Utility of a GFP-expressing Barley yellow mosaic virus for analyzing disease resistance genes.

The soil-borne plasmodiophorid Polymyxa graminis is a vector for Barley yellow mosaic virus (BaYMV), which can severely damage barley plants. Although 22 disease resistance genes have been identified, only a few have been used for breeding virus-resistant cultivars. Recently, BaYMV strains capable of overcoming the effects of some of these genes have been detected. In this study, green fluorescent protein (GFP)-expressing BaYMV was constructed and used to examine viral dynamics in inoculated barley plants. Leaf inoculations resulted in higher infection rates than root or crown inoculations. Additionally, inoculations of some resistant cultivars produced infections that were similar to those observed in a field test. The results of this study indicate that the GFP-expressing virus is a useful tool for visualizing virus replication and dynamics, and for understanding resistance mechanisms.

[A case of meningeal melanomatosis diagnosed by immunostaining of cerebrospinal fluid].

A 49-year-old woman was admitted to our hospital with suspected hypertensive encephalopathy. On the basis of MRI showing leptomeningeal enhancement and Class V cytology of the CSF, she was diagnosed as having leptomeningeal carcinomatosis. Although no primary site was detected, a few melanin granules were observed at the third CSF examination. The atypical cells in the CSF demonstrated immunoreactivity for HMB-45 and S-100, which are specific markers of malignant melanoma. There have been few reports of meningeal melanomatosis in Japan. This case illustrates that immunostaining is diagnostically useful in patients with leptomeningeal carcinomatosis from neoplasms with unknown primary sites.

Immortalization of normal human gingival keratinocytes and cytological and cytogenetic characterization of the cells.

Most in vitro studies of oral carcinogenesis in human cells are carried out with oral keratinocytes immortalized by human papillomavirus type 16 DNA. However, because various etiological factors for oral cancer are known, it is important to establish new human keratinocyte cell lines useful for studying the mechanism of oral carcinogenesis. Normal human gingival keratinocytes in secondary cultures grown in serum-free medium were either transfected with origin (-) SV40 DNA or sequentially transfected with origin (-) SV40 DNA and human c-fos. The transfected cells were continually passaged and analyzed for cytological and cytogenetic characterizations. Four immortal cell lines were grown for over 1100 days in culture and maintained a vigorous growth for over 250 population doublings. They expressed SV40 T antigen, cytokeratins 8 and 18, and E-cadherin, and overexpressed the c-Fos protein. The immortal cell lines had telomerase activity but lacked transformed phenotypes on soft agar or in nude mice. Each cell line had nonrandom chromosomal abnormalities and minisatellite alterations. One of the immortal cell lines, NDUSD-1, retained the capability to deposit calcium, which was also demonstrated in normal human gingival keratinocytes by alizarin red staining, indicating the possibility that NDUSD-1 cells may retain some natural characteristics of normal gingival keratinocytes. Because the oral ectoderm plays an important role in tooth development, these immortal cell lines may be useful in various experimental models for investigations of oral biology and oral carcinogenesis.

An outbreak and isolation of drug-resistant Pseudomonas aeruginosa at Niigata University Hospital, Japan.

Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in patients with impaired host defenses; it is often a cause of life-threatening nosocomial infection in critically ill and immunocompromised patients. An increase in the prevalence of multiple-drug-resistant Pseudomonas aeruginosa (MDRP) in hospitals is thus a worldwide problem. These increases are frequently related to the high selective pressure of antimicrobials commonly used in hospitalized patients, particularly extended-spectrum cephalosporins, beta-lactamase-inhibitor combinations, carbapenems, fluoroquinolones, and aminoglycosides. We evaluated the clinical and microbiological characteristics of drug-resistant P. aeruginosa and MDRP strains that were isolated at Niigata University Hospital, Japan, from 2000 to 2004. We experienced an outbreak of MDRP in 2000, but colonization only was the main feature of the outbreak. Also, the isolation rate of MDRP has decreased since 2004; this reduction in the isolation rate seems to be a result of a move to newly built ward sections in 2001 and the establishment of an infection control team (ICT) in 2003.

Stereoselectivity control by oxaspiro rings during Diels-Alder cycloadditions to cross-conjugated cyclohexadienones: the syn oxygen phenomenon.

