic pathway and enhance induced apoptosis. Ceramide and sphingosine regulate the activity of enzymes involved in transduction of apoptosis signal (protein kinases. Phosphatases, and proteases) and act as second messengers in transduction of the apoptosis signal.

Efficient factor VIII affinity purification using a small synthetic ligand.

BACKGROUND: Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. OBJECTIVE: Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. METHODS: Peptide ligands were screened for binding of (125)I-plasma-derived-FVIII (pdFVIII) in a microbead assay. A selected ligand-coated Toyopearl resin was then used for pdFVIII purification from cell-conditioned Delbucco's modified Eagle's medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV-MZ-6 cells was assayed. RESULTS: A high-affinity octapeptidic FVIII ligand was modified into the small, highly stable and non-toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4-coated Toyopearl resin, pdFVIII was isolated from cell-conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6 m NaCl at pH 6.8. CONCLUSIONS: Ligand L4 offers a valuable alternative to antibody-based procedures for laboratory and industrial production. Its synthesis by established solid-phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.

Two types of projection neurons in human striatum: peculiarities of their somatodendritic structure in ventral and dorsal striatum.

The somatodendritic structure of projection neurons was morphometrically examined in the nucleus accumbens of human brain. In contrast to reticular neurons, spiny neurons of the nucleus accumbens and dorsal striatum have different somatodendritic structure. In both parts of the striatum, reticular neurons were NADPH-diaphorase-positive.

[Formation of spinal cord cicatrix under various experimental conditions]. / Formirovanie mozgovogo rubtsa v razlichnykh éksperimental'nykh usloviiakh.

Structure of the cat spinal cord scar has been studied by means of light and electron microscopy after its lateral hemisection, complete dissection and hemisection in combination with autotransplantation of the sympathetic ganglion, which keeps its connection with the sympathetic trunk, into the cut of the spinal cord. Three zones are revealed in the scar: central (connective tissue), intermediate (glio-connective tissue) and peripheral (zone of glio-cystous and reactive changes of the nervous tissue). Peculiarities of intercellular reactions are revealed in the process of formation of various zones in the scar and their dependence on the type of the experiment. In the experiments with autotransplantation of the sympathetic ganglion into the spinal cord, a definite possibility to restrict scarry changes of the spinal cord is demonstrated in connection with improving reinnervation and revascularization of the traumatized segment.

[Reparative regeneration of cat spinal cord nerve fibers]. / Reparativnaia regeneratsiia nervnykh volokon spinnogo mozga koshki.

Morphology of the regenerative process of certain neural elements at the place where the medullar scar was forming after a lateral hemisection was studied in the cat spinal cord at the level of the 11th thoracic segment. The observations were performed for 3 days--1 year. Methods of light and electron microscopy were applied. It was states that most of conductive tracts are capable for regeneration, nevertheless, the restoration of their integrity does not occur, since regenerating fibres have a marked tendency to form collaterals of the terminal type. The regeneration is accompanied by the formation of axodendritic and axo-axonic synapses whose role is not clear. The neural fibres regeneration is completed for 1 month on the background of a decreased neuroglial reactivity and contributes to its activation and purposeful proliferation which prevents the connective tissue growth. The neural fibres and terminals forming in the regenerative process are preserved for a year without any definite sign of degeneration if a well pronounced glial membrane is present.

Effect of bilirubin on lipid peroxidation, sphingomyelinase activity, and apoptosis induced by sphingosine and UV irradiation.

The effect of bilirubin (BR) on sphingomyelin cycle activity, lipid peroxidation (LPO), and apoptosis induced by sphingosine and UV irradiation has been studied in vivo. Neutral Mg(2+)-dependent sphingomyelinase (SMase) activity and LPO level were monitored in heart, kidney, and liver of mice after administration of BR. BR inhibited both LPO and SMase activities in heart and kidney. BR induced a mild increase in LPO level and moderate increase in lipid contents in liver, consistent with the functional role of liver in both BR and lipid metabolism. BR injected to mice causes simultaneous and unidirectional alterations in both LPO level and SMase activity with a significant (p < 0.05) positive linear correlation between these two parameters. Sphingosine administration results in increased lipid peroxidation in murine liver. Data on DNA fragmentation indicate that exogenous BR may effectively protect thymus cells against sphingosine- and UV-mediated apoptosis. These results have revealed a biochemical association between oxidative stress and BR on one hand and the sphingomyelin cycle and apoptotic cell death on the other hand. Our data show that BR as an antioxidant, due to its effect on the sphingomyelin cycle, can protect membrane lipids against peroxidation and cells against apoptosis induced by various factors.

[Modulation of the level of sphingomyelin cycle products and expression of the CD3 receptor in immunocompetent organs by fumonisin B1]. / Moduliatsiia fumonizinom B1 soderzhaniia produktov sfingomielinovogo tsikla i ékspressii retseptora CD3 v immunokompetentnykh organakh.

