Clinical and molecular epidemiology of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a long-term study from Japan.

The detection rates of extended-spectrum ß-lactamase (ESBL)-producing bacteria in Japan are very low (∼5%) compared with those obtained worldwide. Further, the current trend of these bacteria in Japan is not known, and few studies with longitudinal observations have been reported. To obtain epidemiologic data on ESBL-producing bacteria, their genotypic features, and their antibiotic resistance patterns in Japan, we analyzed bacterial isolates from hospitalized patients at our institution over the 7-year period from 2003 to 2009. Of 2,304 isolates, 202 (8.8%) were found to be ESBL producers, including Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. The detection rates of the ESBL-producing isolates gradually increased and reached 17.1% and 10.5% for the E. coli and K. pneumoniae strains, respectively, in 2009. Genotyping analysis showed that ∼90% of the ESBL-producing isolates carried the CTX-M genotype, in which the CTX-M-9 group was predominant, although the CTX-M-2 group is considered to be the main genotype in Japan; further, many of the strains produced multiple ß-lactamases. The detection rates of ESBL-producing bacteria may tend to be high within a limited region in Japan. A countrywide survey is required to understand the trend for ESBL-producing bacteria at the national level. In addition, our findings suggest that the genotypes of the detected ESBL producers are similar to those exhibiting a successful nosocomial spread worldwide.

CpG methylation of MGMT and hMLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection.

Inactivations of DNA repair genes, O(6)-methylguanine-DNA methyltransferase (MGMT) and hMLH1, by promoter hypermethylation have been reported in several types of primary human neoplasia. This epigenetic inactivation mechanism remains elusive in hepatocellular carcinoma (HCC). To investigate the relation between the expression of MGMT and hMLH1 and the CpG methylation within their promoters in HCCs with or without hepatitis viral infection, we performed immunohistochemistry and urea/bisulphite sequencing on 46 HCCs, corresponding noncancerous tissues, and 20 normal liver tissues. MGMT- and hMLH1-negative HCCs were 60.9% (28 out of 46) and 21.8% (10 out of 46), respectively. HCCs lacking both proteins were 10.9% (five out of 46). The frequency and extent of CpG methylation in the MGMT promoter increased along with hepatitis viral infection and pathological progression. MGMT-negative tumours showed very frequent and widespread methylation in the promoter compared with MGMT-positive tumours. Half of the hMLH1-negative HCCs showed promoter hypermethylation. These data suggested that MGMT gene silencing in a subset of HCCs was likely caused by epigenetic alteration, such as promoter hypermethylation, and that the promoter hypermethylation silenced the hMLH1 gene in half of the hMLH1-negative tumours. A correlation between the promoter methylation status and viral infection, although it was weak, intimated that hepatitis viral infections could play a role in the CpG methylation of the MGMT promoter.

The role of E-cadherin in the differentiation of gallbladder cancer cells.

Cell adhesion molecules are essential for development and maintenance of epithelial architecture. To clarify the role of these molecules in the morphology of gallbladder cancers, four human gallbladder cancer cell lines (GB-d1, KMG-C, GBK-1, and G-415) were examined in vitro. They showed noticeably different morphologies in our standard gel cultures (SC). GB-dl and KMG-C formed cystic and spheroid structures, respectively, which seemed to represent well-differentiated and moderately differentiated cancers, respectively. GBK-1 and G-415 showed branching and "pseudoglandular" structures, respectively, both of which seemed to indicate original dedifferentiated cancers. In floating gel culture (FC), only GB-d1 showed a highly increased tendency toward cyst formation. Expression of E-cadherin and alpha-catenin in the gallbladder cancer cell lines was investigated by Western-blotting analysis. Expression was detected in GB-d1 and KMG-C, but not in GBK-1 and G-415 cells. Furthermore, E-cadherin expression in GB-dl was 1.82 times greater in FC than in SC, while E-cadherin expression levels of KMG-C did not change. Neither GB-d1 nor KMG-C showed any difference in a-catenin expression between SC and FC. Immunostaining of GB-d1 revealed that these proteins were localized to the cell membrane. In contrast, heterogeneous localization of these proteins was detected in the spheroid structures of KMG-C, in both SC and FC. Electronmicroscopic examination revealed that reestablishment of the junctional complex occurred only in GB-d1 cells cultured in FC. The formation of cystic structures in GB-d1 was completely inhibited by an antibody against human E-cadherin. Both expression of E-cadherin and its membranous localization are required for well-differentiated-type morphogenesis in gallbladder cancer cells.

Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin.

The expression of the UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene has been shown to be critical for anthocyanin biosynthesis in the grape berry. Using white cultivars and bud sports with red skin, we examined the expression of seven anthocyanin biosynthetic genes including the UFGT gene and compared the coding/promoter sequences of the UFGT gene. Northern blot analysis showed that the seven anthocyanin biosynthetic genes were expressed coordinately at higher levels in the red-skin sports than in the white-skin progenitors of the sports. It was especially notable that UFGT gene expression was detected only in the red-skin sports and Kyoho. However, there were no differences in either coding or promoter sequences between Italia (Vitis vinifera) and its red-skin sport Ruby Okuyama, or between Muscat of Alexandria (V. vinifera) and the red-skin sport Flame Muscat. From these findings, the phenotypic change from white to red in the sports is thought to be the result of a mutation in a regulatory gene controlling the expression of UFGT.

Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP.

The human nucleotide pool sanitization enzyme, MTH1, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dATP in addition to 8-hydroxy-dGTP. We report here that human MTH1 is highly specific for 2-hydroxy-ATP, among the cognate ribonucleoside triphosphates. The pyrophosphatase activities for 8-hydroxy-GTP, 2-hydroxy-ATP and 8-hydroxy-ATP were measured by high-performance liquid chromatography. The kinetic parameters thus obtained indicate that the catalytic efficiencies of MTH1 are in the order of 2-hydroxy-dATP > 2-hydroxy-ATP > 8-hydroxy-dGTP > 8-hydroxy-dATP >> dGTP > 8-hydroxy-GTP > 8-hydroxy-ATP. Notably, MTH1 had the highest affinity for 2-hydroxy-ATP among the known substrates. ATP is involved in energy metabolism and signal transduction, and is a precursor in RNA synthesis. We suggest that the 2-hydroxy-ATP hydrolyzing activity of MTH1 might prevent the perturbation of these ATP-related pathways by the oxidized ATP.

Expression and prognostic significance of O6-methylguanine-DNA methyltransferase in hepatocellular, gastric, and breast cancers.

BACKGROUND: O6-Methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs O6-methylguanine, a promutagenic DNA base damaged by endogenous and environmental alkylating agents. There are few reports that describe whether or not abnormal MGMT expression correlates with the prognosis in human solid cancers. METHODS: The expression of MGMT was immunohistochemically evaluated in 60, 62, 105, and 46 paraffin-embedded samples from patients with curatively resected hepatocellular, gastric, colorectal, and breast cancers, respectively. RESULTS: The expression of MGMT was a positive predictive factor for overall survival in hepatocellular (P = .005) and gastric cancers (P < .001) and for relapse-free survival in breast cancers (P < .001). MGMT-positive gastric tumors (n = 42) were correlated with the absence of serosal invasion (P = .045), lymph node metastasis (P = .006), intestinal type (P = .018), and low pathological tumor, node, metastasis stage (P < .001). All breast tumors that recurred locally after operation were MGMT negative (P = .004). The clinicopathologic characteristics of colorectal cancers with respect to MGMT expression did not significantly differ. CONCLUSIONS: The expression of MGMT is a predictive prognostic marker in patients with hepatocellular, gastric, and breast cancers. These findings may help to establish therapeutic strategies for patients with these types of solid cancer.

Establishment and characterization of human hilar bile duct carcinoma cell line and cell strain.

We established and characterized a human hilar bile duct carcinoma (HBDC) cell line from cells isolated from the ascites of a 75-year-old Japanese woman. Histopathological findings were confirmed to be poorly differentiated adenocarcinoma (pat BsrlCm according to the Japanese Society of Biliary Surgery General rules for surgical and pathological studies on cancer of the biliary tract, 4th edn.). Using a semi-agarose method, a daughter-cell strain was also established. Both the cell line and the strain were transplanted into scid or nude mice with a 100% inoculation rate. The population doubling times of the cell line and the strain were 32.25 and 35.78 h, respectively. The cell line and strain strongly expressed human epithelial antigen (HEA)-125 and cytokeratin (CK)-19 but did not express desmin and partly expressed vimentin. High values tumor markers (carbohydrate antigen [CA19-9], Span-1, KMO-1) were detected in culture supernatants from both the cell line and the cell strain and the concentrations paralleled the patient's serum data. DNA analysis revealed that the cell strain was diploid, whereas the cell line was aneuploid, with a DNA index (DI) of 0.85. Chromosomal analysis of the cell line and the strain revealed a range of numerical abnormalities (76-93 and 74-88, respectively) as well as structural abnormalities. The establishment of this HBDC cell line and strain may provide some benefit for fundamental biological research.

Multi-forms of human MTH1 polypeptides produced by alternative translation initiation and single nucleotide polymorphism.

The human MTH1 gene for 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase, produces seven types (types 1, 2A, 2B, 3A, 3B, 4A and 4B) of mRNAs. The B-type mRNAs with exon 2b-2c segments have three additional in-frame AUGs in their 5' regions. We report here that these transcripts produce three forms of MTH1 polypeptides (p22, p21 and p18) in in vitro translation reactions. Three polypeptides were also detected in extracts of human cells, using western blotting. B-type mRNAs with a polymorphic alteration (GU-->GC) at the beginning of exon 2c that converts an in-frame UGA to CGA yielding another in-frame AUG further upstream, produced an additional polypeptide (p26) in vitro. Substitution of each AUG abolished the production of each corresponding polypeptide. Cell lines from individuals with the GC allele contain more B-type mRNAs than do those of GT homozygotes, and the former produce all of four polypeptides but the latter lack p26. Amounts of each polypeptide reflected copy number of the GC allele in each cell line. There is an apparent linkage dis-equilibrium between the two polymorphic sites, GT/GC at exon 2c and Val83/Met83 at codon 83 for p18.

DNA mismatch repair deficiency in curatively resected sextuple primary cancers in different organs: a molecular case report.

A male patient synchronously or metachronously underwent six curative resections after diagnoses of cancers in the rectum, urinary bladder, stomach, colon, liver and lung. Five cancers, excluding early colon cancer, were analyzed for instability in seven microsatellite markers and in transforming growth factor beta type II receptor, insulin-like growth factor II receptor and BAX. All analyzed cancers had replication errors and instability in at least one target gene. These results suggest that abnormal DNA mismatch repair system plays a major role in the occurrence of multiple primary cancers in this case.

The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein.

The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OH-dATP), oxidized forms of dATP, but not (R)-8,5'-cyclo-dATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.

Metabolic fate of oxidized guanine ribonucleotides in mammalian cells.

8-Oxo-7,8-dihydroguanine- (8-oxoguanine-) containing nucleotides are generated in the cellular nucleotide pool by the action of oxygen radicals produced during normal cellular metabolism. We examined the interconversion and metabolic fate of 8-oxoguanine-containing ribonucleotides in mammalian cells. (1) 8-OxoGTP can be generated not only by direct oxidation of GTP but also by phosphorylation of 8-oxoGDP by nucleotide diphosphate kinase, and the 8-oxoGTP thus formed can serve as a substrate for RNA polymerase II to induce transcription errors. (2) MTH1 protein carrying intrinsic 8-oxo-dGTPase activity has the potential to hydrolyze 8-oxoGTP to 8-oxoGMP, thus preventing misincorporation of 8-oxoguanine into RNA. 8-OxoGMP, the degradation product, cannot be reutilized, since guanylate kinase, which has the potential to phosphorylate both GMP and dGMP, is inactive on 8-oxoGMP. (3) Ribonucleotide reductase, which catalyzes reduction of four naturally occurring ribonucleoside diphosphates, cannot convert 8-oxoguanine-containing ribonucleotide to the deoxyribonucleotide. This step appears to serve as a gatekeeper to prevent formation of mutagenic substrates for DNA synthesis from oxidized ribonucleotides.

Localization of vascular endothelial growth factor in synovial membrane mast cells: examination with "multi-labelling subtraction immunostaining".

Mast cells are believed to play a novel part in the development of destructive synovial pannus in rheumatoid arthritis (RA). This study was undertaken to investigate the localization of vascular endothelial growth factor (VEGF) in the synovial membrane using a unique immunostaining technique. Synovial specimens of RA patients were examined immunohistochemically and were compared with specimens from non-RA controls. Multi-labelling subtraction immunostaining, a modification of double- and triple-labelling immunostaining, revealed that the VEGF-positive cells were identical to tryptase-positive cells (mast cells). No other cell types were found to be positive for VEGF. The synovium of RA patients showed a larger number of VEGF-positive mast cells than that of non-RA controls (P<0.001). The study suggests that mast cell-derived VEGF may contribute to the development of synovial pannus in RA.

Sensitivity of Escherichia coli (MutT) and human (MTH1) 8-oxo-dGTPases to in vitro inhibition by the carcinogenic metals, nickel(II), copper(II), cobalt(II) and cadmium(II).

The toxicity of Ni(II), Co(II) and Cu(II) in animals, and that of Cd(II) in cultured cells, has been associated with generation of the promutagenic lesion 8-oxo-7,8-dihydroguanine (8-oxoguanine) in DNA, among other effects. One possible source of this base may be 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool, from which it is incorporated into DNA. To promote such incorporation, the metals would have to inhibit specific cellular 8-oxo-dGTPases that eliminate 8-oxo-dGTP from the nucleotide pool. The present study was designed to test such inhibition in vitro on 8-oxo-dGTPases from two different species, the human MTH1 protein and Escherichia coli MutT protein. In the presence of Mg(II), the natural activator of 8-oxo-dGTPases, all four metals were found to inhibit both enzymes. For MTH1, the IC50 values (+/- SE; n = 3-4) were 17 +/- 2 microM for Cu(II), 30 +/- 8 microM for Cd(II), 376 +/- 71 microM for Co(II) and 801 +/- 97 microM for Ni(II). For MutT, they were 60 +/- 6 microM for Cd(II), 102 +/- 8 microM for Cu(II), 1461 +/- 96 microM for Ni(II) and 8788 +/- 1003 microM for Co(II). Thus, Cu(II) and Cd(II) emerged as much stronger inhibitors than Ni(II) and Co(II), and MTH1 appeared to be generally more sensitive to metal inhibition than MutT. Interestingly, in the absence of Mg(II), the activity of the enzymes could be restored by Co(II) to 73% of that with Mg(II) alone for MutT, and 34% for MTH1, the other metals being much less or non-effective. The difference in sensitivity to metal inhibition between the two enzymes may reflect the differences in the amino acid ligands, especially the cysteine ligand, outside their evolutionarily conserved Mg(II)-binding active sites, which might indicate predominantly non-competitive or uncompetitive mechanism of the inhibition. The overall results suggest that inhibition of 8-oxo-dGTPases may be involved in the mechanisms of induction of the 8-oxoguanine lesion in DNA by the metal ions studied, especially the non-redox-active Cd(II) cation.

Biochemical and physicochemical characterization of normal and variant forms of human MTH1 protein with antimutagenic activity.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during cellular metabolism, and its misincorporation into DNA causes mutation. Human cells possess an enzyme that hydrolyzes 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing misincorporation of 8-oxo-7,8-dihydroguanine into DNA. Sequence analyses of the MTH1 gene, encoding the 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphatase (8-oxo-dGTPase) protein in human cell lines revealed that a G to A base substitution frequently occurs at codon 83, which causes a change of valine to methionine in the MTH1 protein [Wu, C. et al., Biochem. Biophys. Res. Commun. 214 (1995) 1239-1245]. Here we isolated cDNAs for the two types of MTH1 protein and expressed them in Escherichia coli mutT-. cells, devoid of their own 8-oxo-dGTPase activity. The two forms of proteins were purified to physical homogeneity, and amino acid analyses confirmed that the variant protein, Met83-MTH1, indeed carries the corresponding amino acid substitution. Met83-MTH1, but not normal type Val83-MTH1, was separated into two peaks in hydrophobic interacting chromatography. 8-Oxo-dGTPase activity of Met83-MTH1 is more thermolabile than that of Val83-MTH1. Circular dichroism (CD) and fluorescence spectroscopic analyses confirmed this conclusion. CD further indicated that Met83-MTH1 has a higher alpha-helix content.