The diastereofacial selectivity operating in Diels-Alder additions involving spirocyclic cross-conjugated cyclohexadienones with dienes of varying reactivity has been investigated. The study has included the ether series 1a-c as well as the lactone/ketone pair 2a/2b. In all cases, the preferred [4+2] cycloaddition pathway consisted of bonding from that pi-surface syn to the oxygen atom. 4-Substituted-4-methyl-2,5-cyclohexadienones (monocyclic systems) were also examined and found to undergo bond formation preferentially from the face bearing the more electron-withdrawing of the two groups at the 4 position. Kinetic parameters were determined for the cycloaddition of 1a and 2a to cyclopentadiene. The rate acceleration profile of solvents was in the order CF(3)CH(2)OH >> CH(3)CN approximately CH(2)Cl(2) for the production of 9a from 1a and CF(3)CH(2)OH >> CH(2)Cl(2) > CH(3)CN for the production of 21a from 2a, respectively. This spread in polarity had no major impact on product distribution, a phenomenon also reflected in the behavior of 4-substituted-4-methyl-2,5-cyclohexadienones under comparable conditions. Theoretical assessment of these experimental facts was undertaken at the HF/6-31G level. The facial selectivity is understandable in terms of the secondary interaction between the HOMO of the diene and LUMO of the dienophile as well as the effective hyperconjugation between the newly forming bond and the 4-anti-C-C sigma-orbital due to the more electron-donating bond, as defined by the Cieplak model.

Preference for the Diels-Alder addition of dienes syn to the O atom in cross-conjugated spirocyclic cyclohexadienones.

In the Diels-Alder reaction, the preferred addition of dienes syn to the O atom in cross-conjugated cyclohexadienones containing an oxa-spiro ring system is observed. The two structures reported here, namely rel-(1R,4aR,9S,9aS,10R)-4a,9,9a,10-tetrahydro-9,10-diphenylspiro[9,10-epoxyanthracene-1(4H),2'-oxiran]-4-one, C27H20O3, and rel-(1R,4aS,9R,9aS,10S)-4a,9,9a,10-tetrahydro-9,10-diphenylspiro[9,10-epoxyanthracene-1(4H),2'-oxetane]-4-one, C28H22O3, are the minor and sole products, respectively, of the reactions of diphenylisobenzofuran with two slightly different cyclohexadienones. These structures differ in the size of the oxa-spiro ring, by one C atom, and in the relative configuration at the spirocyclic ring C atom, leading to some minor conformational differences between the two compounds.

Inhibitory action of telithromycin against Shiga toxin and endotoxin.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is associated with hemolytic uremic syndrome (HUS). High inflammatory cytokine [interleukin (IL)-6 and IL-8] levels and low anti-inflammatory cytokine (IL-10) levels are indicators of a high risk for developing HUS in STEC-infected children. In this study, we investigated inhibitory action of telithromycin, a ketolide, against STEC and against Stx and lipopolysaccharide (LPS). Telithromycin inhibited in vitro STEC growth without inducing Stx phage, in marked contrast to norfloxacin. Stx markedly induced inflammatory (but not anti-inflammatory) cytokine production in human peripheral blood monocytes, while LPS induced both inflammatory and anti-inflammatory cytokine production. Telithromycin selectively inhibited the IL-6 and IL-8 production from Stx-stimulated (but not LPS-stimulated) monocytes. The drug did not significantly inhibit IL-10 production. Our data suggest that Stx plays a crucial role in the stimulation of inflammatory cytokines and such inflammatory response is inhibited by telithromycin, an anti-bacterial agent.

Immortal, telomerase-negative cell lines derived from a Li-Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities.

Five immortal cell lines derived from a Li-Fraumeni syndrome patient (MDAH 087) with a germline mutant p53 allele were characterized with respect to telomere length and genomic instability. The remaining wild-type p53 allele is lost in the cell lines. Telomerase activity was undetectable in all immortal cell lines. Five subclones of each cell line and five re-subclones of each of the subclones also showed undetectable telomerase activity. All five immortal cell lines exhibited variability in the mean length of terminal restriction fragments (TRFs). Subclones of each cell line, and re-subclones of the subclones also showed TRF variability, indicating that the variability is owing to clonal heterogeneity. Chromosome aberrations were observed at high frequencies in these cell lines including the subclones and re-subclones, and the principal types of aberrations were breaks, double minute chromosomes and dicentric chromosomes. In addition, minisatellite instability detected by DNA fingerprints was observed in the immortal cell lines. However, all of the cell lines were negative for microsatellite instability. As minisatellite sequences are considered recombinogenic in mammalian cells, these results suggest that recombination rates can be increased in these cell lines. Tumor-derived human cell lines, HT1080 cells and HeLa cells that also lack p53 function, exhibited little genomic instability involving chromosomal and minisatellite instabilities, indicating that chromosomal and minisatellite instabilities observed in the immortal cell lines lacking telomerase activity could not result from loss of p53 function.

