Investigation toward multi-epitope vaccine candidates using native chemical ligation

We applied native chemical ligation (NCL) method to the synthesis of highly pure lipid-core peptide (LCP) vaccines to attach various peptide epitopes. In the case of the synthesis of LCP vaccine with two different peptide epitopes, LCP moieties having two free Cys and two protected Cys derivatives (S-acetamidemethyl-Cys, (Cys(Acm)), N-methylsulfonylethyloxycarbonyl-Cys (Msc-Cys), or 1,3-thiazolidine-4-carboxylic acid (Thz)) on oligolysine branches were prepared in order to couple two different epitopes by stepwise NCL. It was found that the difficulty in NCL of first two peptide antigen was associated with the steric hindrance. Using Thz instead of Cys(Acm) and Msc-Cys was important to reduce the steric hindrance and improve NCL yield.     

Coenzyme Q10 as a potent compound that inhibits Cdt1–geminin interaction

A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1–geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1–geminin binding. Using this system, we found that coenzyme Q₁₀ (CoQ₁₀) can inhibit Cdt1–geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ₁₀, having a longer isoprenoid chain, was the strongest inhibitor of Cdt1–geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 μM. Surface plasmon resonance analysis demonstrated that CoQ₁₀ bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ₁₀ had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ₁₀ inhibits Cdt1–geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ₁₀ was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ₁₀ could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1–geminin binding. The application of inhibitors for the formation of Cdt1–geminin complex is discussed.     

CMP substitutions preferentially inhibit polysialic acid synthesis

It is widely reported that derivatives of sugar moieties can be used to metabolically label cell surface carbohydrates or inhibit a particular glycosylation. However, few studies address the effect of substitution of the cytidylmonophosphate (CMP) portion on sialyltransferase activities. Here we first synthesized 2'-O-methyl CMP and 5-methyl CMP and then asked if these CMP derivatives are recognized by alpha2,3-sialyltransferases (ST3Gal-III and ST3Gal-IV), alpha2,6-sialyltransferase (ST6Gal-I), and alpha2,8-sialyltransferase (ST8Sia-II, ST8Sia-III, and ST8Sia-IV). We found that ST3Gal-III and ST3Gal-IV but not ST6Gal-I was inhibited by 2'-O-methyl CMP as potently as by CMP, while ST3Gal-III, ST3Gal-IV, and ST6Gal-I were moderately inhibited by 5-methyl CMP. Previously, it was reported that polysialyltransferase ST8Sia-II but not ST8Sia-IV was inhibited by CMP N-butylneuraminic acid. We found that ST8Sia-IV as well as ST8Sia-II and ST8Sia-III are inhibited by 2'-O-methyl CMP as robustly as by CMP and moderately by 5-methyl CMP. Moreover, the addition of CMP, 2'-O-methyl CMP, and 5-methyl CMP to the culture medium resulted in the decrease of polysialic acid expression on the cell surface and NCAM of Chinese hamster ovary cells. These results suggest that 2'-O-methyl CMP and 5-methyl CMP can be used to preferentially inhibit sialyltransferases, in particular, polysialyltransferases in vitro and in vivo. Such inhibition may be useful to determine the function of a carbohydrate synthesized by a specific sialyltransferase such as polysialyltransferase.     

Discovery of novel series of benzoic acid derivatives containing biphenyl ether moiety as potent and selective human beta(3)-adrenergic receptor agonists: Part IV

Identification and SAR study of novel series of beta(3)-AR agonists with benzoic acid are described. Conversion of ether linkage position of phenoxybenzoic acid derivative 2b led to compound 7b with moderate beta(3)-AR activity. Further modification in right, center and left parts of compound 7b was investigated to improve the beta(3)-AR potency and selectivity. Compounds 7g and 7k, with the bulky aliphatic-substituted group at 2-position of benzoic acid moiety, were identified as potent and selective beta(3)-AR agonists. In addition, in vivo efficacy of compounds 7g and 7k was exhibited on dog OAB model.     

Effect of alendronate on osteoclast differentiation and bone volume in transplanted bone.

