The action of timoptin peptides on the hemostatic system

Repeated introduction of thymus polypeptide drug thymoptine (8 times every 24 hours, 1 microgram/kg) led to the increase in total and non-enzymatic fibrinolytic activity of blood plasma and the decrease in fibrinogen concentration. Thymoptine could lyse non-stabilized fibrin and cause depolymerization of aggregates of fibrin monomer in virto. Highest depolymerization activity was observed at thymoptine concentration of 10 micrograms/ml.     

Electron microscopic study of the depolymerization process on unstabilized fibrin as affected by urea or monochloracetic acid

With the help of the electronmicroscopic method the non-stabilized fibrin depolymerization has been studied. It has been established that under the action of urea or monochloridacetic acid the gradual transition of fibril clot's structure in globular fibrinolike material takes place. These globules have morphological likeness with the fibrin-monomer molecules and have analogy of the morphological properties to the non-stabilized fibrin dissolution products by complex compounds of heparin. The elimination of urea or monochloridacetic acid from media gives possibility to reconstruct the fibrillar fibrin structure.     

The effect of tuftsin on fibrin polymerization in vitro and on the hemostatic system when administered intravenously to healthy animals

Taftsine tetrapeptide has antiprocoagulatory properties in vitro in the presence of plasma. Taftsine exerts depolymerization influence on fibrin monomer in concentrations of 10(-1) to 10(-9) mg/ml. At intravenous injection in doses of 1 mg and 300 ug/kg, taftsine causes the increase in plasma clotting time and significant increase in enzymatic and nonenzymatic fibrinolysis. The effect can be observed during 120 min after injection of the peptide.     

Electronmicroscopic study on the molecular shape of non-stabilized fibrin break-down products produced by complex heparin compounds

Experimental data indicate that products develop from non-stabilized fibrin because of nonfermentative splitting by complex heparin connections. These products have a globular form with a diameter of 10-250 A and are similar to morphological fibrinogen molecules. The identified products show no marked lytic effect towards non-stabilized fibrin. The application of S35 labelled heparin in combination with preparative ultracentrifugation and electron microscopy enabled the determination to be established that heparin complexes will combine with particles of fibrinomeres, thus causing their transfer from the fibrillary to the globular condition. The destruction of the connections of heparin complexes with their globular molecule structures of fibrinmonomere, e.g. by protamine sulfate, guarantees their development and primary polymerization. With factor XIIIa being present, the structurally reconstructed fibrin will form a stabilized coagulum of full value which is similar in its ultrastructure to fibrin obtained in control tests.     

Destabilase: an enzyme of medicinal leech salivary gland secretion hydrolyzes the isopeptide bonds in stabilized fibrin

The salivary gland secretion of the leech Hirudo medicinalis contains an enzyme termed by us as destabilase, which hydrolyzes the epsilon-(gamma-glutamyl)-lysine bonds as a result of fibrin stabilization by factor XIIIa in the presence of Ca2+. This hydrolysis, apart from the original lysine and glutamine, is characterized by an appearance of lysine and glutamic acid residues. The accumulation of glutamic acid residues leads to spontaneous depolymerization of the destabilized fibrin. As a result, fluid "spots" of destabilized fibrin depolymerization (DFD) begin to appear at the sites of leech secretion application on the surface of stabilized fibrin plates. The DFD activity of the leech salivary gland secretion manifests itself only in case of stabilized fibrin and increases with an increase in the stabilization degree. Treatment of leech secretion with diisopropylfluorophosphate does not affect the enzyme activity, which is completely blocked by monoiodoacetate. The mechanism of action of leech salivary gland secretion and the enzyme isolated from it, i. e., destabilase, was studied, using a synthetic chromogenic substrate - p-nitroanilide-gamma-glutamic acid. The amidolytic activity of leech salivary gland secretion is 2.2 +/- 0.18 nkat/ml, Km(app) for destabilase is 0.6 X 10(-5) M, V = 5.4 X 10(-3) mol/min.     