Cell-transforming activity and mutagenicity of 5 phytoestrogens in cultured mammalian cells.

For the simultaneous assessment of in vitro carcinogenicity and mutagenicity of phytoestrogens, the abilities of 5 phytoestrogens, daidzein, genistein, biochanin A, prunetin, and coumestrol, to induce cell transformation and genetic effects were examined using the Syrian hamster embryo (SHE) cell model. Cellular growth was inhibited by all phytoestrogens in a concentration-related manner. The growth inhibitory effect of the compounds was ranked: genistein, prunetin > coumestrol > biochanin A > daidzein, which did not correspond to their apoptosis-inducing abilities. Morphological transformation in SHE cells was elicited by all phytoestrogens, except, prunetin. The transforming activities were ranked as follows: genistein > coumestrol > daidzein > biochanin A. Somatic mutations in SHE cells at the Na(+)/K(+) ATPase and hprt loci were induced only by genistein, coumestrol, or daidzein. Chromosome aberrations were induced by genistein or coumestrol, and aneuploidy in the near diploid range was occurred by genistein or biochanin A. Genistein, biochanin A or daidzein induced DNA adduct formation in SHE cells with the abilities: genistein > biochanin A > daidzein. Prunetin was negative for any of these genetic endpoints. Our results provide evidence that genistein, coumestrol, daidzein and biochanin A induce cell transformation in SHE cells and that the transforming activities of these phytoestrogens correspond to at least 2 of the mutagenic effects by each phytoestrogen, i.e., gene mutations, chromosome aberrations, aneuploidy or DNA adduct formation, suggesting the possible involvement of mutagenicity in the initiation of phytoestrogen-induced carcinogenesis.

Effects of azithromycin on shiga toxin production by Escherichia coli and subsequent host inflammatory response.

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the human intestinal mucosa, produces Stx from phage, and causes the development of hemolytic-uremic syndrome via Stx-induced inflammatory cytokine production. Azithromycin exhibited strong in vitro activity against STEC without inducing Stx-converting phage, in marked contrast to norfloxacin. Azithromycin decreased the tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 production from Stx-treated human peripheral mononuclear cells or monocytes to a greater extent than did clarithromycin. In Stx-injected mice, azithromycin significantly suppressed Stx-induced TNF-alpha, IL-1beta, and IL-6 levels in serum and improved the outcome as assessed by survival rate. In the STEC oral infection experiment using immature mice immediately after weaning (weaned immature-mouse model), all mice died within 7 days postinfection. Azithromycin administration gave the mice 100% protection from killing, while ciprofloxacin administration gave them 67% protection. The data suggest that azithromycin (at least at higher concentrations) has a strong effect on Stx production by STEC and on the Stx-induced inflammatory host response and prevents death in mice. Azithromycin may have a beneficial effect on STEC-associated disease.

Emergence of clarithromycin-resistant Helicobacter pylori (CRHP) with a high prevalence in children compared with their parents.

BACKGROUND: Clarithromycin-resistant Helicobacter pylori (CRHP) is increasing worldwide. Clarithromycin resistance in H. pylori from familial members has not been investigated. MATERIALS AND METHODS: Biopsy specimens were taken from 13 families living in Tokyo, Yokohama, and Niigata between 1998 and 2001. Drug resistance was tested with the replica plating method. The minimum inhibitory concentrations of antimicrobial agents for H. pylori strains were determined by the agar dilution method. Molecular analyses of H. pylori strains were performed by ribosomal RNA gene restriction pattern analysis. The DNA region, associated with clarithromycin resistance, was analyzed by PCR and sequencing. RESULTS: Helicobacter pylori strains isolated from a 5-year-old-son displayed clarithromycin resistance with a mutation (A --> G at position 2143) in the 23S ribosomal RNA, whereas H. pylori strains from his parents did not. DNA analyses revealed that the boy was infected with his father's strain. The boy had repeatedly developed otitis media and received clarithromycin since the age of 2 years. Studies on an additional 12 families demonstrated that clarithromycin resistance in the children's strains reached 42.9% and was significantly higher than those of H. pylori strains from their parents (0%) or from adult patients (11.1%) (p <.05). CONCLUSIONS: The rate of clarithromycin resistance in H. pylori strains from Japanese children was extremely high, in contrast to those from their parents or adult patients. Prior history of clarithromycin usage in a child suggested development of clarithromycin resistance in resident H. pylori, which was originated from a parent.

