Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N′-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H⁺,K⁺-adenosine triphosphatase (H⁺,K⁺-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.     

Genetic Variation and Population Structure of Hair Crab (<Emphasis Type="Italic">Erimacrus isenbeckii</Emphasis> ) in Japan Inferred from Mitochondrial DNA Sequence Analysis

Genetic variation and population structure of hair crab (Erimacrus isenbeckii) were examined using nucleotide sequence analysis of 580 base pairs (bp) in the 3′ portion of the mitochondrial cytochrome c oxidase subunit I gene (COI) of 20 samples collected from 16 locales in Japan (the Hokkaido and Honshu Islands) and one in Korea. A total of 27 haplotypes was defined by 23 variable nucleotide sites in the examined COI region. Pairwise population F _ST estimates and neighbor-joining tree inferred distinct genetic differentiation between the representative samples from the Pacific Ocean off the Eastern Hokkaido Island and the Sea of Japan, while others were intermediate between these two groups. AMOVA also showed a weak but significant differentiation among these three groups. The present results suggest a moderate population structure of hair crab, probably influenced by high gene flow between regional populations due to sea current dependent larval dispersal of this species.     

P450 in wild animals as a biomarker of environmental impact

The impact of environmental pollution on selected animals was tested by monitoring the hepatic content of cytochromes P450 and their enzyme activities or by calculating TEQ values from the concentration of pollutants in the body. Fish-eating Stellars Sea Eagles, Haliaeetus pelagicus, found dead in the northern part of Hokkaido island accumulated high levels of PCBs and DDT and metabolites. The TEQ values calculated from the PCB concentration in the eagles were high enough to cause a significant toxic effect in other birds living in the same environment. Some of these birds were also contaminated with high concentrations of lead. Spotted seals, Phoca largha, captured along the coast-line of Hokkaido accumulated PCBs in their fat at about 100 million times the concentrations in the surface sea water. The levels of expressions of hepatic microsomal CYP 1A1and related enzyme activities in these seals showed good correlation to the levels of PCBs accumulated in the fat. The fresh water crabs, Eriocheir japonicus, were captured from three different rivers with various degrees of pollution. The P450 content and the related enzyme activities showed good correlation to TEQ values obtained from the concentrations of PCBs and PCDDs in the crabs from the rivers. The wild rodents, Clethrionomys rufocanus, were captured from urban, agricultural, and forest areas in Hokkaido. Those from the forest area had the lowest CYP content and related enzyme activities, comparable to those in laboratory-raised animals. Those from the urban areas, presumably contaminated with PAHs from fuel combustion, showed increased CYP 1A1 content and related enzyme activities. Those from the agricultural areas showed increased levels of CYP 1A1, 2B, 2E1. Rats treated with some of the agrochemicals used in the area resulted in a similar pattern of induction. It is concluded that P450 can be a useful biomarker for assessing the environmental impact of chemical pollutants on wild animals.     

Reevaluation of <Emphasis Type="Italic">Pholiota squarrosa</Emphasis> lectin-reactive haptoglobin as a pancreatic cancer biomarker using an improved ELISA system

An increase in Lewis- and core-type fucosylation of haptoglobin has been reported in patients with pancreatic cancer (PC), suggesting that fucosylated haptoglobin is a candidate PC biomarker. Previously, we developed a Pholiota squarrosa lectin antibody enzyme-linked immunosorbent assay (PhoSL-ELISA) system for the detection of core-fucosylated haptoglobin. However, with this methodology, positive results were only obtained for some patients with PC, demonstrating the need for a more sensitive detection system. In the current study, we developed an improved PhoSL-ELISA system with higher sensitivity to detect core-fucosylated haptoglobin using high-concentration urea as a denaturing agent with lectin to facilitate detection. We then reevaluated the performance of PhoSL reactive-core-fucosylated haptoglobin (PhoSL-HP) as a PC biomarker using the improved PhoSL-ELISA system. PhoSL-HP levels in the sera of patients with PC were significantly higher than those in healthy volunteers, with an area under the curve (AUC) value of 0.753. Furthermore, the AUC value of CA19–9 improved from 0.793 to 0.907 when combined with PhoSL-HP. Additionally, several CA19–9-negative cases among the patients with PC were diagnosed as positive for PhoSL-HP. In conclusion, PhoSL-HP detection using our improved ELISA system might allow PhoSL-HP to serve as a potential biomarker for PC and thus might be useful to complement the detection of CA19–9 in PC diagnosis.     

