Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells.

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.

Protective effect of C-reactive protein against the lethality induced by Vibrio vulnificus lipopolysaccharide.

Vibrio vulnificus infection has attracted special interest because of its high mortality. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from V. vulnificus infection. In this study, the effect of C-reactive protein (CRP), a typical hepatogenic acute phase protein, on the lethality induced by V. vulnificus lipopolysaccharide (LPS) was investigated in galactosamine-sensitized mice. The pretreatment of CRP, in a dose of at least 2 mg/kg, 2 hr before the challenge of LPS completely protected mice against the lethality by V. vulnificus LPS. The elevation of serum tumor necrosis factor-alpha (TNF-alpha) induced by LPS administration was not affected by CRP pretreatment. However, the LPS- or TNF-alpha-induced hepatotoxicity was completely prevented by CRP. These results indicate that CRP does not prevent the synthesis, but prevents the hepatotoxic action of TNF-alpha. The possibility that impaired production of acute phase proteins in patients with pre-existing hepatic dysfunction may predispose the higher risk of V. vulnificus infection needs to be evaluated further.

Protective mechanism of glucose against alloxan-induced beta-cell damage: pivotal role of ATP.

Glucose prevents the development of diabetes induced by alloxan. In the present study, the protective mechanism of glucose against alloxan-induced beta-cell damage was investigated using HIT-T 15 cell, a Syrian hamster transformed beta-cell line. Alloxan caused beta-cell damages with DNA fragmentation, inhibition of glucose-stimulated insulin release, and decrease of cellular ATP level, but all of these beta-cell damages by alloxan were prevented by the presence of 20 mM glucose. Oligomycin, a specific inhibitor of ATP synthase, completely abolished the protective effects of glucose against alloxan-induced cell damage. Furthermore, treatment of nuclei isolated from HIT-T15 cells with ATP significantly prevented the DNA fragmentation induced by Ca2+. The results indicate that ATP produced during glucose metabolism plays a pivotal role in the protection of glucose against alloxan-induced beta-cell damage.

Purification and characterization of adenosine diphosphate ribose pyrophosphatase from human erythrocytes.

Free ADP-ribose is a turnover product of NAD+, protein-bound polymeric and monomeric ADP-ribose, and cyclic ADP-ribose. But little is known about the specific cellular roles or metabolism of free ADP-ribose. ADP-ribose pyrophosphatase (EC 3.6.1.13), which hydrolyzes ADP-ribose into AMP and ribose-5'-phosphate, was purified from human erythrocytes. Purification was achieved to homogeneity by successive chromatographic steps, resulting in a final purification of 75,790-fold from the hemolysate. The purified enzyme showed a single band with the molecular weight of 34 kDa on SDS-PAGE both in the presence and absence of 2-mercaptoethanol. The molecular weight of the native enzyme calculated by gel filtration was 68 kDa, indicating that the active enzyme is a dimer of identical subunits. The enzyme requiring Mg2+ showed highest activity toward ADP-ribose, and about 40-70% activities with IDP-ribose, ADP-mannose and GDP-mannose. The enzyme showed a Km of 169 +/- 11 microM for ADP-ribose, broad pH optimum around pH 9.5, and pI of 5.1. ADP was a potent noncompetitive inhibitor with a Ki of 16 +/- 1.2 microM. These results suggest that our enzyme is unique, and different from the other ADP-ribose pyrophosphatases reported. ADP-ribose pyrophosphatase may play an important role in the regulation of intracellular steady-state of free ADP-ribose.

Cytotoxicity of Vibrio vulnificus cytolysin on rat peritoneal mast cells.

Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection. The injection of living bacteria or purified V. vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability. In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC. Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH). At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2-deoxy-D-glucose was rapidly effluxed with the concomitant decrease in cellular ATP. VVC-treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP-gamma-S. These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells. At low concentrations, VVC does not induce the release of stored histamine from damaged cells. The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death. However, no increase in blood histamine level was detected. This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.

A variant of ornithine aminotransferase from mouse small intestine.

