Comparative Evaluation of the Antibacterial and Antitumor Activities of 9-Phenylfascaplysin and Its Analogs.

Based on the results of our own preliminary studies, the derivative of the marine alkaloid fascaplysin containing a phenyl substituent at C-9 was selected to evaluate the therapeutic potential in vivo and in vitro. It was shown that this compound has outstandingly high antimicrobial activity against Gram-positive bacteria, including antibiotic-resistant strains in vitro. The presence of a substituent at C-9 of the framework is of fundamental importance, since its replacement to neighboring positions leads to a sharp decrease in the selectivity of the antibacterial action, which indicates the presence of a specific therapeutic target in bacterial cells. On a model of the acute bacterial sepsis in mice, it was shown that the lead compound was more effective than the reference antibiotic vancomycin seven out of nine times. However, ED50 value for 9-phenylfascaplysin (7) was similar for the unsubstituted fascaplysin (1) in vivo, despite the former being significantly more active than the latter in vitro. Similarly, assessments of the anticancer activity of compound 7 against various variants of Ehrlich carcinoma in mice demonstrated its substantial efficacy. To conduct a structure-activity relationship (SAR) analysis and searches of new candidate compounds, we synthesized a series of analogs of 9-phenylfascaplysin with varying aryl substituents. However, these modifications led to the reduced aqueous solubility of fascaplysin derivatives or caused a loss of their antibacterial activity. As a result, further research is required to explore new avenues for enhancing its pharmacokinetic characteristics, the modification of the heterocyclic framework, and optimizing of treatment regimens to harness the remarkable antimicrobial potential of fascaplysin for practical usage.

Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity.

Serine proteases regulate cell functions through G protein-coupled protease-activated receptors (PARs). Cleavage of one peptide bond of the receptor amino terminus results in the formation of a new N-terminus ("tethered ligand") that can specifically interact with the second extracellular loop of the PAR receptor and activate it. Activation of PAR1 by thrombin (canonical agonist) and activated protein C (APC, noncanonical agonist) was described as a biased agonism. Here, we have supposed that synthetic peptide analogs to the PAR1 tethered ligand liberated by APC could have neuroprotective effects like APC. To verify this hypothesis, a model of the ischemic brain impairment based on glutamate (Glu) excitotoxicity in primary neuronal cultures of neonatal rats has been used. It was shown that the nanopeptide NPNDKYEPF-NH2 (AP9) effectively reduced the neuronal death induced by Glu. The influence of AP9 on cell survival was comparable to that of APC. Both APC and AP9 reduced the dysregulation of intracellular calcium homeostasis in cultured neurons induced by excitotoxic Glu (100 µM) or NMDA (200 µM) concentrations. PAR1 agonist synthetic peptides might be noncanonical PAR1 agonists and a basis for novel neuroprotective drugs for disorders related to Glu excitotoxicity such as brain ischemia, trauma and some neurodegenerative diseases.

A New Mild Method for Synthesis of Marine Alkaloid Fascaplysin and Its Therapeutically Promising Derivatives.

Fascaplysin is a marine alkaloid which is considered to be a lead drug candidate due to its diverse and potent biological activity. As an anticancer agent, fascaplysin holds a great potential due to the multiple targets affected by this alkaloid in cancer cells, including inhibition of cyclin-dependent kinase 4 (CDK4) and induction of intrinsic apoptosis. At the same time, the studies on structural optimization are hampered by its rather high toxicity, mainly caused by DNA intercalation. In addition, the number of methods for the syntheses of its derivatives is limited. In the current study, we report a new two-step method of synthesis of fascaplysin derivatives based on low temperature UV quaternization for the synthesis of thermolabile 9-benzyloxyfascaplysin and 6-tert-butylfascaplysin. 9-Benzyloxyfascaplysin was used as the starting compound to obtain 9-hydroxyfascaplysin. However, the latter was found to be chemically highly unstable. 6-tert-Butylfascaplysin revealed a significant decrease in DNA intercalation when compared to fascaplysin, while cytotoxicity was only slightly reduced. Therefore, the impact of DNA intercalation for the cytotoxic effects of fascaplysin and its derivatives needs to be questioned.

Enriched Environment Induces Sex-Specific Changes in the Adult Neurogenesis, Cytokine and miRNA Expression in Rat Hippocampus.

An enriched environment stimulates adult hippocampal plasticity, but the exact cellular and molecular mechanisms are complex, and thus a matter of debate. We studied the behavior and hippocampal neurogenesis in adult male and female Wistar rats that were housed in an enriched environment (EE) for two months. Both EE males and females performed better than control animals in a Barnes maze, meaning that EE enhances spatial memory. However, the expression levels of neurogenesis markers KI67, DCX, Nestin, and Syn1 increased only in EE females, while in EE males only KI67 and BDNF were higher than in the corresponding control. The number of DCX+ neurons on brain slices increased in the dentate gyrus of EE females only, i.e., the level of adult hippocampal neurogenesis was increased in female but not in male rats. The level of anti-inflammatory IL-10 and signaling pathway components was upregulated in EE females. Of 84 miRNAs tested, in the hippocampi of EE female rats we detected upregulation in the expression levels of 12 miRNAs related to neuronal differentiation and morphogenesis, while in EE males four miRNAs were upregulated and involved in the regulation of cell proliferation/differentiation, and one was downregulated and associated with the stimulation of proliferation. Taken altogether, our results point to sex-specific differences in adult hippocampal plasticity, IL-10 expression, and miRNA profiles induced by an enriched environment.

Exogenous GalR2-specific peptide agonist as a tool for treating myocardial ischemia/reperfusion injury.

OBJECTIVES: The aim of this work was to elucidate the role of GalR2 receptor activation in protecting the rat heart in vivo from ischemia/reperfusion (I/R) damage by a pharmacological peptide agonist WTLNSAGYLLGPßAH-OH (G1) and full-length rat galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2 (G2) using M871, a selective inhibitor of GalR2. METHODS: The peptides were prepared by the automatic solid-phase synthesis using the Fmoc-strategy and purified by high-performance liquid chromatography (HPLC). A 40-min left anterior descending (LAD) coronary artery occlusion followed by a 60-min reperfusion was performed. The criteria for damage/protection of the heart were the infarct size (IS) and plasma activity of creatine kinase-MB (CK-MB) at the end of reperfusion. RESULTS: Intravenous injection of G1 or G2 at an optimal dose of 1 mg/kg at the fifth minute of reperfusion significantly reduced the IS (by 35% and 32%, respectively) and activity of CK-MB at the end of reperfusion (by 43% and 38%, respectively) compared with the control. Administration of M871 (8 mg/kg) 5 min before the onset of reperfusion abolished the effects of G1 on IS and CK-MB activity, returning them to control values. Co-administration of M871 (8 mg/kg) with G2 attenuated protective effect of G2 on both IS and plasma СK-MB activity. However, differences in these parameters between the M871+G2 and G2 groups did not reach statistical significance (P = 0.139 and P = 0.121, respectively). CONCLUSION: Thus, GalR2 is the principal receptor subtype that transduces the protective effects of galanin and ligand G1 in myocardial I/R injury. This suggests that GalR2-specific peptide agonists could be used as drug candidates for treating ischemic heart disease.

Single Prolonged Stress Decreases the Level of Adult Hippocampal Neurogenesis in C57BL/6, but Not in House Mice.