Atorvastatin has hypolipidemic and anti-inflammatory effects in apoE/LDL receptor-double-knockout mice

Statins are first-line pharmacotherapeutic agents for hypercholesterolemia treatment in humans. However the effects of statins in animal models of atherosclerosis are not very consistent. Thus we wanted to evaluate whether atorvastatin possesses hypolipidemic and anti-inflammatory effects in mice lacking apolipoprotein E/low-density lipoprotein receptor (apoE/LDLR-deficient mice). Two-month-old female apoE/LDLR-deficient mice (n=24) were randomly subdivided into 3 groups. The control group of animals (n=8) was fed with the western type diet (atherogenic diet) and in other two groups atorvastatin was added to the atherogenic diet at the dosage of either 10 mg/kg or 100 mg/kg per day for a period of 2 months. Biochemical analysis of lipids, ELISA analysis of monocyte chemotactic protein-1 (MCP-1) in blood, quantification of lesion size and expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) in the atherosclerotic lesion by means of immunohistochemistry and Western blot analysis were performed. The biochemical analysis showed that administration of atorvastatin (100 mg/kg/day) significantly decreased level of total cholesterol, lipoproteins (VLDL and LDL), triacylglycerol, and moreover significantly increased level of HDL. ELISA analysis showed that atorvastatin significantly decreased levels of MCP-1 in blood and immunohistochemical and Western blot analysis showed significant reduction of VCAM-1 and ICAM-1 expression in the vessel wall after atorvastatin treatment (100 mg/kg/day). In conclusion, we demonstrated here for the first time strong hypolipidemic and anti-inflammatory effects of atorvastatin in apoE/LDLR-deficient mice. Thus, we propose that apoE/LDLR-deficient mice might be a good animal model for the study of statin effects on potential novel markers involved in atherogenesis and for the testing of potential combination treatment of new hypolipidemic substances with statins.     

Date Seeds (Phoenix dactylifera): Antioxidant Potential and Profile of Free and Bound Polyphenols from Different Cultivars

In this study, the antioxidant activities and detailed phenolic profiling of extracts from seven cultivars of date seeds were investigated. Significant differences were detected among cultivars. Total phenolic content (TP) ranged between 135.9±12.1 and 284.86±21.9 mg GAE/g DM. The total flavonoid value varied between 34.20±0.34 and 94.46±1.04 mg RE/g DM. The condensed tannin ranged from 24.17±1.13 to 201.60±9.95 mg CTE/g DM. Phloroglucinolysis was used to depolymerize the bound polyphenols. Results show the presence of phenolic acids: Hydroxybenzoic acid, hydroxycinnamic acid, and a high amount of flavan-3ols (monomers, dimers, and trimers). Before depolymerization, the highest amount of total polyphenols was identified in Kenta (8.48 g/kg) and the lowest was detected in Hessa (4.74 g/kg). After depolymerization, the flavan-3-ols increased significantly, ranging between 46.91g/kg in Hessa and 72.38 g/kg in Deglet Nour, with a high degree of polymerization (DP) in all cultivars. It can be concluded that date seeds represent a good source of bioactive compounds.     

Veratric acid ameliorates hyperlipidemia and oxidative stress in Wistar rats fed an atherogenic diet

An investigation was made to reveal the protective effects of veratric acid (VA), a phenolic acid against atherogenic diet-induced hyperlipidemic rats. Male albino Wistar rats were fed with atherogenic diet (4% cholesterol, 1% cholic acid, and 0.5% 2-thiouracil) daily for 30 days and treated with VA (40 mg/kg body weight) daily for a period of 30 days. Rats fed with atherogenic diet showed significant (P < 0.05) elevation in the level of plasma lipids, systolic and diastolic blood pressure, oxidative stress markers (thiobarbituric acid reactive substances, lipid peroxides) and significant (P < 0.05) reduction in the activities of enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic (vitamin C, vitamin E, and reduced glutathione) antioxidants in erythrocytes, plasma, and tissues (liver, kidney, and aorta). Oral administration of VA (40 mg/kg body weight) for 30 days to atherogenic diet fed rats markedly attenuates systolic, diastolic blood pressure and lipid peroxidation products. Further, VA treatment significantly improved enzymatic and non-enzymatic antioxidants levels and showed beneficial effects on lipid profile in atherogenic diet rats. All the above alterations were supported by histopathological observations. These results indicate that oral administration of VA ameliorates atherogenic diet-induced hyperlipidemia in rats by its free radical scavenging; improving the antioxidants and lipid lowering properties.     

