DNA methylation of GJA1, BMP2 and BMP4 in a human cementoblast cell line induced by lipopolysaccharide.

AIM: To examine DNA methylation of GJA1, BMP2 and BMP4 in human cementoblasts (HCEM) induced by lipopolysaccharide (LPS). METHODOLOGY: HCEM were cultured in osteoinduction medium. After 24 h, Escherichia coli LPS (1 µg/mL) was added to the medium, which was changed every 2-3 days. Untreated samples were used as controls. Messenger RNA was extracted after 4 weeks, and quantitative real-time polymerase chain reaction (qRT-PCR) for GJA1, BMP2, BMP4 and DNMT1 was performed. Genomic DNA was extracted after 4 weeks, and quantitative methylation-specific polymerase chain reaction was carried out for GJA1, BMP2 and BMP4. To detect mineralization, alizarin red and alkaline phosphatase staining were performed. The cells were also treated with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza) and examined. The significance of differences amongst groups was assessed using a two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test with P < 0.05 being significant. RESULTS: Decreased expression of mRNA was seen in GJA1, BMP2 and BMP4 after 4 weeks (P < 0.05). DNA hypermethylation was detected in GJA1, BMP2 and BMP4 (P < 0.05). Alizarin red staining and alkaline phosphatase staining revealed decreased mineralization levels in HCEM stimulated with LPS. 5Aza abolished the effects of DNA methylation in HCEM stimulated with LPS. CONCLUSIONS: These results suggest that long-term LPS stimulation induces DNA methylation of GJA1, BMP2 and BMP4 in HCEM.

The Great East Japan Earthquake (2011): using the One Health approach to minimise the impact on the livestock industry and human health.

Supplying safe livestock products made from healthy animals is the primary purpose of the agriculture industry, making it essential to include agriculture in the One Health approach to disaster preparedness and response. After the Great East Japan Earthquake of 11 March 2011, and the following crisis at the Fukushima Nuclear Complex, producing and supplying safe livestock products became a challenging issue, because the area was highly polluted with radiation leaks from the nuclear plants. To produce livestock products that satisfied the safety standards for radioactive materials in food for humans, it was necessary to create feeding management guidelines and set standard limits for radioactive materials in animal feeds. The Ministry of Agriculture, Forestry and Fisheries (MAFF) established provisional maximum limits on radioactive caesium in feeds in order to secure safe food for the nation. Furthermore, there were other issues that Japan's livestock industry had to tackle. The authors outline key measures taken by the Livestock Industry Department of the MAFF to reconstruct the livestock industry, which was a small but important part of the whole reconstruction plan. They also discuss the measures implemented to protect companion animals.

La vocation première du secteur de l'élevage est de fournir au consommateur des produits d'origine animale sans danger et issus d'animaux en bonne santé, ce qui suppose d'intégrer le secteur agricole dans les activités de préparation et de réponse aux catastrophes naturelles sur la base d'une approche Une seule santé. Après le grand séisme survenu le 11 mars 2011 sur la côte Pacifique du Tohoku au Japon et la catastrophe du complexe nucléaire de Fukushima qui a suivi, la question de l'innocuité de la production et distribution d'animaux et de produits d'origine animale est devenue un enjeu majeur en raison du taux élevé de contamination de la zone par les émissions radioactives qui s'étaient échappées des centrales nucléaires. Afin de garantir la conformité des produits de l'élevage aux normes de sécurité au regard du risque de contamination radioactive, il a fallu élaborer des lignes directrices en matière de gestion de l'alimentation animale et fixer une valeur limite à la contamination radioactive des aliments destinés aux animaux. Le ministère japonais en charge de l'agriculture, des forêts et des pêches (MAFF) a fixé à titre provisoire une limite de concentration maximale admissible pour le césium radioactif dans les aliments destinés aux animaux afin de garantir l'innocuité des denrées alimentaires distribuées à la nation. Ce problème n'est pas le seul auquel a dû faire face le secteur japonais de l'élevage suite au séisme de 2011. Les auteurs font le point sur les principales mesures adoptées par le département de l'élevage du MAFF pour restaurer le secteur, volet mineur mais néanmoins essentiel du plan de reconstruction nationale. Les mesures adoptées pour protéger les animaux de compagnie sont également présentées.

