Search

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 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.

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.

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.

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.

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-α), interleukin-1β (IL-1β), 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-α, IL-1β, 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.

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.

Ceftazidime-resistant Enterobacter aerogenes was isolated from blood cultures of three patients with fever. DNA analysis using pulsed-field gel electrophoresis and ribosomal RNA gene restriction digest pattern analysis revealed that the strains were clonally similar to each other with a 79.3-96.0% homology. The same strain of E. aerogenes was isolated from a three-way stopcock connected to the indwelling catheter in one of the patients at a concentration of 45 cfu/mL. A similar strain was also isolated from the urine of one other patient on the same floor. The data suggest that E. aerogenes caused septicaemia via low bacterial contamination of a three-way stopcock in a peripheral drip intravenous infusion system in at least one patient, and that the outbreak of E. aerogenes infections was due to clonally-related strains.

Biopsy specimens of the antrum and corpus were obtained from four Helicobacter pylori -infected members of a family and from the same boy (son 1) in whom the infection reappeared after simultaneous successful eradication treatment of three family members, excluding the mother. A total of 18 to 60 H. pylori isolates were obtained from each specimen and subjected to rRNA gene restriction pattern analysis. The father's isolates and the initial isolates from son 1 showed the same Hin dIII type, which was divided into three Hae III subtypes. Isolates from the mother and a brother (son 2) and posttreatment isolates from son 1 showed a distinct Hin dIII type (with one minor subtype), which was divided into six Hae III subtypes. All subtypes of the initial isolates from son 1 were present in the father's isolates, and all subtypes of the posttreatment isolates from son 1 were present in the mother's isolates but not in son 2's. Electron microscopic analysis of the biopsy specimens demonstrated extremely high levels of H. pylori colonization in the father's gastric mucosa. H. pylori adherence with a ruffle formation was also demonstrated. The findings suggest that son 1 was infected initially with the H. pylori strain of the father and son 2 was infected with the H. pylori strain of the mother and that after eradication therapy son 1 was reinfected with the H. pylori strain of the mother, who did not undergo eradication therapy.

BACKGROUND: Clarithromycin-resistant Helicobacter pylori (CRHP) has increasingly been isolated from patients in Japan. The aim of our study was to test whether proton pump inhibitors (PPIs) and their thioether derivatives, which are secreted into the gastric mucosa, could inhibit the growth and motility (a factor in colonization) of CRHP. MATERIALS AND METHODS: CRHP was isolated from patients who had experienced gastritis or peptic ulcers in Tokyo and Niigata. Drugs and related agents tested were omeprazole, lansoprazole, rabeprazole, the thioether derivative of rabeprazole (rabeprazole-TH), clarithromycin, amoxicillin and metronidazole. The MICs of the drugs and agents for H. pylori strains were determined by the agar dilution METHOD: Bacterial swimming in a liquid layer was examined under an inverted, phase-contrast microscope. RESULTS: The PPIs and rabeprazole-TH, but not the anti-H. pylori agents, inhibited the motility of CRHP at both pH 7.4 and 6.0. The concentrations (microg/ml) necessary to inhibit 50% of the motility at pH 7.4 were 0.25-0.5, 8-32, 8-16 and 128-256 for rabeprazole-TH, rabeprazole, lansoprazole and omeprazole, respectively. Rabeprazole-TH exhibited the strongest inhibitory effect against the growth of CRPH (MIC, 0.5 microg/ml). CONCLUSION: Rabeprazole-TH, which is secreted into the gastric mucosa, had the strongest inhibitory action against both the growth and motility of CRHP, suggesting that it is a potential novel agent for CRHP eradication.

