Cytokine expression in rat molar gingival periodontal tissues after topical application of lipopolysaccharide

It is well known that proinflammatory cytokines produced by host cells play an important role in periodontal tissue destruction. However, the localization of the cytokines in in vivo periodontal tissues during development of periodontal disease has not been determined. Immunohistochemical expression of proinflammatory cytokines including IL-1!, IL-1#, and TNF-! was examined at 1 and 3 h, and 1, 2, 3, and 7 days after topical application of lipopolysaccharide (LPS; 5 mg/ml in physiological saline) from E. coli into the rat molar gingival sulcus. In the normal periodontal tissues, a small number of cytokine-positive epithelial cells were seen in the junctional epithelium (JE), oral sulcular and oral gingival epithelium, in addition to macrophages infiltrating in the subjunctional epithelial area and osteoblasts lining the alveolar bone surface. Epithelial remnants of Malassez existing throughout periodontal ligament were intensely positive for IL-1# but negative for the other two cytokines. At 3 h after the LPS treatment, almost all cells in the JE were strongly positive for the cytokines examined. In addition, several cytokine-positive cells, including neutrophils, macrophages, and fibroblasts, were seen in the subjunctional epithelial connective tissue. At day 2, expression of the cytokines in the JE gradually decreased, while cytokine-positive cells in the connective tissue increased in number. Positive staining of the cytokines was seen in osteoclasts and preosteoclasts which appeared along the alveolar bone margin in this period. The number of cytokine-positive cells decreased by day 7. These findings indicate that, in addition to macrophages, neutrophils, and fibroblasts, the JE cells are a potent source of TNF-!, IL-1!, and IL-1# reacting to LPS application, and suggest that JE cells may play an important role in the first line of defense against LPS challenge, and the proinflammatory cytokines transiently produced by various host cells may be involved in the initiation of inflammation and subsequent periodontal tissue destruction.     

Triplex-forming DNAs in the human interphase nucleus visualized in situ by polypurine/polypyrimidine DNA probes and antitriplex antibodies

The polypurine/polypyrimidine (PuPy) tracts present in the human genome are known to be scattered among and within chromosomes. In PuPy tract sequences, triplex formation occurs readily under physiological conditions, leaving single-stranded DNAs capable of hybridization with complementary single-stranded DNAs and RNAs. The formation of single-strands and transmolecular triplexes is thought to enable sequences spaced distantly along the genome to associate with each other and organize nuclear DNA into ordered configurations. Triplex-forming DNAs in the human interphase nucleus were analyzed by combining fluorescence in situ "nondenaturing" hybridization employing PuPy tract probes and immunodetection by antitriplex antibodies. The nondenaturing hybridization technique, which has been used to detect RNA, may detect single-stranded DNAs in nondenatured nuclei, if present. Probes such as (GA/TC)(n) and (GAA/TTC)(n) sequences gave sequence-specific signals that overlapped with or were closely associated with triplexes immunolocalized by using known antitriplex antibodies. Pretreatment of nuclei with antitriplex antibodies blocked probe signal formation. Signal formation was resistant to pretreatment of nuclei with RNases but sensitive to single strand-specific nucleases. Triplexes visualized differentially with distinct PuPy tract probes were associated spatially with centromeric sequences in the interphase nucleus in a sequence-specific manner.     

Causal relationship between the loss of RUNX3 expression and gastric cancer

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.     

Enzymatic synthesis of a capsinoid by the acylation of vanillyl alcohol with fatty acid derivatives catalyzed by lipases

Capsinoids are a novel group of compounds produced by the Capsicum plant. We synthesized a capsinoid by the lipase-catalyzed esterification of vanillyl alcohol with fatty acid derivatives in an organic solvent. The use of seven out of 17 commercially available lipases, especially Novozym 435, was applicable to the synthesis of vanillyl nonanoate, a model compound of capsinoids. The yield of vanillyl nonanoate under the optimum conditions of 50 mM vanillyl alcohol and 50 mM methyl nonanoate in 500 microl of dioxane, using 20 mg of Novozym 435 and 50 mg of 4 A molecular sieves at 25 degrees C, was 86% in 20 h. Several capsinoid homologues having various acyl chain lengths (C6-C18) were synthesized at 64-86% yields from the corresponding fatty acid methyl ester. The natural capsinoids, capsiate and dihydrocapsiate, were obtained by a 400-fold-scale reaction at these optimum conditions in 60% and 59% isolated yields, respectively.     

Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase,that needs product of upstream gene,sldB, for activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.     

The role of calcium channels in osteocyte function

Osteocytic response to stretching, which is potentiated by PTH, is distinct from that of osteoblast to high frequency strain. A MAPK dependent signaling pathway is suggested in the osteoblast response. At least two different types of mechanotransduction pathways are present in bone cells of osteoblastic lineage.     

Role of BMP-4 in the inducing ability of the head organizer in Xenopus laevis

BMP-4 has been implicated in the patterning of the Dorsal-Ventral axis of mesoderm and ectoderm. In this study, we describe the posteriorizing effect of BMP-4 on the neural inducing ability of dorsal mesoderm (dorsal lip region) in Xenopus gastrulae. Dorsal lip explants dissected from stage 10.25 embryos retained anterior inducing ability when precultured for 6 hrs until sibling embryos reach stage 12. When the dorsal lips from stage 10.25 embryos were treated with a range of BMP-4 concentrations, posterior tissues were induced in adjacent ectoderm in a dose-dependent manner. Thus activin-treated explants able to act as head inducers can also induce posterior structures in the presence of BMP-4. To investigate whether BMP-4 directly affects the inducing ability of dorsal mesoderm, we blocked the BMP-4 signaling pathway by injection of mRNA encoding a truncated form of the BMP-4 receptor (tBR) mRNA. Under these conditions, activin-treated explants induced anterior tissues following BMP-4 treatment. Taken together, these results indicate that BMP-4 may affect the head inducing ability of dorsal mesoderm and confer trunk-tail inducing ability during Xenopus gastrulation.     

Anti-bovine rhodopsin monoclonal antibody recognizing light-dependent structural change

The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh 29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2-39 in the amino acid sequence. Further analysis suggested that the antigenic determinant included at least residues 21-25. These results were consistent with the structural model for membrane topology of rhodopsin. The antigenicity of the rhodopsin was compared among several states. The antibody bound to both ammonyx LO-solubilized unbleached and bleached rhodopsin. In contrast, upon membrane-embedded rhodopsin, unbleached one was 100-times less antigenic than bleached one. The results suggested that the segment around the determinant of membrane-embedded rhodopsin should undergo a structural change upon absorption of light. Rh 29 detected a band corresponding to bovine, porcine and octopus opsins in immunoblotting. Protein blot of crayfish rhabdome did not show any reactive band. These bands except for crayfish reacted with concanavalin A as well. The N-terminal structure may, therefore, conserved between mammal and erthropoda and diverge between them and cepharopoda.     

Interaction of plexin-B1 with PDZ domain-containing Rho guanine nucleotide exchange factors

The Rho family GTPase has been implicated in plexin-B1, a receptor for Semaphorin 4D (Sema4D), mediating signal transduction. Rho may also play a function in this signaling pathway as well as Rac, but the mechanisms for Rho regulation are poorly understood. In this study, we have identified two kinds of PDZ domain-containing Rho-specific guanine nucleotide exchange factors (RhoGEFs) as proteins interacting with plexin-B1 cytoplasmic domain. These PDZ domain-containing RhoGEFs showed significant homology to human KIAA0380 (PDZ-RhoGEF) and LARG (KIAA0382), respectively. Both KIAA0380 and LARG could bind plexin-B1 and a deletion mutant analysis of plexin-B1, KIAA0380 and LARG revealed that KIAA0380 and LARG bound plexin-B1 cytoplasmic tail through their PDZ domains. The tissue distribution analysis indicated that plexin-B1 was co-localized with KIAA0380 and LARG in various tissues. Immunocytochemical analysis showed that LARG was recruited to plasma membrane by plexin-B1. These results suggest that PDZ domain-containing RhoGEFs play a role in Sema4D-plexin-B1 mediating signal transduction.