Structural aspects of nucleic acid-sensing Toll-like receptors

Invading pathogens elicit potent immune responses in cells through interactions between structurally conserved molecules derived from the pathogens and specialized innate immune receptors such as the Toll-like receptors (TLRs). Nucleic acid is one of the principal TLR ligands. Nucleic acid-sensing TLRs recognize an array of nucleic acids, including double-stranded RNA, single-stranded RNA, and DNAs with specific sequence motifs. Although ligand-induced dimerization is commonly observed followed by TLR activation, both the specific recognition mechanisms and the ligand–receptor interactions vary among different TLRs. In this review, we highlight our current understanding of how these receptors recognize their cognate ligands based on the recent advances in structural biology.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

Pyridoxine 5'-phosphate oxidase is a candidate gene responsible for hypertension in Dahl-S rats

To identify candidate genes responsible for hypertension in Dahl salt-sensitive rats (Dahl-S), an oligonucleotide microarray analysis was performed to find differentially expressed genes in kidneys of Dahl-S and Lewis rats. We obtained 101 F2 male rats from Dahl-S and Lewis rats and performed precise measurements of blood pressure (BP) and heart rate by telemetric monitoring at 14 weeks of age after 9 weeks of salt-loading. The correlation analysis between genotypes of differentially expressed genes and BP in F2 rats indicated that pyridoxine 5'-phosphate oxidase (Pnpo) and catecholamine-O-methyltransferease (Comt) showed a highly significant association with BP. However, in the case of Comt, the Dahl-S genotype correlated with low BP. Short/branched chain acyl-CoA dehydrogenase and Sah also showed a significant association with systolic blood pressure. The present study provided evidence that Pnpo is a candidate gene responsible for hypertension in Dahl-S rats.     

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol

Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3α-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD⁺-dependent 3α-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3α-hydroxysteroids and bile acids using NAD⁺ as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3α-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD⁺-dependent type of cytosolic 3α-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3α-HSD resulted in a switch in favor of NADP⁺ specificity, suggesting their key roles in coenzyme specificity.     

Suppression of matrix metalloproteinase production in nasal fibroblasts by tranilast, an antiallergic agent, in vitro

Allergic rhinitis is an inflammatory disease characterized by nasal wall remodeling with intense infiltration of eosinophils and mast cells/basophils. Matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are the major proteolytic enzymes that induce airway remodeling. These enzymes are also important in the migration of inflammatory cells through basement membrane components. We evaluated whether tranilast (TR) could inhibit MMP production from nasal fibroblasts in response to tumor necrosis factor-alpha (TNF-alpha) stimulation in vitro. Nasal fibroblasts (NF) were established from nasal polyp tissues taken from patients with allergic rhinitis. NF (2 x 10(5) cells/mL) were stimulated with TNF-alpha in the presence of various concentrations of TR. After 24 hours, the culture supernatants were obtained and assayed for MMP-2, MMP-9, TIMP-1, and TIMP-2 levels by ELISA. The influence of TR on mRNA expression of MMPs and TIMPs in cells cultured for 12 hours was also evaluated by RT-PCR. TR at more than 5 x 10(-5) M inhibited the production of MMP-2 and MMP-9 from NF in response to TNF-alpha stimulation, whereas TIMP-1 and TIMP-2 production was scarcely affected. TR also inhibited MMP mRNA expression in NF after TNF-alpha stimulation. The present data suggest that the attenuating effect of TR on MMP-2 and MMP-9 production from NF induced by inflammatory stimulation may underlie the therapeutic mode of action of the agent in patients with allergic diseases, including allergic rhinitis.     

Characterization of the cells in the repair tissue of full-thickness articular cartilage defects

 It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro. Accepted: 3 November 1997     

Inulavosin and its benzo‐derivatives,melanogenesis inhibitors,target the copper loading mechanism to the active site of tyrosinase

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure–activity relationship of inulavosin and its benzo‐derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper‐binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo‐derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo‐tyrosinase that has a conformational defect.     

Development of a microbioreactor for glycoconjugate synthesis

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-β-N-acetylglucosaminidase derived from Mucor hiemalis (Endo-M), was constructed and used for glycoconjugate synthesis. The transglycosylation was performed with a reaction mixture containing an oxazoline derivative of sialo complex-type glycoside (SG), which was prepared from a sialo complex-type glycopeptide SGP derived from hen egg yolk, as a glycosyl donor and N-Fmoc-N-acetylglucosaminyl-l-asparagine [Fmoc-Asn(GlcNAc)-OH] as an acceptor. The reaction mixture was injected into a glycosynthase microbioreactor at a constant flow rate. Highly efficient and nearly stoichiometric transglycosylation occurred in the microbioreactor, and the transglycosylation product was eluted from the other end of the reactor. The glycosynthase microbioreactor was stable and could be used repeatedly for a long time.