Yoshimasa Morino

Protective effect of lipoproteins containing apoprotein A-I on Cu2+ - catalyzed oxidation of human low density lipoprotein

Two apoprotein A‐I (apoA‐I)‐containing lipoproteins, one containing apoA‐I and apoA‐II (LpA‐I/A‐II) and the other containing only apoA‐I (LpA‐I), were examined for their effect on Cu2+‐mediated oxidation of low density lipoprotein (LDL). The presence of LpA‐I or LpA‐I/A‐II prevented LDL oxidation when assessed by the electrophoretic mobility, apoprotein B fragmentation and amounts of thiobarbituric acid‐reactive substances. The protection of LDL oxidation by these lipoproteins was effective for up to 6 h, with LpA‐I being more active than LpA‐I/A‐II. Results from these in vitro model experiments raise a possibility that LpA‐I mayplay a role in protecting LDL from Cu2+ ‐mediated oxidation.

Inhibition of stress-induced gastric injury in the rat by glutathione

Glutathione metabolism occurs via interorgan cycles in which hepatic synthesis of reduced glutathione and its transfer to extrahepatic tissues play an important role. To elucidate the physiologic significance of the cycles and tissue thiol status during stress-induced gastric mucosal injury, dynamic aspects of glutathione metabolism were analyzed in rats that were treated with water-immersion restraint. This treatment induced gastric mucosal lesion with concomitant decrease in the levels of perchloric acid-soluble thiols in various tissues, particularly in the liver and stomach. During the treatment, glutathione levels markedly decreased in the liver but not in other tissues. Depletion of hepatic glutathione by buthionine sulfoximine, a specific inhibitor for gamma-glutamyl cysteine synthetase, markedly decreased hepatic glutathione levels and increased the gastric injury. Intraperitoneal injection of reduced glutathione significantly increased plasma levels of glutathione and inhibited the occurrence of gastric injury without affecting intracellular glutathione levels. These results indicate that extracellular glutathione and its interorgan metabolism might play a critical role in the protection of gastric mucosa particularly when animals were challenged with various stress.

Two reactions are simultaneously catalyzed by a single enzyme: The arginine-dependent simultaneous formation of two products, ethylene and succinate, from 2-oxoglutarate by an enzyme from Pseudomonas syringae

A single enzyme isolated from Pseudomonas syringae pv. phaseolicola PK2 simultaneously catalyzed two reactions, namely, the formation of ethylene and succinate from 2-oxoglutarate, at a molar ratio of 2:1. In the main reaction, 2-oxoglutarate was dioxygenated to produce one molecule of ethylene and three molecules of carbon dioxide. In the sub-reaction, both 2-oxoglutarate and L-arginine were mono-oxygenated to yield succinate plus carbon dioxide and L-hydroxyarginine, respectively, the latter being further transformed to guanidine and L-delta 1-pyrroline-5-carboxylate. We propose a dual-circuit mechanism for the entire reaction, in which the binding of L-arginine and 2-oxoglutarate in a Schiff-base structure generates a common intermediate for two reactions.

High density lipoprotein mediates selective reduction in cholesteryl esters from macrophage foam cells

To elucidate an anti-atherogenic nature of high density lipoprotein (HDL) at cellular level, its in vitro effect on macrophage foam cells was examined. Rat peritoneal macrophages were converted to foam cells by incubation with [3H]cholesterol-labeled acetylated LDL. HDL addition to these foam cells resulted in a reduction in cellular radioactive cholesteryl esters (CE) as well as its CE mass. The radioactive free cholesterol (FC) was similarly reduced with time, whereas its FC mass level was unaltered. Other lipoproteins such as very low density lipoprotein and low density lipoprotein also reduced the radioactive FC. However, their CE-reducing capacity was negligibly weak. These results suggest that (i) CE reduction is selective to HDL, (ii) FC transfer from plasma membrane to lipoprotein (cholesterol efflux) expressed by reduction in radioactive FC is not selective to HDL but occurs to other lipoproteins, (iii) the CE-reducing capacity of HDL became weaker when cellular binding of HDL was reduced by chemical modification with tetranitromethane or a chemical cross-linker, dithiobis-succinimidylpropionate, suggesting an importance of the specific binding in the HDL-mediated CE reduction. These in vitro results gave an experimental support to a definite role of HDL as an anti-atherogenic lipoprotein in vivo.