Conebulization of surfactant and urokinase restores gas exchange in perfused lungs with alveolar fibrin formation

Alveolar fibrin generation has been suggested to possess strong surfactant-inhibitory potency. In perfused rabbit lungs, fibrin formation in the alveolar space was induced by sequential ultrasonic aerosolization of fibrinogen and thrombin, and the efficacy of rescue administration of surfactant and urokinase was investigated. Ventilation-perfusion (VA/Q) distribution was assessed by the multiple inert gas elimination technique. Aerosolization of fibrinogen (approximately 20 mg/kg body wt) increased shunt flow to approximately 7%. Sequential nebulization of fibrinogen and thrombin (1.3 U/kg body wt) caused alveolar fibrin deposition, documented immunohistologically, and provoked marked shunt flow, progressing to approximately 22% at the end of the experiments. The hemodynamics were virtually unchanged. Rescue aerosolization of natural bovine surfactant (15 mg/kg body wt) or urokinase-type plasminogen activator (4,500 U/kg body wt), undertaken after fibrin formation, improved gas exchange but progressive shunt flow still occurred (efficacy, surfactant > urokinase). In contrast, conebulization of surfactant and urokinase reversed shunt flow to approximately 7%, with an increased appearance of normal VA/Q matching. We conclude that alveolar fibrin formation is a potent surfactant-inhibitory mechanism in intact lungs, provoking severe VA/Q mismatch with a predominance of shunt flow, and that rescue aerosolization of surfactant plus urokinase may offer restoration of gas exchange under these conditions.     

Fibrinolytic properties of a heparin-ocrase complex]

The obtained heparin-ocrase complex includes properties of inhibiting coagulation and a high fibrinolytic activity in vitro and in vivo. The fibrinolytic activity of the heparin-ocrase complex is higher than the equivalent amount of ocrase. The fibrinolytic activity of the complex is not only evident on non-stabilized fibrin plates, but likewise on stabilized ones and at the presence of inhibitors of enzymatic fibrinolysis, such as Trasylol and epsilon-aminocaproic acid. The complex formation between heparin and protease was confirmed by means of cross-paper electrophoresis and spectrophotometry.     

Endothelial expression of endoglin in normocholesterolemic and hypercholesterolemic C57BL/6J mice before and after atorvastatin treatment

Endoglin (CD105) is a homodimeric transmembrane glycoprotein strongly related to transforming growth factor (TGF)-beta signaling and many pathological states. In this study, we wanted to evaluate whether endoglin is expressed in normocholesterolemic and hypercholesterolemic C57BL/6J mice as well as whether it is affected by atorvastatin treatment in these mice. C57BL/6J mice were fed with chow diet or an atherogenic diet for 12 weeks after weaning. In 2 atorvastatin-treated groups, mice were fed the same diets (chow or atherogenic) as described above except atorvastatin was added at the dosage of 10 mg x kg(-1) x day(-1) for the last 8 weeks before euthanasia. Biochemical analysis of blood samples revealed that administration of atherogenic diet significantly increased levels of total cholesterol, VLDL, LDL, and decreased levels of HDL. Atorvastatin treatment resulted in a significant decrease in total cholesterol and VLDL only in mice fed by atherogenic diet. Quantitative stereological analysis revealed that atorvastatin significantly decreased endothelial expression of endoglin in C57BL/6J mice fed the atherogenic diet. In conclusion, we demonstrated that endothelial expression of endoglin is upregulated by hypercholesterolemia and decreased by the hypolipidemic effect of atorvastatin in C57BL/6J mice, suggesting that endoglin expression could be involved in atherogenesis.     

Genistein suppresses leptin‐induced proliferation and migration of vascular smooth muscle cells and neointima formation

