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.

DNA hypermethylation of extracellular matrix-related genes in human periodontal fibroblasts induced by stimulation for a prolonged period with lipopolysaccharide derived from Porphyromonas gingivalis.

OBJECTIVES AND BACKGROUND: The involvement of DNA methylation in periodontal disease is not clear. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis is involved in the progression of periodontal disease. We recently developed an in vitro model of LPS infection in human periodontal fibroblast cells (HPdLFs) for a prolonged period. In this study, we examined genome-wide analysis of DNA methylation in HPdLFs stimulated with LPS derived from P. gingivalis for a prolonged period. We noted the hypermethylation of extracellular matrix (ECM)-related genes and examined whether hypermethylation affected their transcription levels. MATERIAL AND METHODS: HPdLFs were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The culture was repeated, alternating 3 d with LPS derived from P. gingivalis and 3 d without LPS for 1 mo. Untreated samples were used as controls. DNA was analyzed using the human CpG island microarray. Quantitative methylation-specific polymerase chain reaction was carried out to confirm reproducibility of the microarray data. The expression levels of mRNA of the selected ECM-related genes from the data were analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS: We found 25 ECM-related genes with hypermethylation at the CpG island of the promoter region, which exhibited a fourfold greater hypermethylation than controls. Among these genes, hypermethylation of nine ECM-related genes, FANK1, COL4A1-A2, 12A1 and 15A1, LAMA5 and B1, MMP25, POMT1 and EMILIN3, induced a significantly downregulated expression of their mRNA. CONCLUSION: These results indicate that LPS derived from P. gingivalis may cause DNA hypermethylation of some ECM-related genes followed by downregulated expression of their transcriptional levels.

[Right ventricular outflow tract reconstruction for pulmonary atresia with ventricular septal defect: the possibility of ventriculo-arterial direct anastomosis].

Between September 1997 and December 2000, 29 patients with pulmonary atresia associated with ventricular septal defect were undergone corrective surgery. The age at operation ranged from 24 days to 14.7 years. The 5 patients were younger than 3 months. The body weight at operation ranged from 1.5 kg to 35.8 kg. 10 patients had MAPCAs. 21 patients had been undergone modified Blolock-Taussig shunt and/or uniforcalization of pulmonary artery (PA). We could perform right ventricular outflow tract reconstruction with ventriculo-arterial direct anastomosis in 19 patients (65%) (4/10; patients with MAPCAs, 3/5; younger than 3 months). We performed pulmonary angioplasty on stenotic and/or underdeveloped leasions as a concomitant procedure in 13/19 cases. Our technique is composed of 4 points, (1) fully mobilizing central and distal pulmonary artery (PA), (2) dissecting the PA with connective tissue together, (3) pulmonary arterial wall flap, (4) right ventricular outflow tract reconstruction during cardiac arrest.

Clinical characteristics in non-insulin-dependent diabetic patients with long duration in Japan--relation to risk factors for vascular complications.

In this study, we evaluated the control state of body weight, blood pressure, and blood glucose during the recent 10 years in 82 patients of non-insulin-dependent diabetes mellitus (NIDDM) who had long duration of diabetes for 20-25 years without ischemic heart disease (IHD) and diabetic nephropathy (Neph). The patients without either IHD or Neph showed significantly lower incidences of obese (11 vs. 40%, p < 0.05), hypertensive (0 vs. 20%, p < 0.05) and poor glycemic control state (7 vs. 27%, p < 0.05) for 10 years than the patients with IHD alone, and showed significantly lower incidences of hypertensive (0 vs. 20%, p < 0.05) and poor glycemic control state (7 vs. 33%, p < 0.01) than the patients with Neph alone. The control state of body weight was similar between the patients without either IHD or Neph and with Neph alone. In addition, the patients without either IHD or Neph showed significantly lower incidences of obese (11 vs. 56%, p < 0.01) and hypertensive control state (0 vs. 40%, p < 0.01) for 10 years than the patients with both IHD and Neph. The control state of blood glucose was similar between the two groups. These results suggest that for long survival of NIDDM patients without development or progression of IHD and Neph, non-obese and non-hypertensive state as well as good glycemic control should be maintained for long time.

Chronic administration of aqueous extract of Stevia rebaudiana (Bert.) Bertoni in rats: endocrine effects.

1. The effects of the active principles of S. rebaudiana (SR) on endocrine parameters of male rats were studied upon chronic administrations (60 days) of a concentrated, crude extract of its leaves, starting at prepubertal age (25-30 days old). 2. The following determinations were made: glycemia; serum levels of T3 and T4; available binding sites in thyroid hormone-binding proteins (T3R index); binding of [3H]R 1881 to prostate cytosol; zinc content in prostate, testis, submandibular salivary gland (SMG) and pancreas; water content in testis and prostate. The body weight gain and the final weight of testis, prostate, seminal vesicle, SMG and adrenal were also studied. 3. Results showed that the SR-treated group did not significantly differ from the control group, with exception to the seminal vesicle weight, which fell by about 60%. 4. It is concluded that if the SR extract does have some potential to decrease rat fertility at all, this effect is almost certainly not exerted on the male.

[Clinical study of carumonam].

To evaluate the clinical efficacy of carumonam (CRMN, AMA-1080), the drug was used in the treatment of 10 patients including 4 with pneumonia and each with acute tonsillitis, chronic bronchitis, Mycoplasma pneumonia, primary atypical pneumonia (PAP), chronic pyelitis, and acute cystitis. Since beta-lactam antibiotics were not active against Mycoplasma pneumonia and PAP, these diseases were excluded from the clinical efficacy evaluation of CRMN. Responses were excellent in 1 patient and good in 7. Side effects were not observed. The laboratory test recognized slight elevations of GOT, GPT and eosinophilia in 1 patient and a slight leucopenia in another upon the administration of CRMN.