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Biomedical subjects

M Inoue

Publications and source records attributed to M Inoue.

At least 109 records · Page 6Linked to original sources
PubMed2000

Measurement of isotopes of light rare earth elements in the form of oxide ions: a new development in thermal ionization mass spectrometry

An isobaric interference free thermal ionization mass spectrometry (TIMS) method is presented here for the precise isotope measurements of light rare earth elements (LREEs). LREE isotopes can be measured in the form of monoxide ion (MO+), but the previous determinations of LREE isotopes in the form of oxide were hindered, notably by the 17O and 18O related isobaric interferences. In the present study, 16O-enriched oxygen was introduced into the ion source chamber via a specially designed gas inlet system. It was observed that the isobaric interferences caused by 17O and 18O were remarkably reduced by up to 6 and 70 times, respectively, compared to those caused by natural oxygen bleeding. Isotopic ratios of La, Ce, and Nd obtained without isobaric interference corrections show the excellent precision and accuracy in the present study. The results also shed some light on the formation mechanism of the measured oxide species in TIMS. Furthermore it promises more precise determination of LREE abundances and La-Ce and Sm-Nd ages in geochemistry and cosmochemistry.

Journal Article↗
PubMed2000

A novel telomerase-specific gene therapy: gene transfer of caspase-8 utilizing the human telomerase catalytic subunit gene promoter.

Apoptosis is a genetically encoded cell death process and is a pathway that may be disrupted in tumor cells. Therefore, therapies that restore the ability to undergo apoptosis are promising for the treatment of tumor cells. We have demonstrated that the transfer of apoptosis-inducible genes inhibits the growth of tumors in vitro and in vivo through induction of apoptosis. However, to restrict induction of apoptosis to tumor cells, we need to explore a tumor-specific expression system of these genes. In the present study, we developed the telomerase-specific transfer system of apoptosis-inducible genes, utilizing the promoter of the human telomerase catalytic subunit (hTERT) gene. Approximately 90% of tumors have telomerase activity whereas most normal cells do not express the activity. These observations indicate that telomerase is a particularly attractive target for the tumor-specific expression system of vectors. We demonstrate here that by using the hTERT promoter-driven caspase-8 expression vector (hTERT/caspase-8), apoptosis is restricted to telomerase-positive tumor cells of wide range, and is not seen in normal fibroblast cells without telomerase activity. Furthermore, treatment of subcutaneous tumors in nude mice with the hTERT/caspase-8 construct inhibited tumor growth significantly because of induction of apoptosis (p < 0.01). The telomerase-specific expression of apoptosis-inducible genes afforded by the hTERT promoter, therefore, may be a novel and promising targeting approach for the treatment of tumors with telomerase activity.

Animals↗
PubMed2000

Identification and characterization of negative regulatory elements of the human telomerase catalytic subunit (hTERT) gene promoter: possible role of MZF-2 in transcriptional repression of hTERT.

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of human telomerase and is a critical determinant of the enzymatic activity of telomerase. Expression of hTERT is known to be regulated mainly at the transcriptional level. In the present study, using transient expression assays, we identified a 400 bp silencer of the hTERT promoter between -776 and -378 upstream of the proximal core promoter. The inhibitory effects of this silencer were enhanced with cellular differentiation. A computer-assisted homology search identified multiple binding motifs for myeloid-specific zinc finger protein 2 (MZF-2) within this region. Mutation introduced in these sites resulted in significant activation of hTERT transcription. Gel shift assays demonstrated that MZF-2 proteins specifically bound to these sites. Overexpression of MZF-2 in cells led to down-regulation of hTERT transcription as well as telomerase activity. These findings suggest that the 400 bp region upstream of the hTERT core promoter that we identified functions as a negative regulatory region and that MZF-2 may be an effector of negative regulation of hTERT.

Base Sequence↗
PubMed2000

Phonon screening in high-temperature superconductors

In good conductors optical phonons are usually screened, and therefore not observed. However, sharp features due to infrared-active modes in the copper-oxygen planes are observed in the optical conductivity of Pr1.85Ce0.15CuO4 and YBa2Cu3O6.95. Oscillator strengths indicate that the screening of these modes is poor or totally absent. These materials are compared with eta-Mo4O11, in which lattice modes appear suddenly below the charge-density wave transition. It is proposed that poor screening in the cuprates originates from fluctuating charge inhomogeneities in the copper-oxygen planes.