Obesity is a strong risk factor for the development of cardiovascular diseases and is associated with a marked increase in circulating leptin concentration. Leptin is a peptide hormone mainly produced by adipose tissue and is regulated by energy level, hormones and various inflammatory mediators. Genistein is an isoflavone that exhibits diverse health‐promoting effects. Here, we investigated whether genistein suppressed the atherogenic effect induced by leptin. The A10 cells were treated with leptin and/or genistein, and then the cell proliferation and migration were analysed. The reactive oxygen species (ROS) and proteins levels were also measured, such as p44/42MAPK, cell cycle‐related protein (cyclin D1 and p21) and matrix metalloproteinase‐2 (MMP‐2). Immunohistochemistry and morphometric analysis were used for the neointima formation in a rat carotid artery injury model. Genistein (5 μM) significantly inhibited both the proliferation and migration of leptin (10 ng/ml)‐stimulated A10 cells. In accordance with these finding, genistein decreased the leptin‐stimulated ROS production and phosphorylation of the p44/42MAPK signal transduction pathway. Meanwhile, genistein reversed the leptin‐induced expression of cyclin D1, and cyclin‐dependent kinase inhibitor, p21. Genistein attenuated leptin‐induced A10 cell migration by inhibiting MMP‐2 activity. Furthermore, the leptin (0.25 mg/kg)‐augmented neointima formation in a rat carotid artery injury model was attenuated in the genistein (5 mg/kg body weight)‐treated group when compared with the balloon injury plus leptin group. Genistein was capable of suppressing the atherogenic effects of leptin in vitro and in vivo, and may be a promising candidate drug in the clinical setting.     

Atherogenic effect of beta-blocker propranolol revealed in the rabbit denudated aorta

The influence of beta-blocker propranolol onto atherogenic properties of blood serum and to formation of myointimal thickening in rabbit aorta, which was caused by denudation, were investigated. The preparation was introduced per os in the dose 6 mg/kg. Culture of mouse peritoneal macrophages was used to estimate the atherogenic properties of the serum. Serum of propranolol-treated rabbits induced accumulation of cholesterol in cultivated cells. Propranolol also induced an increase of the thickness of aortic intima, lipid accumulation and increasing of cell's number in myointimal thickening. Thus, atherogenic effect of beta-blocker propranolol was found both in vitro and in vivo.     

The influence of an increased cobalt supply on ruminal parameters and microbial vitamin B12 synthesis in the rumen of dairy cows

The aim of the study was to examine the effects of an elevated dietary cobalt supply to dairy cows on rumen fermentation parameters and microbial vitamin B12 synthesis in the rumen. Five lactating dairy cows fitted with a ruminal and a duodenal cannula were subsequently fed either a ration containing only the native cobalt content (0.17 mg Co/ kg DM) or a ration supplemented with cobalt sulphate (0.29 mg Co/kg DM). The pH-value, the ammonia concentration as well as the concentration and the molar proportions of short chain fatty acids in the rumen were not significantly influenced by feeding the ration with the higher cobalt content. While there was no difference in microbial protein flow, the cobalamin flow at the duodenum was significantly elevated in supplemented animals (3.67 +/- 0.69 vs. 8.63 +/- 2.22 mg B12/d). The efficiency of cobalt utilisation for ruminal vitamin B12 synthesis was calculated to be 7.1 +/- 1.3% for the unsupplemented and 9.5 +/- 2.4% for the supplemented ration. Further investigation has to prove if there are any benefits for cows resulting from the elevated cobalamin synthesis measured, caused by feeding higher amounts of dietary cobalt.     

The effects of an infusion of prostacyclin on their endotoxin shock in rabbits.

30 rabbits received an infusion of lipopolysaccharide B (75 micrograms/kg.h) over 4 hours (groups E, EI, EA; n = 10 each). Saline was given to a control group (C; n = 8). In group EI, prostacyclin (PGI2; 500 ng/kg.min) was given simultaneously to endotoxin. Into group EA animals, aspirin (20 mg/kg) was injected before the endotoxin infusion was started. PGI2 and aspirin both improved survival of animals (6/10 each vs. 2/10 in group E). The drop of platelet counts was significantly reduced by PGI2, while leukocyte depletion was similar in all endotoxin groups. PGI2 preserved the functional capacity of platelets as indicated by collagen stimulated aggregation and thromboxane formation. PGI2 but not aspirin significantly reduced renal fibrin deposition.     

Effect of rosuvastatin on obesity-induced cardiac oxidative stress in Wistar rats--a preliminary study

The prevalence of obesity has been rising alarmingly and it has now become a global concern causing an enormous economic burden on the health care system. Obesity is generally linked to complications in lipid metabolism and oxidative stress. The aim of the present study was to investigate the effect of rosuvastatin (10 mg/kg, po) on obesity-induced oxidative stress in high fat-fed Wistar rats. Oral administration of rosuvastatin (10 mg/kg) for 21 days along with high fat diet brought about significant elevation in serum high density lipoprotein and cardiac antioxidant enzymes levels (superoxide dismutase, catalase, glutathione, glutathione peroxidase, glutathione peroxidase-, glutathione reductase- and glutathione-S-transferase) while decreasing in serum lactate dehydrogenase, apolipoprotein-B, lipids (triglycerides, total cholesterol, low density lipoprotein-cholesterol, very low density lipoprotein-cholesterol and atherogenic index) and cardiac thiobarbituric acid reactive substances levels. The results were comparable with orlistat, a standard antiobesity drug. These preliminary results for the first time demonstrate that administration of rosuvastatin can be beneficial for the suppression of obesity-induced oxidative stress and dyslipidemia in high fat-fed Wistar rats.     

