Yoshiko Sameshima

Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress.

We detected platelet‐activating substance in gastrointestinal areas, which was confirmed to be platelet‐activating factor (PAF) on the basis of the following findings: 1) it comigrated with authentic PAF on thin‐layer chromatography; 2) it did not aggregate PAF‐desensitized platelets; and 3) its activity was completely antagonized by the receptor antagonists CV3988 and L‐652,731. The level of PAF was determined with a bioassay method based on the release of [3H] serotonin from washed rabbit platelets. In the normal rat stomach, the level of PAF was high in the antrum (940 ± 200 nmol PAF/mol phosphorus of original phospholipids), especially in the antral mucosa (1801 ± 426 nmol/mol phosphorus of original phospholipids). The stomach PAF level was significantly altered by water immersion stress. Stress for a period of 1 h was associated with a decrease in the antral PAF level to 39 ± 7% of that of untreated controls. This low PAF level persisted during stress. On the other hand, in the corpus, stress for periods of 1 and 3 h was associated with decreases in the PAF content, and further stress (7 h) resulted in restoration of the PAF level to normal. Furthermore, 7 h of stress was associated with distinct hemorrhagic lesions, which were prevented by CV3988 infused i.v. before the stress. This is the first report of an association between a decrease of the endogenous PAF level in animal tissues and tissue damage.—Sugatani, J.; Fujimura, K.; Miwa, M.; Mizuno, T.; Sameshima, Y.; Saito, K. Occurrence of platelet‐activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress. FASEB J. 3: 65‐70; 1989.

Characterization of ferrochelatase in kidney and erythroleukemia cells

Ferrochelatase from bovine kidney mitochondria has been purified 1600-fold with a 6.5% yield, exhibiting a specific activity of 490 nmol mesoheme formed/mg of protein per min. The Km values for mesoporphyrin IX and protoporphyrin IX with iron were 12.5 and 12.7 microM, respectively. The Km values for iron and zinc with mesoporphyrin IX were 3.51 and 3.17 microM, respectively. The purified enzyme showed a single band with an apparent molecular mass of 42,000 daltons (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbit antibody against the purified enzyme markedly inhibited activities of the enzyme from both the kidney and liver. Immunoblot analysis showed that the antibody reacted with the renal as well as the hepatic enzymes showing the same molecular weight. Peptide mapping with trypsin or alpha-chymotrypsin showed that digested peptides of renal enzyme were similar to those of hepatic enzyme. Ferrochelatase activity in mouse erythroleukemia (MEL) cells increased in parallel with an increase of heme synthesis by treatment with dimethylsulfoxide. Using immunoblotting techniques, the amount of the enzyme in the MEL cells has been shown to increase by the induction, showing a molecular mass of 41 kDa which was the same as that of the mouse hepatic enzyme. Comparative structural analysis of the enzyme of MEL cells and that of mouse liver by peptide mapping showed that the partial digestive peptides of both enzymes exhibited a similar pattern. These results strongly suggest that ferrochelatase in kidney, liver and erythroid cells can be of one type.

Properties and localization of phospholipase A2 activity in rat stomach.

In the glandular stomach of rat, phospholipase A2 activity was detected and characterized by the use of sonicated 1-acyl-2-[1-14C]oleoyl-sn-glycero-3-phosphocholine (1 mM in 200 mM glycylglycine buffer/2 mM CaCl2) as substrate. The specific activity was 4.9 X 10(-2) mumol/min per mg protein in the whole glandular stomach homogenate (n = 8). It showed individual variation among rats. The optimal pH was 8.0. The activity was relatively heat-resistant and calcium-dependent. More than 90% of phospholipase A2 activity was located in the corpus and less than 10% was in the antrum. In the corpus wall, which consists of mucosa, submucosa, muscularis externa and serosa, the mucosal part (mucosa and submucosa) contained 80-90% of total activity. The effect of some clinical agents (ulcerogenic and anti-ulcer agents) on the phospholipase A2 activity was examined. The activity in the corpus was inhibited by cimetidine (50% inhibition at 5 X 10(-3) M) and stimulated by indomethacin (50% stimulation at 5 X 10(-5) M). Cetraxate hydrochloride (10(-6)-10(-3) M), 16,16-dimethylprostaglandin E2 (10(-7)-10(-4) M) and dexamethasone (10(-7)-10(-3) M) had no effect.