El objetivo primordial de la industria agropecuaria estriba en abastecer a la población de productos inocuos, obtenidos a partir de animales sanos. Ello hace indispensable incluir la agricultura en toda labor de preparación y respuesta para casos de catástrofe que se efectúe desde los planteamientos de Una sola salud. Después del llamado Gran terremoto del Japón Oriental que tuvo lugar el 11 de marzo de 2011, y tras la crisis del complejo nuclear de Fukushima, la producción y el suministro de productos ganaderos inocuos pasaron a ser una cuestión muy espinosa, pues la zona estaba sumamente contaminada por fugas radiactivas de las centrales nucleares. Para obtener productos ganaderos que cumplieran las normas de inocuidad relativas a la presencia de material radiactivo en los alimentos destinados al consumo humano fue necesario establecer directrices de gestión de alimentos y fijar límites normalizados de material radiactivo en los piensos animales. A fin de garantizar que el país gozara de un suministro de alimentos inocuos, el Ministerio de Agricultura, Bosques y Pesca estableció límites máximos provisionales de cesio radiactivo en los piensos. Pero este no era el único problema con que debía lidiar la industria ganadera del Japón. Los autores exponen a grandes líneas las principales medidas adoptadas por el Departamento de Industria Ganadera del Ministerio para reconstruir la industria pecuaria, en lo que representaba una parte pequeña, pero importante, del plan de reconstrucción global. Los autores examinan asimismo las medidas instituidas para proteger a los animales de compañía.

Alkaline extracellular conditions promote the proliferation and mineralization of a human cementoblast cell line.

AIM: To investigate the proliferation and mineralization of a human cementoblast cell line under alkaline conditions. METHODOLOGY: A human cementoblast cell line was cultured in alkaline media with several pHs (pH 7.6, 8.0 and 8.4) without CO2 . Cell numbers, phospho-p44/42 expression, alkaline phosphatase (ALP) activity and mineralization were evaluated. The significance of differences between groups was assessed using two-way analysis of variance 15 (ANOVA) followed by Bonferroni's multiple comparison test (α = 0.01). RESULTS: Cell numbers increased in a time-dependent manner in the high pH medium groups. Western blot analysis revealed the upregulated expression of phospho-p44/42 under alkaline conditions. ALP activity was also increased at pH 8.0 and 8.4. Alizarin red staining revealed increased mineralization in the high pH medium groups. The incorporation of the transient receptor potential ankyrin subfamily member 1 (TRPA1) antagonist HC030031 markedly negated the effect on proliferation and mineralization. CONCLUSIONS: Extracellular alkaline conditions promoted the proliferation and mineralization of human cementoblasts in vitro via TRPA1.

Value of 100 kVp scan with sinogram-affirmed iterative reconstruction algorithm on a single-source CT system during whole-body CT for radiation and contrast medium dose reduction: an intra-individual feasibility study.

AIM: To perform an intra-individual investigation of the usefulness of a contrast medium (CM) and radiation dose-reduction protocol using single-source computed tomography (CT) combined with 100 kVp and sinogram-affirmed iterative reconstruction (SAFIRE) for whole-body CT (WBCT; chest-abdomen-pelvis CT) in oncology patients. MATERIALS AND METHODS: Forty-three oncology patients who had undergone WBCT under both 120 and 100 kVp protocols at different time points (mean interscan intervals: 98 days) were included retrospectively. The CM doses for the 120 and 100 kVp protocols were 600 and 480 mg iodine/kg, respectively; 120 kVp images were reconstructed with filtered back-projection (FBP), whereas 100 kVp images were reconstructed with FBP (100 kVp-F) and the SAFIRE (100 kVp-S). The size-specific dose estimate (SSDE), iodine load and image quality of each protocol were compared. RESULTS: The SSDE and iodine load of 100 kVp protocol were 34% and 21%, respectively, lower than of 120 kVp protocol (SSDE: 10.6±1.1 versus 16.1±1.8 mGy; iodine load: 24.8±4versus 31.5±5.5 g iodine, p<0.01). Contrast enhancement, objective image noise, contrast-to-noise-ratio, and visual score of 100 kVp-S were similar to or better than of 120 kVp protocol. CONCLUSION: Compared with the 120 kVp protocol, the combined use of 100 kVp and SAFIRE in WBCT for oncology assessment with an SSCT facilitated substantial reduction in the CM and radiation dose while maintaining image quality.

