Seiichi Himeno

Activation of the β-catenin gene by interstitial deletions involving exon 3 as an early event in colorectal tumorigenesis

beta-Catenin has been identified as an oncogene in several tumors including colorectal cancers. beta-Catenin gene is activated by interstitial deletions involving exon 3 in colorectal carcinomas of Japanese population, in contrast to amino acid substitutions detected among Caucasian population. The aim of this study was to examine the type and frequency of beta-catenin gene mutation during early stages of colorectal tumorigenesis. We screened 100 colorectal adenomas for somatic mutations in the beta-catenin gene by single-strand conformation polymorphism method, as well as polymerase chain reaction amplification. In cases with mutations, sequencing analyses and immunohistochemical staining were also performed. Somatic interstitial deletions of 272-413 bp, each of which included all parts of exon 3, were detected in three tumors. However, no adenoma carried missense mutations. We confirmed accumulation of aberrant beta-catenin protein in cytoplasm and nuclei of adenoma cells by immunohistochemical analysis. Our results suggested that activation of the beta-catenin gene by interstitial deletions involving exon 3 might be less frequent compared with frequent alterations of adenomatous polyposis coli (APC) gene, but could be an early event in colorectal tumorigenesis equivalent to APC gene alterations in the Japanese population.

Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.

Effect of synthetic trypsin inhibitor on plasma immunoreactive cholecystokinin in rats.

SummaryA sensitive and specific radioimmunoassay system for rat plasma cholecystoldnin (CCK) was employed to study the effect of trypsin inhibitor on plasma CCK levels. Feeding the trypsin inhibitor, FOY-305 200 mg/kg, to rats for 10 days stimulated the pancreatic growth. However, there was no significant difference in fasting plasma concentrations and duodenal contents of CCK and secretin. On the other hand, acute ingestion of same dosage of FOY-305 caused a marked (20-fold) and sustained elevation of plasma CCK levels. The principal form of circulating CCK was CCK-22 like on Sephadex G-50, in contrast to that of the cerebrum and duodenum where CCK-8 like form, or CCK-8 and CCK-22 like forms were main components, respectively. A significant rise in plasma levels of immunoreactive secretin was also found. These results suggest that the pancreatic growth observed by feeding the trypsin inhibitor to rats is caused by an excessive amount of CCK in conjunction with secretin.

Forskolin-induced cyclic AMP production and gastric acid secretion in dispersed rabbit parietal cells: Novel evidence for a major role of cyclic AMP in acid release

Forskolin, a unique diterpine which is a direct activator of cyclic AMP-generating systems, stimulated both cyclic AMP and acid production in dispersed rabbit parietal cells. This agent was also capable of augmenting the action of histamine on both cyclic AMP and acid production at a low concentration. These findings provided novel evidence for a major role of cyclic AMP in gastric acid secretion.

Purification and structural determination of urinary NH2-terminal big gastrin fragments.

We previously demonstrated that there existed extremely abundant NH2-terminal big gastrin immunoreactivity (NT G-34-IR) in human urine. This report describes the purification and sequence of NT G-34-IR from the urine of an achlorhydric patient. The purification was carried out by a combination of Sep-Pak C18 cartridges, Sephadex G-25, and HPLC steps using a radioimmunoassay specific for NH2-terminus of G-34 and ultraviolet absorption at 214 nm as monitors. Three peptides were isolated. The amino acid analysis, mass spectrometry, and sequence analysis confirmed the structures of urinary NT G-34 fragments being less than Glu-Leu-Gly-Pro-Gln-Gly-Pro-Pro, less than Glu-Leu-Gly-Pro-Gln-Gly- Pro-Pro-His, and less than Glu- Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu. NH2-terminal octapeptide of G-34 was the main component of urinary NT G-34-IR.

Prostaglandins D2 diminishes transmucosal potential difference in rat colonic mucosa in vitro in contrast to the increasing effect of prostaglandin E1

The effect of Prostaglandin D2 (PGD2) on ion transport was investigated in the rat colon in vitro. Ion transport across the intestinal mucosa was estimated by transmucosal potential difference (PD) and short circuit current (Isc) in the Ussing chamber. PGD2 added to the serosal reservoir induced a sustained reduction in PD and Isc at the concentration of higher than 10(-7)M, producing the maximal decrease at 10(-5)M. PGD2 at 10(-5)M completely blocked the increase in PD elicited by prostaglandin E1 (PGE1), theophylline, dibutyryl cAMP or serotonin. Adenylate cyclase activity was determined in the colonic mucosal homogenates after addition of PGD2 and PGE1. Treatment with PGD2 or PGE1 caused a significant increase in the enzyme activity. Combined treatment with both prostaglandins induced no more increase than that elicited by PGE1 alone. These results suggest that PGD2 has an anti-secretory effect on the rat colon and it may regulate the ion transport process through other mechanism than the modification of cyclic AMP concentration in mucosal cells.

Marked secretion of pyruvate in human duodenal juice stimulated with pancreozymin or secretin

Pyruvate and lactate in duodenal aspirates were investigated to determine whether they are excreted from human pancreas as substrates for alkaline secretion as is bicarbonate. Secretion of these acids was compared with that of another organic acid, citrate, which is thought to be excreted in close relationship to digestive enzymes. All acids were assayed in the fluid obtained from 11 subjects without pancreatic diseases, before and after sequential intravenous injections of 1 unit/kg pancreozymin and 1 unit/kg secretin. Pyruvate concentrations were markedly increased by each stimulation, especially by secretin, and the cumulative excretions of pyruvate and bicarbonate after secretin stimulation were significantly correlated among the subjects. In contrast, lactate concentrations, although high just after administration of pancreozymin, declined to a considerable extent following each injection, rather similar to those of protein or citrate. These data suggest that pyruvate may be secreted from human pancreatic duct cells similar to bicarbonate secretion through mechanisms related to alkaline secretion.

Family study of hereditary xanthinuria--decreased duodenal xanthine oxidase activity and increased urinary excretion of xanthine and hypoxanthine in heterozygotes.

Hereditary xanthinuria is caused by a defect of xanthine oxidase (EC. 1.2.3.2) and inherited with an autosomal recessive manner (Auscher et al., 1977; Wilson et al., 1974). It is characterized by decrease of urate and increase of xanthine and hypoxanthine in blood and urine (Holmes et al., 1983).

71 FAMILY STUDY OF HEREDITARY XANTHINURIA -DECREASED DUODENAL XANTHINE OXIDASE ACTIVITY AND INCREASED URINARY EXCRETION OF XANTHINE AND HYPOXANTHINE IN HETEROZYGOTES

We studied two brothers with hereditary xanthinuria (xanthine oxidase deficiency) and their family members. The two brothers had extremely low concentrations of urate but markedly high concentrations of xanthine and hypoxanthine in plasma and urine. Xanthine oxidase activities were virtually absent in the duodenal mucosa. In their parents (presumed obligate heterozygotes), the activities of xanthine oxidase were about half that of normal subjects. Although plasma xanthine and hypoxanthine concentrations of the parents were normal, urinary xanthine and hypoxanthine excretions were significantly higher than those of normal subjects (xanthine, father 17.1 mg/g creatinine and mother 27.4 vs. normal controls 5.7 to 11.0; hypoxanthine, father 14.0 and mother 27.3 vs. controls 4.0 to 8.4). Similar changes in the metabolite concentrations were seen in at least 6 other relatives, suggesting they were heterozygotes. This study indicates that the presumed obligate heterozygotes of xanthine oxidase deficiency retained about half normal enzyme activities causing the partial metabolic blockage in vivo at this enzyme step.