Inhibition of ischemia and reflow-induced liver injury by an SOD derivative that circulates bound to albumin

Ischemia followed by reflow often results in tissue injury. Although reactive oxygens seem to play an important role in the pathogenesis of postischemic reflow-induced tissue injury, the mechanism and an efficient way to inhibit oxidative injury are not known. We studied the mechanism by which hepatic transport function was inhibited by a transient occlusion followed by reflow of the portal vein and hepatic artery by using a superoxide dismutase (SOD) derivative (SM-SOD) which circulates bound to albumin with a half-life of 6 h. Occlusion of the hepatic vessels for 20 min followed by reflow for 60 min significantly inhibited transhepatic transport of cholephilic ligands, such as bromosulfophthalein (BSP) and taurocholic acid. Intravenous administration of SM-SOD markedly inhibited the reflow-induced decrease in transhepatic transport of these ligands. Thiobarbituric acid - reactive metabolites (TBAR) in the liver and plasma remained unchanged during occlusion and reflow, while TBAR in the bile increased significantly. Intravenous injection of SM-SOD inhibited the reflow-induced increase in biliary TBAR. Xanthine oxidase activity in plasma also increased during occlusion and reflow by an SM-SOD-inhibitable mechanism. Polymorphonuclear leukocyte-dependent chemiluminescence of the peripheral blood remained unchanged during occlusion, but increased markedly with time after reflow. SM-SOD also inhibited the increase in chemiluminescence almost completely. These and other results suggested that the superoxide radical and/or its metabolite(s) might play an important role in the pathogenesis of the reflow-induced liver injury and that SM-SOD might be useful for studying the mechanism for tissue injury caused by oxygen toxicity.

Inhibition of oxygen toxicity by targeting Superoxide dismutase to endothelial cell surface

Since enzymes that degrade reactive oxygens, such as Superoxide dismutase (SOD), are significantly lower in plasma than in intracellular compartments, cell surface membranes should be protected against hazardous oxygens particularly when animals are challenged with oxidative stress. To minimize oxygen toxicity on endothelial cell surface, a fusion gene consisting of cDNA coding human Cu2+/Zn2+‐SOD and heparin‐binding peptide was constructed and expressed in yeast. The resulting enzyme (HB‐SOD) bound to a heparin‐Sepharose column and cultured endothelial cells; binding was inhibited either by high NaCl concentrations or heparin. When injected intravenously, HB‐SOD predominantly bound to vascular endothelial cell surface. Carrageenin‐induced paw edema and cold‐induced brain edema of the rat were markedly inhibited by a single dose of HB‐SOD. These results suggest that Superoxide radical and/or its metabolite(s) occurring at or near the outer surface of vascular endothelial cells might play a critical role in the pathogenesis of vasogenic edema.

Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2

The gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 was found to be encoded by an indigenous plasmid, designated pPSP1. The gene for the ethylene-forming enzyme was cloned and expressed in Escherichia coli JM109. Nucleotide sequence analysis of the clone revealed an open reading frame that encodes 350 amino acids (mol. wt. 39,444). In a comparison with other proteins, the homology score for the entire amino-acid sequence of the ethylene-forming enzyme of Pseudomonas syringae versus ethylene-forming enzymes from plants and 2-oxoglutarate-dependent dioxygenases was low. However, functionally significant regions are conserved.

Effect of a superoxide dismutase derivative on cold-induced brain edema

Although the involvement of reactive oxygen species has been suggested in the pathogenesis of brain edema, direct evidence supporting this concept is lacking. To elucidate a critical role of oxygen radicals, effect of a superoxide dismutase (SOD) derivative that circulated bound to albumin with a half-life of 6 h on the occurrence of cold-induced brain edema was studied in the rat. When animals were challenged with brain injury by applying a liquid-nitrogen-cold probe to one side of the cerebral hemisphere over the bony skull for 20 s, the vascular permeability of the underlying tissue increased significantly and unilateral brain edema occurred as determined by the accumulation of intravenously injected Evans blue and the increase in brain weight. Intravenous administration of the SOD derivative markedly suppressed the increase in vascular permeability and the occurrence of brain edema, particularly at their early stages. These and other results suggest that superoxide anion and/or its metabolite(s) might play a critical role in the pathogenesis of traumatic brain injury.

Immunohistochemical localization of apolipoprotein E in human glial neoplasms.

Immunocytochemical analyses revealed the presence and distribution of apolipoprotein E (apo E) in normal human brain tissue as well as in 77 human intracranial neoplasms. In normal brain tissues, the perikarya of astrocytes exhibited a strong positive reaction, whereas the Bergmann glia were stained to a moderate degree. However, no immunoreactivity was observed with neurons, oligodendrocytes, ependymal cells, and choroidal epithelium. Among the intracranial neoplasms, oligodendroglioma, choroid plexus papilloma, hemangioblastoma, primary malignant lymphoma, neurinoma, meningioma, pituitary adenoma, and craniopharyngioma were all negative. Immunoreactivity in the peripheral neuroblastoma was nil. However, the perikarya of astrocytomas and glioblastomas showed a positive reaction. Analyses on the degree of anaplasia and the amount of apo-E as an intensity of immunostaining showed a negative correlation. The astrocytic elements were stained in mixed oligoastrocytomas and medulloblastomas with glial differentiation. A few cases of ependymomas showed weak perikaryal immunostaining. Western blot analyses with anti-apo E antibody of a freshly prepared surgical specimen with astrocytomas revealed a single band with a molecular weight of approximately 37,000. The well differentiated cultured human astrocytoma cells secreted apo E into the medium. These lines of evidence suggest that apo E may serve as a potential marker specific for astrocytomas and glioblastomas, as well as an indicator of astrocytic tumor cell differentiation. The apo E localization in human brain tumors could be clinically relevant and diagnostically useful.

Chemical cross-linking alters high-density lipoprotein to be recognized by a scavenger receptor in rat peritoneal macrophages

Rat peritoneal macrophages possess a surface receptor for high-density lipoprotein (HDL). To obtain the functional aspect of the HDL receptor, the present study was undertaken to modify HDL with three different cross-linkers; dimethylsuberimidate, disuccinimidylsuberate and dithiobissuccinimidylpropionate (DSP) and determine their effect on the ligand activity for the HDL receptor. Upon modification at a low reagent concentration, DSP was found to be most effective in cross-linking of HDL apolipoproteins. The ligand activity of DSP-HDL for the HDL receptor was reduced by greater than 60%. Experiments with these macrophages at 37 degrees C showed; (i) the amounts of the cell-associated [125I]DSP-HDL as 3.5-fold higher than [125I]HDL; (ii) the cell-association of [125I]DSP-HDL was effectively (greater than 70%) inhibited by unlabeled DSP-HDL, whereas HDL showed a partial inhibition (30%); (iii) [125I]DSP-HDL underwent chloroquine-sensitive intracellular degradation; and (iv) DSP-HDL induced a 3-fold increase in the incorporation of [14C]oleic acid into cholesteryl oleate when compared with unmodified HDL. Experiments at 0 degrees C showed that the cellular binding of [125I]DSP-HDL was competed by acetylated low-density lipoprotein and dextran sulfate. These findings indicate that DSP-HDL is recognized as a ligand by a scavenger receptor of rat peritoneal macrophages, a notion consistent with HDL modified with tetranitromethane (Kleinherenbrink-Stins, M.F. et al. (1989) J. Lipid Res. 39, 511-520).