Discovery of pyrrolo[3,2-c]quinoline-4-one derivatives as novel hedgehog signaling inhibitors

The Hedgehog (Hh) signaling pathway plays a significant role in the regulation of cell growth and differentiation during embryonic development. Since activation of the Hh signaling pathway is implicated in several types of human cancers, inhibitors of this pathway could be promising anticancer agents. Using high throughput screening, thieno[3,2-c]quinoline-4-one derivative 9a was identified as a compound of interest with potent in vitro activity but poor metabolic stability. Our efforts focused on enhancement of in vitro inhibitory activity and metabolic stability, including core ring conversion and side chain optimization. This led to the discovery of pyrrolo[3,2-c]quinoline-4-one derivative 12b, which has a structure distinct from previously reported Hh signaling inhibitors. Compound 12b suppressed stromal Gli1 mRNA expression in a murine model and demonstrated antitumor activity in a murine medulloblastoma allograft model.     

Synthesis and application of an Nδ-acetyl-Nδ-hydroxyornithine analog: identification of novel metal complexes of deferriferrichrysin

Synthesis of Fmoc-protected N(δ)-acetyl-N(δ)-(tert-butoxy)-l-ornithine has revealed it to be a metal-chelating amino-acid precursor. This protected amino acid was compatible with the preparation of ferrichrome peptides by standard Fmoc-based solid-phase peptide synthesis. Evaluation of deferriferrichrysin for metal ion chelation revealed that zirconium(IV) and titanium(IV) formed complexes with deferriferrichrysin.     

Influence of dental treatment on physical performance in community-dwelling elderly persons

doi: 10.1111/j.1741‐2358.2011.00563.x
Influence of dental treatment on physical performance in community‐dwelling elderly persons Objective: The aim of the study was to investigate the influence of dental treatment on physical performance (muscle strength and balance function) among the elderly. Background: Oral conditions have been associated with physical performance. We hypothesised that improved oral conditions by dental treatment would lead to improved physical performance. Methods: A total of 154 persons aged 65 years or over were judged to be in need of dental treatment as a result of dental examination; of these, 121 persons underwent dental treatment. After 1 year, differences in each parameter of physical performance before and after the intervention were evaluated using the Wilcoxon signed rank sum test. The Spearman rank correlation coefficient was calculated to examine correlations between changes in self‐assessed masticatory ability (masticatory ability) and each parameter of physical performance, and multivariate logistic regression analysis was performed using changes in each parameter of physical performance as the dependent variable and changes in masticatory ability as the principal independent variable. Results: Improved physical performance was not observed for the total study population with dental treatment; however, in subjects with improved masticatory ability, one‐leg standing times with eyes open increased significantly. A significant correlation was established between changes in masticatory ability and each parameter of physical performance. These relationships were not found in those without dental treatment. A significant relationship was also established for one‐leg standing time after adjusting for age, gender, dentition status and needs of dental treatments. Conclusion: Chewing ability may be a positive contributing factor to balance function among the elderly.     

Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71

Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.     

Sphingosylphosphorylcholine induces stress fiber formation via activation of Fyn-RhoA-ROCK signaling pathway in fibroblasts

Sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, has recently been reported to modulate actin cytoskeleton rearrangement. We have previously demonstrated Fyn tyrosine kinase is involved in SPC-induced actin stress fiber formation in fibroblasts. However, Fyn-dependent signaling pathway remains to be elucidated. The present study demonstrates that RhoA-ROCK signaling downstream of Fyn controls stress fiber formation in SPC-treated fibroblasts. Here, we found that SPC-induced stress fiber formation was inhibited by C3 transferase, dominant negative RhoA or ROCK. SPC activated RhoA, which was blocked by pharmacological inhibition of Fyn activity or dominant negative Fyn. Constitutively active Fyn (ca-Fyn) stimulated stress fiber formation and localized with F-actin at the both ends of stress fibers, both of which were prevented by Fyn translocation inhibitor eicosapentaenoic acid (EPA). In contrast, inhibition of ROCK abolished only the formation of stress fibers, without affecting the localization of ca-Fyn. These results allow the identification of the molecular events downstream SPC in stress fiber formation for a better understanding of stress fiber formation involving Fyn.     

Effects of etoposide on the proliferation of hexaploid H1 (ES) cells

Etoposide is a specific inhibitor of topoisomerase II, which is an enzyme that enables double-stranded DNA to pass through another double-stranded DNA. Topoisomerase II is a major constituent of chromosome scaffold, existing at appreciable amounts in cells. To examine the effects of etoposide on the cell cycle, hexaploid H1 (ES) cells (6H1 cells) were used with diploid H1 (ES) cells (2H1 cells) as a control. Exponentially growing 2H1 and 6H1 cells were exposed to etoposide at various concentrations, and cultured for about 60 days in L15F10 medium with leukemia inhibitory factor. With a high concentration of etoposide (1 μM), the DNA histograms showed G(2)/M accumulation, suggesting that etoposide arrested the cell cycle at the G(2)/M phase. With a low concentration of etoposide (50 nM), the cell proliferation was suppressed with a doubling time of 98.4 h for 2H1 cells and 51.6 h for 6H1 cells, and without significant alteration in DNA histograms. Time-lapse videography revealed that 6H1 cells survived in the medium containing 50 nM etoposide had a cell cycle time of 18.8 h, which was equivalent to 19.2 h of the doubling time for the 6H1 cell population in drug-free medium, suggesting that a part of the cell population died and was excluded from the cell system. It was concluded that etoposide affected the cell cycle at a wide range of concentrations.     