Response of porcine epithelial rests of Malassez to stimulation by interleukin-6.

AIM: To investigate the proliferation and migration of epithelial cell rests of Malassez (ERM) after stimulation with IL-6. METHODOLOGY: Porcine-derived ERM were seeded on Dulbecco's modified Eagle's Medium, and IL-6 (100 pg mL-1 ) was incorporated into the culture medium. The WST-1 assay was performed to evaluate cell proliferation, and absorption was measured at 450 nm. A wound-healing assay and immunofluorescence assay for integrin α3 were conducted to investigate migration. The Kruskal-Wallis test and the Mann-Whitney U-test with Bonferroni correction were used to analyse data of WST-1 and wound-healing assays. RESULTS: Cell proliferation following the stimulation by IL-6 increased over time, with a significant increase being observed at 6 h (P < 0.05), but not in a concentration-dependent manner. Cell proliferation was significantly greater in IL-6-treated ERM than in nontreated ERM (P < 0.05). The results of the wound-healing assay revealed earlier closure in IL-6-treated ERM (P < 0.05). In the immunofluorescence assay, integrin α3 was detected at the edge of cell processes adjacent to the wound area. A neutralized antibody abrogated the effects of the IL-6 stimulation in cell proliferation and migration. CONCLUSION: IL-6 promoted the proliferation and migration of porcine ERM in vitro.

Actin stabilization induces apoptosis in cultured porcine epithelial cell rests of Malassez.

AIM: To test whether actin stabilization by jasplakinolide induces inhibition of cell viability and apoptosis in epithelial cell rests of Malassez (ERM). METHODOLOGY: ERM derived from porcine were spread in a 96-well dish (5 × 10(4) /well) using Dulbecco's modified Eagle's medium. The actin-specific stabilization reagent, jasplakinolide, was incorporated into the culture medium and incubated for 24 h. To evaluate cell viability, the WST-1 assay was carried out and absorption (450 nm) was measured. To detect apoptotic cells, monoclonal antibody to single-strand DNA (ssDNA) was used and absorption (405 nm) was measured. Actin stabilization and apoptosis induced by jasplakinolide were morphologically investigated by staining with Alexa Fluor 568 phalloidin and observed under a fluorescent microscope. As a negative control, DMSO was used instead of jasplakinolide. Differences between the jasplakinolide-treated group and the control group were analysed statistically using the Student's t-test. RESULTS: Cell viability decreased in a concentration-dependent manner, and cell viability in the jasplakinolide-treated ERM was lower than that in nontreated ERM (n = 16, P < 0.01). Apoptotic cells in the jasplakinolide-treated ERM were more frequently detected compared to that in nontreated ERM (n = 16, P < 0.01). Morphologically, shrinkage, irregular forms and fragmentation of nuclei suggesting apoptotic bodies were observed in jasplakinolide-treated ERM, whilst actin filaments were extended in non-treated ERM. CONCLUSION: Actin stabilization by jasplakinolide inhibited cell viability and induced apoptosis in epithelial cell rests of Malassez.

Measures against increased environmental radiation dose by the TEPCO Fukushima Dai-ichi NPP accident in some local governments in the Tokyo metropolitan area: focusing on examples of both Kashiwa and Nagareyama cities in Chiba prefecture.

The accident of the Fukushima Dai-ichi nuclear power plant of Tokyo Electric Power Cooperation (TEPCO) after the great east Japan earthquake (11 March 2011) elevated the background level of environmental radiation in Eastern Japan. Around the Tokyo metropolitan area, especially around Kashiwa and Nagareyama cities, the ambient dose equivalent rate has been significantly increased after the accident. Responding to strong requests from citizens, the local governments started to monitor the ambient dose equivalent rate precisely and officially, about 3 months after the accident had occurred. The two cities in cooperation with each other also organised a local forum supported by three radiation specialists. In this article, the activities of the local governments are introduced, with main focus on radiation monitoring and measurements. Topics are standardisation of environmental radiation measurements for ambient dose rate, dose mapping activity, investigation of foodstuff and drinking water, lending survey meters to citizens, etc. Based on the data and facts mainly gained by radiation monitoring, risk management and relating activity have been organised. 'Small consultation meetings in kindergartens', 'health consultation service for citizens', 'education meeting on radiation protection for teachers, medical staffs, local government staffs, and leaders of active volunteer parties' and 'decontamination activity', etc. are present key activities of the risk management and restoration around the Tokyo metropolitan area.

Acute antibody-mediated rejection in living ABO-incompatible kidney transplantation: long-term impact and risk factors.

The impact of acute antibody-mediated rejection (AAMR) on the long-term outcome on ABO-incompatible (ABOI) kidney transplantation is not well understood. We retrospectively analyzed the long-term impact of AAMR and risk factors for AAMR in 57 consecutive recipients performed between 1999 and 2004. Nineteen patients (33%) who developed AAMR within 3 months posttransplantation constituted of the AMR group. The graft survival rate was significantly lower in the AMR group (AMR vs. non-AMR, respectively; 5 years: 84% vs. 95%; 8 years: 45% vs. 95%; p = 0.009). The prevalence of transplant glomerulopathy at 1 year posttransplantation was significantly higher in the AMR group (AMR 64% vs. non-AMR 3%, p < 0.001). Multivariate analysis demonstrated that anti-blood group IgG antibody titers of 1:32 at the time of transplantation (OR, 9.52; p = 0.041) and donor-specific anti-HLA antibodies (DSHA) detected by Luminex single bead method (OR, 5.68; p = 0.015) were independent risk factors for AAMR regardless of baseline anti-blood group IgG antibody titers. Our results indicate that AAMR has a heavy impact on the long-term outcome and preoperative DSHA appears to have a more significant association with poor graft outcomes than anti-blood group antibodies, even in ABOI kidney transplantation.

Allograft glomerulitis: histologic characteristics to detect chronic humoral rejection.

The aim of this study was to clarify the histopathologic significance of allograft glomerulitis in chronic allograft nephropathy (CAN). Review of our renal allograft biopsy files revealed 140 specimens with CAN among 115 selected patients. They were classified into two groups: one had CAN with glomerulitis (group G), and the other was free of this finding (group NG). We evaluated the clinicopathologic parameters as follows: levels of serum creatinine and proteinuria in the biopsy; presence of circulating anti-donor antibodies; allograft failure rate; history of biopsy-proven acute cellular rejection (ACR) and acute humoral rejection (AHR); complications of ACR and chronic rejection (CR); and results of immunofluorescence studies for C4d and HLA-DR. The glomerulitis group showed a significantly greater incidence of CR complications, the presence of circulating anti-donor antibodies, and C4d deposition in peritubular and glomerular capillaries. This group also showed higher levels of serum creatinine and proteinuria, higher graft loss rate, and increased AHR incidence, although the differences were not significant. There was also no statistical significance in the HLA-DR expression on tubular epithelial cells. The present results strongly suggest that humoral factors may play an important role in the progression of glomerulitis in CAN. Therefore, we suspect that glomerulitis in CAN is one of the main histologic markers for CR. The presence of glomerulitis may represent humoral factor-dependent inflammation. It should be considered an important diagnostic criterion for CR in addition to double-contour formation and elastica disruptions with or without subendothelial inflammation (Banff '97).

Efficacy of IgM anti-blood type antibody monitoring by enzyme-linked immunosorbent assay after renal transplantation across the blood barrier: high-dose immunoglobulin administration blocks IgM rather than IgG anti-blood type antibodies.

We used an enzyme linked immunosorbent assay (ELISA) to investigate the presence of subtypes of anti-blood-type antibodies in patients with biopsy-proven humoral rejection after ABO-incompatible renal transplantation. High agglutinin IgG and IgM anti-blood type antibodies from 12 ABO-incompatible recipients with vascular rejection were separately assessed using an ELISA. Patients who exhibited excellent renal function despite high agglutinin titers of anti-blood-type antibodies(n = 8) were also examined. All 12 rejection patients exhibited highly elevated titers of IgG and IgM, while the eight stable patients exhibited only slightly elevated IgG titers, but not IgM. IgG and IgM titers did not change after plasmapheresis and steroid pulse therapy, whereas IVIg treatment significantly blocked both IgG and IgM, with IgM being blocked to a larger extent than IgG. Blocking of IgM seems to play an important role in improving ABO-incompatible grafts.

