DNA repair defect in AT cells and their hypersensitivity to low-dose-rate radiation

Ataxia telangiectasia (AT) and normal cells immortalized with the human telomerase gene were irradiated in non-proliferative conditions with high- (2 Gy/min) or low-dose-rate (0.3 mGy/min) radiation. While normal cells showed a higher resistance after irradiation at a low dose rate than a high dose rate, AT cells showed virtually the same survival after low- and high-dose-rate irradiation. Although the frequency of micronuclei induced by low-dose-rate radiation was greatly reduced in normal cells, it was not reduced significantly in AT cells. The number of gamma-H2AX foci increased in proportion to the dose in both AT and normal cells after high-dose-rate irradiation. Although few gamma-H2AX foci were observed after low-dose-rate irradiation in normal cells, significant and dose-dependent numbers of gamma-H2AX foci were observed in AT cells even after low-dose-rate irradiation, indicating that DNA damage was not completely repaired during low-dose-rate irradiation. Significant phosphorylation of ATM proteins was detected in normal cells after low-dose-rate irradiation, suggesting that the activation of ATM plays an important role in the repair of DNA damage during low-dose-rate irradiation. In conclusion, AT cells may not be able to repair some fraction of DNA damage and are severely affected by low-dose-rate radiation.     

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.     

Novel matrix metalloproteinase inhibitors: generation of lead compounds by the in silico fragment-based approach

Generation of structurally new matrix metalloproteinase inhibitors was successfully carried out using an in silico technique. In order to identify the small fragment interacting with residues in the S1' pocket of MMP-1 through hydrogen bonds, we performed in silico screening using the LUDI program. As a result, acetyl-L-alanyl-(N-methyl)amide (Ac-L-Ala-NHMe) was selected to link with another fragment, hydroxamic acid that interacted with catalytic zinc. By this approach, the L-glutamic acid derivative 2b was discovered to be a new type of matrix metalloproteinase inhibitor. Further transformation to reduce its peptidic nature and improve activity yielded nonpeptidic lead compounds as inhibitors of MMP-1, -2, -3, and -9.     

Heme oxygenase activity in placenta: direct dependence on oxygen availability

Carbon monoxide (CO), which is formed endogenously from heme catalyzed by heme oxygenase (HO), is proposed to play a role in vascular control. The mRNA and protein expression of the inducible isoform of HO (HO-1) increases in response to hypoxia, and it has been assumed that HO activity also increases. This assumption requires evaluation because the catalytic activity of HO requires three molecules of O(2) for each molecule of CO formed from heme, and HO activity may be limited by O(2) availability. To test the hypothesis that low physiological O(2) concentrations limit HO activity, heme-derived CO formation by microsomal fractions of homogenates of chorionic villi of human placentas was determined after exposure to 0, 1, 5, or 21% O(2). Results revealed that HO activity was directly dependent on O(2) concentration. Thus, although hypoxia may increase HO protein and mRNA expression, there is a progressive decrease in HO activity with decreasing O(2) concentration and the dependence of HO activity on O(2) concentration is similar in chorionic villi from noninfarcted areas of preeclamptic and normotensive placenta.     

Expression of a sweet cherry DREB1/CBF ortholog in Arabidopsis confers salt and freezing tolerance

Dehydration responsive element binding protein 1 (DREB1)/C-repeat binding factor (CBF) induces the expression of many stress-inducible genes in Arabidopsis. We have previously reported the identification of three DREB1/ICBF homologs from sweet cherry (Prunus avium). To identify the function of these homologs, one of the genes, CIG-B, was transformed into Arabidopsis. In one of the transgenic plant lines, the DREB1/CBF target gene cor15a was induced in the absence of stress treatment. The cor15a-overexpressing transgenic plant exhibited mild growth retardation and had greater salt and freezing tolerance than did the wild-type and the transgenic lines in which cor15a was not induced. These results suggest that this sweet cherry DREB1/CBF homolog has a function similar to that of DREB1/CBF.     

Determination of a new immunosuppressant, mycophenolate mofetil, and its active metabolite, mycophenolic acid, in rat and human body fluids by high-performance liquid chromatography

Mycophenolate mofetil, a new immunosuppressant, is a morpholinoethyl ester of mycophenolic acid. A new selective, sensitive and simple high-performance liquid chromatographic method was developed for the determination of mycophenolic acid and mycophenolate mofetil in biological samples. The preparation of samples was based on liquid—liquid extraction. The compounds were separated on a CN column using acetonitrile—0.01 M phosphate buffer (1:4, v/v) as the mobile phase. UV detection was used at wavelengths 215 and 304 nm. The detection limit was 5 ng per injection volume. This method enabled pharmacokinetic and pharmacodynamic studies in humans and rats.     

L-lactic acid secreted from gastric mucosal cells enhances growth of Helicobacter pylori

BACKGROUND: Helicobacter pylori mainly inhabit the mucus layer in the gastric mucosa. However, mechanisms involving H. pylori colonization and proliferation in gastric mucosa are not well established. This study focuses on elucidating the role of gastric mucosal cells on growth of H. pylori. MATERIALS AND METHODS: H. pylori was co-cultured with the murine gastric surface mucosal cells (GSM06), and the growth of H. pylori on the cells was assessed by enumerating the colony-forming units (CFU). The H. pylori growth factor in the culture media conditioned by GSM06 cell was purified by HPLC, and the chemical structure of the growth factor was identified by analyses of (1)H- and (13)C-NMR spectra. RESULTS: A marked increase in the number of CFU of H. pylori was observed in the GSM06 cells. The enhanced H. pylori growth was also observed when indirectly incubated with GSM06 cells through semi-permeable membrane. In addition, culture media conditioned by GSM06 cell stimulated H. pylori growth approximately one thousand-fold. By bioassay-guided purification, the H. pylori growth factor was isolated from the conditioned medium of GSM06 cells and identified as L-lactic acid. The H. pylori growth-enhancing activity under microaerobic condition was well correlated with L-lactic acid concentrations in the conditioned media. CONCLUSIONS: This study demonstrates that L-lactic acid secreted by gastric mucosal cells enhances the growth of H. pylori, and this L-lactic acid-dependent growth of H. pylori may be important to the long-term colonization of H. pylori in the stomach.     

Rapid, extensive and reversible vacuolation of Schizosaccharomyces pombe induced by amphotericin B

Abstract The fission yeast Schizosaccharomyces pombe has no large vacuoles under normal growth conditions, although budding yeasts usually have large central vacuoles. The minimum inhibitory concentration of amphotericin B to S. pombe was 0.5 μg ml⁻¹; treatment with 0.2 μg ml⁻¹ for 20 min induced rapid and extensive vacuolation in S. pombe exponential phase cells. Growth rate of the cells with 0.2 μg ml⁻¹ amphotericin B was much reduced for 6 h, showing extensive vacuolation. Vacuolation in itself was not fatal: on removal of the drug, most cells recovered gradually and eventually multiplied.