The fluoroquinolone ofloxacin (OFLX) is one of the candidates of antibacterial agents to be topically used against periodontitis. To estimate the maximum concentration of OFLX which exerts little or no adverse effect on the periodontal ligament, cytological and cytogenetic effects of OFLX on human periodontal ligament fibroblasts (Pel cells) were examined. Treatment of Pel cells with < or =0.3 mM OFLX for 24 or 48 h had little inhibitory effect on cellular growth and survival. DNA, RNA and protein syntheses in Pel cells did not decrease in response to treatment with < or =0.3 mM OFLX. The constitutive level of alkaline phosphatase mRNA was retained in cells treated with < or = 0.03 mM OFLX for 24 or 48 h. The level of type I procollagen mRNA was not affected by treatment with < or = 0.003 mM OFLX for 24 or 48 h. Cytogenetic effects of OFLX were evaluated by the ability of OFLX to induce chromosome aberrations in Pel cells. Treatment with OFLX at 0.3-3.0 mM for 6, 24, or 48 h failed to induce chromosome aberrations in Pel cells. The failure of OFLX to induce chromosome aberrations was seen even in the presence of exogenous metabolic activation using a 5% rat liver post-mitochondrial supernatant mixture. These results indicate that treatment of Pel cells with < or =0.003 mM OFLX has few or no adverse effects on the cytological and cytogenetic endpoints examined, suggesting that there would be little adverse effect on growth and differentiation of the periodontal ligament, as well as little cytogenetic activity, if OFLX were to be topically administered to the gingival crevice at the minimal inhibitory concentration (MIC90) against periodontopathic bacteria (< or = 0.0027 mM). It is important to note, however, that extrapolation of these findings to in vivo conditions has yet to be undertaken.

Bisphenol-A (BP-A), a monomer of plastics used in numerous consumer products and a xenoestrogen, induces cellular transformation and aneuploidy in Syrian hamster embryo (SHE) cells. In this study, the abilities of 4 other bisphenols to induce cellular transformation and genetic effects in SHE cells were examined and compared to BP-A. Cellular growth was inhibited by all bisphenols in a concentration-related manner. The growth inhibitory effect of the bisphenols ranked: BP-5 > BP-4 > BP-3 > BP-2 or BP-A. Morphological transformation of SHE cells was induced by BP-A, BP-3, BP-4 and BP-5, and the induced-transformation frequencies were highest with BP-4. None of the bisphenols induced gene mutations at the Na(+)/K(+) ATPase locus or the hprt locus, or chromosomal aberrations in SHE cells. By contrast, aneuploidy induction in the near-diploid range was exhibited by BP-A, BP-3, BP-4 or BP-5, corresponding to the transforming activity of each compound. The results indicate that BP-A, BP-3, BP-4 and BP-5 exhibit transforming activity in SHE cells, while BP-2 does not, and that aneuploidy induction may be a causal mechanism of the transforming activity.

2-methoxyestradiol (2-MeOE(2)) is an endogenous metabolite of 17beta-estradiol and a proposed inhibitor of tumor growth and angiogenesis. However, 2-MeOE(2) is also an inhibitor of microtubule assembly and other microtubule inhibitors, e.g. colcemid and diethylstilbestrol, induce aneuploidy and cell transformation in cultured mammalian cells. To assess the in vitro carcinogenicity and related activity of 2-MeOE(2), the abilities of this metabolite to induce cell transformation and genetic effects were studied simultaneously using Syrian hamster embryo (SHE) fibroblasts. Growth of these cells was reduced by treatment with 2-MeOE(2) at 0.1-1.0 microg/ml in a concentration-dependent manner. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 2-48 h also resulted in a concentration- and treatment time-related increase in the mitotic index and the percentage of multinucleated cells. Treatment with 2-MeOE(2) at 0.1-1.0 microg/ml for 48 h induced a statistically significant increase in the frequencies of morphological transformation of SHE cells in a concentration-dependent manner. A statistically significant increase in the frequencies of somatic mutations at the Na(+)/K(+) ATPase or hprt locus was also observed in cells treated with 2-MeOE(2) for 48 h at 0.1 or 0.3 microg/ml, respectively. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 24 h induced chromosome aberrations, mainly breaks, exchanges and chromosome pulverization. The incidence of chromosome aberrations was not affected by co-treatment with alpha-naphthoflavone, an inhibitor of 2-hydroxylase that inhibits oxidative conversion of 2-MeOE(2) to 2-hydroxyestradiol, but the incidence was slightly increased by co-treatment with L-ascorbic acid. Numerical chromosomal changes in the near diploid range and in the tetraploid and near tetraploid ranges were also detected in 2-MeOE(2)-treated cells. These findings indicate that 2-MeOE(2) has cell transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity.