Clinical application of the measurement of serum asialoglycoproteins to estimate residual liver function in patients with chronic liver diseases with or without hepatocellular carcinoma

SummaryThe correlation between the amount of asialoglycoproteins and results of conventional liver function tests was studied in patients with chronic liver diseases, with or without hepatocellular carcinoma. The objective was to determine the clinical significance of the measurement of levels of serum asialoglycoproteins. The levels were elevated in accordance with the progress of liver diseases, and correlated with the decrease in albumin content, cholinesterase activity, the ratio of esterified cholesterol to total cholesterol and to the increase of indocyanine green retention at 15 min (p<0.001). There was no correlation with values of glutamic oxaloacetic and pyruvic transaminases. The amount of serum asialoglycoproteins also correlated with survival time in fatal cases of cirrhosis and/or hepatocellular carcinoma.Bilirubin and bile acids did not interfere with the measurement of serum asialoglycoproteins in cases of hyperbilirubinemia.Serum asialoglycoprotein levels are a good indicator of hepatic functional reserve in patients with chronic liver diseases, with or without hepatocellular carcinoma.

Serum platelet-activating factor acetylhydrolase activity in rats with gastric ulcers induced by water-immersion stress.

Platelet-activating factor (PAF) acetylhydrolase is an enzyme which hydrolyzes PAF to yield inactive lysoPAF. This study focused on the influence of water-immersion stress on serum PAF acetylhydrolase activity. The enzyme activity was determined by measurement of [3H]acetate produced from 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine upon precipitation of the complex of the radioactive substrate and albumin with trichloroacetic acid. The onset of water-immersion stress caused the development of gastric lesions associated with a significant increase in serum PAF acetylhydrolase activity. Serum PAF acetylhydrolase may leak into the blood from some tissues in rats with gastric injury induced by water-immersion stress and might control the action of PAF.

Gastric Hemodynamic Disturbance Induced by Hemorrhagic Shock in Rats: Role of Platelet-Activating Factor

The role of platelet-activating factor (PAF) in the induction of rat gastric mucosal damage by ischemia-reperfusion was examined with reference to hemodynamic characteristics. Gastric mucosal hemodynamics was measured continuously, using laser-Doppler flowmetry and reflectance spectrophotometry. Not only gastric mucosal damage but also characteristic hemodynamic change--that is, ischemia with congestion--was observed in the PAF infusion and reperfusion periods of the ischemia-reperfusion model. CV-6209, a specific PAF antagonist, significantly alleviated gastric mucosal damage and hemodynamic disturbance induced by ischemia-reperfusion. These results strongly suggest that PAF is a potent mediator of gastric mucosal damage during reperfusion in the ischemia-reperfusion model.

DIFFUSE CALCIFICATION IN GASTRIC CANCER

The progressive expansion of calcification into the wall of the stomach and peritoneal metastatic foci was observed in a 31-year-old female with Borrmann type 4 calcified advanced gastric cancer. Despite treatment with systemic lentinan, uracil tegaful and mitomycin C, together with intraperitoneal injections of mitomycin C, OK-432 and prednisolone, the patient died 27 months after first presentation. The case provided a useful means of studying the mechanism of calcification.

