Kumiyo Ashida

Direct measurement of nitric oxide release in gastric mucosa during ischemia-reperfusion in rats

Nitric oxide (NO) generation in the rat gastric mucosa during ischemia-reperfusion was measured using an NO-sensitive electrode. Under pentobarbital sodium anesthesia, an electrode was inserted into the submucosa from the serous membrane side in the fundus. After steady-state baseline recording, the celiac artery was clamped for 30 min, and then ischemia-reperfusion was achieved by removing the clamp. The clamping of the celiac artery caused a decrease in blood flow and an increase in NO level in the gastric tissue. Just after the removal of the clamp, the NO level rapidly fell and returned to the baseline level. Administration of N G-nitro-l-arginine methyl ester (an NO synthase inhibitor, 30 mg/kg ip) before ischemia significantly attenuated both the increase in NO level during ischemia and the formation of acute gastric mucosal lesions observed after 60 min reperfusion. Administration of superoxide dismutase (a superoxide radical scavenger, 10,000 U/kg iv) at the end of ischemia inhibited both the rapid decrease in NO level during the reperfusion and the gastric mucosal erosions. Because NO and superoxide radical produce a highly reactive peroxynitrite, it can be argued that NO has an important pathological role in acute gastric mucosal injury induced by ischemia-reperfusion. Our conclusion was strongly supported by immunohistochemical staining of nitrotyrosine residues, an indication of peroxynitrite formation.

Lipopolysaccharide-induced increase in plasma nitrotyrosine concentrations in rats.

Since the production of peroxynitrite may contribute to the pathophysiology of endotoxemia or sepsis, the quantities of the produced peroxynitrite were evaluated in rats after lipopolysaccharide (LPS) treatment by measuring plasma nitrotyrosine concentrations with a new method. The intraperitoneal administration of LPS caused a persistent increase in plasma nitrotyrosine concentrations, which reached a maximum with 6-fold level of the base line (105 pmol ml-1) at 24 h and gradually declined to 3-fold level of the base line at 7 days. However, plasma concentrations of nitrite and nitrate peaked at 18 h, returning to base line within 48 h. The effect of LPS on the increase in plasma concentration of nitrotyrosine was dose-dependent and consistent with that of nitrite and nitrate concentrations. On the other hand, intravenous injection of nitrotyrosine revealed a rapid clearance with a plasma half-life of 1.67 h. These results indicate that the elevation of plasma nitrotyrosine concentrations may persist for more than a week after LPS treatment, and that the determination of plasma nitrotyrosine concentrations may be useful to detect the previous peroxynitrite-dependent oxidative damages.

Quantitative analysis of cyclooxygenase-2 gene expression on acute gastric injury induced by ischemia-reperfusion in rats

The rat model of acute gastric damage induced by ischemia-reperfusion (I-R) has been used to evaluate the protective effect of various drugs on gastric injury. However, the quantitative expression state of cyclooxygenase-2 (COX-2), a protein which induces cytoprotective prostaglandins during inflammation, is still unknown in acute gastric injury induced by I-R. Therefore, we have quantitatively investigated the level of expression of COX-2 mRNA in injured gastric tissue of this model using the reverse transcription-competitive polymerase chain reaction method. The mRNA for COX-2 was expressed at low or undetectable levels in the normal gastric tissues in control rats, which were fasted for 18 hrs without I-R. The mRNA levels of COX-2 in injured gastric tissues were higher than those of control tissues between 6 hrs and 48 hrs after I-R. The maximum expression of COX-2 mRNA was recorded at 24 hrs (approximately a 200-fold increase). The expression state of COX-2, which has been ascertained in this study, should be useful in evaluating the effect of various drugs on the expression of COX-2 in acute gastric damage.

Loss of heterozygosity of the retinoblastoma gene in liver cirrhosis accompanying hepatocellular carcinoma

Carcinogenesis is a multistep process. Most hepatocellular carcinoma (HCC) is preceded by liver cirrhosis, but the genetic changes involved in cirrhosis are not known well. The present study was conducted to evaluate aberration of the retinoblastoma (RB) gene in HCC and adjacent non-tumorous liver using 22 patients with chronic liver damage accompanying HCC. The specimens obtained by microdissection from paraffinembedded tissues were analyzed using an assay based on the polymerase chain reaction for highly polymorphic nucleotide sequences of microsatellites in theRB gene. Out of 22 cases, 15 showed constitutional heterozygosity for the microsatellite markers. In 11 (73.3%) of these 15 informative cases, the primary HCC foci showed loss of heterozygosity (LOH). In 8 of these 11 doubly informative (informative and LOH-positive in primary HCC) cases, LOH was found in 20 (64.5%) of 31 microdissected non-tumorous foci. All of the non-tumorous foci showingRB loss were cirrhotic lesions but there were no foci of chronic hepatitis. The remaining 4 cases without LOH in HCC foci showed no LOH in non-tumorous lesions. In our study, LOH of theRB gene was frequently observed in liver cirrhosis surrounding tumor.

Circulating p53 Antibody in Patients with Colorectal Cancer

The presence of serum anti-p53 antibody has been reported to be associated with survival of patients with breast cancer, ovarian cancer, and hepatocellular carcinoma. To clarify prognostic significance of p53 antibody in colorectal cancer, serum p53 antibody was measured in patients with colorectal cancer. The 89 patients included 71 with colorectal cancer and 18 with colon polyp. An enzyme-linked immunosorbent assay was used to detect p53 antibodies in serum. Clinicopathological parameters such as age, sex, degree of differentiation of cancer, location of tumor, liver metastasis, stage classification, Dukes classification, CEA, CA19-9, and immunostaining of p53 and anti-p53 antibody were evaluated as prognostic factors of colorectal cancer. p53 antibody was positive in 18 of 71 (25%) with colorectal cancer, whereas it was positive in only 1 of 18 (6%) with colon polyp. The patients with p53 antibody had higher CEA and CA19-9 levels, higher positive rates of p53 protein expression in cancer cells, and higher liver metastasis rates. The p53 antibody positivity at stage classification I–IIIb/Dukes classification A–C was significantly lower than that at stage classification IV/Dukes classification D. Overall survival in colorectal cancer patients with p53 antibody was significantly shorter than in those without p53 antibody. A Cox regression analysis showed that liver metastasis, stage classification, Dukes classification, CA19-9, and p53 antibody were significant prognostic factors in colorectal cancer. Serum anti-p53 antibody could serve as one of the prognostic factors in patients with colorectal cancer.

Effect of Plaunotol on Gastric Injury Induced by Ischaemia‐Reperfusion in Rats

Plaunatol, an anti‐ulcer drug, increases prostaglandin content in gastric tissue but its effect on radical‐mediated gastric damage or activity against reactive oxygen species is unknown. We examined the effects of oral administration of plaunotol (Kelnac) on the acute gastric mucosal lesion and its progression to ulcer lesion induced by ischaemia‐reperfusion in rats.

Substances in the aqueous fraction of cigarette smoke inhibit lipid peroxidation in synaptosomes of rat cerebral cortex.

The effects of water‐soluble substances in cigarette smoke on lipid peroxidation were investigated using nerve terminals prepared from the rat cerebral cortex. The prepared smoke‐substances significantly reduced the spontaneous increase in thiobarbituric acid‐reactive substances in synaptosomes in a dilution factor‐dependent manner. Furthermore, the aqueous extract also inhibited the elevation of lipid peroxidation induced by 2,2′‐azobis (2‐amidinopropane) dihydrochloride, a peroxyl radical generator. Smoke‐substances scavenged superoxide radicals generated from stimulated human leukocytes and from the xanthine/xanthine oxidase system. These effects were not mimicked by nicotine. The antioxidant effects of smoke‐substances were preserved for several days at 5 ° or ‐80 °C. The results suggest that the smoke‐substances may possess long half‐lives and scavenge the radicals which cause lipid‐peroxidation in synaptosome membranes.

Polymerase chain reaction-based non-RI approaches for detection of allelic loss in the p53 tumor suppressor gene in hepatocellular carcinoma for clinical use

Abstract Inactivation of the p53 tumor suppressor gene is considered to occur as a late event involved in hepatocarcinogenesis. Its detection is thought to provide a useful information for the clinical management of hepatocellular carcinomas (HCCs). A rapid screening test was devised for allelic loss of the p53 gene in samples obtained by needle biopsy using non-radioisotopic (RI) microsatellite analysis combined with the microdissection method for clinical laboratory. Biopsy materials from 23 HCCs were examined to detect loss of heterozygosity (LOH) of the p53 gene after use for a histopathological diagnosis. Two microsatellite loci in the p53 gene were amplified by the polymerase chain reaction (PCR) using DNA extracted from microdissected cells, and amplified DNA fragments were subjected to non-RI detection using silver staining. More than 40–50 microdissected cells from a formalin-fixed paraffin-embedded tissue are enough to amplify both alleles of the p53 gene during the PCR. The combination of the two polymorphic microsatellite markers encompassed 60% of Japanese patients as informative. This method has detected LOH of the p53 locus in 54% of informative primary HCCs. Furthermore, LOH of the p53 gene was frequently detected in more advanced HCC as to the histological grade and clinical stage as shown in the previous report. The system for rapid and safe detection of the p53 gene allelic loss should provide useful information on the strategy for the treatment of HCC in the clinical laboratory.