Biomedical subjects
M Mori
Publications and source records attributed to M Mori.
Toll-like receptor 2 and 4 surface expressions on human monocytes are modulated by interferon-gamma and macrophage colony-stimulating factor.
Human Toll-like receptor 2 (TLR2) and TLR4 are recently identified receptors. TLR4 was shown to be the main protein involved in recognizing Gram-negative bacteria, whereas TLR2 is apparently the key factor in responses to other types of microbial pathogens. We examined regulations of TLR2 and TLR4 surface expressions in human peripheral blood monocytes by interferon-gamma (IFN-gamma) and macrophage colony-stimulating factor (M-CSF). IFN-gamma up-regulated both TLR2 and TLR4, but enhanced the surface expression, on human peripheral blood monocytes of TLR4 more than that of TLR2. On the other hand, M-CSF up-regulated both TLR2 and TLR4 surface expression on human peripheral blood monocytes, with no change in the ratio of TLR2/TLR4 surface expression. These results indicate that IFN-gamma enhances receptors involved in the response to Gram-negative bacteria more than those involved in responses to other types of microbial pathogens, whereas M-CSF enhances the receptor response to Gram-negative bacteria in the same manner as to other types of microbial pathogens.
Expression of multiple cancer-testis antigen genes in gastrointestinal and breast carcinomas.
Cancer-testis antigens (CTAs) such as MAGE are selectively expressed in various types of human neoplasms but not in normal tissues other than testis. This characteristic feature of CTAs makes them promising antigens for cancer-specific immunotherapy. A critical requirement for this therapy is identification of promising antigens. In this study, we investigated the expression of 6 genes recently identified by serological analysis of antigens by recombinant expression (SEREX) libraries: NY-ESO-1, LAGE-1, SCP-1, SSX-1, SSX-2, and SSX-4, in many surgical samples of gastrointestinal and breast carcinomas using reverse transcription-polymerase chain reaction. We found relatively high expression of SCP-1 (23.5%) and SSX-4 (20.6%) in gastric carcinoma, LAGE-1 (39.1%) and NY-ESO-1 (23.9%) in oesophageal carcinoma, and SCP-1 (34.1%) in breast carcinoma. We also found frequent synchronous expression with MAGE, including LAGE-1 (46.2%) in oesophageal carcinoma, SSX-4 (46.7%) in gastric carcinoma, and SCP-1 (38.3%) in breast carcinoma. Immunohistochemical analysis of the tumour samples expressing both MAGE-4 and NY-ESO-1 genes demonstrated differences in distribution between MAGE-4 and NY-ESO-1 in serial sections. We concluded that NY-ESO-1, LAGE-1, SCP-1 and SSX-4 genes may be promising candidates for cancer-specific immunotherapy in addition to MAGE, and that polyvalent cancer vaccines may be useful in cases of heterogeneous expressions of CTA genes in gastrointestinal and breast carcinomas.
Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway.
Excessive nitric oxide (NO) production in cytokine-activated beta cells has been implicated in beta cell disruption in type 1 diabetes. beta cells are very vulnerable to NO-induced apoptosis. However, the mechanism underlying this phenomenon is unclear. Low concentrations of NO that lead to apoptosis apparently do not cause severe DNA damage in mouse MIN6 beta cells. CHOP, a C/EBP homologous protein that is induced by endoplasmic reticulum (ER) stress and plays a role in growth arrest and cell death, was induced by a NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP). SNAP increased cytosolic Ca(2+), and only agents depleting ER Ca(2+) induced CHOP expression and led to apoptosis, suggesting that NO depletes ER Ca(2+). Overexpression of calreticulin increased the Ca(2+) content of ER and afforded protection to cells against NO-mediated apoptosis. Furthermore, pancreatic islets from CHOP knockout mice showed resistance to NO. We conclude that NO depletes ER Ca(2+), causes ER stress, and leads to apoptosis. Thus, ER Ca(2+) stores are a new target of NO, and the ER stress pathway is a major mechanism of NO-mediated beta cell apoptosis.
Long-term treatment with neutral endopeptidase inhibitor improves cardiac function and reduces natriuretic peptides in rats with chronic heart failure.
