Melanocortin Derivatives Induced Vascularization and Neuroglial Proliferation in the Rat Brain under Conditions of Cerebral Ischemia.

Stroke remains the second leading cause of death worldwide. The development of new therapeutic agents focused on restoring vascular function and neuroprotection of viable tissues is required. In this study the neuroprotective activity of melanocortin-like ACTH(4-7)PGP and ACTH(6-9)PGP peptides was investigated in rat brain at 24 h after transient middle cerebral artery occlusion (tMCAO). The severity of ischemic damage, changes in the proliferative activity of neuroglial cells and vascularization of rat brain tissue were analyzed. The administration of peptides resulted in a significant increase in the volume density of neurons in the perifocal zone of infarction compared to rats subjected to ischemia and receiving saline. Immunohistochemical analysis of the proliferative activity of neuroglia cells using PCNA antibodies showed a significant increase in the number of proliferating cells in the penumbra and in the intact cerebral cortex of rats receiving peptide treatment. The effect of peptides on vascularization was examined using CD31 antibodies under tMCAO conditions, revealing a significant increase in the volume density of vessels and their sizes in the penumbra after administration of ACTH(4-7)PGP and ACTH(6-9)PGP. These findings confirm the neuroprotective effect of peptides due to the activation of neuroglia proliferation and the enhancement of collateral blood flow.

The Ability of the Nitric Oxide Synthases Inhibitor T1023 to Selectively Protect the Non-Malignant Tissues.

Previously, we showed that a nitric oxide synthase (NOS) inhibitor, compound T1023, induces transient hypoxia and prevents acute radiation syndrome (ARS) in mice. Significant efficacy (according to various tests, dose modifying factor (DMF)-1.6-1.9 against H-ARS/G-ARS) and safety in radioprotective doses (1/5-1/4 LD10) became the reason for testing its ability to prevent complications of tumor radiation therapy (RT). Research methods included studying T1023 effects on skin acute radiation reactions (RSR) in rats and mice without tumors and in tumor-bearing animals. The effects were evaluated using clinical, morphological and histological techniques as well as RTOG classification. T1023 administration prior to irradiation significantly limited the severity of acute RSR. This was due to a decrease in radiation alteration of the skin and underlying tissues, and the preservation of the functional activity of cell populations that are critical in the pathogenesis of radiation burn. The DMF values for T1023 for skin protection were 1.4-1.7. Moreover, its radioprotective effect was fully selective to normal tissues in RT models of solid tumors-T1023 reduced the severity of acute RSR and did not modify the antitumor effects of γ-radiation. The results indicate that T1023 can selectively protect the non-malignant tissues against γ-radiation due to hypoxic mechanism of action and potentiate opportunities of NOS inhibitors in RT complications prevention.

Brain Protein Expression Profile Confirms the Protective Effect of the ACTH(4-7)PGP Peptide (Semax) in a Rat Model of Cerebral Ischemia-Reperfusion.

The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

Novel Insights into the Protective Properties of ACTH(4-7)PGP (Semax) Peptide at the Transcriptome Level Following Cerebral Ischaemia-Reperfusion in Rats.

Cerebral ischaemia is the most common cause of impaired brain function. Biologically active peptides represent potential drugs for reducing the damage that occurs after ischaemia. The synthetic melanocortin derivative, ACTH(4-7)PGP (Semax), has been used successfully in the treatment of patients with severe impairment of cerebral blood circulation. However, its molecular mechanisms of action within the brain are not yet fully understood. Previously, we used the transient middle cerebral artery occlusion (tMCAO) model to study the damaging effects of ischaemia-reperfusion on the brain transcriptome in rats. Here, using RNA-Seq analysis, we investigated the protective properties of the Semax peptide at the transcriptome level under tMCAO conditions. We have identified 394 differentially expressed genes (DEGs) (>1.5-fold change) in the brains of rats at 24 h after tMCAO treated with Semax relative to saline. Following tMCAO, we found that Semax suppressed the expression of genes related to inflammatory processes and activated the expression of genes related to neurotransmission. In contrast, ischaemia-reperfusion alone activated the expression of inflammation-related genes and suppressed the expression of neurotransmission-related genes. Therefore, the neuroprotective action of Semax may be associated with a compensation of mRNA expression patterns that are disrupted during ischaemia-reperfusion conditions.

Genome-wide transcriptome analysis using RNA-Seq reveals a large number of differentially expressed genes in a transient MCAO rat model.

