A chemical synthesis of UDP-LacNAc and its regioisomer for finding ‘oligosaccharide transferases’

A chemical synthesis of uridine 5’-diphospho-N-acetyllactosamine (Galβ(1→4)GlcNAc-UDP; UDP-LacNAc) and Galβ(1→3)GlcNAc-UDP is described. Coupling of the disaccharide imidate derivatives with dibenzylphosphate gave the corresponding 1-phosphates, which were condensed with UMP-imidazolate to give the target UDP-oligosaccharides after purification by anion exchange HPLC and gel filtration column chromatography. Using this methodology a variety of oligosaccharide nucleotide analogues can be synthesized. These UDP-oligosaccharides may be useful for finding so-called `oligosaccharide transferases’, the glycosyltransferases which transfer the oligosaccharide moiety onto glycosyl acceptors.     

NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens

The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.     

Contribution of complement factor H Y402H polymorphism to sudden sensorineural hearing loss risk and possible interaction with diabetes

Sudden sensorineural hearing loss (SSNHL) is one of the most common diseases encountered by otolaryngologists; however, the etiology is unclear. The aim of this study was to assess the association between SSNHL and polymorphism of complement factor H (CFH) Y402H, which is implicated in age-related macular degeneration. We conducted a case-control study, in which the cases were 72 SSNHL patients and the controls were 2161 residents selected randomly from the resident register. The odds ratio (OR) for SSNHL risk was determined using the additive-genetic model of CFH Y402H polymorphism. The OR for SSNHL risk was 1.788 (95% confidence interval [CI]: 1.008-3.172) with no adjustments and 1.820 (CI: 1.025-3.232) after adjusting for age and sex. Of the three lifestyle-related diseases hypertension, dyslipidemia, and diabetes, only diabetes was significantly associated with SSNHL risk. We classified both the controls and SSNHL patients into those with or without diabetes, and the OR for SSNHL risk was 6.326 (CI: 1.885-21.225) in diabetic subjects and 1.214 (CI: 0.581-2.538) in nondiabetic subjects. We conclude that CFH Y402H polymorphism and SSNHL risk are significantly related, and that diabetic CFH Y402H minor allele carriers may be susceptible to SSNHL.     

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Therefore, we studied the genetic pathway of LR asymmetric development in Drosophila. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. In this study, we found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 (fz2) and Wnt4, which encode a receptor and ligand of Wnt signaling, respectively, were required for the LR asymmetric development of the AMG. arrow (arr), an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Our genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 were expressed in the visceral muscles of the midgut. Consistent with these results, we observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. Our results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates.     

Novel reactions of 5-bromouracil derivatives with active methylene compounds

5-Bromouracil derivatives (1) reacted with carbanions at room temperature to give 6-substituted uracils (6), 2,4-diazabicyclo[4,1,0] heptanes (8) and 2,4-diazabicyclo[4,3,0]nonane derivative (9), which depend largely upon the structure of the active methylene compounds employed for the generation of carbanions.     

Novel deprotection method of Fmoc group under neutral hydrogenation conditions

Novel deprotection method of the Fmoc (9-flurorenylmethoxycarbonyl) protective group under Pd/C-catalyzed hydrogenation conditions at room temperature was developed. The addition of CH₃CN accelerated the deprotection of the Fmoc group, and also the application of H₂ pressure (3 atm) shows notable rate enhancement.     

Functional roles of the tissue inhibitor of metalloproteinase 3 (TIMP-3) during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells

The tissue inhibitor of the metalloproteinase-3 (TIMP-3) gene was isolated as a gene involved in the process of ascorbate-induced differentiation of mouse MC3T3-E1 cells by the differential display method. The functional roles of TIMP-3 were characterized by establishing stable cell lines, which constitutively expressed the TIMP-3 gene. The TIMP-3 transfectants produced type I collagen at the same level as that of normal cells in response to ascorbic acid 2-phosphate (AscP). However, the expression of the other osteoblastic marker proteins such as alkaline phosphatase (ALPase), osteopontin (OP), osteocalcin (OC), osteonectin (ON) and matrix metalloproteinases (MMPs) remained at a low level even in the presence of AscP. Furthermore, no mineralization of the extracellular matrix (ECM) occurred with the transfectants. Remodeling ECM through TIMPs and MMPs is concluded to be required for osteoblastic differentiation.     

Synthesis and structure-activity relationships of 2-substituted-8-hydroxyadenine derivatives as orally available interferon inducers without emetic side effects

Recently we reported the adenine derivatives (2-4) as new interferon (IFN) inducers. In the present study, we conducted a detailed structure and activity relationship study of 4 and its related derivatives on IFN inducing activity. From this study, we found that compound 4 exhibited the most potent IFN inducing activity in vitro with a minimum effective concentration of 0.01 microM, and 4 also showed strong IFN-inducing activity at doses of more than 0.3mg/kg by oral administration in mice. This potency was 10-fold stronger than that of Imiquimod. Moreover, 4 did not cause emesis in ferrets even at doses as high as 10mg/kg, whereas, 80% of animals were emetic when orally administered with the same dose of Imiquimod. These results indicate that compound 4 is superior to Imiquimod with respect to efficacy and safety.     

Mechanisms of factor Xa-catalyzed cleavage of the factor VIIIa A1 subunit resulting in cofactor inactivation

Activation of factor VIII by factor Xa is followed by proteolytic inactivation resulting from cleavage within the A1 subunit (residues 1-372) of factor VIIIa. Factor Xa attacks two sites in A1, Arg(336), which precedes the highly acidic C-terminal region, and a recently identified site at Lys(36). By using isolated A1 subunit as substrate for proteolysis, production of the terminal fragment, A1(37-336), was shown to proceed via two pathways identified by the intermediates A1(1-336) and A1(37-372) and generated by initial cleavage at Arg(336) and Lys(36), respectively. Appearance of the terminal product by the former pathway was 7-8-fold slower than the product obtained by the latter pathway. The isolated A1 subunit was cleaved slowly, independent of the presence of phospholipid. The A1/A3-C1-C2 dimer demonstrated an approximately 3-fold increased cleavage rate constant, and inclusion of phospholipid further enhanced this value by approximately 2-fold. Although association of A1 or A1(37-372) with A3-C1-C2 enhanced the rate of cleavage at Arg(336), inclusion of A3-C1-C2 did not affect the cleavage at Lys(36) in A1(1-336). A synthetic peptide 337-372 blocked the cleavage at Lys(36) (IC(50) = 230 microm) while showing little if any effect on cleavage at Arg(336). Proteolysis at Lys(36), and to a lesser extent Arg(336), was inhibited in a dose-dependent manner by heparin. These results suggest that inactivating cleavages catalyzed by factor Xa at Lys(36) and Arg(336) are regulated in part by the A3-C1-C2 subunit. Furthermore, cleavage at Lys(36) appears to be selectively modulated by the C-terminal acidic region of A1, a region that may interact with factor Xa via its heparin-binding exosite.