The ornithine aminotransferase (OAT) activity of mouse was found to be highest in the small intestine. The mitochondrial OAT from mouse small intestine was purified to homogeneity by the procedures including heart treatment, ammonium sulfate fractionation, octyl-Sepharose chromatography, and Sephadex G-150 gel filtration. Comparing to the amino acid sequence of mouse hepatic OAT, six N-terminal amino acid residues have been deleted in intestinal OAT. However, the subsequent sequence was identical with that of hepatic OAT. The molecular weights of both intestinal and hepatic OAT were estimated as 46 kDa by SDS-gel electrophoresis and as 92 kDa by gel filtration, indicating that both native OATs are dimeric. Biochemical properties of intestinal OAT, such as molecular weight, pH optimum and K(m) values for L-ornithine and alpha-ketoglutarate, were similar to those of hepatic OAT. However, intestinal OAT was more labile than hepatic OAT to tryptic digestion.

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase.

Lipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 b of recovery period after the depletion of LPL, with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.

Glycosylation of lipoprotein lipase in human subcutaneous lipomas.

Glycosylation of lipoprotein lipase (LPL) was studied in human subcutaneous lipomas. Heparin-releasable LPL activities were higher in lipomas than those in adjacent normal adipose tissues, and showed good correlation with cellular LPL protein mass. Molecular weight of LPL subunit was 57 kDa in both tissues. After endoglycosidase H-digestion, two types of LPL subunits were found in normal adipose tissues; partially sensitive (55 kDa) and totally sensitive (52 kDa) form. In lipoma tissues, the fraction of partially sensitive form (55 kDa) was increased comparing with control adipose tissues. These results suggest that partially sensitive subunits constitute the major secretable form of LPL in human subcutaneous lipomas.

Protective mechanism of glucose against alloxan-induced pancreatic beta-cell damage.

Glucose prevented the alloxan- or H2O2-induced inhibition of insulin secretion in rat pancreatic islets. Hydrogen peroxide was detected during the incubation of islets with alloxan, and this generation of hydrogen peroxide was not affected by glucose. Treatment of beta-cells with alloxan or H2O2 caused elevation of cytosolic free Ca2+ and decrease of cellular NAD+. Glucose blocked the decrease of cellular NAD+ level, but did not abolish the increase of cytosolic Ca2+. These results indicate that glucose protected pancreatic beta-cell damage after the H2O2 generation and Ca2+ influx on a chain of reactions in the diabetogenesis of alloxan.

Glycosylation, dimerization, and heparin affinity of lipoprotein lipase in 3T3-L1 adipocytes.

The relationship between glycosylation, dimerization, and heparin affinity of lipoprotein lipase (LPL) was studied in 3T3-L1 adipocytes. Three forms of LPL subunits were found in normal cells; totally endo H-resistant (57 kDa), partially sensitive (54 kDa), and totally sensitive (51 kDa) forms. LPL in normal cells was active, dimeric, and showed high affinity for heparin. LPL in cells treated with tunicamycin, preventing the transfer of N-linked oligosaccharide chain, was unglycosylated (51 kDa) and inactive. LPL proteins were found as an aggregate, and had low affinity for heparin. After treatment with castanospermine, an inhibitor of ER glucosidase I, 80% of LPL activity was inhibited. Most of LPL proteins were totally endo H-sensitive, present as an aggregate, and had low affinity for heparin. LPL in cells treated with deoxymannojirimycin, an inhibitor of Golgi mannosidase I, was active, dimeric, and had high affinity for heparin as in normal cells. But LPL subunits were all endo H-sensitive. These results suggest that core glycosylation and subsequent removal of glucose residue is required, but processing after Golgi mannosidase I is not necessary for dimerization and acquisition of high heparin affinity of LPL.

Assay of ornithine aminotransferase with ninhydrin.

We developed an assay system for ornithine aminotransferase (EC 2.6.1.13) using ninhydrin. Pyrroline 5-carboxylate, a product of enzymatic transamination, reacts with ninhydrin under hot acidic conditions to form a reddish pigment soluble in ethanol. The millimolar extinction coefficient of reaction product dissolved in ethanol was 16.5 at 510 nm. Acidification with perchloric acid effectively abolished the interfering color development by L-ornithine and L-glutamate. The paired activity measurement in mouse tissues by ninhydrin and o-aminobenzaldehyde methods showed a good correlation (gamma = 0.985). In our ninhydrin method, stable ninhydrin replaced unstable o-aminobenzaldehyde, and sensitivity was much higher than that with the conventional o-aminobenzaldehyde method.