Histological analysis of late renal allografts of antidonor antibody positive patients with C4d deposits in peritubular capillaries.

The association of humoral immunity with late renal allograft dysfunction has recently been recognized, and many reports have revealed C4d deposits in peritubular capillaries (C4d in PTC), and the presence of serum antidonor HLA antibody in patients suffering from graft dysfunction, long time after transplantation. In this study, morphological changes in renal allograft biopsies more than 1 year after transplantation in 14 patients with C4d in PTC and serum antidonor antibody were investigated for the presence of chronic rejection (CR). In addition to the light microscope study, an electron microscope study was done to evaluate the multilayering of the peritubular capillary basement membrane (MLPTC). Histologically, only seven of 14 patients met the criteria of CR, and 71.4% (5/7) of CR patients had episodes of acute humoral rejection (AHR), coexisting with acute tubulointerstitial rejection. Peritubular capillaritis was observed in all patients, although it differed in severity. Transplant glomerulitis and interstitial inflammation were also observed in many patients: 71.4% (10/14) and 92.9% (13/14) respectively. MLPTC was observed in 12 patients (85.7%), but the severity of the MLPTC did not reflect the severity of peritubular capillaritis or any other histological features. The long-term outcomes of the patients CR, especially those with episodes of AHR, were poor, and two of them lost their graft functions. On the other hand, patients without CR had relatively favourable outcomes. In conclusion, we confirmed the diverse morphological changes of late renal allografts, which cannot be categorized as chronic humoral rejection (CHR), and such patients who do not have typical morphological changes such as CHR, should be followed-up on a long-term basis in order to clarify the significance of C4d on PTC in late renal allografts.

AMY-1, a c-Myc-binding protein, is localized in the mitochondria of sperm by association with S-AKAP84, an anchor protein of cAMP-dependent protein kinase.

We have reported that a novel c-Myc-binding protein, AMY-1 (associate of Myc-1), stimulated the transcription activity of c-Myc. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human HeLa cDNA library, and a clone encoding cAMP-dependent protein kinase anchor protein 149 (AKAP149), was obtained. AMY-1 was found to bind in vitro and in vivo to the regulatory subunit II binding region of AKAP149 and S-AKAP84, a splicing variant of AKAP149 expressed in the testis. AMY-1 was expressed postmeiotically in the testis, as S-AKAP84 was expressed. Furthermore, S-AKAP84 and regulatory subunit II, a regulatory subunit of cAMP-dependent protein kinase, made a ternary complex in cells, and AMY-1 was localized in the mitochondria of HeLa and sperm in association with AKAP149 and S-AKAP84, respectively. These results suggest that AMY-1 plays a role in spermatogenesis.

Stilbenoids in lianas of Gnetum parvifolium.

Five new stilbene dimers were isolated from the lianas of Gnetum parvifolium in addition to known stilbenoids. The structures of the compounds were established on the basis of spectroscopic evidence, including long-range coupling and nuclear Overhauser effect experiments, in NMR spectrum. Among the isolates, 2b-hydroxyampelopsin F showed potent inhibitory activity in the Maillard reaction.

Promotion of evolution by intracellular coexistence of mutator and normal DNA polymerases.

The efficient evolution of a population requires both genetic diversity and stable reproduction of advantageous genotypes. The accuracy of DNA replication guarantees the stable reproduction, while errors during DNA replication produce the genetic diversity. Thus, one key to the promotion of evolution is inherent in DNA replication. In bacteria, replication forks progress bidirectionally from the single origin of replication on a genome. One replication fork contains two DNA polymerase molecules so that four DNA polymerases simultaneously carry out the replication of a genome. It is generally believed that the fidelity of the intracellular DNA polymerases is identical (parity strategy). To test this, we examined the effects of the intracellular coexistence of a mutator polymerase with low fidelity and a normal polymerase with high fidelity on adaptive evolution (disparity strategy). From the analysis using genetic algorithms based on the bacterial replication, it was found that the population using the disparity strategy could further expand its genetic diversity and preserve the advantageous genotypes more profoundly than the parity population. This strongly suggests that bacteria replicating with a disparity strategy may undergo rapid evolution, particularly during severe environmental changes. The implications of the conspicuous adaptability of Escherichia coli mutator strains are discussed in this context.

Expression of xenoantigen transformed human cancer cells to be susceptible to antibody-mediated cell killing.

The carbohydrate epitope, alphaGal epitope, is known as a major xenoantigen. The epitope exists abundantly in non-primate mammals and has recently been found on C-type retroviruses. Humans and Old World monkeys have anti-alphaGal antibody, a natural antibody. The present study was performed to examine if the alphaGal epitope could be used as a new target of gene therapy against human cancer. Bovine alpha1-3 galactosyltransferase (alpha1-3 GT) cDNA which produces the alphaGal epitope was electrophoretically transfected into the human pancreatic cancer cell line, MIA PaCa-2 and the human hepatocellular carcinoma cell line, huH7. The expression of the alphaGal epitope was confirmed by flow cytometry, using specific binding with IB4 lectin conjugated with fluorescein isothiocyanate. Transfected MIA PaCa-2 cells and huH7 cells showed a positive log shift for the alphaGal epitope. Next, we examined whether human cancer cells expressing the alphaGal epitope could be lysed by natural antibodies using the complement-dependent cytotoxic cross-match test. The results showed that transfected MIA PaCa-2 cells and huH7 cells were effectively lysed by human natural antibodies, and the killing was observed with any serum irrelevant of blood type. The results indicate that transfection of the functional alpha1-3 GT gene into human cancer cells can lead to their transformation, making them susceptible to lysis by natural antibodies, and, thus, useful for gene therapy.

Dynamic helical CT of T1 and T2 glottic carcinomas: predictive value for local control with radiation therapy.

BACKGROUND AND PURPOSE: Tumor volume and cartilage invasion have been suggested as prognostic factors of glottic carcinomas following definitive radiation therapy. Radiologic examinations provide additional information regarding the deep extension of tumor. We determined whether dynamic helical CT can predict local control of early (T1 and T2 stage) glottic carcinomas treated with definitive radiation therapy. METHODS: Sixty-eight patients with early glottic carcinoma evaluated on pretreatment dynamic helical CT were treated with definitive radiation therapy. Tumor detectability, maximum dimension, tumor volume, and involvement of anatomic subsites (anterior commissure, ventricle, subglottic region, and thyroid and arytenoid cartilages) were determined by consensus by three radiologists without previous knowledge of the clinical information. The CT findings were correlated with local control. RESULTS: The two-year local control rate was 76%; 91% for T1 and 60% for T2 lesions. Univariate analysis revealed clinical T stage, tumor detectability, maximum dimension, tumor volume, anterior commissure involvement, ventricle involvement, and thyroid cartilage involvement as significant prognostic factors. Thyroid cartilage involvement was an independent predictor by multivariate analysis. The lesions separate from the thyroid cartilage had a 95% probability of local control, whereas the lesions adjacent to the cartilage had only a 42% control rate. CONCLUSION: Dynamic helical CT provides prognostic information for the results of definitive radiation therapy. Patients with a tumor adjacent to the thyroid cartilage had an increased risk of local failure.

Early glottic squamous cell carcinoma. Predictive value of MR imaging for the rate of 5-year local control with radiation therapy.