Efficient synthesis of a 4,5-epoxy-2-cyclohexen-1-one derivative bearing a spirolactone via a Diels-Alder reaction with high pi-facial selectivity: a synthetic study towards scyphostatin.

The Diels-Alder reaction of spirolactones with cyclopentadiene afforded the adduct with high pi-facial selectivity; a hydrophilic analogue of scyphostatin was synthesized from the Diels-Alder adduct.

Quantitative comparison of the cytocidal effect of seven macrolide antibiotics on human periodontal ligament fibroblasts.

The cytocidal effect of seven macrolide antibiotics on human periodontal ligament fibroblasts (Pel cells) was studied. Pel cells were exposed for 48 h to erythromycin (EM), clarithromycin (CAM), roxithromycin (RXM), azithromycin (AZM), josamycin (JM), midecamycin (MDM), and rokitamycin (RKM), and allowed to form colonies. The cytocidal effect of the macrolides was measured as a decrease in colony-forming efficiency and was found to increase with the concentration. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration that decreases colony-forming efficiency 50% relative to control cells, was extrapolated from the concentration-response curves. The rank of the macrolides according to their cytocidal effect (LD50) was RKM > RXM > CAM > AZM > JM > MDM approximately EM. RKM, RXM, CAM, AZM, and JM were at least 1.7-12.2 times more cytocidal than MDM or EM. When extrapolated from the concentration-response curves, the relative survival of the Pel cells exposed to each of the macrolides at the MIC90 concentrations for periodontopathic bacteria was estimated to be: > or = 53.8% for RKM, > or = 92.7% for RXM, > or = 94.6% for CAM, > or = 97.1% for AZM, and > or = 86.2% for EM. The effect of the antibiotics on the mRNA expression of alkaline phosphatase (ALP) and type I procollagen (COL) was examined in Pel cells exposed for 48 h to RXM, CAM, AZM, and EM, which exhibited strong, moderate, and weak cytocidal activity. The constitutive levels of both ALP and COL mRNA were retained in cells exposed to RXM at < or = 3 microM, CAM at < or = 10 microM, and AZM or EM at < or = 3 microM. The MIC90 against periodontopathic bacteria is < or = 4.8 microM for RXM, 5.3 microM for CAM, 2.7 microM for AZM, and 21.8 microM for EM. These results suggest that topical administration of CAM or AZM to the gingival crevice at their MIC90 concentration for periodontopathic bacteria would have little adverse effect on the growth and differentiation of the periodontal ligament. It is important to note, however, that these findings have yet to be extrapolated to in vivo conditions.

Association of p16(INK4a) and pRb inactivation with immortalization of human cells.

To examine the association of cell cycle regulatory gene inactivation with human cell immortalization, we determined the expression status of INK4a, Rb, and WAF1/ CIP1, in eleven in vitro immortalized human cell lines, including fibroblasts and keratinocytes. Two human papillomavirus type 16 E6 expressing cell lines with telomerase activity, including a fibroblast cell line and a keratinocyte cell line, expressed no detectable p16(INK4a). These cell lines had a hyperphosphorylated pRb and reduced expression of p21(WAF1/CIP1). All of seven fibroblast cell lines immortalized either spontaneously or by (60)Co, X-rays, 4-nitroquinoline 1-oxide or aflatoxin B(1), maintaining their telomeres by the ALT (alternative lengthening of telomeres) pathway, displayed loss of expression of p16(INK4a) and hyperphosphorylation of pRb. Levels of p21(WAF1/CIP1) expression varied among the cell lines. Two fibroblast cell lines that became immortalized following infection with a retrovirus vector encoding human telomerase catalytic subunit (hTERT) cDNA were also accompanied by inactivation of p16(INK4a) and pRb pathways. Acquisition of telomerase activity alone was not sufficient for immortalization of these cell lines. Taken together, all the cell lines including fibroblasts and keratinocytes, with either telomerase activity or the ALT pathway for telomere maintenance showed loss of expression of p16(INK4a) and hyperphosphorylation of pRb. These demonstrate the association of inactivation of both p16(INK4a) and pRb with immortalization of human cells including fibroblasts and epithelial cells and telomerase-positive cells and ALT-positive cells.