Alendronate, one of the bisphosphonates, is known to have an inhibitory effect on bone resorption. The purpose of this study was to investigate the effects of alendronate on ectopic bone graft resorption and to determine the optimal dose in the mouse. The grafted bone in the control group disappeared due to resorption by osteoclasts within 5 weeks. In the experimental groups, the area of bone tissue decreased by only 20-40% at 5 weeks post-operatively. At 8 and 9 weeks after surgery, the decreased area of bone structure was significantly less in all the 10(-4) M injected alendronate-immersed groups than in the 10(-4) M non-injected alendronate-immersed. At 9 weeks after surgery, the number of osteoclasts were significantly less in the 10(-4) M injected alendronate-treated groups than in the 10(-4) M non-injected alendronate-treated groups. These results suggest that alendronate inhibits resorption of ectopic bone graft at concentrations of 10(-4) and 10(-6) M.     

High monospore-producing mutants obtained by treatment with MNNG in Porphyra yezoensis Ueda (Bangiales,Rhodophyta)

Hydrobiologia - Two high monospore-producing pigmentation mutants were obtained by treatment with MNNG in Porphyra yezoensis Ueda. The mutants produced many monospores in young gametophytic blades...     

Albumin suppresses vascular endothelial growth factor via alteration of hypoxia-inducible factor/hypoxia-responsive element pathway

Reduction of vascular endothelial growth factor (VEGF) expression plays a crucial role in chronic kidney disease (CKD). In order to clarify a cause of VEGF suppression in CKD, we examined an interaction between proteinuria and VEGF. Rat proximal tubular cells were subjected to hypoxia with or without albumin to mimic proteinuric conditions, and VEGF expression was assessed by real-time quantitative PCR and enzyme-linked immunosorbent assays. Albumin significantly reduced VEGF expression under hypoxia. Luciferase activity controlled by hypoxia-responsive element (HRE) was suppressed by albumin, demonstrating suppression of the hypoxia-inducible factor (HIF)/HRE pathway. Studies utilizing a proteasome inhibitor and a prolyl hydroxylase inhibitor showed that mechanisms of HIF/HRE pathway suppression by albumin load did not involve degradation of HIF protein levels. Further, albumin did not change HIF mRNA levels. Our data, for the first time, suggest a clear ‘link’ between proteinuria and hypoxia, the two principal pathogenic factors for CKD progression.     

Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes.

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4, a member of the TLR family that participates in pathogen recognition. TLRs recruit a cytoplasmic protein, MyD88, upon pathogen recognition, mediating its function for immune responses. Two major pathways for LPS have been suggested in recent studies, which are referred to as MyD88-dependent and -independent pathways. We report in this study the characterization of the MyD88-independent pathway via TLR4. MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS, whereas they responded to LPS by activating IFN-regulatory factor 3 as well as inducing the genes containing IFN-stimulated regulatory elements such as IP-10. In contrast, a lipopeptide that activates TLR2 had no ability to activate IFN-regulatory factor 3. The MyD88-independent pathway was also activated in cells lacking both MyD88 and TNFR-associated factor 6. Thus, TLR4 signaling is composed of at least two distinct pathways, a MyD88-dependent pathway that is critical to the induction of inflammatory cytokines and a MyD88/TNFR-associated factor 6-independent pathway that regulates induction of IP-10.     

Embryogenomics: developmental biology meets genomics

Fundamental questions in developmental biology are: what genes are expressed, where and when they are expressed, what is the level of expression and how are these programs changed by the functional and structural alteration of genes? These questions have been addressed by studying one gene at a time, but a new research field that handles many genes in parallel is emerging. The methodology is at the interface of large-scale genomics approaches and developmental biology. Genomics needs developmental biology because one of the goals of genomics – collection and analysis of all genes in an organism – cannot be completed without working on embryonic tissues in which many genes are uniquely expressed. However, developmental biology needs genomics – the high-throughput approaches of genomics generate information about genes and pathways that can give an integrated view of complex processes. This article discusses these new approaches and their applications to mammalian developmental biology.