Apoptosis-inducing neurotoxicity of dopamine and its metabolites via reactive quinone generation in neuroblastoma cells

Neurotoxic properties of L-dopa and dopamine (DA)-related compounds were assessed in human neuroblastoma SH-SY5Y cells with reference to their structural relationship. L-Dopa and its metabolites containing two free hydroxyl residues on their benzene ring showed toxicity in the cell, which was prevented by superoxide dismutase (SOD) and reduced glutathione (GSH), but not by catalase. Furthermore, a synthetic derivative of DA, 3-hydroxy-4-methoxyphenethylamine (HMPE) containing methoxy residue at position 4 in the benzene ring, exerted partial cytotoxicity, which was not prevented by SOD, GSH or catalase. However, the metabolites containing methoxy residue at position 3 failed to show a toxic effect in the SH-SY5Y cells. Moreover, DA induced apoptotic cell death, which was observed by nuclear and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and measurement of caspase-3 activity; this compound up-regulated apoptotic factor p53 while down-regulating anti-apoptotic factor Bcl-2. In the cell-free in vitro electron spin resonance (ESR) spectrometry, DA possessing two hydroxyl groups showed generation of DA-semiquinone radicals, which were markedly prevented by addition of SOD or GSH but not by catalase. On the other hand, methylation of one of the hydroxyl residues on the benzene ring of DA converted DA to an unoxidizable compound (3-MT or HMPE), and caused it to lose the property to produce semiquinone radicals. It has been previously reported that SOD acting as a superoxide:semiquinone oxidoreductase prevents quinone formation, and that reduced GSH through forming a complex with DA-quinone prevents quinone binding to the thiol group of the intact protein. Therefore, the present results suggest that DA and its metabolites containing two hydroxyl residues exert cytotoxicity mainly due to generation of highly reactive quinones.     

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type I receptor kinase

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC(50)=8.2nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50)=32nM).     

DISC1 localizes to the centrosome by binding to kendrin

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation that segregated with major mental disorders in a Scottish family. Using the yeast two-hybrid system, we screened a human brain cDNA library for interactors of the DISC1 protein. One of the positive clones encoded kendrin/pericentrin-B, a giant protein known to localize specifically to the centrosome. The interaction between DISC1 and kendrin in mammalian cells was demonstrated by an immunoprecipitation assay. Residues 446-533 of DISC1 were essential for the interaction with kendrin. Immunocytochemical analysis revealed the colocalization of DISC1 and kendrin to the centrosome. These data indicate that DISC1 localizes to the centrosome by binding to kendrin. Kendrin has been reported to anchor the gamma-tubulin complex to the centrosome, providing microtubule nucleation sites. The present study suggests the possible involvement of DISC1 in the pathophysiology of mental disorders due to its putative effect on centrosomal function.     

Ovarian Cancer Stem Cells Are Enriched in Side Population and Aldehyde Dehydrogenase Bright Overlapping Population

Cancer stem-like cells (CSCs)/cancer-initiaiting cells (CICs) are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP) analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDH^Br) cells from ovarian cancer cells. Both SP cells and ALDH^Br cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2), than those of main population (MP) cells and ALDH^Low cells, respectively. We analyzed an SP and ALDH^Br overlapping population (SP/ALDH^Br), and the SP/ALDH^Br population exhibited higher tumor-initiating ability than that of SP cells or ALDH^Br cells, enabling initiation of tumor with as few as 10² cells. Furthermore, SP/ADLH^Br population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDH^Low, MP/ALDH^Br and MP/ALDH^Low cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDH^Br population was detected in several gynecological cancer cells with ratios of 0.1% for HEC—1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDH^Br overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs.     

Human immune responses elicited by an intranasal inactivated H5 influenza vaccine

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.     

Characterization and Transcription of the Genes Involved in Butyrate Production in Butyrivibrio fibrisolvens TypeI and II Strains

The genes in Butyrivibrio fibrisolvens that encode the enzymes involved in butyrate production were sequenced. In a type I strain (ATCC 19171(T)), the genes coding for the enzymes that catalyze the conversion from acetyl-CoA to butyryl-CoA, thl (thiolase), crt (crotonase), hbd (beta-hydroxybutyryl-CoA dehydrogenase), bcd (butyryl-CoA dehydrogenase), etfB (electron transfer flavoprotein [ETF]-beta), and etfA (ETF-alpha), were found to be clustered and arranged in this order. A type II strain (ATCC 51255) had the same clustered genes with the same arrangement, except that crt was not present in the clustered genes. The deduced amino acid sequences of these enzymes did not greatly differ between the two strains, and even between B. fibrisolvens and clostridia. Amino acid identity appeared to be higher within the same type than between types I and II. The clustered genes were shown to be cotranscribed, and constitutively transcribed without being affected significantly by culture conditions.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.