Many people experience traumatic events during their lives, but not all of them develop severe mental pathologies, characterized by high levels of anxiety that persists for more than a month after psychological trauma, such as posttraumatic stress disorder (PTSD). We used a single prolonged stress protocol in order to model PTSD in long-inbred C57BL/6 and wild-derived (house) female mice. The susceptibility of mice to single prolonged stress was assessed by behavior phenotyping in the Open Field and Elevated Plus Maze, the level of neuroinflammation in the hippocampus was estimated by real-time PCR to TNFα, IL-1ß, IL-6, IL-10, Iba1 and GFAP, as well as immunohistochemical analysis of microglial morphology and mean fluorescence intensity for GFAP+ cells. The level of neurogenesis was analyzed by real-time PCR to Ki67, Sox2 and DCX as well as immunohistochemistry to Ki67. We showed that long-inbread C57BL/6 mice are more susceptible to a single prolonged stress protocol compared to wild-derived (house) mice. Stressed C57BL/6 mice demonstrated elevated expression levels of proinflammatory cytokines TNFα, IL-1ß, and IL-6 in the hippocampus, while in house mice no differences in cytokine expression were detected. Expression levels of Iba1 in the hippocampus did not change significantly after single prolonged stress, however GFAP expression increased substantially in stressed C57BL/6 mice. The number of Iba+ cells in the dentate gyrus also did not change after stress, but the morphology of Iba+ microglia in C57BL/6 animals allowed us to suggest that it was activated; house mice also had significantly more microglia than C57BL/6 animals. We suppose that decreased microglia levels in the hippocampus of C57BL/6 compared to house mice might be one of the reasons for their sensitivity to a single prolonged stress. Single prolonged stress reduced the number of Ki67+ proliferating cells in the dentate gyrus of the hippocampus but only in C57BL/6 mice, not in house mice, with the majority of cells detected in the dorsal (septal) hippocampus in both. The increase in the expression level of DCX might be a compensatory reaction to stress; however, it does not necessarily mean that these immature neurons will be functionally integrated, and this issue needs to be investigated further.

Chimeric Agonist of Galanin Receptor GALR2 Reduces Heart Damage in Rats with Streptozotocin-Induced Diabetes.

Neuropeptide galanin and its N-terminal fragments reduce the generation of reactive oxygen species and normalize metabolic and antioxidant states of myocardium in experimental cardiomyopathy and ischemia/reperfusion injury. The aim of this study was to elucidate the effect of WTLNSAGYLLGPßAH-OH (peptide G), a pharmacological agonist of the galanin receptor GalR2, on the cardiac injury induced by administration of streptozotocin (STZ) in rats. Peptide G was prepared by solid phase peptide synthesis using the Fmoc strategy and purified by preparative HPLC; its structure was confirmed by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. Experimental animals were randomly distributed into five groups: C, control; S, STZ-treated; SG10, STZ + peptide G (10 nmol/kg/day); SG50, STZ + peptide G (50 nmol/kg/day); G, peptide G (50 nmol/kg/day). Administration of peptide G prevented hyperglycemia in SG50 rats. By the end of the experiment, the ATP content, total pool of adenine nucleotides, phosphocreatine (PCr) content, and PCr/ATP ratio in the myocardium of animals of the SG50 group were significantly higher than in rats of the S group. In the SG50 and SG10 groups, the content of lactate and lactate/pyruvate ratio in the myocardium were reduced, while the glucose content was increased vs. the S group. Both doses of peptide G reduced the activation of creatine kinase-MB and lactate dehydrogenase, as well as the concentration of thiobarbituric acid reactive products in the blood plasma of STZ-treated rats to the control values. Taken together, these results suggest that peptide G has cardioprotective properties in type 1 diabetes mellitus. Possible mechanisms of peptide G action in the STZ-induced diabetes are discussed.

Study of Structure-Activity Relationships of the Marine Alkaloid Fascaplysin and Its Derivatives as Potent Anticancer Agents.

Marine alkaloid fascaplysin and its derivatives are known to exhibit promising anticancer properties in vitro and in vivo. However, toxicity of these molecules to non-cancer cells was identified as a main limitation for their clinical use. Here, for the very first time, we synthesized a library of fascaplysin derivatives covering all possible substituent introduction sites, i.e., cycles A, C and E of the 12H-pyrido[1-2-a:3,4-b']diindole system. Their selectivity towards human prostate cancer versus non-cancer cells, as well as the effects on cellular metabolism, membrane integrity, cell cycle progression, apoptosis induction and their ability to intercalate into DNA were investigated. A pronounced selectivity for cancer cells was observed for the family of di- and trisubstituted halogen derivatives (modification of cycles A and E), while a modification of cycle C resulted in a stronger activity in therapy-resistant PC-3 cells. Among others, 3,10-dibromofascaplysin exhibited the highest selectivity, presumably due to the cytostatic effects executed via the targeting of cellular metabolism. Moreover, an introduction of radical substituents at C-9, C-10 or C-10 plus C-3 resulted in a notable reduction in DNA intercalating activity and improved selectivity. Taken together, our research contributes to understanding the structure-activity relationships of fascaplysin alkaloids and defines further directions of the structural optimization.

212Pb: Production Approaches and Targeted Therapy Applications.

Over the last decade, targeted alpha therapy has demonstrated its high effectiveness in treating various oncological diseases. Lead-212, with a convenient half-life of 10.64 h, and daughter alpha-emitter short-lived 212Bi (T1/2 = 1 h), provides the possibility for the synthesis and purification of complex radiopharmaceuticals with minimum loss of radioactivity during preparation. As a benefit for clinical implementation, it can be milked from a radionuclide generator in different ways. The main approaches applied for these purposes are considered and described in this review, including chromatographic, solution, and other techniques to isolate 212Pb from its parent radionuclide. Furthermore, molecules used for lead's binding and radiochemical features of preparation and stability of compounds labeled with 212Pb are discussed. The results of preclinical studies with an estimation of therapeutic and tolerant doses as well as recently initiated clinical trials of targeted radiopharmaceuticals are presented.

Enduring Effects of Conditional Brain Serotonin Knockdown, Followed by Recovery, on Adult Rat Neurogenesis and Behavior.

Serotonin (5-hydroxytryptamine, 5-HT) is a crucial signal in the neurogenic niche of the hippocampus, where it is involved in antidepressant action. Here, we utilized a new transgenic rat model (TetO-shTPH2), where brain 5-HT levels can be acutely altered based on doxycycline (Dox)-inducible shRNA-expression. On/off stimulations of 5-HT concentrations might uniquely mirror the clinical course of major depression (e.g., relapse after discontinuation of antidepressants) in humans. Specifically, we measured 5-HT levels, and 5-HT metabolite 5-HIAA, in various brain areas following acute tryptophan hydroxylase 2 (Tph2) knockdown, and replenishment, and examined behavior and proliferation and survival of newly generated cells in the dentate gyrus. We found that decreased 5-HT levels in the prefrontal cortex and raphe nuclei, but not in the hippocampus of TetO-shTPH2 rats, lead to an enduring anxious phenotype. Surprisingly, the reduction in 5-HT synthesis is associated with increased numbers of BrdU-labeled cells in the dentate gyrus. At 3 weeks of Tph2 replenishment, 5-HT levels return to baseline and survival of newly generated cells is unaffected. We speculate that the acutely induced decrease in 5-HT concentrations and increased neurogenesis might represent a compensatory mechanism.

New PAR1 Agonist Peptide Demonstrates Protective Action in a Mouse Model of Photothrombosis-Induced Brain Ischemia.

Protease-activated receptors (PARs) are involved not only in hemostasis but also in the development of ischemic brain injury. In the present work, we examined in vivo effects of a new peptide (AP9) composing Asn47-Phen55 of PAR1 "tethered ligand" generated by activated protein C. We chose a mouse model of photothrombosis (PT)-induced ischemia to assess AP9 effects in vivo. To reveal the molecular mechanism of AP9 action, mice lacking ß-arrestin-2 were used. AP9 was injected intravenously once 10 min before PT at doses of 0.2, 2, or 20 mg/kg, or twice, that is, 10 min before and 1 h after PT at a dose of 20 mg/kg. Lesion volume was measured by magnetic resonance imaging and staining of brain sections with tetrazolium salt. Neurologic deficit was estimated using the cylinder and the grid-walk tests. Blood-brain barrier (BBB) disruption was assessed by Evans blue dye extraction. Eosin-hematoxylin staining and immunohistochemical staining were applied to evaluate the number of undamaged neurons and activated glial cells in the penumbra. A single administration of AP9 (20 mg/kg), as well as its two injections (20 mg/kg), decreased brain lesion volume. A double administration of AP9 also reduced BBB disruption and neurological deficit in mice. We did not observe the protective effect of AP9 in mice lacking ß-arrestin-2 after PT. Thus, we demonstrated for the first time protective properties of a PAR1 agonist peptide, AP9, in vivo. ß-Arrestin-2 was required for the protective action of AP9 in PT-induced brain ischemia.

[MeArg1, NLe10]-apelin-12: Optimization of solid-phase synthesis and evaluation of biological properties in vitro and in vivo.

