Endoplasmic Reticulum Stress-Related Inflammation and Cardiovascular Diseases

The endoplasmic reticulum (ER) is the site of synthesis and maturation of proteins designed for secretion or for localization on the cell membrane. Various types of stress from both inside and outside cells disturb ER function, thus causing unfolded or misfolded proteins to accumulate in the ER. To improve and maintain the ER functions against such stresses, the ER stress response pathway is activated. However, when the stress is prolonged or severe, apoptosis pathways are activated to remove damaged cells. It was recently reported that the ER stress pathway is also involved in the inflammatory response, whereby inflammation induces ER stress, and ER stress induces an inflammatory response. Therefore, the ER stress response pathway is involved in various diseases, including cardiovascular diseases such as atherosclerosis and ischemic diseases, in various ways. The ER stress pathway may represent a novel target for the treatment of these diseases

Antibacterial Effects of Disulfiram in Helicobacter pylori

Background: Helicobacter pylori infection poses a risk of the occurrence of gastrointestinal diseases, such as gastric cancer. Its incidence rate is significantly reduced by eradication, and thereby, eradication therapy is generally performed. Disulfiram is an oral prescription drug mainly used for the treatment of alcohol dependence. In recent years, reports have been made on its anticancer and antibacterial effects, and thus, it has recently become an interesting subject. This study aimed to examine the antibacterial activity of disulfiram, investigate the presence or absence of its antibacterial activity on H. pylori, and determine whether it could be a new bactericidal drug against drug-resistant H. pylori. Materials and Methods: Drug-sensitive strains of H. pylori and amoxicillin-resistant, clarithromycin-resistant, and metronidazole-resistant strains were used, and a growth inhibition test of H. pylori using disulfiram was performed. Furthermore, the expression of urease, vacuolating cytotoxin A (VacA), and CagA, the virulence proteins of H. pylori, was quantitatively analyzed using the Western blotting method. In addition, for H. pylori used in this study, the 16SrDNA sequence, a ribosomal gene involved in protein production, was analyzed to examine the presence or absence of gene mutation. Results: Disulfiram suppressed the growth of 7 out of 12 H. pylori strains at 1 mu g/mL, and no correlation was observed between their susceptibility/resistance to current eradication antimicrobial drugs and disulfiram resistance. Disulfiram reduced the expression levels of urease, VacA, and CagA proteins. H. pylori, which showed resistance to disulfiram, tended to have fewer gene deletions/insertions in the 16S rDNA sequence; however, no specific mutation was detected. Conclusion: Disulfiram has a bactericidal effect on H. pylori at low concentrations, suggesting that it can be used as a supplement for current H. pylori eradication drugs

Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice

INTRODUCTION: The role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR). METHODS: Peritoneal macrophages from female BALB/c mice bearing a 7-day LMM3 tumor were inoculated intradermally (3 × 10(5 )cells per site) into syngeneic mice. Before inoculation, TMps were stimulated with the muscarinic agonist carbachol in the absence or presence of different muscarinic antagonists or enzyme inhibitors. Angiogenesis was evaluated by counting vessels per square millimeter of skin. The expression of mAchR, arginase and cyclo-oxygenase (COX) isoforms was analyzed by Western blotting. Arginase and COX activities were evaluated by urea and prostaglandin E(2 )(PGE(2)) production, respectively. RESULTS: TMps, which stimulate neovascularization, express functional mAchR, because carbachol-treated TMps potently increased new blood vessels formation. This response was completely blocked by preincubating TMps with pirenzepine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), M(1 )and M(3 )receptor antagonists, and partly by the M(2 )receptor antagonist methoctramine. M(1 )receptor activation by carbachol in TMps triggers neovascularization through arginase products because N(ω)-hydroxy-L-arginine reversed the agonist action. Preincubation of TMps with methoctramine partly prevented carbachol-stimulated urea formation. In addition, COX-derived liberation of PGE(2 )is responsible for the promotion of TMps angiogenic activity by M(3 )receptor. We also detected a higher expression of vascular endothelial growth factor (VEGF) in TMps than in macrophages from normal mice. Carbachol significantly increased VEGF expression in TMps, and this effect was totally reversed by methoctramine and pirenzepine. Arginase and COX inhibitors partly decreased VEGF derived from TMps. CONCLUSION: TMps themselves induce a potent angiogenic response that is augmented by carbachol action. mAchR activation triggers arginine metabolism, PGE(2 )synthesis and VEGF production, promoting neovascularization

Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage

To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP‐Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud‐specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11‐13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle‐shaped structure required to generate five digits

尿路上皮癌微小環境内におけるDisabled Homolog 2 (DAB2) は腫瘍細胞上皮間葉転換を介して遊走能・浸潤能を高める

Disabled homolog-2 (DAB2) has been reported to be a tumor suppressor gene. However, a number of contrary studies suggested that DAB2 promotes tumor invasion in urothelial carcinoma of the bladder (UCB). Here, we investigated the clinical role and biological function of DAB2 in human UCB. Immunohistochemical staining analysis for DAB2 was carried out on UCB tissue specimens. DAB2 expression levels were compared with clinicopathological factors. DAB2 was knocked-down by small interfering RNA (siRNA) transfection, and then its effects on cell proliferation, invasion, and migration, and changes to epithelial-mesenchymal transition (EMT)-related proteins were evaluated. In our in vivo assays, tumor-bearing athymic nude mice subcutaneously inoculated with human UCB cells (MGH-U-3 or UM-UC-3) were treated by DAB2-targeting siRNA. Higher expression of DAB2 was associated with higher clinical T category, high tumor grade, and poor oncological outcome. The knock-down of DAB2 decreased both invasion and migration ability and expression of EMT-related proteins. Significant inhibitory effects on tumor growth and invasion were observed in xenograft tumors of UM-UC-3 treated by DAB2-targeting siRNA. Our findings suggested that DAB2 expression was associated with poor prognosis through increased oncogenic properties including tumor proliferation, migration, invasion, and enhancement of EMT in human UCB.博士（医学）・甲第768号・令和3年3月15日© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

Mitochondrial dysfunction and increased reactive oxygen species impair insulin secretion in sphingomyelin synthase 1-null Mice

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic β-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic β-cells.Masato Yano, Ken Watanabe, Tadashi Yamamoto, Kazutaka Ikeda, Takafumi Senokuchi, Meihong Lu, Tsuyoshi Kadomatsu, Hiroto Tsukano, Masahito Ikawa, Masaru Okabe, Shohei Yamaoka, Toshiro Okazaki, Hisanori Umehara, Tomomi Gotoh, Wen-Jie Song, Koichi Node, Ryo Taguchi, Kazuya Yamagata, Yuichi Oike, Mitochondrial Dysfunction and Increased Reactive Oxygen Species Impair Insulin Secretion in Sphingomyelin Synthase 1-null Mice, Journal of Biological Chemistry, Volume 286, Issue 5, 2011, Pages 3992-4002, ISSN 0021-9258, https://doi.org/10.1074/jbc.M110.179176

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 is required for normal cartilage development

CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage