Hiroyuki Sugimoto

Sequence, expression in Escherichia coli , and characterization of lysophospholipase II

Here we report the sequence, expression in Escherichia coli cells, and characterization of a new small-form lysophospholipase named lysophospholipase II from mouse embryo. The cDNA clone was found and identified among mouse expressed sequence tags in the database search for the homologue of lysophospholipase I previously cloned from rat liver (H. Sugimoto et al., J. Biol. Chem. 271 (1996) 7705-7711). The predicted amino acids sequence contained 231 residues with a calculated molecular weight of 24794, and showed 64% identity to that of lysophospholipase I with the Gly-X-Ser-X-Gly esterase/lipase consensus. The lacZ fusion protein expressed in E. coli cells exhibited lysophospholipase activity and reacted with antibody raised against previously purified pig gastric lysophospholipase II (H. Sunaga et al., Biochem. J. 308 (1995) 551-557), but not with antibody against rat liver lysophospholipase I. The expressed enzyme was purified to a specific activity of 0.15 micromol/min per mg by DEAE-Sepharose A-500 chromatography. The enzyme preferentially utilized zwitterionic lysophospholipids in the order of lysophosphatidylcholine>lysophosphatidylethanolamine, but poorly acidic lysophospholipids, such as lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid. Not only the 1-acyl isomer, but also the 2-acyl isomer were deacylated. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that lysophospholipase II transcript as well as lysophospholipase I transcript was widely distributed in mouse tissues.

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.

Effects of eicosanoids on lipopolysaccharide-induced ornithine decarboxylase activity and polyamine metabolism in the mouse liver

BACKGROUND/AIMS During endotoxic shock, arachidonic acid is released from the inflammatory cell membranes and is metabolized to form eicosanoids, which modify the deleterious effects of lipopolysaccharide (LPS) on liver function. However, it is not known which prostaglandins (PGs) or leukotrienes (LTs) are produced or how they affect the LPS-treated liver. As LPS treatment elevates hepatic ornithine decarboxylase (ODC) activity and affects the polyamine levels of the mouse liver, this study was carried out to examine the effects of eicosanoids and their inhibitors on the induction of ODC activity and polyamine levels in the LPS-treated mouse liver. METHODS LPS in the presence or absence of other drugs was intraperitoneally administered to 6-week-old mice and the livers were then removed. The hepatic ODC activity, polyamine levels, and level of ODC mRNA were determined. RESULTS The levels of LPS-induced ODC activity, the putrescine (PUT) and N1-acetylspermidine (A-SPD) were reduced by the administration of PGE1. ODC activity was enhanced by the administration of corticosterone, AA-2414 (an antagonist of thromboxane (TX) A2) and TXB2, whereas the A-SPD level was reduced by corticosterone and AA-2414 treatment. The level of ODC mRNA changed in parallel with the change in ODC activity. CONCLUSIONS PGE1 may reduce the LPS-induced production of inflammation-accelerating cytokines and reduce the level of ODC activation. Corticosterone and AA-2414 treatment may attenuate the LPS-induced production of eicosanoids, and enhance the LPS-induced ODC activation. It is possible that the eicosanoids produced by LPS treatment inhibit ODC activation during endotoxic shock.

Elevation of N1-acetylspermidine and putrescine in hepatic tissues of patients with fulminant hepatitis and liver cirrhosis

Hepatic polyamines were assayed in 15 patients with liver diseases, 5 with fulminant hepatitis (FH); 3 with exacerbated liver cirrhosis (ELC); and 7 with liver cirrhosis (LC). Hepatic putrescine and N1-acetylspermidine, as percentages of total polyamines, were elevated and spermine was decreased in all 15 patients. The increase in hepatic N1-acetylspermidine levels appeared to be greater in patients with FH and ELC than in those with LC. These results suggest that the production of N1-acetylspermidine in human liver is closely associated with the induction of spermidine/spermine N1-acetyltransferase activity in liver diseases.

