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.

Isolation and analysis of expression mechanisms of a rice gene, EL5, which shows structural similarity to ATL family from Arabidopsis, in response to N-acetylchitooligosaccharide elicitor.

Two rice cDNAs, EL5 and RRF1, were isolated and characterized. EL5 was responsive to N-acetylchitooligosaccharide, a biotic elicitor active in suspension-cultured rice cells. The structural specificity of the elicitor required for the expression of EL5 was consistent with other defense reactions observed in the experimental system, indicating that the elicitor signal to EL5 is transmitted through a single class of receptor-mediated recognition events. However, the intracellular signaling pathway to EL5 was distinct from those to other elicitor-responsive genes. Sequence analysis and alignment showed that a genomic sequence stored in rice genome databases in addition to EL5 and RRF1 belongs to the ATL family of RING-H2 finger motif proteins first isolated from Arabidopsis.

Cryopreservation of isolated fish blastomeres: effects of cell stage, cryoprotectant concentration, and cooling rate on postthawing survival

The toxicity of the cryoprotectant dimethyl sulfoxide (Me(2)SO) to isolated blastomeres was examined in three fish species representative of distinct environments: marine (whiting, Sillago japonica); estuarine (pejerrey, Odontesthes bonariensis); and freshwater (medaka, Oryzias latipes). The effects of embryonic stage, Me(2)SO concentration, and cooling rate on the cryopreservation of blastomeres were also studied. Whiting sheds small planktonic eggs whereas the other two species shed large demersal eggs. Isolated blastomeres from the three species tolerated Me(2)SO concentrations up to 9% relatively well for over 5 h but lost viability rapidly at 18%. Cells from later embryonic stages (512 or 1024 cells) were more tolerant of Me(2)SO than those from earlier stages (128 or 256 cells). The three factors examined, alone or in combination, had a significant effect on the survival of blastomeres after freezing and thawing, but the extent of the effect and the optimum conditions varied with the species. In general, the highest rates of successful cryopreservation were observed with older rather than younger blastomeres, slower rather than faster cooling, and with 9-18% rather than 0% Me(2)SO. Survival rates for blastomeres cryopreserved under the most effective combination of the three factors examined for each species were 19.9 +/- 10.1% for whiting, 34.1 +/- 8.5% for medaka, and 67.4 +/- 12.8% for pejerrey. Copyright 1999 Academic Press.

Nucleotide sequence of oat (Avena sativa L.) cDNA encoding an auxin-binding protein (ABP1).

We isolated and determined a nucleotide sequence of a cDNA clone encoding a protein homologous to maize major auxin-binding protein (ABP1) from a cDNA library of oat coleoptiles. The deduced amino acid sequence of this clone contained an N-linked glycosylation signal and an ER-retention signal. Furthermore, two domains that were important to interact with auxins, were conserved in this clone at amino acid level.

Inhibitory effects of caffeine on Ca2+ influx and histamine secretion independent of cAMP in rat peritoneal mast cells.

1. Caffeine did not evoke Ca2+ mobilization and histamine secretion. 2. Caffeine, as well as other methylxanthines but not forskolin or 8 bromo-cAMP, inhibited Ca2+ responses from compound 48/80. 3. Evoked histamine secretion was severely reduced by caffeine but not by cAMP analogs. 4. In beta-escin-permeabilized cells, caffeine did not affect resting and IP3-stimulated 45Ca2+ release, but it inhibited Ca(2+)-induced histamine secretion. 5. These results indicate that caffeine inhibits Ca2+ influx and Ca2+ efficacy in the secretory apparatus independent of cAMP, resulting in the inhibition of secretagogs-evoked histamine secretion from rat mast cells.

Inositol 1,4,5-trisphosphate- and caffeine-sensitive Ca(2+)-storing organelle in bovine adrenal chromaffin cells.

The uptake and release properties of Ca2+ by several subcellular fractions of the bovine adrenal medulla were investigated. Investigation by the 45Ca2+ tracer method showed that permeabilized cells and the fractions of mitochondria (MT) and microsomes (MC) caused ATP-dependent Ca2+ uptake in a Ca2+ concentration-dependent manner (pCa 8-4), whereas permeabilized cells and the fractions of secretory granules (SG) were able to accumulate a significant amount of Ca2+ even in the absence of ATP, which was completed by the addition of hexokinase and glucose. In these organelle fractions, Ca2+ uptake in the presence of ATP at pCa 7 and pCa 5.8 was well-correlated with the activity of the NADPH cytochrome c reductase (marker enzyme for the endoplasmic reticulum) and cytochrome c oxidase (marker enzyme for mitochondria), respectively. As detected by Fura-2 ratiometry, both inositol 1,4,5-trisphosphate (IP3) and caffeine caused concentration-dependent Ca2+ releases from permeabilized cells and MC, but not from MT and SG. In an ATP-depleted condition, homogenates still took up a significant amount of Ca2+ but was not able to respond to IP3 and caffeine. These results suggest that the endoplasmic reticulum is a major Ca(2+)-storing organelle, which releases Ca2+ in response to IP3 and caffeine in bovine adrenal chromaffin cells.