Chemically modified peptide apelin-12 ([MeArg1, NLe10]-apelin12, peptide M) is able to reduce reactive oxygen species (ROS) formation, cell death, and metabolic and ionic homeostasis disorders in experimental myocardial ischemia-reperfusion injury. These beneficial effects indicate the therapeutic potential of this compound in cardiovascular diseases. The goals of this work were to optimize the synthesis of peptide M, and to study its proteolytic stability and effect on the heart function of rabbits with doxorubicin (Dox) cardiomyopathy. We have developed a rational method of solid-phase synthesis of peptide M using the Fmoc methodology in combination with the temporary protection of the guanidine function of arginine residues by protonation (salt formation) during the formation of the amide bond. It avoids the formation of by-products, and simplifies the post-synthetic procedures, providing an increase in the yield of the final product of higher purity. Comparative evaluation of the proteolytic stability of peptide M and apelin-12 in human blood plasma was carried out using 1H NMR spectroscopy. It was shown that the half-life of peptide M in plasma is approximately three times longer than that of apelin-12. Intravenous infusion of increasing doses of peptide M caused a gradual increase in left ventricular (LV) fractional shortening and ejection fraction in rabbits after 8 weeks of Dox administration (2 mg/kg weekly). The effect of the modified peptide on LV systolic dysfunction was significantly more pronounced than the effect of apelin-12, which suggests the promise of using this pharmacological agonist of the APJ receptor in patients with heart failure.

Galanin receptors activation modulates myocardial metabolic and antioxidant responses to ischaemia/reperfusion stress.

The mechanisms of protective action of the neuropeptide galanin and its N-terminal fragments against myocardial ischaemia/reperfusion (I/R) injury remain obscure. The aim of this work was to study effects of a novel peptide agonist of galanin receptors [ßAla14, His15]-galanin (2-15) (G1) and the full-length galanin (G2) on energy and antioxidant status of the heart with acute infarction. The peptides were synthesized by the automatic solid phase method using Fmoc technology. Their structure was identified by 1 H-NMR spectroscopy and MALDI-TOF mass spectrometry. Experiments were performed on anaesthetized open-chest rats subjected to myocardial regional ischaemia and reperfusion. Intravenous (iv) administration of optimal doses of peptides G1 and G2 (1.0 and 0.5 mg/kg, respectively, at the onset of reperfusion significantly reduced infarct size (on average by 40% compared with control) and the plasma activity of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). These effects were associated with augmented preservation of aerobic energy metabolism, increased activity of Cu,Zn superoxide dismutase (Cu,Zn-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and decreased lipid peroxidation in the area at risk (AAR) at the end of reperfusion. Peptide G1 showed more efficient recovery of the majority of metabolic and antioxidant parameters. The results provide evidence that the galaninergic system can be considered a promising target to reduce energy dysregulation and oxidative damage in myocardial I/R injury.

Lack of Brain Serotonin Affects Feeding and Differentiation of Newborn Cells in the Adult Hypothalamus.

Serotonin (5-HT) is a crucial signal in the neurogenic niche microenvironment. Dysregulation of the 5-HT system leads to mood disorders but also to changes in appetite and metabolic rate. Tryptophan hydroxylase 2-deficient (Tph2-/- ) mice depleted of brain 5-HT display alterations in these parameters, e.g., increased food consumption, modest impairment of sleep and respiration accompanied by a less anxious phenotype. The newly discovered neural stem cell niche of the adult hypothalamus has potential implications of mediating stress responses and homeostatic functions. Using Tph2-/- mice, we explore stem cell behavior and cell genesis in the adult hypothalamus. Specifically, we examine precursor cell proliferation and survival in Tph2-/- mice at baseline and following Western-type diet (WD). Our results show a decline in BrdU numbers with aging in the absence of 5-HT. Furthermore, wild type mice under dietary challenge decrease cell proliferation and survival in the hypothalamic niche. In contrast, increased high-calorie food intake by Tph2-/- mice does not come along with alterations in cell numbers. However, lack of brain 5-HT results in a shift of cell phenotypes that was abolished under WD. We conclude that precursor cells in the hypothalamus retain fate plasticity and respond to environmental challenges. A novel link between 5-HT signaling and cell genesis in the hypothalamus could be exploited as therapeutic target in metabolic disease.

Galanin and its N-terminal fragments reduce acute myocardial infarction in rats.

Agonists and antagonists for galanin receptor subtypes GalR1-3 can be used as putative therapeutics targets for the treatment of various human diseases. However, effects of galanin and its N-terminal fragments on myocardial ischemia/reperfusion injury remain unclear. This study was designed to assess the ability of the full-length galanin (GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2, G1), the natural fragments WTLNSAGYLL-NH2 (G2) and WTLNSAGYLLGPHA (G3), and their modified analogs WTLNAAGYLL (G4) and WTLNSAGYLLGPßAH (G5) to limit acute myocardial infarction in rats in vivo. The peptides G2-5 were synthesized by the automatic solid phase method using Fmoc technology, purified by preparative HPLC and identified by 1H NMR spectroscopy and MALDI -TOF mass spectrometry. The peptides G1-5 were administered by i.v. bolus injection at the onset of reperfusion at doses of 0.25, 0.50, 1.0, 2.0 or 3.0 mg/kg. The optimal doses of the peptides G1-5 significantly reduced the infarction area and decreased the activity of CK-MB and LDH in blood plasma at the end of reperfusion compared with the control. Among the peptides studied, G5 showed high efficacy in reducing the infarct size and the activity of necrosis markers in blood plasma with no significant effect on hemodynamic parameters. The results suggest that a novel agonist for galanin receptors G5 may be a promising tool for the treatment of myocardial ischemia/reperfusion (I/R) injury. Further studies are warranted to explore the stability of this peptide in blood plasma and mechanisms that contribute to its cardioprotective effects.

Galanin/GalR1-3 system: A promising therapeutic target for myocardial ischemia/reperfusion injury.

N-terminal fragments of galanin (2-11) and (2-15) are critical for binding to GalR1-3 receptors, members of the G-protein-coupled receptor superfamily, and are involved in myocardial protection against ischemia/reperfusion (I/R) injury. This study was designed to synthesize novel GalR1-3 agonists with improved properties and evaluate their efficiency as cardioprotective agents. Peptide agonists were synthesized by the automatic solid phase method using Fmoc technology and purified by preparative HPLC. Their chemical structure was identified by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. Novel ligands of galanin receptors have greater solubility in water than natural galanin fragments. Cardiac function indices, myocardial infarct size and plasma activity of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were measured to assess the peptide bioactivity. Infusion of optimal concentrations of the peptides (210-240 µM) after global ischemia enhanced functional recovery of isolated rat heart during reperfusion. Intravenous administration of the peptides in a dose range of 1-2 mg/kg at the onset of reperfusion significantly reduced infarct size and plasma levels of CK-MB and LDH in rats in vivo. The chimeric ligand [ßAla14, His15]-galanin (2-15) exhibited the most beneficial effect on both models of I/R injury. The results suggest that pharmacological agonists of GalR1-3 receptors can be a rational basis for drug developments in the field of cardiovascular diseases.

Protective Effects of a Novel Agonist of Galanin Receptors Against Doxorubicin-Induced Cardiotoxicity in Rats.

The clinical use of antineoplastic agent doxorubicin (DOX) is limited due to its cardiotoxic action. [ßAla14, His15]-galanine (2-15) (G) is a novel synthetic agonist of galanin receptors GalR1-3 having cardioprotective properties in animal models in vivo. The aim of the present study was to explore effects of G on DOX-induced cardiotoxicity. Wistar rats were divided into four groups and treated with DOX (D group), DOX and G (D + G group), G (G group), and saline (control). Before treatment and at the end of the study, concentration of thiobarbituric acid reactive substances (TBARS) and activity of creatine kinase-MB (CK-MB) were determined in blood plasma, the animals were weighed, and cardiac function was evaluated by echocardiography. At the end of experiments, the hearts were used to determine energy metabolites and mitochondrial respiration in permeabilized fibers. After an 8-week study, D group exhibited a pronounced cardiac failure, the absence of weight gain, an increased plasma TBARS concentration, and CK-MB activity. These disorders were accompanied by a reduced myocardial content of high-energy phosphates and mitochondrial respiratory parameters. Co-administration of G with DOX significantly decreased plasma TBARS level and prevented an increase in plasma CK-MB activity. In D + G group, myocardial contents of ATP, PCr, total adenine nucleotides, and total creatine as well as myocardial PCr/ATP ratio and the respiratory control index were higher than in D group at the end of the experiments. Peptide G significantly improved parameters of left ventricular (LV) function and caused weight gain in animals of D + G group. These results suggest that peptide G may be a potential pharmacological agent that attenuates the cardiotoxic effects of DOX.