Role of Ca2+ in alloxan-induced pancreatic beta-cell damage.

Pretreatment of rats with verapamil, a Ca(2+)-antagonist, completely prevented alloxan-induced hyperglycemia. Verapamil also abolished the inhibition of insulin secretion by alloxan and H2O2 in isolated rat pancreatic islets. H2O2 generation from alloxan was not affected by verapamil, but alloxan- and H2O2-induced DNA strand breaks were completely prevented. Treatment of beta-cells with alloxan and H2O2 caused elevation of cytosolic free Ca2+, and this increase of Ca2+ was also abolished by verapamil. These results suggest that alloxan-derived oxygen radicals may disturb intracellular Ca2+ homeostasis by increasing Ca2+ influx, which results in secondary reactions ultimately leading to DNA strand breaks and cytotoxicity of beta-cells.

Inhibitory mechanism of Ca2+ on the hemolysis caused by Vibrio vulnificus cytolysin.

Calcium in millimolar concentrations protected mouse erythrocytes from hemolysis caused by Vibrio vulnificus cytolysin without affecting the release of intracellular K+ from the cells. This effect was maximal at 25 mM CaCl2. The protection was not absolute and could be partially overcome by increased concentrations of cytolysin. Calcium failed to block both the binding and oligomer formation of cytolysins on the erythrocyte membrane. After pore formation, the continued presence of calcium is required for the prevention of hemolysis. There was hardly any inflow of calcium into the erythrocytes through pores as measured by 45Ca2+ uptake. The presence of calcium after the abolition of Ca2+ gradient by ionomycin cannot inhibit the hemolysis caused by cytolysin. These results suggest that calcium exerts its major inhibitory effect on V. vulnificus cytolysin-induced hemolysis as an osmotic protectant, and that cytolysin may become an useful tool for permeabilizing cells selectively for small ions such as potassium or sodium while preventing the Ca2+ flow.

Hemolytic mechanism of cytolysin produced from V. vulnificus.

The characteristics of hemolytic action of cytolysin produced from V. vulnificus were investigated in mouse erythrocytes. The cytolysin bound erythrocyte membranes in temperature-independent manner and then lysed cells temperature-dependently. Hemoglobin release by the cytolysin was completely inhibited by the presence of raffinose or melezitose, but K+ release was not affected. The cytolysin-induced hemolysis was always accompanied with the conversion of membrane-bound cytolysin into an oligomer of 210 kDa, corresponding to a tetramer of native cytolysins. Nonesterified cholesterol inactivated the cytolysin by converting active monomeric cytolysin into inactive oligomer. The results suggest that the cytolysin lyses erythrocytes due to the formation of small pores on erythrocyte membrane by cholesterol-mediated oligomerization of the cytolysin.

CD3 stimulation causes phosphorylation of phospholipase C-gamma 1 on serine and tyrosine residues in a human T-cell line.

The human T-cell line Jurkat was found to contain at least two immunologically distinct isoforms of inositol phospholipid-specific phospholipase C (PLC), PLC-beta 1 and PLC-gamma 1. Treatment of Jurkat cells with antibody to CD3 led to phosphorylation of PLC-gamma 1 but not of PLC-beta 1. The phosphorylation of PLC-gamma 1 occurred rapidly and transiently on both serine and tyrosine residues; tyrosine phosphorylation reached a maximum level less than 1 min after stimulation and decreased rapidly, both in the presence and in the absence of orthovanadate. Two-dimensional phosphopeptide map analysis revealed that the major sites of tyrosine and serine phosphorylation in PLC-gamma 1 from activated Jurkat cells are the same as those in PLC-gamma 1 from cells treated with peptide growth factors such as epidermal growth factor and platelet-derived growth factor. Previously, it has been shown that multiple phosphorylation of PLC-gamma 1 by the growth factor receptor tyrosine kinases leads to activation of PLC-gamma 1. Thus, the current data suggest that inositol phospholipid hydrolysis triggered by the T-cell antigen receptor-CD3 complex is due, at least in part, to activation of PLC-gamma 1 and that the mechanism by which this activation is achieved involves phosphorylation of multiple tyrosine residues on PLC-gamma 1 by a nonreceptor tyrosine kinase coupled to the T-cell antigen receptor-CD3 complex.