BACKGROUND: The transient middle cerebral artery occlusion (tMCAO) model is used for studying the molecular mechanisms of ischemic damage and neuroprotection. Numerous studies have demonstrated the role of individual genes and associated signaling pathways in the pathogenesis of ischemic stroke. Here, the tMCAO model was used to investigate the genome-wide response of the transcriptome of rat brain tissues to the damaging effect of ischemia and subsequent reperfusion. RESULTS: Magnetic resonance imaging and histological examination showed that the model of focal ischemia based on endovascular occlusion of the right middle cerebral artery for 90 min using a monofilament, followed by restoration of the blood flow, led to reproducible localization of ischemic damage in the subcortical structures of the brain. High-throughput RNA sequencing (RNA-Seq) revealed the presence of differentially expressed genes (DEGs) in subcortical structures of rat brains resulting from hemisphere damage by ischemia after tMCAO, as well as in the corresponding parts of the brains of sham-operated animals. Real-time reverse transcription polymerase chain reaction expression analysis of 20 genes confirmed the RNA-Seq results. We identified 469 and 1939 genes that exhibited changes in expression of > 1.5-fold at 4.5 and 24 h after tMCAO, respectively. Interestingly, we found 2741 and 752 DEGs under ischemia-reperfusion and sham-operation conditions at 24 h vs. 4.5 h after tMCAO, respectively. The activation of a large number of genes involved in inflammatory, immune and stress responses, apoptosis, ribosome function, DNA replication and other processes was observed in ischemia-reperfusion conditions. Simultaneously, massive down-regulation of the mRNA levels of genes involved in the functioning of neurotransmitter systems was recorded. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that dozens of signaling pathways were associated with DEGs in ischemia-reperfusion conditions. CONCLUSIONS: The data obtained revealed a global profile of gene expression in the rat brain sub-cortex under tMCAO conditions that can be used to identify potential therapeutic targets in the development of new strategies for the prevention and treatment of ischemic stroke.

Synthesis and Investigation of Photophysical and Biological Properties of Novel S-Containing Bacteriopurpurinimides.

Novel hybrid molecule containing 2-mercaptoethylamine was synthesized starting from O-propyloxime-N-propoxy bacteriopurpurinimide (dipropoxy-BPI), which was readily oxidized in oxygen atmosphere yielding the corresponding disulfide analogue (disulfide-BPI). Spectral, photophysical, photodynamic, and biological properties of compound were properly evaluated. Compounds bearing disulfide moiety can directly interact with glutathione (GSH), thereby reducing its intracellular concentration. Indeed, mice sarcoma S37 cell line was treated in vitro with disulfide-BPI, yielding a CC50 value of 0.05 ± 0.005 µM. A relatively high level of singlet oxygen was detected. It was demonstrated (by fluorescence) that the PS was rapidly accumulated in a cancer nest (S37) at a relatively high level after 2 h upon intravenous administration. After 24 h, no traces of the molecule were detected in the tumor mass. Moreover, high photodynamic efficiency was demonstrated at doses of 150-300 J/cm2 against two different in vivo tumor models, achieving 100% regression of cancer growth.

The effect of Semax and its C-end peptide PGP on the morphology and proliferative activity of rat brain cells during experimental ischemia: a pilot study.

The neuropeptide preparation Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has been employed successfully in clinical practice for treating patients with severe brain blood circulation disorders. In spite of numerous studies, many aspects of the therapeutic effects of this preparation remain unknown. In this context, the effects of Semax and its C-end tripeptide PGP on the functional morphology of nervous tissue cells were studied in the normal rat brain and in a model of incomplete global rat brain ischemia. In control animals, both peptides activated the capillary network and caused similar morphological changes to neurons and the neuropil regions. We show here for the first time at the histological level that Semax and PGP increased proliferation of the neuroglia, blood vessel endothelium, and progenitor cells in the subventricular zone. In these experimental conditions, only Semax abated the manifestation of ischemic damage to the nervous tissue. This was probably attributable to a decrease in vascular stasis symptoms as well as the trophic effect of the peptide.

Expression of sphingomyelin synthase 1 gene in rat brain focal ischemia.

Metabolites of the sphingomyelin cycle are reported to play an important role in neuronal death after ischemia. To elucidate the involvement of the key enzyme of this cycle, sphingomyelin synthase (SMS), in the mechanism underlying cerebral ischemia, we, for the first time, investigated changes in the mRNA expression of the SMS1 gene in rats after focal cerebral ischemia. According to our histological analysis, the damaged area is localized only in the ipsilateral cortex. In the ischemic cortex, the level of SMS1 transcripts was decreased at 3 and 24 h after occlusion, and at 72 h it had returned to the control level. A reduced level of SMS1 mRNA expression in the subcortex of rats with occlusion and sham-operated animals also was appeared during the first 24 h after surgery. This could be attributed to the effect of surgical stress. Seventy-two hours after occlusion, SMS1 mRNA expression in subcortex of ischemic rats was still at a decreased level; this may be considered to be a somewhat distant extended effect. Our results show the early response of the SMS1 gene that can be induced by both ischemia and stress. The results also suggest that inhibition of SMS1 mRNA expression may contribute to ceramide accumulation in a damaged cortex.

