Genotoxicity of quinone pigments from pathogenic fungi

The genotoxicity and mutagenicity of several kinds of quinone pigments from pathogenic fungi were examined by means of the hepatocyte primary culture (HPC)/DNA repair test and of Ames test with TA98 and TA100. Clear genotoxicity of the two quinone chemicals, xanthomegnin and luteosporin were observed in the HPC/DNA repair test, though definite mutagenicity was not detected in the Salmonella microsome test. These two pigments are thus suspected to be genotoxic carcinogens.     

Enhancing effect of wortmannin on muscarinic stimulation of phospholipase D in rat pheochromocytoma PC12 cells.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK), enhanced carbachol-induced formation of [3H]phosphatidylethanol ([3H]PEt), a marker of phospholipase D (PLD) activity, in [3H]palmitic acid-labeled PC12 cells. The apparent EC50 value was 1.5 microM, and the effect was maximal at 3 microM and slightly attenuated at higher concentration. Wortmannin alone had no significant effect on [3H]PEt formation. The enhancing effect of wortmannin was observed at the initial increasing phase of [3H]PEt formation but not at the subsequent plateau phase. Wortmannin enhanced also phorbol ester-induced PLD activation. Although the precise mechanism remains to be clarified, these results suggest that MLCK may be involved in PLD regulation in PC12 cells.     

Bioconversion of 7-Aminocephalosporanic Acid by Intact Rhodotorula glutinis Cells

When 7-aminocephalosporanic acid (7-ACA) was used as a single carbon source in the enrichment culture medium for screening 7-ACA-degrading microorganisms, pink yeast colonies appeared frequently, and these were identified as Rhodotorula glutinis. These intact R. glutinis cells converted (i) 7-ACA to deacetyl-7-ACA (7-ADACA) and (ii) monochloroacetyl-7-ACA to monochloroacetyl-7-ADACA at sufficiently high levels to be of commercial interest. Acetylation of 7-ADACA to 7-ACA, the reverse reaction of hydrolysis in an organic medium with methyl acetate as an acetyl donor, was also demonstrated.     

Kinetics and collagenolytic role of eosinophils in chronic gastric ulcer in the rat

 An association between eosinophils and tissue damage has been observed in numerous disorders. However, few reports have addressed the role of infiltrating eosinophils in gastric ulcer healing. The aim of this study was to investigate the kinetics and role of eosinophils infiltrating experimental chronic gastric ulcers in the rat. We developed a monoclonal antibody against human matrix metalloproteinase 1 (MMP1) purified from conditioned culture medium of human skin fibroblasts. Acetic acid-induced gastric ulcers were resected from rats on days 1, 3, 5, 10, 20, 40, and 180 after the days of induction (day 0). Tissue specimens were immunostained with this antibody and examined with an electron microscope. Few eosinophils were observed in the granulation tissue until day 20. By days 40 and 180, MMP1-positive eosinophils had increased in the granulation tissue of open ulcers. Azan staining revealed dispersed collagen fibers around infiltrating eosinophils. In contrast, scars demonstrated few eosinophils in fibrous tissue on days 40 and 180. Eosinophils which express MMP1 infiltrate granulation tissue at the chronic stage of gastric ulceration. The results suggest that eosinophils may play a role in tissue remodeling and deterioration of ulceration. Accepted: 18 March 1997     

The ret oncogene can induce melanogenesis and melanocyte development in Wv/Wv mice.

We recently reported the establishment of transgenic mouse lines carrying the mouse metallothionein/ret fusion gene in which severe melanosis and melanocytic tumors developed. In the present study, we demonstrate that a significant number of pigmented hairs developed in Wv/Wv mice crossed to one of the transgenic mouse lines. The pigmented hair of Wv/Wv mice carrying the ret oncogene did not lose color during aging and reappeared after shaving, indicating that the melanocytes in the hair follicle function. The melanocytic tumors also developed in these mice, although the incidence was lower than that in the wild transgenic mice. Furthermore, the neutral tube culture of mouse embryos indicated that neural crest cells of the transgenic mice gave rise to a cell population that autonomously produced melanin even in the absence of melanocyte stimulating hormone. These results strongly suggested that the introduced ret oncogene could compensate for the defect of c-kit in Wv mice during both embryogenesis and postnatal life and induce a high level of melanin synthesis in the process of melanocyte development.     

The ret oncogene can induce melanogenesis and melanocyte development in W/W mice

We recently reported the establishment of transgenic mouse lines carrying the mouse metallothionein/ret fusion gene in which severe melanosis and melanocytic tumors developed. In the present study, we demonstrate that a significant number of pigmented hairs developed in W^v/W^v mice crossed to one of the transgenic mouse lines. The pigmented hair of W^v/W^v mice carrying the ret oncogene did not lose color during aging and reappeared after shaving, indicating that the melanocytes in the hair follicle function. The melanocytic tumors also developed in these mice, although the incidence was lower than that in the wild transgenic mice. Furthermore, the neural tube culture of mouse embryos indicated that neural crest cells of the transgenic mice gave rise to a cell population that autonomously produced melanin even in the absence of melanocyte stimulating hormone. These results strongly suggested that the introduced ret oncogene could compensate for the defect of c-kit in W^v mice during both embryogenesis and postnatal life and induce a high level of melanin synthesis in the process of melanocyte development.     

Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts

Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.     

Development and characterisation of microsatellite loci in Farfugium japonicum (Asteraceae)

Eight microsatellite loci for the perennial herb Farfugium japonicum, including the rheophytic variety luchuense endemic to riparian areas of the Ryukyu Islands, Japan, were isolated and characterised. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.344–0.885 and 0.121–0.754, respectively, from 69 individuals in one population. Six loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). The primers amplifying microsatellite sequences in F. japonicum may provide a population genetics tool useful in the establishment of a conservation strategy.     

High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris

Hen egg lysozyme (HEL) is one of the sweet-tasting proteins. To understand why lysozyme is sweet, the enzyme was synthesized at high yields by a recombinant method. The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia pastoris expression and secretion vector pPIC6alpha. This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast. Expression of HEL was carried out in fermenter cultures. Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography. Approximately 400 mgL-1 of recombinant HEL was obtained. The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans. The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus. These results demonstrate that the P. pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield.