Screening and incorporation of rust resistance from Allium cepa into bunching onion (Allium fistulosum) via alien chromosome addition.

Bunching onion (Allium fistulosum L.; 2n = 16), bulb onion (Allium cepa L. Common onion group), and shallot (Allium cepa L. Aggregatum group) cultivars were inoculated with rust fungus, Puccinia allii, isolated from bunching onion. Bulb onions and shallots are highly resistant to rust, suggesting they would serve as useful resources for breeding rust resistant bunching onions. To identify the A. cepa chromosome(s) related to rust resistance, a complete set of eight A. fistulosum - shallot monosomic alien addition lines (MAALs) were inoculated with P. allii. At the seedling stage, FF+1A showed a high level of resistance in controlled-environment experiments, suggesting that the genes related to rust resistance could be located on shallot chromosome 1A. While MAAL, multi-chromosome addition line, and hypoallotriploid adult plants did not exhibit strong resistance to rust. In contrast to the high resistance of shallot, the addition line FF+1A+5A showed reproducibly high levels of rust resistance.

The usefulness of an autologous blood clot combined with gelatin for regeneration of periodontal tissue.

Several growth factors have been used in tissue regeneration therapy. Here we describe the use of an autologous blood clot combined with gelatin for teeth affected by severe periodontitis and vertical root fracture treated using three oral surgical procedures: periodontal flap surgery, intentional tooth replantation (IR), and tooth autotransplantation. Treatment with a blood clot improved the condition of the periodontal tissue, including reduction of pocket depth. Radiographical images demonstrated no evidence of ankylosis, and revealed the presence of alveolar bone regeneration. Our successful clinical outcome suggests that use of an autologous blood clot combined with gelatin is clinically effective for regeneration of lost periodontal tissue.

The usefulness of three-dimensional imaging for prognostication in cases of intentional tooth replantation.

This paper describes the utility of three-dimensional (3D) images obtained with cone beam computed tomography (CBCT) for prediction of successful clinical outcome in two cases of intentional tooth replantation (IR). IR was performed for teeth affected by vertical root fracture and root perforation with local application of blood clot and oxy-tetracycline antibiotic. High-resolution 3D images demonstrated no evidence of ankylosis, but did reveal the presence of alveolar bone regeneration, suggesting a good long-term prognosis. Our observations in these cases suggested that local application of the above two materials might help to induce the regeneration of lost periodontal tissues in IR.

Oxidative stress caused by a low concentration of hydrogen peroxide induces senescence-like changes in mouse gingival fibroblasts.

Periodontal tissue deteriorates under persistent oxidative stress induced by inflammatory reactions in the microflora of the oral cavity. This study aimed to evaluate the cellular properties of mouse gingival fibroblasts (MGFs) in the presence of oxidative stress. MGFs from 10-, 30- and 52-week-old mice were used to evaluate the changes in the cellular properties with aging. The study investigated the effects of oxidative stress on the cellular properties of MGFs from 10-week-old mice. The expression of p53, p21 and murine double minute 2 (Mdm2) in the MGFs in response to oxidative stress was also examined. By day 8, the number of MGFs increased in culture. However, the increase was markedly lower in MGFs derived from aged mice. Oxidative stress due to hydrogen peroxide (H2O2)-induced morphological changes characterized by a round shape with enlarged nuclei and expanded cytoplasm. The cell number of MGFs was decreased subsequent to treatment with 50 µM or a higher concentration of H2O2. MGFs treated with H2O2 at 20 µM showed a similar cell growth curve as the one seen in 52-week-old mice. Phosphorylated p53 protein was increased in MGFs subsequent to treatment with 20 µM H2O2, along with an upregulated transcription of p21 and Mdm2 mRNAs. These results suggest that treatment with a lower concentration of H2O2 in MGFs induces cell cycle arrest, resulting in stress-induced premature senescence, possibly correlated with the development of periodontal diseases.

Antimicrobial and antifungal effects of tissue conditioners containing a photocatalyst.

This study examined the antimicrobial/antifungal ability of a tissue conditioner containing a photocatalyst for Escherichia coli, Streptococcus mutans, Staphylococcus aureus and Candida albicans. The photocatalyst was mixed with tissue conditioners powders at concentrations of 0, 10, 15, and 20 wt%. Tissue conditioners powders containing a photocatalyst were mixed with liquid to make test specimens. Test specimens inoculated by each microorganism were irradiated by ultraviolet light for 0-, 2- and 4 hours. The antimicrobial/antifungal effects were evaluated by the CFU technique. The CFU values of each microorganism for tissue conditioners containing a photocatalyst showed significant decrease following UV-irradiation. The improvement in antimicrobial/antifungal effects was concomitant with the increase of the mixing ratio and the irradiation time. Therefore, the results indicated that tissue conditioners containing a photocatalyst might have photocatalytic ability.

An EST-SSR linkage map of Raphanus sativus and comparative genomics of the Brassicaceae.

Raphanus sativus (2n = 2x = 18) is a widely cultivated member of the family Brassicaceae, for which genomic resources are available only to a limited extent in comparison to many other members of the family. To promote more genetic and genomic studies and to enhance breeding programmes of R. sativus, we have prepared genetic resources such as complementary DNA libraries, expressed sequences tags (ESTs), simple sequence repeat (SSR) markers and a genetic linkage map. A total of 26 606 ESTs have been collected from seedlings, roots, leaves, and flowers, and clustered into 10 381 unigenes. Similarities were observed between the expression patterns of transcripts from R. sativus and those from representative members of the genera Arabidopsis and Brassica, indicating their functional relatedness. The EST sequence data were used to design 3800 SSR markers and consequently 630 polymorphic SSR loci and 213 reported marker loci have been mapped onto nine linkage groups, covering 1129.2 cM with an average distance of 1.3 cM between loci. Comparison of the mapped EST-SSR marker positions in R. sativus with the genome sequence of A. thaliana indicated that the Brassicaceae members have evolved from a common ancestor. It appears that genomic fragments corresponding to those of A. thaliana have been doubled and tripled in R. sativus. The genetic map developed here is expected to provide a standard map for the genetics, genomics, and molecular breeding of R. sativus as well as of related species. The resources are available at http://marker.kazusa.or.jp/Daikon.

Protogenin, a new member of the immunoglobulin superfamily, is implicated in the development of the mouse lower first molar.

BACKGROUND: Protogenin (Prtg) has been identified as a gene which is highly expressed in the mouse mandible at embryonic day 10.5 (E10.5) by a cDNA subtraction method between mandibles at E10.5 and E12.0. Prtg is a new member of the deleted in colorectal carcinoma (DCC) family, which is composed of DCC, Neogenin, Punc and Nope. Although these members play an important role in the development of the embryonic central nervous system, recent research has also shed on the non-neuronal organization. However, very little is known regarding the fetal requirement of the non-neuronal organization for Prtg and how this may be associated with the tooth germ development. This study examined the functional implications of Prtg in the developing tooth germ of the mouse lower first molar. RESULTS: Ptrg is preferentially expressed in the early stage of organogenesis. Prtg mRNA and protein were widely expressed in the mesenchymal cells in the mandible at E10.5. The oral epithelial cells were also positive for Prtg. The expression intensity of Prtg after E12.0 was markedly reduced in the mesenchymal cells of the mandible, and was restricted to the area where the tooth bud was likely to be formed. Signals were also observed in the epithelial cells of the tooth germ. Weak signals were observed in the inner enamel epithelial cells at E16.0 and E18.0. An inhibition assay using a hemagglutinating virus of Japan-liposome containing Prtg antisense-phosphorothioated-oligodeoxynucleotide (AS-S-ODN) in cultured mandibles at E10.5 showed a significant growth inhibition in the tooth germ. The relationship between Prtg and the odontogenesis-related genes was examined in mouse E10.5 mandible, and we verified that the Bmp-4 expression had significantly been decreased in the mouse E10.5 mandible 24 hr after treatment with Prtg AS-S-ODN. CONCLUSION: These results indicated that the Prtg might be related to the initial morphogenesis of the tooth germ leading to the differentiation of the inner enamel epithelial cells in the mouse lower first molar. A better understanding of the Prtg function might thus play a critical role in revealing a precious mechanism in tooth germ development.

Self-cleaning ability of a photocatalyst-containing denture base material.

This study examined the ability of a photocatalyst mixed in a denture base resin to decompose organic substances which adhered to the denture base resin surface. The photocatalyst was mixed with denture base resin at concentrations of 0, 5, 10, and 15% (w/w). Decomposition test, bending test, and surface roughness measurement were performed at 1, 7, 30, 90, and 180 days after polymerization. Decomposition ability was evaluated based on the residual amount of methylene blue (MB) dissolved in ethanol after UV irradiation for 12 hours. As the mixing ratio increased, the amount of MB in the solution decreased. Meanwhile, no changes in the amount of MB in the immersion solution were observed in the photocatalyst-free resin specimen. Therefore, the results indicated that a denture base resin containing a photocatalyst might have a photocatalytic ability.

Age-dependent changes in cell proliferation and cell death in the periodontal tissue and the submandibular gland in mice: a comparison with other tissues and organs.

This study investigated the age-dependent changes in the number of BrdU- and TUNEL-positive cells in murine gingival tissue and submandibular gland, and compared the findings with those in other tissues and organs. The cell proliferative activity was decreased after 20 weeks of age in epithelial cells of the gingiva, tongue, buccal mucosa and skin. A decreased cell proliferative activity was also associated with aging in the liver and kidney parenchymal cells. Meanwhile, cell death showed peculiar changes in gingival subepithelial tissue, and mucous and serous acini of the submandibular gland. An increase of TUNEL-positive cells was demonstrated in gingival subepithelial tissue after 20-week-old of age. A significant increase of TUNEL-positive cells was also found in the mucous acinar cells in the 20-week-old mice and in the serous acini after 20 weeks. The fluctuation in the number of TUNEL-positive cells in the subepithelial tissue of the skin, and BrdU- and TUNEL-positive staining ratios in the liver was smaller than that in other tissue and organs throughout life. This study may provide useful information for better understanding the influence of aging on the functional alteration that occurs in the gingival tissue and submandibular gland of the elderly.

High expression of PKC-MAPK pathway mRNAs correlates with glomerular lesions in human diabetic nephropathy.

BACKGROUND: Activation of protein kinase C (PKC) is a major signaling pathway for transforming growth factor (TGF)-beta to induce extracellular matrix (ECM) production in diabetic nephropathy (DN). PKC also activates mitogen-activated protein kinase (MAPK), which is called the PKC-MAPK pathway. The PKC-MAPK pathway is probably responsible for PKC-related abnormalities in diabetic glomeruli. To confirm the involvement of this pathway, we determined the localization and expression of mRNAs in glomeruli by in situ hybridization method. METHODS: In the present study, we examined expression of PKCbeta1, MAPK/ERK kinase (MEK) 1, MEK2, extracellular signal-regulated protein kinase (ERK) 1, ERK2, and TGF-beta1 mRNAs using renal tissue samples from kidneys affected by DN (N= 21) and from normal human kidney (NHK; N= 6). We also performed an immunohistochemical study using anti-phosphorylated MEK1/2 (P-MEK) and ERK1/2 (P-ERK) antibodies. The glomerular severity of DN was classified into three groups according to mesangial expansion: D1 (N= 4), D2 (N= 13), and D3 (N= 4). We analyzed differences and correlations between variables. RESULTS: In the glomeruli, the number of cells that stained for these mRNAs in DN was significantly higher than in NHK. The expression of PKC-MAPK pathway mRNAs tended to be inversely proportional to the degree of mesangial expansion. The P-MEK and P-ERK signal intensity were parallel to its mRNA expression pattern. Furthermore, there were significant correlations among the P-MEK, P-ERK signal intensity, PKCbeta1 mRNA expression. CONCLUSION: Our results suggest that high expression of PKC-MAPK pathway mRNAs plays an important role in the development and/or progression of early tissue damage in DN.

Expression of human nephrin mRNA in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is associated with functional changes in the filtration barrier, and microalbuminuria is a strong predictor of the development of overt DN. Nephrin is a novel podocyte-specific protein which localizes at the slit diaphragm. This study examines the expression of nephrin mRNA in the kidneys of type 2 diabetics with DN. METHODS: Renal tissues were obtained from 13 type 2 diabetics with DN. We also examined samples from five patients with minimal change nephrotic syndrome (MCNS) and five normal kidneys (normals) as control. The severity of DN was classified into two grades based on histopathological findings. DN grade 1 (DN1 = seven patients) presented mild mesangial expansion, and DN grade 2 (DN2 = six patients) moderate mesangial expansion. Nephrin mRNA was quantitated and localized by in situ hybridization. RESULTS: Cells positive for nephrin mRNA were detected exclusively in glomerular epithelial cells. The percentage of cells positive for nephrin mRNA in DN2 was significantly lower than in MCNS and normal kidneys. Furthermore, there was an inverse correlation between the percentage of cells positive for nephrin mRNA and extent of proteinuria. CONCLUSION: The low expression of nephrin mRNA may be closely linked to development and/or progression of proteinuria in human diabetic nephropathy.

Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish.

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.

Glomerular expression of connective tissue growth factor mRNA in various renal diseases.

Connective tissue growth factor (CTGF) is a cysteine-rich member of a new family of growth regulators. It is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis. The present study was designed to elucidate the role of CTGF in diabetic nephropathy (DN), immunoglobulin A nephropathy (IgA-N), membranous nephropathy (MN), and minimal change nephrotic syndrome (MCNS). We evaluated the expression and localization of CTGF mRNA in surgically excised renal tissue samples from 10 patients with DN, 10 with IgA-N, 10 with MN, 10 with MCNS, and 10 normal human kidney (NHK) tissue samples, by using high-resolution in situ hybridization with digoxigenin-labelled oligonucleotide. To quantify CTGF mRNA expression, we counted all nuclei, and nuclei surrounded by CTGF-positive cytoplasm, in at least 10 randomly selected cross-sections of non-sclerotic glomeruli, and expressed the results as a percentage of total glomerular cells. In all glomeruli, CTGF mRNA was expressed mainly in glomerular intrinsic cells, including glomerular mesangial and epithelial cells and some cells of Bowman's capsule. The percentage of cells positive for CTGF mRNA was significantly higher in DN and IgA-N than in MN, MCNS and NHK. However, there was no significant difference in the percentage of CTGF mRNA-positive cells between DN and IgA-N. Our study indicates that CTGF may play an important role in the development and progression of glomerulosclerosis in DN and IgA-N, which are both accompanied by mesangial matrix expansion and comprise two major causes of end-stage renal failure.

In situ expression of heat shock proteins, Hsc73, Hsj2 and Hsp86 in the developing tooth germ of mouse lower first molar.

This study examined the detailed gene expression pattern of three different heat shock proteins (HSPs), Hsc73, Hsj2, and Hsp86, by means of an in situ hybridization method. Hsc73, Hsj2, and Hsp86 were shown in our previous study to be differentially expressed in the mouse embryonic mandible at day 10.5 (E10.5) gestational age. These HSP genes showed similar expression patterns during development of the mouse lower first molar. HSPs-expressing cells were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5, and then were slightly localized at E12 in an area where the tooth germ of the lower first molar is estimated to be formed. A strong expression of HSPs was observed in the tooth germ at E13.5. At the cap stage, HSPs were expressed in the enamel organ and dental papilla. At the bell stage, HSPs were distinctly expressed in the inner enamel epithelium and dental papilla cells facing the inner enamel epithelial layer, which later differentiate into ameloblasts and odontoblasts, respectively. This study is the first report in which Hsc73, Hsj2, and Hsp86 were distinctly expressed in the developing tooth germ, thus suggesting these HSPs are related to the development and differentiation of odontogenic cells.