Development of selective inhibitors of transglutaminase. Phenylthiourea derivatives

For the purpose of developing a transglutaminase inhibitor which could be effective in physiological and pharmacological studies, a series of phenylthiourea derivatives of alpha, omega-diaminoalkanes were designed, synthesized, and evaluated kinetically as inhibitors of transglutaminases. A homologous series of compounds of the structure phenylthiourea-(CH2)n-NH2, where n = 2, 3, 4, 5, and 6, were tested for the inhibition of both guinea pig liver transglutaminase-catalyzed amine incorporation into various glutamine-containing substrates and plasma transglutaminase (factor XIIIa)-catalyzed amine incorporation into fibrin and fibrin cross-linking. It was found that the inhibitory activity of the compounds increases with increasing number of methylene groups in the side chain up to a maximum of n = 5. A further increase in the length of the methylene side chain to n = 6 results in decreased activity. The Ki value (4.9 X 10(-5) M) of 1-(5-aminopentyl)-3-phenylthiourea (PPTU) (n = 5) for the inhibition of guinea pig transglutaminase-catalyzed amine incorporation into the B chain of oxidized insulin is in close agreement to its Km(app) value (7.1 X 10(-5) M) obtained using 14C-labeled PPTU. PPTU was also found to be a potent inhibitor of plasma transglutaminase-catalyzed fibrin cross-linking. The finding that the specificity of the alkylamines for inhibition is correlated with the length of their methyl side chains is compatible with those reported for aliphatic amines and monodansylcadaverine analogues (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl). The phenylthiourea derivatives, however, are far less toxic in mice than monodansylcadaverine as indicated by their LD50 values: PPTU, 400 +/- 25 mg/kg; and monodansylcadaverine, 160 +/- 20 mg/kg.     

Anti-obese activity of Butea monosperma (Lam) bark extract in experimentally induced obese rats

To study the efficacy of ethanolic extract of B. monosperma bark in cafeteria and atherogenic diet fed rats and monosodium glutamate (MSG) obese rats, different doses (200, 400 and 800 mg/kg) of ethanolic extract of B. monosperma bark showed dose dependent decrease in body weight, daily food intake, glucose, lipids, internal organs' weight and fat pad weight in cafeteria and atherogenic diet fed rats and monosodium glutamate obese rats. The results suggested that B. monosperma has significant anti-obese activity.     

Effect of coenzyme Q10 on catalase activity and other antioxidant parameters in streptozotocin-induced diabetic rats

Although coenzyme Q10 (CoQ10) is a component of the oxidative phosphorylation process in mitochondria that converts the energy in carbohydrates and fatty acids into ATP to drive cellular machinery and synthesis, its effect in type I diabetes is not clear. We have studied the effect of 4 wk of treatment with CoQ10 (10 mg/kg, ip, daily) in streptozotocin (STZ)-induced (40 mg/kg, iv in adult rats) type I diabetes rat models. Treatment with CoQ10 produced a significant decrease in elevated levels of glucose, cholesterol, triglycerides, very-low-density lipoprotein, lowdensity lipoprotein, and atherogenic index and increased high-density lipoprotein cholesterol levels in diabetic rats. CoQ10 treatment significantly decreased the area under the curve over 120 min for glucose in diabetic rats, without affecting serum insulin levels and the area under the curve over 120 min for insulin in diabetic rats. CoQ10 treatment also reduced lipid peroxidation and increased antioxidant parameters like superoxide dismutase, catalase, and glutathione in the liver homogenates of diabetic rats. CoQ10 also lowered the elevated blood pressure in diabetic rats. In conclusion, CoQ10 treatment significantly improved deranged carbohydrate and lipid metabolism of experimental chemically induced diabetes in rats. The mechanism of its beneficial effect appears to be its antioxidant property.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.