Lipopolysaccharide enhances the production of nicotine-induced prostaglandin E2 by an increase in cyclooxygenase-2 expression in osteoblasts

Previous studies have indicated that lipopolysaccharide(LPS)from Gram-negative bacteria inplaque induces the release of prostaglandin E_2(PGE_2),which promotes alveolar bone resorption in periodontitis,and that tobacco smoking might be an important risk factor for the development and severity of periodontitis.We determined the effect of nicotine and LPS on alkaline phosphatase(ALPase)activity,PGE_2 production,and the expression of cyclooxygenase(COX-1,COX-2),PGE_2 receptors Ep1-4,and macrophage colonystimulating factor(M-CSF)in human osteoblastic Saos-2 cells.The cells were cultured with 10~(-3)M nicotinein the presence of 0,1,or 10μg/ml LPS,or with LPS alone.ALPase activity decreased in cells cultured withnicotine or LPS alone,and decreased further in those cultured with both nicotine and LPS,whereas PGE_2production significantly increased in the former and increased further in the latter.By itself,nicotine did notaffect expression of COX-1,COX-2,any of the PGE_2 receptors,or M-CSF,but when both nicotine and LPSwere present,expression of COX-2,Ep3,Ep4,and M-CSF increased significantly.Simultaneous addition of10~(-4)M indomethacin eliminated the effects of nicotine and LPS on ALPase activity,PGE_2 production,and M-CSF expression.Phosphorylation of protein kinase A was high in cells cultured with nicotine and LPS.Theseresults suggest that LPS enhances the production of nicotine-induced PGE_2 by an increase in COX-2 expres-sion in osteoblasts,that nicotine-LPS-induced PGE_2 interacts with the osteoblast Ep4 receptor primarily inautocrine or paracrine mode,and that the nicotine-LPS-induced PGE_2 then decreases ALPase activity andincreases M-CSF expression.     

Gene Systems Network Inferred from Expression Profiles in Hepatocellular Carcinogenesis by Graphical Gaussian Model

Hepatocellular carcinoma (HCC) in a liver with advanced-stage chronic hepatitis C (CHC) is induced by hepatitis C virus, which chronically infects about 170 million people worldwide. To elucidate the associations between gene groups in hepatocellular carcinogenesis, we analyzed the profiles of the genes characteristically expressed in the CHC and HCC cell stages by a statistical method for inferring the network between gene systems based on the graphical Gaussian model. A systematic evaluation of the inferred network in terms of the biological knowledge revealed that the inferred network was strongly involved in the known gene-gene interactions with high significance Open image in new window , and that the clusters characterized by different cancer-related responses were associated with those of the gene groups related to metabolic pathways and morphological events. Although some relationships in the network remain to be interpreted, the analyses revealed a snapshot of the orchestrated expression of cancer-related groups and some pathways related with metabolisms and morphological events in hepatocellular carcinogenesis, and thus provide possible clues on the disease mechanism and insights that address the gap between molecular and clinical assessments.     

Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans

Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.     

Competitive Chemiluminescent Immunoassay for Estradiol Using an N-Functionalized Acridinium Ester

We have compared three competitive chemiluminescent immunoassays (CLIA) for estradiol (E2) using an N-functionalized acridinium ester (AE). The assays were a standard competitive assay using immobilized antibody and directly labeled antigen (type A), an immobilized antibody and indirectly labeled antigen (type B), and an immobilized antigen and labeled antibody (type C). In an antibody-immobilized system, the assay using both AE- and E2-labeled thyroglobulin as a tracer (type B) was more sensitive than that using AE directly coupled with E2 (type A). Subsequently, a comparison of the antibody-immobilized system (type B) and an antigen-immobilized system (type C) showed that the latter was slightly more sensitive than the former. The sensitivity of the CLIA (type C) was similar or superior to commercially available CLIA or radioimmunoassays for E2. Thus, the N-functionalized AE proved to be a useful labeling reagent for a competitive CLIA with high sensitivity.     

Cloning and Sequencing of the Spore Germination Gene of Bacillus megaterium ATCC 12872: Similarities to the NaH-Antiporter Gene of Enterococcus hirae

The germination mutant TM-31 of Bacillus megaterium ATCC 12872, was isolated by transposon Tn917 insertional mutagenesis. Glucose, L -proline, L -leucine and KNO₃ germinated TM-31 poorly. The DNA in the region of the Tn917 insertion was cloned, and its nucleotide sequence determined. One major open reading frame was present on the cloned DNA. The hydrophobic protein encoded is presumably membrane-associated. A homology search revealed that the gene encoded in the region of the Tn917 insertion is homologous to napA of Enterococcus hirae. napA codes for the NaH-antiporter. It is hypothesized that transport of cations must play an important role in spore germination in B. megaterium ATCC 12872.     

Ion permeation properties of a cloned human 5-HT3 receptor transiently expressed in HEK 293 cells

Summary Human 5-HT₃ receptors expressed in HEK 293 cells were studied using patch-clamp techniques. The permeability ratios of cations to Na⁺ were Li⁺, 1.16; K⁺, 1.04; Rb⁺, 1.11; Cs⁺ 1.11; NMDG⁺, 0.04; Ca²⁺, 0.49, and Mg²⁺, 0.37. The permeability sequence of the alkali metal cations was Lⁱ⁺ > Rb⁺ = Cs⁺ > K⁺ > Na⁺. Increased external concentrations of Ca²⁺ or Mg²⁺ decreased 5-HT-induced currents at all potentials tested in a voltage-independent manner. The single-channel conductance of human 5-HT₃ receptors measured by fluctuation analysis of whole-cell currents was 790 ± 100fS. Differences in the basic properties of 5-HT₃ receptors between species may explain interspecies differences in pharmacological properties.     

Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement. Received: 14 July 1998 / Accepted: 2 September 1998     

Plagiochilines C,D, E and F,four novel secoaromadendrane-type sesquiterpene hemiacetals from Plagiochila asplenioides and Plagiochila semidecurrens

Four novel secoaromadendrane-type sesquiterpene hemiacetals, plagiochilines C, D, E and F, together with the previously known (?)-bicyclogermacrene, have been isolated from the liverwort, Plagiochila asplenioides and their structures have been spectroscopically elucidated. From Plagiochila semidecurrens plagiochilines A and C have been obtained along with (?)-bicyclogermacrene and its related sesquiterpene hydrocarbons.     

Heavy charged particles produce a bystander effect via cell-cell junctions.

Radiation-induced damage to living cells results from either a direct hit to cellular DNA, or from indirect action which leads to DNA damage from radiation produced radicals. However, in recent years there is evidence that biological effects such as cell killing, mutation induction, chromosomal damage and modification of gene expression can occur in a cell population exposed to low doses of alpha particles. In fact these doses are so low that not all cells in the population will be hit directly by the radiation. Using a precision alpha-particle microbeam, it has been recently demonstrated that irradiated target cells can induce a bystander mutagenic response in neighboring "bystander" cells which were not directly hit by alpha particles. Furthermore, these results suggest that gap-junction mediated cell-to-cell communication plays a critical role in this bystander phenomenon. The purpose of this section is to describe recent studies on bystander biological effects. The recent work described here utilized heavy charged particles for irradiation, and investigated the role of gap-junction mediated cell-cell communication in this phenomenon.