leupeptins and Hemolysis

High glucose can lead to serious phosphatidylserine exposure of erythrocytes which may influence the protective effect of glucose on lyophilization of erythrocytes. In this study, caspase activation has not occurred during phosphatidylserine exposure of erythrocytes. However, leupeptin can efficiently inhibit phosphatidylserine exposure of erythrocytes induced by high glucose. With increase of the leupeptin concentrations, the percentages of cells with exposed phosphatidylserine were decreased steadily. In addition, trehalose and sucrose can significantly inhibit phosphatidylserine exposure and cell shrinkage of erythrocytes induced by high glucose through increasing tolerance to osmotic shock. When the disaccharide concentrations were more than 100 mM, the percentages of cells with exposed phosphatidylserine were similar to those of control cells. Moreover, addition of disaccharides in the glucose buffer can result in high osmotic pressure which may facilitate uptake of glucose and disaccharides into erythrocytes and higher cellular glucose and disaccharide concentrations can provide more protection for lyophilized erythrocytes. Although disaccharides can increase the osmotolerance and decrease the phosphatidylserine exposure of erythrocytes exposed to high glucose, whether disaccharides can prevent phosphatidylserine exposure of lyophilized erythrocytes still needs further researches.

Topics: Caspase 3; Caspase 8; Cell Size; Erythrocytes; Freeze Drying; Glucose; Hemolysis; Humans; Leupeptins; Lipid Peroxidation; Phosphatidylserines; Sucrose; Trehalose

The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar K(i) was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens.

Topics: Adhesins, Bacterial; Animals; Bacteroidaceae Infections; Cysteine Endopeptidases; Disease Models, Animal; Gingipain Cysteine Endopeptidases; Hemagglutinins; Hemolysis; Humans; Leupeptins; Mice; Mice, Inbred BALB C; Mutation; Pigments, Biological; Porphyromonas gingivalis; Protease Inhibitors; Virulence

The biological effects of EGF and PDGF growth factors on A172 and hEGFr-3T3 cell lines were studied using RBC induced cytolysis and polyacrylamide-gelatin gel electrophoresis assays. The authors report that growth factor-induced cytotoxicity in these cells is mediated by proteolytic enzymes. Treatment of A172 cells with either EGF or PDGF resulted in marked increase of their cytotoxicity (Release Index = 150%). Similarly, RBC induced release index by hEGFr-3T3 cells was elevated to 420% in the presence of 3.4 pM of EGF. However, in A172 cells, PDGF did not have a significant effect on DNA and protein synthesis indicating that stimulation of proteolytic activity is independent of the growth factor signaling pathway. Growth factor induced cytotoxicity was significantly reduced by protease inhibitors in both cell lines. Using EDTA and leupeptin several proteolytic species were identified and localized to cellular membranes as evidenced by polyacrylamide-gelatin electrophoresis assay. These data suggest that growth factors regulate the activation or secretion of proteolytic enzymes in cancer cells and may mediate the invasive and metastatic behavior of these cells.

Topics: Amiloride; Animals; Cell Line; Cell Survival; Cells, Cultured; Cycloheximide; Enzyme Activation; Epidermal Growth Factor; Hemolysis; Humans; Leupeptins; Mice; Peptide Hydrolases; Platelet-Derived Growth Factor; Tosyllysine Chloromethyl Ketone; Tumor Cells, Cultured

Inhibitory effects of nafamostat mesilate (nafamostat) on various enzymes were investigated, and they were compared with those of gabexate mesilate (gabexate), leupeptin, aprotinin and urinastatin in vitro. Nafamostat inhibited trypsin, plasmin, thrombin, pancreatic kallikrein, Clr and Cls more potently than gabexate and leupeptin. Gabexate and leupeptin did not inhibit pancreatic kallikrein and thrombin, respectively. Aprotinin inhibited trypsin, plasmin, pancreatic kallikrein and chymotrypsin. Urinastatin inhibited trypsin and chymotrypsin. Nafamostat inhibited the complement-mediated hemolysis in diluted serum more potently than gabexate and leupeptin, but aprotinin and urinastatin did not. Nafamostat, furthermore, inhibited the complement-mediated hemolysis in undiluted serum, but gabexate did not. Unlike aprotinin and urinastatin, nafamostat and gabexate inhibited alpha 2-macroglobulin bound trypsin as well as free trypsin to the same extent. The inhibitory effect of gabexate toward trypsin was reduced more markedly than that of nafamostat after incubation with plasma at 37 degrees C. These results show that nafamostat is more useful than other inhibitors such as gabexate, leupeptin, aprotinin and urinastatin.

Topics: alpha-Macroglobulins; Aprotinin; Benzamidines; Complement Inactivator Proteins; Gabexate; Glycoproteins; Guanidines; Hemolysis; In Vitro Techniques; Leupeptins; Protease Inhibitors; Protein Binding

Effects of various aminoacids and their derivatives on the classical pathway and alternative pathway of the complement were studied. Leupeptin, acetyl-leucyl-leucyl-arginal, inhibited CH50 and Cl-esterase, but did not inhibit the alternative pathway. When aminoacids of carbon chains of the order of seven were used, arginine and lysine had stronger effects than trans-aminomethyl cyclohexane carboxylic acid (t-AMCHA), cis-aminomethyl cyclohexane carboxylic acid (cis-AMCHA) and epsilon aminocaproic acid (EACA). SH-compounds, cysteine, homocysteine and glutathione, had the strongest inhibitory effects among these aminoacids on both classical and alternative pathways. When effects on Cl esterase were compared, arginine, lysine, t-AMCHA, cis-AMCHA and EACA had weak inhibition while SH-compounds showed strong inhibition. Poly-L-lysine, which had extremely strong inhibition of CH50, had no inhibition of Cl esterase. The inhibitory effects of antifibrinolytic agents, EACA and t-AMCHA, were weak but when effects on early parts of the classical pathway, C(1,4,2)H50 were tested, some inhibitory activities were recognized. Thus inhibitory effects of these agents were due to their activities on the early parts of the classical pathway.

Topics: Amino Acids; Aminocaproic Acid; Complement C1 Inactivator Proteins; Complement C1s; Complement Inactivator Proteins; Hemolysis; Leupeptins

Normal human red cells incubated with saline extracts of tea develop paroxysmal nocturnal hemoglobinuria-like defects as demonstrated by positive acid and sucrose hemolysis tests. All of a variety of tea preparations tested provoked a sensitivity to complement-dependent hemolysis and, with one exception, a moderate decrease in red cell acetylcholinesterase activity. Complement-dependent hemolysis in teaincubated red cells was inhibited by antisera to C3 and C3 activator, but not by antisera to C4. This suggests that incubation with tea may alter the red cell membrane in a way that specifically potentiates the lytic effects of the alternate pathway of complement, but not the classic pathway. Leupeptin, a protease inhibitor, also prevented complement-dependent hemolysis of red cells incubated with tea. Although the clinical consequences of these observations are unknown, the study was initiated following a report of a young male who had developed an acute limited intravascular hemolytic episode following ingestion of large quantities of a herbal tea.

Topics: Acetylcholinesterase; Adult; Complement System Proteins; Erythrocytes; Hemoglobinuria, Paroxysmal; Hemolysis; Humans; Leupeptins; Male; Tea