Function of RNH-1/14-3-3 beta gene in cellular differentiation and proliferation.

It has recently been reported that the members of 14-3-3 protein family participate in cell cycle control and associate with Raf, Bcr, Bcr-Abl, and polyomavirus middle tumor antigen (MT) as modulators of signal transduction. During cDNA cloning for the 17-kDa neuronal differentiation factor (K2 factor) secreted from rat hepatoma Kagura-2 (K2) cells from a K2 cDNA library using rat prepronerve growth factor (prepro NGF) cDNA as a probe, we obtained RNH-1 (rat NGF homologue) clone, which was identified as 14-3-3 beta cDNA, but not K2 factor, although no significant homology is present between 14-3-3 beta and prepro NGF cDNAs. RNH-1/14-3-3 beta gene was markedly expressed as a 2.9-kb mRNA in K2 cells and in newborn rat brain tissue. In PC12 cells the expression of this gene was down-regulated during the neuronal differentiation primed by NGF. The enforced expression of RNH-1/14-3-3 beta in PC12 and K2 cells conferred on them a higher sensitivity to NGF for neuronal differentiation and an intense growth ability in low serum medium, respectively. These results provide additional evidence that RNH-1/14-3-3 beta protein participates in cellular differentiation, proliferation and transformation through the signal transduction pathways of various growth factors.

Minor knee trauma as a possible cause of asymmetrical proximal tibial physis closure. A case report.

An unusual case is presented in which minor soft tissue injury may have caused premature asymmetrical closure of the proximal tibial physis resulting in a 30 degrees genu recurvatum that necessitated corrective osteotomy. Awareness of the possibility of a hidden physeal injury in the presence of soft tissue injury and a normal radiograph may lead to its early recognition and treatment.

[A case of oligomeganephronia--quantitative measurement of the glomerular surface area and the number of glomeruli with a color image analyzer].

We report here a nine-year-old boy with oligomeganephronia. The patient had proteinuria and morphometric analysis of the renal biopsy specimen confirmed the diagnosis of oligomeganephronia. We employed a color image analyzer (OLYMPUS Corp.) to determine the glomerular surface area and the number of glomeruli per mm2 of the renal cortex of the patient. All the scanned glomeruli showed marked hypertrophy; the mean maximal surface area for the 5 glomeruli was 46 x 10(3) microns 2, which is approximately 4 times larger than that of normal children of the same age. The number of glomeruli per mm2 of the renal cortex of the patient had decreased to 1. 11 per mm2, which is approximately one fourth that of normal controls with IgA glomerulonephritis. The color image analyzer proved to be useful for the diagnosis of oligomeganephronia enabling the quantitative measurement of the glomerular surface area and the number of glomeruli per mm2 of the renal cortex.

Chromatographic profiles of urinary isoenzymes in healthy children.

Urinary excretion of alkaline phosphatase, gamma-glutamyltransferase and lactate dehydrogenase was studied in a carefully selected group of 155 healthy children, 83 females and 72 males. Enzyme activity was assayed in randomly collected urine samples after gel filtration of the urine specimens. On chromatograms, urinary enzymes of alkaline phosphatase, gamma-glutamyltransferase and lactate dehydrogenase were separated into 4, 2 and 5 isoenzymes, respectively. Mean values of alkaline phosphatase, gamma-glutamyltransferase and lactate dehydrogenase activity were 4.59, 21.6 and 10.0 U/g creatinine. There were no sex-related differences besides lactate dehydrogenase which showed a higher excretion in females than in males. The excretion of urinary enzymes clearly decreased with increasing age.

A method for measuring specific activities of 14C-labelled compounds by gas chromatography-mass spectrometry-computer system.

A method for measuring specific activities of 14C-labelled compounds by gas chromatography-mass spectrometry-computer system (GC-MS-CPU) was developed. This method was proved to provide practically precise and accurate specific activities of various 14C-labelled compounds with such merits as requirement of small amount of samples, being applicable to volatile compounds, and convenience. The C.V. percent obtained for tested compounds was within 3.9 and the reliable sensitivity should be over 37 MBq/mM (1 mCi/mM). This method was also useful for obtaining information on the labelling pattern and the synthetic procedures applied.