Down regulation effect of Rosmarinus officinalis polyphenols on cellular stress proteins in rat pheochromocytoma PC12 cells

Polyphenols are known to exhibit wide spectrum of benefit for brain health and to protect from several neurodegenerative diseases. The present study was sought to determine the neuroprotective effects of Rosmarinus officinalis' polyphenols (luteolin, carnosic acid, and rosmarinic acid) through the investigation of stress-related proteins. We carried out measurement of the expression of heat-shock protein (Hsp) 47 promoter in heat stressed Chinese hamster ovary transfected cells. We performed proteomic analysis and confirmed gene expression by real time PCR in PC12 cells. Results showed that these compounds modulated significant and different effects on the expression of 4 stress-related proteins: heat shock protein 90 α (Hsp90), Transitional endoplasmic reticulum ATPase (VCP/p97), Nucleoside diphosphate kinase (NDK), and Hypoxia up-regulated protein 1 (HYOU1)) at translational and post translational levels in PC12 cells and they downregulated the expression of Hsp47 activity in Chinese hamster transformed cells. These findings suggest that luteolin, carnosic acid, and rosmarinic acid may modulate the neuroprotective defense system against cellular stress insults and increase neuro-thermotolerance.     

Effects of salt and ligand concentrations on the thermal unfolding and refolding of halophilic starch-binding domain from Kocuria varians α-amylase

The starch binding domain of α-amlylase from moderate halophile was expressed in E. coli with His tag (His- SBD12) and characterized for its halophilic properties. His-SBD12 was stable up to 35°C and showed binding activity, although at reduced level, to amylose even in the absence of NaCl. Both NaCl and specific ligands exhibited insignificant influence on the secondary structure of His-SBD12, but showed significant stabilization effects against thermal unfolding concentration-dependently, showing its halophilic properties. NaCl increased thermal stability of His-SBD12 by 4°C at 0.2 M and 15°C at 2 M, and enhanced refolding rate by ~7-fold at 0.2 M and ~170-fold at 2 M. Its specific ligands, β- cyclodextrin (at 3 mM) and maltose (at 470 mM), also stabilized the protein by 11° C, most likely reflecting affinity difference between these two ligands. However, they showed marginal effects on refolding rate. These observations suggest that although binding of NaCl and specific ligands to the native structure can explain their stabilization effects on His- SBD12, it is not a sole factor for modulating their effects on folding of His-SBD12.     

Comparison of effects of a selective 5-HT reuptake inhibitor versus a 5-HT4 receptor agonist on in vivo neurogenesis at the rectal anastomosis in rats

It was recently reported that activation of enteric neural 5-HT(4) receptors (SR4) promotes reconstruction of enteric neural circuit injury in distal gut of guinea pigs and that this reconstruction involves neural stem cells. We aimed to explore a novel approach using a selective serotonin reuptake inhibitor (SSRI), which increases endogenous 5-HT, to repair enteric nerve fiber injury in the rat distal gut. Enteric nerve fiber injury was performed by rectal transection and subsequent end-to-end one-layer anastomosis. The SSRI fluvoxamine maleate (100 μmol/l) was applied locally at the anastomotic site to compare with the 5-HT(4) agonist mosapride citrate (100 μmol/l) (applied for patent) applied locally and orally. Unlike mosapride, fluvoxamine failed to promote the regeneration of the nerve fiber tract across the anastomosis. Furthermore, fluvoxamine did not generate anti-distal-less homeobox 2 (DLX2)- and anti-SR4-positive cells (neural stem cells) and/or anti-neurofilament (NF)-positive cells (neural cells) in newly formed granulation tissue at the anastomosis, whereas these cell types were observed in mosapride-treated preparations. In contrast to its effects in guinea pigs, mosapride generated 5-bromo-2'-deoxyuridine (BrdU)-positive neural cells in ganglia sites 3 mm oral and anal from the anastomosis 2 wk after nerve fiber injury. All actions of mosapride were observed after local and or oral applications. These findings indicate that local SSRI treatment does not induce in vivo nerve fiber tract growth across the anastomosis in the rat distal gut. Mosapride induces nerve fiber tract growth across the anastomosis, mediated through enteric neural stem cells possibly from neural crest-derived stem cells or mesenchymal stem cells in the bone marrow.