PURPOSE: To evaluate MR findings in early (T1 and T2 stages) glottic carcinomas and the predictive value of MR imaging for the rate of 5-year local control with radiation therapy. MATERIAL AND METHODS: Eighty-three patients with early glottic carcinomas were prospectively examined with MR at 1.5 T. MR investigation included unenhanced T1-weighted, T2-weighted, dynamic and contrast-enhanced T1-weighted images. Three patients with presumed advanced diseases on MR were initially treated with total laryngectomy and were excluded from the study. The remaining 80 patients were treated with radiation therapy with curative intent. Tumor detectability, size and relationship to the thyroid cartilage were determined on MR images. The MR findings were then correlated with the rate of local control. RESULTS: Forty-eight of 80 lesions (60%) were detected on MR imaging. All detected lesions but 1 demonstrated increased signal on T2-weighted images. The lesions were best delineated on dynamic images (statistically significant). The 5-year local control rate with radiation therapy was 72%. Univariate analysis revealed clinical T stage, MR detectability, tumor size and relationship to the thyroid cartilage as significant predictors. Multivariate analysis revealed that the relationship to the thyroid cartilage was an independent factor. CONCLUSION: MR provides prognostic information about the results of definitive radiation therapy. To evaluate the tumor extension in lesions detected on precontrast MR images, contrast-enhanced dynamic images should be obtained.

AMY-1 is a trigger for the erythrocyte differentiation of K562 cells.

We have reported that a novel c-Myc binding protein, AMY-1, stimulated the transcription activity of c-Myc and was translocated from cytoplasm to nuclei in a c-Myc-dependent manner. Here, the role of AMY-1 in cell differentiation was examined. AMY-1 expression was up-regulated after differentiation induction of human K562 cells to erythrocyte cells by AraC, while c-Myc expression was rapidly down-regulated. K562 cell lines expressing exogenous AMY-1 were established, and these cells expressed a high level of epsilon-globin mRNA, a marker gene necessary for erythrocyte cell differentiation, without differentiation induction. The addition of AraC rapidly initiated differentiation in these cell lines, which continued to differentiate to erythrocyte cells possessing a high level of hemoglobin even after the decrease in AMY-1 expression. These results suggest that AMY-1 is a trigger for K562 cells to differentiate to erythrocyte cells and that AMY-1 may have a function independent of or different from c-Myc.

Establishment and characterization of a colonic epithelial cell line MCE301 from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

We produced an immortalized colonic epithelial cell line, MCE301, using fetal mice transgenic for the temperature-sensitive simian virus 40 large T-antigen gene. MCE301 cells showed epithelial-like morphology and maintained tight connections with neighboring cells. The cells grew at a permissive temperature (33 degrees C), but the growth of the cells was significantly prevented at the nonpermissive temperature (39 degrees C). The cells expressed large T-antigen at 33 degrees C but not at 39 degrees C. MCE301 cells were not transformed, as judged by the absence of anchorage-independent growth in soft agar gel and lack of tumor formation in nude mice. Electron microscopic studies showed that the cells formed microvilli-like structures on the cell surface and junctional complexes such as tight junctions and desmosomes between the cells. The cells expressed cytosketal (acidic cytokeratins and actin), basement membrane (laminin and collagen type IV) and junctional complex proteins (ZO-1 and desmoplakin I + II), as judged by specific antibodies. Fetal bovine serum, epidermal growth factor, insulin-like growth factor and insulin significantly increased the cell growth at 33 degrees C. Moreover, MCE301 cells expressed colonic mucin Muc2 mRNA as demonstrated by reverse transcriptase-polymerase chain reaction, indicating that the cells originate from mucus-secreting cells. Alkaline phosphatase, a brush border-associated enzyme, was detected in the cells. Sodium butyrate (2 mM), an inducer of cellular differentiation, markedly elevated alkaline phosphatase activity. Thus, the present mouse colonic epithelial cell line MCE301 possessing these unique characteristics should provide a useful in vitro model of colonic epithelium.

A conspicuous adaptability to antibiotics in the Escherichia coli mutator strain, dnaQ49.

By repeating the cycle of mutagenesis and selection, the Escherichia coli dnaQ49 mutator acquired high level resistance to ampicillin (30,000 micrograms ml-1), streptomycin (26,000 micrograms ml-1) and ofloxacin (3000 micrograms ml-1). Under the strong pressure of ofloxacin, dnaQ49 also followed the history of mutations in the gyrase and topoisomerase i.v. genes previously observed in clinical isolates of quinolone-resistant E. coli. The results of these in vitro experiments suggest that naturally existing mutators may participate in the rapid acquisition of resistance to various antibiotics in patients. A possible mechanism for the occurrence of this adaptability is discussed with special reference to the property of mutagenesis accompanying DNA replication.