Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.     

Development of chemically stable solid phases for the target isolation with reduced nonspecific binding proteins

Poly(methacrylate) matrices for affinity resins were designed and synthesized based on our previous results that nonspecific protein absorption on affinity resins strongly depended on their hydrophobic property. The novel affinity resins bearing FK506 (6a, 6b) captured specific binding protein, FKBP12, with a small amount of nonspecific binding proteins. The amount of nonspecific binding proteins on 6a-6b was much reduced compared to that on commercially available poly(methacrylate) resins, Toyopearl (8), and was almost the same as that on one of the most popular resins, Affigel (9). Interestingly, 6a and 6b could isolate FKBP52 as a specific binding protein as well, although 8 and 9 could not.     

Oxidative damage of periodontal tissue in the rat periodontitis model: effects of a high-cholesterol diet

Studies suggest an association between consumption of a high-cholesterol diet and periodontitis. We addressed the mechanism by which high dietary cholesterol could be detrimental to periodontal health in a rat model. Feeding a high-cholesterol diet augmented the effects of bacterial pathogens and their products (e.g., lipopolysaccharide and proteases) on production of pro-inflammatory cytokines in fibroblasts. High dietary cholesterol also increased mitochondrial 8-hydroxydeoxyguanosine in the periodontal tissues. These results suggest that excessive tissue oxidative damage induced by high dietary cholesterol could potentiate pro-inflammatory cytokine production by fibroblasts stimulated with bacterial pathogens.     

Phylogenetic relationships within parrots (Psittacidae) inferred from mitochondrial cytochrome-b gene sequences

Blood and tissue samples of 40 individuals including 27 parrot species (15 genera; 3 subfamilies) were collected in Indonesia. Their phylogenetic relationships were inferred from 907 bp of the mitochondrial cytochrome-b gene, using the maximum-parsimony method, the maximum-likelihood method and the neighbor-joining method with Kimura two-parameter distance. The phylogenetic analysis revealed that (1) cockatoos (subfamily Cacatuinae) form a monophyletic sister group to other parrot groups; (2) within the genus Cacatua, C. goffini and C. sanguinea form a sister group to a clade containing other congeners; (3) subfamily Psittacinae emerged as paraphyletic, consisting of three clades, with a clade of Psittaculirostris grouping with subfamily Loriinae rather than with other Psittacinae; (4) lories and lorikeets (subfamily Loriinae) emerged as monophyletic, with Charmosyna placentis a basal sister group to other Loriinae, which comprised the subclades Lorius; Trichoglossus+Eos; and Chalcopsitta+ Pseudeos.     

UV-B radiation induces epithelial tumors in mice lacking DNA polymerase eta and mesenchymal tumors in mice deficient for DNA polymerase iota

DNA polymerase eta (Pol eta) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlight-induced skin cancer. We recently reported in a study of Polh mutant mice that Pol eta is involved in the somatic hypermutation of immunoglobulin genes, but the cancer predisposition of Polh-/- mice has not been examined until very recently. Another translesion synthesis polymerase, Pol iota, a Pol eta paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol iota is enigmatic. Here, we generated Polh Poli double-deficient mice and compared the tumor susceptibility of them with Polh- or Poli-deficient animals under the same genetic background. While Pol iota deficiency does not influence the UV sensitivity of mouse fibroblasts irrespective of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histological diagnosis after chronic treatment with UV light reveals that Pol iota deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not observed in Polh(-/-) mice. These results suggest the involvement of the Pol eta and Pol iota proteins in UV-induced skin carcinogenesis.     

A free radical scavenger but not FGF-2-mediated angiogenic therapy rescues myonephropathic metabolic syndrome in severe hindlimb ischemia

The therapeutic use of angiogenic factors shows promise in the treatment of critical limb ischemia; however, its potential for myonephropathic metabolic syndrome (MNMS), a fatal complication caused by arterial reconstruction, has not been elucidated. The objective of this study was to evaluate the effectiveness of recombinant Sendai virus-mediated gene transfer of fibroblast growth factor-2 (FGF-2) directly compared with that of a radical scavenger, MCI-186, in a rat model of MNMS. MNMS was surgically induced by aortic occlusion below renal arteries for 4 h, followed by 6 h of reperfusion. Administration of MCI-186 (twice; iv 5 min before induced ischemia and ip 5 min before reperfusion; 10 mg/kg, respectively), but not FGF-2 gene transfer (once, 48 h before induced ischemia), dramatically prevented the increase of serum biochemical markers as well as the edema of the gastrocnemius muscle. The effect of MCI-186 was accompanied by the marked suppression of the neutrophilic infiltration into the local (muscle) and remote (lung) organs. Although serum and muscular levels of a neutrophil-chemoattractant (growth-related oncogene/cytokine-induced neutrophil chemoattractant-1) were not affected by any treatment, the serum level of soluble intercellular adhesion molecule-1 was decreased by treatment with MCI-186 but not by treatment with FGF-2. These results suggest the distinct mechanism of MNMS from critical limb ischemia without reperfusion. Therefore, radical scavenging should be paid more attention than therapeutic angiogenesis when arterial circulation is reconstructed.     

Glutaredoxin modulates platelet-derived growth factor-dependent cell signaling by regulating the redox status of low molecular weight protein-tyrosine phosphatase

Glutaredoxin (GRX) is a glutathione-disulfide oxidoreductase involved in various cellular functions, including the redox-dependent regulation of certain integral proteins. Here we demonstrated that overexpression of GRX suppressed the proliferation of myocardiac H9c2 cells treated with platelet-derived growth factor (PDGF)-BB. After stimulation with PDGF-BB, the phosphorylation of PDGF receptor (PDGFR) beta was suppressed in GRX gene-transfected cells, compared with controls. Conversely, the phosphorylation was enhanced by depletion of GRX by RNA interference. In this study we focused on the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in the dephosphorylation of PDGFRbeta via a redox-dependent mechanism. We found that depletion of LMW-PTP using RNA interference enhanced the PDGF-BB-induced phosphorylation of PDGFRbeta, indicating that LMW-PTP works for PDGFRbeta. The enhancement of the phosphorylation of PDGFRbeta was well correlated with inactivation of LMW-PTP by cellular peroxide generated in the cells stimulated with PDGF-BB. In vitro, with hydrogen peroxide treatment, LMW-PTP showed decreased activity with the concomitant formation of dithiothreitol-reducible oligomers. GRX protected LMW-PTP from hydrogen peroxide-induced oxidation and inactivation in concert with glutathione, NADPH, and glutathione disulfide reductase. This strongly suggests that retention of activity of LMW-PTP by enhanced GRX expression suppresses the proliferation of cells treated with PDGF-BB via enhanced dephosphorylation of PDGFRbeta. Thus, GRX plays an important role in PDGF-BB-dependent cell proliferation by regulating the redox state of LMW-PTP.     

Modification of protein with BGL06, a novel branched oligoglycerol derivative

A branched oligoglycerol derivative, BGL06, with a cascade-like structure of glycerol units was used as a novel reagent for protein modification. Modification reaction with a recombinant human granulocyte-colony stimulating factor derivative, ND28, was carried out successfully in aqueous conditions to obtain a coupled form, BGL06-ND28. Characterization of the modified ND28 suggests that two types of products were obtained by controlling the reaction; one was H(BGL06)-ND28, a highly modified version coupled with 4.34 molecules of BGL06 units on average, and the other was L(BGL06)-ND28, a moderately modified version coupled with 2.58 molecules of BGL06 units on average, respectively. The properties of these products were compared to the known polyethylene glycol (PEG)-modified ND28. In the cell proliferation assays, unlike PEGylation, modification with BGL06 did not produce a significant loss of biological activity even when the modification extent was elevated. Under such conditions, 76.0% of the activity was in fact maintained for H(BGL06)-ND28, while PEGylated ND28 retained only 24.6% of biological activity in vitro even though the extent of modification was smaller. In addition, H(BGL06)-ND28 showed comparable thermostability to a 20 kDa PEG-modified counterpart. Therefore, the BGL06 derivative will be a useful alternative as a protein modification reagent where PEGylation is not effective.     

Purification and characterization of a carbohydrate: acceptor oxidoreductase from Paraconiothyrium sp. that produces lactobionic acid efficiently

A carbohydrate:acceptor oxidoreductase from Paraconiothyrium sp. was purified and characterized. The enzyme efficiently oxidized beta-(1-->4) linked sugars, such as lactose, xylobiose, and cellooligosaccharides. The enzyme also oxidized maltooligosaccharides, D-glucose, D-xylose, D-galactose, L-arabinose, and 6-deoxy-D-glucose. It specifically oxidized the beta-anomer of lactose. Molecular oxygen and 2,6-dichlorophenol indophenol were reduced by the enzyme as electron acceptors. The Paraconiothyrium enzyme was identified as a carbohydrate:acceptor oxidoreductase according to its specificity for electron donors and acceptors, and its molecular properties, as well as the N-terminal amino acid sequence. Further comparison of the amino acid sequences of lactose oxidizing enzymes indicated that carbohydrate:acceptor oxidoreductases belong to the same group as glucooligosaccharide oxidase, while they differ from cellobiose dehydrogenases and cellobiose:quinone oxidoreductases.     

Prominent down-regulation of storage protein genes after bacterial challenge in eri-silkworm, Samia cynthia ricini

We constructed two independent cDNA libraries from the fat body of Escherichia coli- or Candida albicans-challenged eri-silkworm Samia cynthia ricini larvae. We performed comparative expressed sequence tag (EST) analysis of the two cDNA libraries and found that two putative storage protein genes, ScSP1 and ScSP2, were markedly repressed by E. coli injection as compared with C. albicans injection. By quantitative real-time RT-PCR analysis, we showed that ScSP1 mRNA significantly reduced to 1/32-1/3 in the fat body of the female larvae, and ScSP2 mRNA reduced to 1/7-1/3 and 1/22-1/5 in the females and males, respectively, 12-36 h after E. coli injection as compared with PBS injection. In addition, SDS-PAGE analysis revealed that the accumulation of both the ScSP proteins in the larval hemolymph apparently decreased up to 36 h after E. coli injection. However, the amounts of the two ScSP proteins returned to the same level as those in the larvae injected with PBS by 48 h after injection, showing that the reduction in ScSPs caused by the bacterial challenge was transient. Moreover, potential binding sites for the Drosophila Rel/NF-kappaB protein Dorsal were found in the 5' upstream regulatory regions of ScSP1 and ScSP2, suggesting the participation of the Rel/NF-kappaB proteins in controlling the bacterial suppression of the ScSP genes. These results suggested the hypothesis that S. c. ricini has a genetic program to shut down temporarily dispensable gene expression in order to induce an acute and efficient expression of immune-related genes. These findings may provide new insight into the innate immune system in lepidopteran insects.