Hormonal function and proliferative activity of thymic cells in humans: immunocytochemical correlations.

OBJECTIVES: The immunocytochemical study of the localization of hormones in thymic cells has been performed to clarify possible correlations of their expression with proliferative activity of thymocytes. METHODS: We used commercial antibodies to serotonin, melatonin, somatostatin, glucagon, gastrin, beta-endorphin and histamine, and ABP or BSP kits for visualization of reaction. Computer image analysis was used to find correlations between hormone production and proliferative activity of thymocytes. RESULTS: Different subpopulations of thymocytes are able to produce hormones: precursors of T-lymphocytes (CD4-CD8-) contain serotonin and melatonin, immature cortical cells (CD4+CD8+) produce only serotonin, mature medullar cells (CD4+CD8-) show immunoreactivity to serotonin, melatonin, beta-endorphin and histamine. The expression of serotonin, somatostatin and gastrin is localized in thymic epithelial cells, formatting Gassal's bodies. Proliferative activity of thymocytes depends from the expression of serotonin and somatostatin in thymic cells. CONCLUSION: The data received testify the expression of different hormones in human thymic cells and showing by this fact high endocrine activity of thymus. The presence of correlations between hormonal expression and cell proliferative activity could be considered as the bright illustration of important role of neuroimmunoendocrine mechanisms in the regulation of local thymic homeostasis.

Gastrointestinal melatonin: cellular identification and biological role.

Melatonin, a pineal hormone, because of its wide activity spectrum, is a subject of much current interest for biologists and physicians. It has been demonstrated that pineal gland is not an exclusive source of melatonin synthesis. Melatonin synthesis has been found in different sites of the organism, and a major source of extrapineal melatonin is the gastrointestinal tract. The role of melatonin in gastrointestinal functions is considered in the present review.

Tau-protein expression in human blood lymphocytes: a promising marker and suitable sample for life-time diagnosis of Alzheimer's disease.

OBJECTIVES: Taking into account the hypothesis that Alzheimer's disease (AD) might be a systemic disease that affects several tissues in the body, the aim of this study was to try to detect the expression of tau-protein in human peripheral blood lymphocytes (PBL) in patients with AD. MATERIAL AND METHODS: Blood samples were obtained from patients with AD (n=16, age 67-98) and from volunteers without psychoneurological pathology (n=10, age 65-78). PBL were isolated on Ficoll-Paque gradient centrifugation. For cell fixation and permeabilization we used a fixative solution (4% formaldehyde and 0.1% glutaraldehyde) and 0.03% Triton X-100. Immunocytochemical detection of tau-protein was carried out by biotin-streptavidin complex method with tau monoclonal antibody (1:100, clone TAU-2, ICN) and universal immunostaining kit IMMU-MARK (ICN). RESULTS: The expression of tau-protein was shown in PBL in absolute majority of AD patients studied. Only in two healthy volunteers a single lymphocyte from many cells (i.e. a smear) demonstrated a very weak-positive immunostaining to tau-protein CONCLUSION: This first demonstration of clear difference in localization of tau-protein in blood lymphocytes between healthy and sick people testifies to the fact that tau-protein could be considered as a promising marker and blood lymphocytes as a suitable sample for life-time diagnosis of AD.

Pattern selection during electropolishing due to double-layer effects.

We extend our earlier study of nanoscale pattern formation during electropolishing [Nanotechnology 7, 360 (1996); Phys. Rev. B 56, 12 608 (1997)]. The patterns are attributed to preferential adsorption of organic molecules on the convex portion of the electrode due to its enhanced electric field. This local enhancement occurs because of the effect of surface curvature on the double-layer potential drop. By allowing for transport correction to the double-layer potential drop at thermodynamic equilibrium, we estimate this anodic overpotential to be in the realistic mV range and hence verify the Debye-Huckel approximation used in our model. This small anodic overpotential suggests that pattern formation is a generic electropolishing phenomenon whose only requirement is that the polarizability of the organic additive relative to water must lie within a range specified by our theory. We verify this prediction experimentally with a variety of electrolyte solutions. The voltage ranges for specific hexagonal and ridge patterns are well correlated by our model with only a single parameter. (c) 1999 American Institute of Physics.