To examine a direct involvement of genotoxic effects of estrogens in the initiation of hormonal carcinogenesis, the abilities of 17beta-estradiol (E2) and 8 of its metabolites to induce cellular transformation and genetic effects were studied using the Syrian hamster embryo (SHE) cell model. Treatment with E2, estrone (E1), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-methoxyestrone (2-MeOE1), 16alpha-hydroxyestrone (16alpha-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) or estriol (E3) for I to 3 days inhibited SHE cell growth in a concentration-dependent manner. Concentration-dependent increases in the frequency of morphological transformation in SHE cells were exhibited by treatment for 48 hr with each of all estrogens examined, except for E3. The transforming activities of the estrogens, determined by the induced transformation frequencies, were ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 > or = E2 or E1 > 2-MeOE1 or 16alpha-OHE1 > E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced only when the cells were treated with 4-OHE1, 2-MeOE1 or 4-OHE2 for 48 hr. Some estrogen metabolites induced chromosome aberrations in SHE cells following treatment for 24 hr. The rank order of the clastogenic activities of the estrogens that induced chromosome aberrations was 4-OHE1 > 2-OHE1 or 4-OHE2 > 2-OHE2 > E1. Significant increases in the percentage of aneuploid cells in the near diploid range were exhibited in SHE cells treated for 48 hr or 72 hr with each of the estrogens, except for 4-OHE1 and E3. Our results indicate that the transforming activities of all estrogens tested correspond to at least one of the genotoxic effects by each estrogen, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the possible involvement of genotoxicity in the initiation of estrogen-induced carcinogenesis.

Evaluation of disorders constitutes an important part of work in rehabilitation medicine. But all of the objective evaluation methods for quantifying ADL have both advantages and disadvantages, and they are often difficult to use. Evaluation and judgment of ADL should be carried out in terms of movements simplified as much as possible. An ADL Score Study Group, consisting of medical co-workers, has been organized, and they have proposed a tentative plan for patients with paraplegia due to spinal cord injury. Method; ADL items were limited to 10, with sub-items limited to two to three per item, to simplify the ADL score as much as possible. The evaluation was carried out in accordance with the followings; 0 point, requiring full assistance, 1 point, partial assistance, 2 points, possible to do activities with insufficient perfectness, 3 points, possible to do activities independently, with a maximum of 99 points. Subjects were 35 patients with complete paralysis due to spinal cord injury. Thirty four persons were males and one was female. Results; Evaluation scores were 81 points for Group A (Spinal cord injury T1-7), 85 points for Group B (Spinal cord injury T8-L1), 95 points for Group C (Spinal cord injury L2 or lower). It was demonstrated that the evaluation of two items, reach and walking, was sufficient for independent patients with paraplegia due to spinal cord injury.

Simultaneous determination of glucose and sucrose was achieved with an apparatus containing two enzyme reactors and an octadecylsilica column positioned between the two reactors in tandem in a single flow line. The enzymes used were mutarotase and glucose dehydrogenase for glucose analysis, and sucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate dehydrogenase for sucrose analysis. The pH and the concentration of the phosphate buffer used as the medium for immobilization of mutarotase and glucose dehydrogenase to activated aminopropyl glass were studied. Several aqueous buffers and their pH values were also examined for use as the carrier medium, and HEPES buffer (pH 7.5) was the most favorable. In this apparatus under these conditions, NADH due to glucose and that due to sucrose were observed separately. The calibration curve for glucose was linear in the range of 1 - 500 μM, and that for sucrose, 5 - 500 μM. This method was applied to the analyses of glucose and sucrose in several ...