Cardioprotective properties of N-terminal galanin fragment (2-15) in experimental ischemia/reperfusion injury.

BACKGROUND AND PURPOSE: Galanin is an endogenous peptide involved in diverse physiological functions in the central nervous system including central cardiovascular regulation. The present study was designed to evaluate the potential effects of the short N-terminal galanin fragment 2-15 (G) on cardiac ischemia/reperfusion (I/R) injury. EXPERIMENTAL APPROACH: Peptide G was synthesized by the automatic solid phase method and identified by 1H-NMR spectroscopy and mass spectrometry. Experiments were performed on cultured rat cardiomyoblast (H9C2) cells, isolated perfused working rat hearts and anaesthetized open-chest rats. KEY RESULTS: Cell viability increased significantly after treatment with 10 and 50 nM of G peptide. In hypoxia and reoxygenation conditions, exposure of H9C2 cells to G peptide decreased cell apoptosis and mitochondrial reactive oxygen species (ROS) production. Postischemic infusion of G peptide reduced cell membrane damage and improved functional recovery in isolated hearts during reperfusion. These effects were accompanied by enhanced restoration of myocardial metabolic state. Treatment with G peptide at the onset of reperfusion induced minor changes in hemodynamic variables but significantly reduced infarct size and plasma levels of necrosis markers. CONCLUSION AND IMPLICATIONS: These findings suggest that G peptide is effective in mitigating cardiac I/R injury, thereby providing a rationale for promising tool for the treatment of cardiovascular diseases.

Impact of Atherosclerosis- and Diabetes-Related Dicarbonyls on Vascular Endothelial Permeability: A Comparative Assessment.

BACKGROUND: Malondialdehyde (MDA), glyoxal (GO), and methylglyoxal (MGO) levels increase in atherosclerosis and diabetes patients. Recent reports demonstrate that GO and MGO cause vascular endothelial barrier dysfunction whereas no evidence is available for MDA. METHODS: To compare the effects of MDA, GO, or MGO on endothelial permeability, we used human EA.hy926 endothelial cells as a standard model. To study cortical cytoplasm motility and cytoskeletal organization in endothelial cells, we utilized time-lapse microscopy and fluorescent microscopy. To compare dicarbonyl-modified protein band profiles in these cells, we applied Western blotting with antibodies against MDA- or MGO-labelled proteins. RESULTS: MDA (150-250 µM) irreversibly suppressed the endothelial cell barrier, reduced lamellipodial activity, and prevented intercellular contact formation. The motile deficiency of MDA-challenged cells was accompanied by alterations in microtubule and microfilament organization. These detrimental effects were not observed after GO or MGO (250 µM) administration regardless of confirmed modification of cellular proteins by MGO. CONCLUSIONS: Our comparative study demonstrates that MDA is more damaging to the endothelial barrier than GO or MGO. Considering that MDA endogenous levels exceed those of GO or MGO and tend to increase further during lipoperoxidation, it appears important to reduce oxidative stress and, in particular, MDA levels in order to prevent sustained vascular hyperpermeability in atherosclerosis and diabetes patients.

Determination of the equilibrium constant of C60 fullerene binding with drug molecules.

We report a new analytical method that allows the determination of the magnitude of the equilibrium constant of complexation, Kh, of small molecules to C60 fullerene in aqueous solution. The developed method is based on the up-scaled model of C60 fullerene-ligand complexation and contains the full set of equations needed to fit titration datasets arising from different experimental methods (UV-Vis spectroscopy, 1H NMR spectroscopy, diffusion ordered NMR spectroscopy, DLS). The up-scaled model takes into consideration the specificity of C60 fullerene aggregation in aqueous solution and allows the highly dispersed nature of C60 fullerene cluster distribution to be accounted for. It also takes into consideration the complexity of fullerene-ligand dynamic equilibrium in solution, formed by various types of self- and hetero-complexes. These features make the suggested method superior to standard Langmuir-type analysis, the approach used to date for obtaining quantitative information on ligand binding with different nanoparticles.