The fungi (Fusarium moniliforme) residing on cereals produce a broad range of mycotoxins, among which fumonisins display a high toxicity alongside with carcinogenic and teratogenic activities. Taking into account the ability of fumonisins to inhibit sphingolipid synthesis, the role of sphingomyelin cycle products in immune reactions was studied with the view of establishing the correlation between the expression of the surface receptor CD3 in immunocompetent organs (spleen, thymus) (T-cell mediated immunity) and the degree of sphingomyelin cycle activation (changes in the activities of sphingomyelinase and sphingomyelin and ceramide content) in the spleen, thymus and liver 2.5 hours after intraperitoneal injection of fumonisin B1 (FB1) (5 and 20 micrograms/animal). Significant sphingomyelinase activation was found in the thymus of animals injected with 20 micrograms of fumonisin. It coincides with a loss of the sphingomyelin and ceramide content. The changes in the sphingomyelinase activity and sphingomyelin content in the spleen and in the liver caused by fumonisin were insignificant, while the ceramide content dropped drastically. Fumonisin decreased the receptor CD3 expression on the surface of thymus cells "in vitro" and "in vivo", which is consistent with the sharp decrease of the ceramide content in this organ. Ceramide accumulation in thymus and spleen cells treated with sphingomyelinase in vitro correlates with the increased affinity of receptor CD3. The putative role of ceramide in the expression of receptors modulating T-cell mediated immunity under the influence of fumonisin is discussed.

Relation of biological activity of mutant forms of recombinant human tumor necrosis factor alpha and accumulation of sphingosine in murine liver.

TNF-alpha induced sphingomyelin hydrolysis by sphingomyelinase and both sphingosine and ceramide generation have been reported to be implicated in a number of TNF-alpha responses, including cytotoxicity and apoptosis. We found that sphingosine, a highly cytotoxic product of enzymatic degradation of sphingomyelin, is accumulated in liver of mice treated with TNF-alpha. To determine the role of sphingosine in TNF-alpha toxicity, TNF-alpha mutants differing in their cytotoxicity to L929 cells as well as haemorrhagic tumor necrosis, tumor regression and lethal toxicity in mice were used in our experiments. The mutants with highest toxicity and tumor-necrotizing activity caused accumulation of sphingosine exceeded its control level 5,5 times in murine liver cells. TNF-alpha variants which caused moderate increase in sphingosine content were significantly less toxic. The observed relationship between toxicity of TNF-alpha mutants, the toxicity of sphingosine, and the extent of its accumulation in murine liver provides evidence to suggest that this sphingomyelin metabolite may be mediator of TNF-alpha-induced cell damage and death.

Haemophilia A: effects of inhibitory antibodies on factor VIII functional interactions and approaches to prevent their action.

Factor VIII (FVIII) is an essential component of the intrinsic pathway of blood coagulation. Normal functioning of FVIII requires its interactions with other components of the coagulation cascade. In the circulation, it exists as a complex with von Willebrand factor (vWF). Upon activation by thrombin or activated factor X (FXa), activated FVIII (FVIIIa) functions as a cofactor for the serine protease factor IXa. Their complex assembled on the phospholipid surface activates FX to FXa, which consequently participates in formation of thrombin, the key protease of the coagulation cascade. Genetic deficiency in FVIII results in a coagulation disorder haemophilia A, which is treated by infusions of FVIII products. Approximately 25-30% of patients develop antibodies inhibiting FVIII activity (FVIII inhibitors). The major epitopes of inhibitors are located within the A2, C2 and A3 domains of the FVIII molecule. The inhibitory effects of antibodies are manifested at various stages of the FVIII functional pathway, including FVIII binding to vWF, activation of FVIII by thrombin, and FVIIIa incorporation into the Xase complex. We summarize the current knowledge of the FVIII sites involved in interaction with its physiological ligands and different classes of inhibitory antibodies and describe their inhibitory mechanisms. We outline the strategies aimed to overcome the effects of inhibitory antibodies such as development of human/porcine FVIII molecules, resistant to inhibitors. We also discuss approaches to modulate the antibody response, as well as efforts to develop a long-term immunotolerance to FVIII protein.

Role of endogenous TNF-alpha and sphingosine in induced DNA synthesis in regenerating rat liver after partial hepatectomy.

Cytokine-stimulated metabolism of sphingomyelin results in the accumulation of ceramide and sphingosine which play a part in the regulation of cell proliferation, differentiation, and reception, as well as in oncogenesis. Formation of TNF-alpha (a member of the cytokine family), accumulation of sphingosine, and DNA synthesis (measured by immunoblotting, HPLC, and [3H]thymidine incorporation, respectively) were studied in rat liver after partial hepatectomy. The content of TNF-alpha was found to increase during 12 h following hepatectomy. The maximum of sphingomyelinase activity and accumulation of sphingosine precede the maximum of DNA synthesis. Sphingosine is known to inhibit protein kinase C. On the other hand, it stimulates the metabolism of phosphatidylinositol, thus causing accumulation of diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate protein kinase C. Hence, the release of TNF-alpha in regenerating liver may modulate DNA synthesis through the accumulation of sphingosine which is involved in regulation of protein kinase C activity and of phosphatidylinositol turnover.