Hydration is an essential process in skincare treatments. Low water content in the skin can lead to skin problems, including reduction in skin elasticity and an increase in skin ripples or wrinkles. Consumers use skincare products to reduce age- and environment-related skin changes. Based on our company beauty theory, we proposed a unique skincare routine coupled with a specially formulated moisturizer that improves skin complexion and has the potential to enhance our customer affection, thus generating an overall high customer satisfaction. By using a scientific approach as well as Nagamachi (Applied Ergon 33:289–294, 2002) [1], the differences in skin physiology parameters and affective values, attributed to the various skincare routines, were assessed in the study subjects who underwent a home-use test.

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.

Catechol estrogens (CEs) are considered critical intermediates in estrogen (E)-induced carcinogenesis. Previously, we demonstrated that estradiol (E2), estrone (E1), and four of their catechol estrogens, 2- and 4-OHE2 and 2- and 4-OHE1 induced morphological transformation in Syrian hamster embryo (SHE) cells, and their transforming activities varied as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 ≧ E1, E2, which are consistent with the genetic effects, i.e., chromosome aberrations and DNA adduct formation, of each E. To further elucidate the mechanism of hormonal carcinogenesis, we studied the effect of the catechol-O-methyltransferase (COMT) inhibitor Ro41-0960 on the transforming and clastogenic activities of the CEs using SHE cells. The frequencies of transformation and chromosome aberrations induced by 4-OHE1 were not affected by co-treatment with Ro-41-0960, but those induced by 2-OHE1 were markedly enhanced. The frequency of transformation induced by 4-OHE1 was markedly decreased by E2 in a concentration dependent manner, but this decrease was not inhibited by Ro41-0960. Cell treatment with E2, 2-OHE1, or 4-OHE1 alone induced apoptosis as detected by the TUNEL method. Additive effect on the induction of apoptosis was observed in cells treated with E2 + 2-OHE1 or 4-OHE1. The % apoptotic cells induced by E2 and 4-OHE1 decreased in the presence of Ro41-0960, while those induced by E2 and 2-OHE1 did not. These results suggest an important role of both the substrate specificity of COMT and the induction of apoptosis in CE-induced carcinogenesis.

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.

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)OHCH(3)CN approximately CH(2)Cl(2) for the production of 9a from 1a and CF(3)CH(2)OHCH(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.

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.

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

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.

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 RKMRXMCAMAZMJMMDM 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, andor = 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 ator = 3 microM, CAM ator = 10 microM, and AZM or EM ator = 3 microM. The MIC90 against periodontopathic bacteria isor = 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.

The abilities of 17β-estradiol (E2) and its eight metabolites to induce cellular transformation and genetic effects were studied simultaneously using the Syrian hamster embryo (SHE) cell model. Treatment for 1–3 days with E2, estrone (E1), 2-hydroxyestrone (2- OHE1), 4-hydroxyestrone (4-OHEi), 2-methoxyestrone (2-MeOE1), 16α-hydroxyestrone (16α-OHE1), 2-hydroxyestradiol (2-OHE2), 4- hydroxyestradiol (4-OHE2), or estriol (E3) inhibited SHE cell growth in a dose-dependent manner. Morphological transformation in SHE cells were induced by exposure for 48 hr to each estrogen (E), except for E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced following treatment of cells with 4-OHE1, 2-MeOE1 or 4-OHE2 for 48 hr. Chromosome aberrations were elicited only in cells treated with 4-OHE1, 2- OHE1, 4-OHE2, 2-OHE2 or E1 for 24 hr. Aneuploidy induction in the near diploid range was observed in SHE cells treated with each E, except for 4-OHE1 and E3. The results indicate that the transforming activities of all Es tested correspond to at least one of genotoxic effects by each E, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the involvement of genotoxicity in the initiation of E-induced carcinogenesis.