OBJECTIVE: Increased secretion of atrial and brain natriuretic peptide (ANP and BNP) from hearts is known to exhibit favorable effects in patients and animals with heart failure, and inhibition of neutral endopeptidase (NEP), an enzyme that degrades ANP and BNP, may further increase these peptide levels. However, it is still unknown whether such elevation of the ANP and BNP may offer a therapeutic benefit to the progression of chronic heart failure (CHF). We examined the effects of ONO-9902, a novel NEP inhibitor, on changes in hemodynamic parameters, NEP activity and neurohumoral factors in rats with CHF induced by left coronary artery ligation (CAL). METHODS: Male Wistar rats (220-240 g) were subjected to induction of acute myocardial infarction by CAL. Rats were orally treated with ONO-9902 (300 mg/kg/day) from the 1st to 6th week after the operation. Hemodynamic and/or biochemical assessments were performed at the 1st and 6th weeks after the operation. RESULTS: A single administration of ONO-9902 inhibited the plasma and kidney NEP activities and thereby further augmented the elevation of plasma ANP concentration in rats with CAL at the 1st week after the operation. In rats with CAL at the 6th week after the operation, the left ventricular end-diastolic pressure (LVEDP) increased and cardiac output index (COI) decreased as compared with those of sham-operated rats. These changes were accompanied by marked increases in the plasma ANP, BNP and endothelin-1 (ET-1). Chronic treatment with ONO-9902 attenuated the increase in LVEDP and the decrease in COI. These changes were associated with a decrease in plasma ANP, BNP and ET-1 concentrations. CONCLUSIONS: The results suggest that chronic treatment with NEP inhibitor improves depressed cardiac function in rats with CHF. ONO-9902 may offer a new and possible therapeutic approach in patients with CHF.
Fast synaptic transmission mediated by P2X receptors in CA3 pyramidal cells of rat hippocampal slice cultures.
1. A fast ATP-mediated synaptic current was identified in CA3 pyramidal cells in organotypic hippocampal slice cultures. In the presence of inhibitors for ionotropic glutamate and GABA receptors, extracellular stimulation in the pyramidal cell layer evoked fast synaptic currents that reversed near 0 mV, reflecting an increase in a non-selective cationic conductance. This response was mimicked by focal application of ATP. Antagonists of ionotropic P2X receptors reduced both synaptic and ATP-induced currents. 2. Using a pharmacological approach, the source of synaptically released ATP was determined. Synaptic ATP responses were insensitive to presynaptic blockade of GABAergic transmission between interneurons and CA3 pyramidal cells with the mu-opioid receptor agonist D-Ala(2),MePhe(4),Met(O)(5)-ol-enkephalin (FK33-824), but were blocked by adenosine, which inhibits glutamate release from synaptic terminals in the hippocampus. However, selective inhibition of mossy fibre glutamatergic transmission with the metabotropic glutamate receptor group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) did not affect the response. This result points to the associational fibres as the source of the ATP-mediated synaptic response. 3. These results suggest that ATP, coreleased with glutamate, induces a synaptic response in CA3 pyramidal cells that is observed mainly under conditions of synchronous discharge from multiple presynaptic inputs.
Sensitized mast cells migrate toward the antigen: a response regulated by p38 mitogen-activated protein kinase and Rho-associated coiled-coil-forming protein kinase.
Although mast cells accumulate within the mucosal epithelial layer of patients with allergic rhinitis and bronchial asthma, the responsible chemotactic factors are undefined. We investigated whether mast cells sensitized with Ag-specific IgE migrate toward the Ag. MC/9 mast cells sensitized with anti-DNP IgE migrated toward DNP-conjugated human serum albumin. This migration was directional, and the degree was stronger than that induced by stem cell factor. IL-3 and stem cell factor-dependent cultured mast cells derived from mouse bone marrow also migrated toward the Ag. Subsequent migration mediated by the Fc(epsilon)RI was significantly inhibited by incubating the cells with Y-27632, a Rho-associated coiled-coil-forming protein kinase inhibitor, or with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Both p38 MAPK and MAPK-activated protein kinase (MAPKAPK)2 were activated following Fc(epsilon)RI aggregation, and activation of MAPKAPK2 was almost completely inhibited by 10 microM SB203580. Wortmannin or a low concentration of SB203580 partially inhibited MAPKAPK2, but did not block mast cell migration. In contrast, Y-27632 did not affect the activation of MAPKAPK2. These results indicate that Ag works not only as a stimulant for allergic mediators from IgE-sensitized mast cells, but also as a chemotactic factor for mast cells. Both p38 MAPK activation and Rho-dependent activation of Rho-associated coiled-coil-forming protein kinase may be required for Fc(epsilon)RI-mediated cell migration.