Significance of the conserved amino acid sequence for human MTH1 protein with antimutator activity.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during normal cellular metabolism, and incorporation into DNA causes transversion mutation. Organisms possess an enzyme, 8-oxo-dGTPase, which catalyzes the hydrolysis of 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing the occurrence of mutation. There are highly conserved amino acid sequences in prokaryotic and eukaryotic proteins containing this and related enzyme activities. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site- directed mutagenesis of the cloned cDNA for human 8-oxo-dGTPase, and the activity and stability of mutant forms of the enzyme were examined. When lysine-38 was replaced by other amino acids, all of the mutants isolated carried the 8-oxo-dGTPase-negative phenotype. 8-Oxo-dGTPase-positive revertants, isolated from one of the negative mutants, carried the codon for lysine. Using the same procedure, the analysis was extended to other residues within the conserved sequence. At the glutamic acid-43, arginine-51 and glutamic acid-52 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild type protein. We propose that Lys-38, Glu-43, Arg-51 and Glu-52 residues in the conserved region are essential to exert 8-oxo-dGTPase activity.

[A comparative study on the serum and tissue 5-FU concentrations in patients with hepatocellular carcinoma after preoperative oral administration of UFT and 5'-DFUR].

UFT or 5'-DFUR was orally administered to the patients with hepatocellular carcinoma preoperatively and the concentrations of these drugs and 5-FU in the serum, liver tissue and cancer tissue obtained at the time of operation were measured. The unchanged 5'-DFUR was not detected in any of these samples. The concentration of 5-FU in cancer tissue was significantly higher in UFT treated group (0.409 microgram/g) than that in 5'-DFUR group (0.040 microgram/g). However, the 5-FU levels in the serum and noncancerous liver tissue were also higher than those in the patients with other organ cancers. Although UFT is a useful drug for the adjuvant chemotherapy of hepatocellular carcinoma, the dose was considered to be minimized to avoid the side effects since the activity of drug-metabolizing enzymes may be decreased in hepatocellular carcinoma complicated with liver cirrhosis.

[Intermittent intra-arterial infusion chemotherapy using implantable reservoir for the treatment of hepatic metastasis].

Intermittent intra-arterial infusion chemotherapy using implantable reservoir was performed for hepatic metastases and the therapeutic effects were evaluated. We treated 21 patients with hepatic metastases of gastric cancer in 8 cases, rectal cancer in 6 cases, colon cancer in 5 cases and breast cancer in 2 cases. The reduction rate of the tumor diameter as seen by CT scan was used as a criteria for antitumor effectiveness. Only 1 case was PR, for an efficacy rate of 5%. Changes in serum CEA level were related to antitumor effectiveness.

Changes in mouse brain monoamine oxidase activity in the first, second and fourth generations after manganese administration.

The present study was conducted to explore whether or not manganese effect on brain monoamine oxidase (EC 1.4.3.4) is subject to hereditary genetic amplification. Mice of both sexes were given manganese through four generations, and the enzyme activity was measured in the cerebral cortex, cerebellum, hypothalamus and hippocampus of each of the generations except for the third, whose activity we were not in a position to measure. Intrinsic enzyme activity was highest in the cerebellum, and was followed by those in the cerebral cortex and hypothalamus. The activity in the hippocampus was the lowest. Manganese administration greatly stimulated the activity in the cerebellum. However, as generation succeeded, the level of susceptibility to manganese gradually declined. Manganese concentration in pooled suborgan fractions proved to be, in every case, higher in the cerebral cortex, cerebellum and hippocampus and lower in the hypothalamus. No indication was found that the manganese effect is genetically inherited.