Bisphenol-A (BP-A) is a major component of epoxy, polycarbonate and other resins. For an assessment of in vitro carcinogenicity and related activity of BP-A, the abilities of this compound to induce cellular transformation and genetic effects were examined simultaneously using the Syrian hamster embryo (SHE) cell model. Cellular growth was reduced by continuous treatment with BP-A at doses > or = 100 microM. However, colony-forming efficiencies were not decreased significantly following treatment with up to 200 microM BP-A for 48 hr. Morphological transformation of SHE cells was induced by treatment of cells with BP-A at 50 to 200 microM for 48 hr. BP-A exhibited transforming activity at doses > or = 50 microM but was less active than the benzo[alpha]pyrene used as a positive control. Over the dose range that resulted in cellular transformation, treatment of SHE cells with BP-A failed to induce gene mutations at the Na+/K+ ATPase locus or the hprt locus. No statistically significant numbers of chromosomal aberrations were detected in SHE cells treated with BP-A. However, treatment of cells with BP-A induced numerical chromosomal changes in the near diploid range at doses that induced cellular transformation. 32P-Postlabeling analysis revealed that exposure of cells to BP-A also elicited DNA adduct formation in a dose-dependent fashion. Our results indicate that BP-A has cell-transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity.

Phenolphthalein is a cathartic agent widely used in non-prescription laxatives. For the simultaneous assessment of in vitro carcinogenicity and mutagenicity of phenolphthalein, the ability of this chemical to induce cell transformation and genetic effects was examined using the Syrian hamster embryo (SHE) cell model. Cell growth was reduced by treatment with phenolphthalein at 10-40 microM in a dose-related manner. Treatment with phenolphthalein for 48 hr induced a dose-dependent increase in morphological transformation of SHE cells. Over the dose range that resulted in cell transformation ( 10-40 microM), treatment of SHE cells with phenolphthalein induced gene mutations at the hprt locus but not at the Na+/K+ ATPase locus. A statistically significant level of chromosomal aberrations was elicited in SHE cells treated with phenolphthalein at the highest dose (40 microM). Meanwhile, neither numerical chromosomal changes nor DNA adduct formation, analyzed by the nuclease P1 enhancement version of 32P-post-labeling, were induced by treatment with phenolphthalein at any concentrations examined. We thus report cell-transforming activity and mutagenicity of phenolphthalein assessed with the same mammalian cells in culture. Our results provide evidence that phenolphthalein has cell-transforming and genotoxic activity in cultured mammalian cells. The mutagenic and clastogenic activities of phenolphthalein could be a causal mechanism for carcinogenicity in rodents.

The Diels-Alder reactions of cross-conjugated ketones bearing an oxa-spiro-ring with some simple sterically undemanding dienes (cyclopentadiene, 2,3-cyclohexadiene, 2,3-dimethyl-1,3-butadiene) afforded the adduct with high π-facial selectivity under mild conditions. The π-facial selectivity can be explained in terms of Cieplak model.

To see the digestibility and the absorption rate of the meatless meat, its main constituent is a defatted soybean (Protein 50.65%, Fat 0.31%, Carbohydrate 29.35%), the investigation was made for 12 days by using mature arbino rats with the diet containing 40% of it.Then, same rats were given non Protein non Fat diets for 10 days, the endogenous N in feces was determined. And by using this value, true digestibility and absorption rate of the meatless meat was calculated.According to the results, apparent absorption rate of this protein is 85.8±3.4%, and the true one is 89.7±3.5%. Though, digestibility and absorption rate of Carbohydrate in this diet is 98.4±0.4%, digestibility and absorption rate of dextrine which is 80% of this Carbohydrate is 99.3%, the absorption rate of the Carbohydrate in meatless meat, which correspond to 20% of the total, is 94.8%.The absorption rate of Fat 96.3% is a digestibility and absorption rate of soybean oil which was added to the diet.

Comparative studies on the effect of lightness and darkness on the birth rate of rats were performed for one year.(1) The lightness group invariably showed a high birth rate, but the darkness group still had reproductive ability after six months of darkness.(2) The average body weight of the offsprings at weaning during the experimental periods showed no significant difference between lightness and darkness groups.(3) Data of reproductive ability of individual rats and litters exhibited large differences and indicated that litter-mates should be used in reproductive studies.