Hepatoma-derived growth factor is associated with reduced sensitivity to irradiation in esophageal cancer.
Radiotherapy is a useful component of treatment for esophageal cancer. Identification of the genes that are differentially expressed between radiosensitive and radioresistant cancer cells is important for predicting clinical effectiveness of radiotherapy. We established human esophageal cancer cell lines resistant to X-ray. Using differential display, we obtained one gene that was expressed in radiosensitive cells but was rarely expressed in radioresistant cells, and that gene was identical with hepatoma-derived growth factor (HDGF), an acidic polypeptide with mitogenic activity for fibroblasts. The semiquantitative reverse transcription-PCR assay confirmed that HDGF mRNA expression was reduced in established radioresistant cells, and its reduction was associated with reduced sensitivity to irradiation. Radiotherapy was more effective in clinical cases with high HDGF mRNA expression compared with cases with low expression (P < 0.05). The findings demonstrate that HDGF may play an important role in radiosensitivity, and it could be a novel marker predicting effectiveness of radiotherapy in clinical cases.
Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction.
Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (Dlk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of Dlk may play a role in the pathogenesis of MDS, the disease specificity of Dlk expression was tested by a quantitative "real-time" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders.
Cationic diazacrown ether for the separation of positional isomers by capillary electrophoresis.
The use of a positively charged diazacrown ether, N,N,N',N'-tetramethyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane dichloride (1), in capillary electrophoresis is reported, for the first time. The addition of the cationic diazacrown 1 to the running buffer permitted the successful separation of positional isomers of aromatic anions such as naphthalenedisulfonate, naphthalenedicarboxylate and phthalate. At a concentration of 1 of 2 mM, all the analyte anions were completely separated in less than 5 min. The ion association constants of the solute anions with diazacrown 1 were evaluated and the findings are discussed.
Differential expression of the cationic amino acid transporter 2(B) in the adult rat brain.
L-Arginine is a substrate for the synthesis of proteins, nitric oxide (NO), creatine, urea, proline, glutamate, polyamines and agmatine. In the central nervous system (CNS), arginine is extracted from the blood and exchanged by cells through carriers called cationic amino acid transporters (CAT) and belonging to the so-called system y+. In order to better understand the arginine transport in the CNS, we studied in detail the regional distribution of the cells expressing the CAT2(B) transcript in the adult rat brain by non-radioisotopic in situ hybridization. We show that CAT2(B) is expressed in neurons and oligodendrocytes throughout the brain, but is not detected in astrocytes. The pattern of localization of CAT2(B) in the normal adult rat brain fits closely that of CRT1, a specific creatine transporter. Our study demonstrates that the in vivo expression of CAT2(B) differs from that reported in vitro, implying that local cellular interactions should be taken into account in studies of gene regulation of the CAT2(B) gene. Our work suggests that CAT2(B) may play a role in case of increased NO production as well as arginine or creatine deficiency in the brain.
Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells.
Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6-12 h. mRNA for argininosuccinate synthetase (AS), a citrulline-arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS/IFN-gamma was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline-arginine recycling are important for high-output production of NO in activated microglial cells.
Quantitative trait locus analysis for chronic pancreatitis and diabetes mellitus in the WBN/Kob rat.
A causative gene mutation is still undefined in approximately half of patients with hereditary pancreatitis, and no genetic factor has been identified in most patients with sporadic chronic pancreatitis. To identify a pancreatitis-associated gene, we performed a quantitative trait locus (QTL) analysis for the traits of chronic pancreatitis and diabetes mellitus in WBN/Kob rats. We identified two highly significant QTLs for chronic pancreatitis and/or hyperinsulinemia on chromosomes 7 and X. These QTLs were located on completely different chromosomal regions from those of causative genes that have been reported for human chronic pancreatitis: PRSS1, CFTR, and SPINK1. For these QTLs, prevalences of the WBN/Kob allele significantly increased in the rats with chronic pancreatitis. These findings indicate that chronic pancreatitis in WBN/Kob rats is controlled by multiple genes, and a genetic analysis in WBN/Kob rats might be useful for gene targeting for human chronic pancreatitis.
A new and general synthetic pathway to strychnos indole alkaloids: total syntheses of (-)-dehydrotubifoline and (-)-tubifoline by palladium-catalyzed asymmetric allylic substitution.
[see reaction]. A novel procedure for the synthesis of an indole skeleton was developed. Treatment of a cyclohexenol derivative having a silyloxymethyl group at the 2-position with N-tosyl-o-bromoaniline in the presence of Pd2dba3*CHCl3 and (S)-BINAPO gave compound 6a with 84% ee in 75% yield. Compound 6a was converted into 11, which was treated with Pd(OAc)2 and Me(2)PPh in the presence of Ag2CO3 to give indoline derivative 12. From 12, we succeeded in the total syntheses of (-)-dehydrotubifoline and (-)-tubifoline.
Antitumor effect of beta2-microglobulin in leukemic cell-bearing mice via apoptosis-inducing activity: activation of caspase-3 and nuclear factor-kappaB.
We have reported previously that beta2-microglobulin (beta2m) induces apoptosis in leukemic cells in vitro, and that an interaction between beta2m and HLA class I antigen induces apoptosis. Here we examined whether beta2m can induce apoptosis in leukemic cells in vivo and whether it has an antitumor effect in tumor-bearing mice. Daily administration of 50 or 250 microg of beta2m induced apoptosis and an antitumor effect on K562 leukemia cell-bearing mice in the same manner as tumor necrosis factor-alpha. In tumor tissues in beta2m-treated mice, both caspase-3 and nuclear factor-kappaB (NF-kappaB) were stained more strongly than in control mice by anti-caspase-3 and anti-NF-kappaB p65/Rel A polyclonal antibodies. We also observed the in vivo immunological effects of beta2m on lymphoid and hematopoietic organs, such as thymus, bone marrow, Peyer's patches, liver, and spleen in normal mice. Using antibodies against caspase-3 and NF-kappaB, immunohistochemical staining showed that no specific tissues were damaged or stained in normal mice. We conclude that beta2m stimulates caspase-3 and NF-kappaB pathways to induce apoptosis, making it a useful approach to a new therapy for leukemia.
Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor.
A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.
Purification, molecular cloning, and functional expression of dog liver microsomal acyl-CoA hydrolase: a member of the carboxylesterase multigene family.
To clarify the reason for the high acyl-CoA hydrolase (ACH) activity found in dog liver microsomes, the ACH was purified to homogeneity using column chromatography. The purified enzyme, named ACH D1, exhibited a subunit molecular weight of 60 KDa. The amino terminal amino acid sequence showed a striking homology with rat liver carboxylesterase (CES) isozymes. ACH D1 possessed hydrolytic activities toward esters containing xenobiotics in addition to acyl-CoA thioesters, and these activities were inhibited by a specific inhibitor of CES or by CES RH1 antibodies. These findings suggest that this protein is a member of the CES multigene family. Since ACH D1 appears to be a protein belonging to the CES family, we cloned the cDNA from a dog liver lambdagt10 library with a CES-specific probe. The clone obtained, designated CES D1, possessed several motifs characterizing CES isozymes, and the deduced amino acid sequences were 100% identical with those of ACH D1 in the first 18 amino acid residues. When it was expressed in V79 cells, it showed high catalytic activities toward acyl-CoA thioesters. In addition, the characteristics of the expressed protein were identical with those of ACH D1 in many cases, suggesting that CES D1 encodes liver microsomal ACH D1.
Spontaneous loss-of-function mutations of the 8-oxoguanine DNA glycosylase gene in mice and exploration of the possible implication of the gene in senescence.
8-Oxoguanine is one of the major premutagenic oxidative base legions in vivo and is suspected to play a crucial role in various pathophysiological processes, such as cancer and aging. Mammalian 8-oxoguanine DNA glycosylase (OGG1) is thought to play a major role in the removal of 8-oxoguanine adducts in vivo. We have identified several inbred mouse strains with a spontaneous mutation, OGG1-R336H or double mutations, OGG1-R304W/R336H. R304W mutation caused a complete loss of OGG1 activity, while the R336H mutation led to disruption of nuclear localization of the enzyme although the activity remained normal. Among the double mutants was SAMP1, which exhibits accelerated senescence and short lifespan. We assessed the possible implication of the mutant OGG1 and 8-oxoguanine in aging utilizing SAMP1 mice. SAMP1 retained 1.5- to 1.9-fold increase in 8-oxoguanine level of hepatic nuclear DNA as compared with normal mice, until at least 12 months of age. A genetic association study, however, indicated that the mutant Ogg1 gene per se is not responsible for the accelerated senescence and short lifespan of SAMP1. Mutant OGG1 may be associated with pathologic conditions in other mouse strains.