Gastric Mucosal Damage Induced in Rats by Intravenous Administration of Platelet-Activating Factor

We examined the time courses for gastric mucosal hemodynamics and lipid peroxides after intravenous infusion of PAF (platelet-activating factor). The gastric mucosal hemodynamics were continuously monitored by laser-Doppler flowmetry and reflectance spectrophotometry during 20 min infusion of PAF and the subsequent 60 min. Gastric mucosal damage was assessed by microscopical scoring and measurement of thiobarbituric acid (TBA) reactants. Intravenous infusion of PAF caused macroscopical gastric mucosal damage, hemodynamic disturbance, a reduction in blood pressure and elevation of TBA reactants. These changes did not recover to control levels, even at 60 min after PAF infusion. The mucosal injury caused by the hemodynamic disturbance (congestion) was related to the elevation of TBA reactants, that reflect lipid peroxidation and oxygen-derived free radicals.

Determination of Free Tryptophan in Plasma and Its Clinical Applications

Free tryptophan in plasma was separated by centrifugation through an Amicon ultrafiltration membrane cone. The value obtained without control of pH was found to be lower than that obtained with control of pH by an improved method ( Hijikata et al. (1981) Anal. Biochem. 118, 10-16). For determination of the total tryptophan concentration in the plasma, high performance liquid chromatography (HPLC) was better than the method of Denckla Dewey as modified by Bloxam & Warren ( (1974) Anal. Biochem. 60, 621-625), as judged on the basis of sensitivity, recovery rate and coefficient of variance. The total tryptophan concentration in the plasma determined by HPLC was lower than that determined by the Bloxam & Warren method. The total tryptophan concentration (t-Trp), free tryptophan concentration (f-Trp) and f-Trp/t-Trp ratio were 55.8 +/- 10.2 mumol/l, 11.6 +/- 1.5 mumol/l and 0.211 +/- 0.03 (mean +/- 1 SD) respectively, in healthy subjects (controls). No significant difference was observed between the values of controls and those of patients with liver cirrhosis, hepatocellular carcinoma, liver cirrhosis with hepatocellular carcinoma and hepatic encephalopathy of liver cirrhosis without bleeding. But in liver cirrhosis with bleeding, free tryptophan concentration (f-Trp, 48.0 +/- 23.3 mumol/l, p less than 0.001) and f-Trp/t-Trp ratio (0.645 +/- 0.289, p less than 0.001) were significantly higher than those of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in Plasma Amino Acids During the Oral Glucose Tolerance Test and the Effect of these Changes on Hepatic Encephalopathy

Changes in amino acid concentrations in plasma during a 100 g oral glucose tolerance test were investigated in patients with liver cirrhosis and in healthy controls. In the controls, almost all amino acid concentrations reached a nadir about 3 hours after glucose loading, then returned to initial levels after 6 hours. Immunoreactive insulin levels reached a peak about 30 minutes after loading, then decreased gradually, reaching initial levels after 6 hours. In the controls, the decrease ratios, defined as maximum decrease during the 3 hours after loading/initial concentration in plasma, were 0.607 and 0.554 for isoleucine (Ile) and leucine (Leu) respectively and 0.382 for valine (Val) which is significantly lower than for Ile or Leu. A similar tendency was recognized in patients with liver cirrhosis. The initial concentration of tyrosine (Tyr) and phenylalanine (Phe) in liver cirrhosis was significantly higher and their decrease ratios were significantly lower than in controls. Though no difference was observed between initial concentrations of tryptophan (Trp) in controls and liver cirrhosis patients, the decrease ratio of Trp in liver cirrhosis was lower (0.061) than that of controls (0.279) (p less than 0.001). The value, t-Trp/BCAA + AAA, i.e. total Trp concentration (mmol/l)/concentration (mmol/l) of branched chain amino acids (BCAA, Ile + Leu + Val) plus aromatic amino acids (AAA, Tyr + Phe), which is known to correlate with the brain Trp concentration of rats (Fernstrom, J. D. & Wurtman, R. J. (1972) Science 178, 414-416), changed significantly from 9.6 +/- 2.4 (mean +/- 1 SD) at the initiation to 12.9 +/- 3.3 at 3 hours after loading in controls (p less than 0.001), and in liver cirrhosis it changed from 10.3 +/- 1.9 to 15.8 +/- 3.1 (p less than 0.001).