Effect of biotin on ammonia intoxication in rats and mice

The effects of biotin on ammonia concentration in blood and brain were evaluated in hyperammonemic rats and mice. Rats were injected with 5 mmol/kg BW of ammonium acetate, and mice were injected with 10 mmol/kg BW. Increases in blood ammonia levels in rats 15–30 min after ammonia loading were prevented by treatment with 0.2 ml/100 g BW of biotin or 0.04 ml/100g BW of arginineglutamate with statistical significance. Blood ammonia levels after ammonia loading were lower, although not significantly, in the arginine glutamate-treated rats than in the biotin-treated animals. In mice also, increases in blood and brain ammonia levels after ammonia loading were prevented by the administration of biotin. The decrease in brain glutamate and aspartate after ammonia loading was lower and the brain glutamine level was higher in biotin-treated mice than in the controls. These findings indicate the protective effect of biotin against ammonia intoxication.

Superoxide dismutase and α-tocopherol suppress the paraquat-induced elevation of N1-acetylspermidine and putrescine in primary culture of adult rat hepatocytes

A transient increase in N1-acetylpolyamines and putrescine (PUT) was observed in hepatocytes at the early stage of primary culture of rat hepatocytes. After pre-culture for 36 hr when the polyamine content returned to constant levels, we tested the effects of superoxide dismutase (SOD) and alpha-tocopherol on the paraquat-induced increase in N1-acetylspermidine (N1-acetyl-SPD) and PUT in the culture. Paraquat increased (N1-acetyl-SPD in a dose-dependent manner. It also increased PUT at doses between 0.1 and 1.3 mM. Both SOD and alpha-tocopherol suppressed the increase in N1-acetyl-SPD and PUT induced by paraquat. These results suggested that superoxide anion is one of the factors which increase N1-acetyl-SPD and PUT in hepatocytes. Lipopolysaccharide (LPS) little affected the polyamine concentration in the cultured hepatocytes, though it increases polyamine in mouse liver when given in vivo. These findings suggested that the formation of superoxide anion after administration of LPS in vivo is mediated by Kupffer cells.

Glucocorticoids suppress and oestrogens enhance the lipopolysaccharide-induced increase in putrescine and N1-acetylspermidine in mouse liver

Previously we reported that administration of lipopolysaccharide (LPS) to mice increased the hepatic levels of putrescine (PUT) and N1-acetylspermidine (N1-acetyl-SPD). In the current study, we examined the in vivo effects of some steroid hormones on the LPS-induced increase in PUT and N1-acetyl-SPD. Corticosterone, hydrocortisone and dexamethasone suppressed the LPS-induced increase in PUT and N1-acetyl-SPD in mouse liver in a dose-dependent manner, dexamethasone being the most effective among them. On the other hand, oestrone and oestradiol-17 beta enhanced the LPS-induced increase in PUT and N1-acetyl-SPD in a dose-dependent manner. Oestradiol-17 alpha and 16 beta-ethyl-oestradiol, as an inactive oestradiol isomer and an antioestrogen, respectively, likewise enhanced the increase in PUT and N1-acetyl-SPD concentrations induced by LPS. 16 alpha-hydroxy-oestradiol (oestriol), 16 alpha-hydroxyestrone, 2-hydroxyoestradiol, 2-hydroxyoesterone, progesterone, testosterone, diethylstilboestrol and nonsteroidal antioestrogens such as tamoxifen and nafoxidine had no effect on the increase. Oestradiol-17 beta enhanced and corticosterone had little effect on the carbon tetrachloride-induced increase in PUT and N1-acetyl-SPD. These results suggest that glucocorticoids suppress the increase by preventing the immunological injury by Kupffer cells on hepatocytes and that the stimulatory effect of oestrogens may not be associated with their oestrogenic activities mediated by the oestrogen receptor system.

A case of familial mediterranean fever with paroxysaml pseudo-obstruction of bowel.

症例は44才,男性. 37才より発熱を伴う腹痛発作を繰り返していた.発作時には著明な腹部膨隆と腹部単純X線写真上腸管内の著明なガス像を示した.消化管を中心に精査したが異常なく,開腹術を施行するも試験開腹に終った.周期熱の一種である家族性地中海熱を疑いレセルピンやコルヒチンを投与したところ発作が消失し,メタラミノール投与により発作が誘発され,この発作はコルヒチンにより抑制された.以上より,家族性地中海熱と診断した.診断にあたって,白血球数, CRPや血沈などの検査値は一定しなかったが,メタラミノール発作誘発試験が陽性で有用であった.