Differences in parasitism of Meloidogyne incognita and two genotypes of M. arenaria on Solanum torvum in Japan

Two genotypes of root‐knot nematode, Meloidogyne arenaria (A2‐O and A2‐J), are found in Japan. They were distinguished from each other based on mitochondrial DNA sequences. The primer set (C2F3/1108) amplified a 1.7‐kb fragment from A2‐J, whereas a 1.1‐kb fragment was amplified from A2‐O. M. arenaria (A2‐O) was detected in local regions of southern Japan, whereas M. arenaria (A2‐J) was widespread from the Kyushu region to the Tohoku region. The distribution of M. arenaria (A2‐J) overlaps with the cultivation area of eggplant. Solanum torvum is used worldwide as a rootstock for eggplant cultivation, and it is resistant to Meloidogyne spp. In particular, it is reported that S. torvum is resistant to M. arenaria outside Japan. In this study, we inoculated S. torvum rootstock cultivars with M. arenaria (A2‐J), M. arenaria (A2‐O) and Meloidogyne incognita populations. Although M. incognita and M. arenaria (A2‐O) produced only a few egg masses on S. torvum, thereby confirming its resistance, the four geographical populations of M. arenaria (A2‐J) produced large numbers of egg masses on S. torvum. This study confirmed that S. torvum is resistant to M. incognita and M. arenaria (A2‐O) populations, but susceptible to populations of M. arenaria (A2‐J) in the eggplant production area of Japan.     

Synthesis and evaluation of 11C-labeled coumarin analog as an imaging probe for detecting monocarboxylate transporters expression

Upregulated monocarboxylate transporters (MCTs) in tumors are considered diagnostic imaging targets. Herein, we synthesized the positron emission tomography probe candidates coumarin analogs 2 and 3, and showed 55 times higher affinity of 2 for MCTs than a representative MCT inhibitor. Whereas [¹¹C]2 showed low tumor accumulation, probably due to adduct formation with plasma proteins, [¹¹C]2 showed high initial brain uptake, suggesting that the scaffold of 2 has properties that are preferable in imaging probes for the astrocyte–neuron lactate shuttle. Although further optimization of 2 is required, our findings can be used to inform the development of MCT-targeted imaging agents.     

Goniodomin A, an antifungal polyether macrolide, exhibits antiangiogenic activities via inhibition of actin reorganization in endothelial cells.

Goniodomin A (GDA) is an antifungal polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax. Previous studies revealed that GDA profoundly affected cytoskeletal reorganization. We examined the effect of GDA on the angiogenic properties of vascular endothelial cells. GDA itself did not affect proliferation of, migration of, and tube formation in type I collagen gels by, bovine aortic endothelial cells (BAECs). Proliferation of BAECs stimulated by bFGF was not affected by GDA at concentrations of up to 10 nM. However, at similar concentrations, GDA significantly inhibited bFGF-induced migration and tube formation in type I collagen gels by BAECs. Actin reorganization is required for cell migration. GDA caused the perinuclear aggregation of filamentous actin and inhibited stress fiber formation in bFGF- or VEGF-stimulated BAECs and lysophosphatidic acid-stimulated HeLa cells. However, GDA did not affect stress fiber structures already formed through Gbetagamma expression or in constitutively active RhoA mutant HeLa cells. Finally, GDA inhibited forming of vasucular system in a chorioallantoic membrane. Our results indicated that GDA suppressed angiogenic properties of ECs at least in part through the inhibition of actin reorganization and inhibited angiogenesis in vivo.