Journal Article↗
PubMed2000

Oxidative cellular damage associated with transformation of Helicobacter pylori from a bacillary to a coccoid form.

Exposure to unfavorable conditions results in the transformation of Helicobacter pylori, a gastric pathogen, from a bacillary form to a coccoid form. The mechanism and pathophysiological significance of this transformation remain unclear. The generation of the superoxide radical by H. pylori has previously been shown to inhibit the bactericidal action of nitric oxide, the concentration of which is relatively high in gastric juice. With the use of chemiluminescence probes, both the quality and quantity of reactive oxygen species generated by H. pylori have now been shown to change markedly during the transformation from the bacillary form to the coccoid form. The transformation of H. pylori was associated with oxidative modification of cellular proteins, including urease, an enzyme required for the survival of this bacterium in acidic gastric juice. Although the cellular abundance of urease protein increased during the transformation, the specific activity of the enzyme decreased and it underwent aggregation. Specific activities of both superoxide dismutase and catalase in H. pylori also decreased markedly during the transformation. The transformation of H. pylori was also associated with oxidative modification of DNA, as revealed by the generation of 8-hydroxyguanine, and subsequent DNA fragment. These observations indicate that oxidative stress elicited by endogenously generated reactive oxygen species might play an important role in the transformation of H. pylori from the bacillary form to the coccoid form.

Bacterial Proteins↗
PubMed2000

Enantioselective photocyclization of amides to beta-lactam derivatives in inclusion crystals with an optically active host

Irradiation of inclusion crystals of 2-(N-acyl-N-alkylamino)cyclohex-2-enones and N,N-dimethylphenylglyoxylamide with chiral host molecules gave the optically active N-alkyl-1-azaspiro[3.5]-nonane-2,5-diones and 3-hydroxy-1-methyl-3-phenylazetidin-2-one, respectively. The crystal structure of the 1:1 inclusion complex of N,N-dimethylphenylglyoxylamide with (-)-trans-1,4-bis[3-(o-chlorophenyl)-3-hydroxy-3-phenylprop-1-ynyl]-2,3,5,6- tetrachloro-2,5-cyclohexadiene-1,4-diol was analyzed by X-ray diffraction.

Journal Article↗
PubMed2000

Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries.

BACKGROUND: Rho-associated kinase (ROCK), an effector of small GTPase Rho, regulates vascular tone via a calcium sensitization mechanism and plays a key role in the pathogenesis of hypertension. However, its role in vascular growth remains unclear. METHODS AND RESULTS: Y-27632, a specific ROCK inhibitor, and the overexpression of dominant-negative ROCK suppressed the mitogen-induced DNA synthesis of cultured vascular smooth muscle cells (VSMCs), which indicates the essential role of ROCK in the control of VSMC proliferation in vitro. Y-27632 also suppressed the chemotaxis of VSMCs. Male Wistar rats were systemically given Y-27632 (35 to 70 mg. kg(-1). day(-1)) through an intraperitoneal infusion. The neointimal formation of balloon-injured carotid arteries was significantly suppressed in Y-27632-treated rats (intima/media ratio, 0.22+/-0.02) compared with vehicle-treated rats (intima/media ratio, 0.92+/-0.21) or hydralazine-treated rats with a similar blood pressure decrease (intima/media ratio, 1.03+/-0.15). The phosphorylation of myosin phosphatase and myosin light chain was elevated in injured arteries in a Y-27632-sensitive manner, indicating the augmentation of ROCK activity in neointimal formation. The downregulation of the cyclin-dependent kinase inhibitor p27(kip1) in injured vessels was reversed by Y-27632 treatment, reflecting the antiproliferative effect of ROCK inhibition in vivo. CONCLUSIONS: We conclude that ROCK plays a key role in the process of neointimal formation after balloon injury. Thus, the inhibition of ROCK may be a potential therapeutic strategy for treating vascular proliferative disorders and hypertension.

Amides↗
PubMed2000

Comparison of lifestyle and risk factors among Japanese with and without gastric cancer family history.

To find specific risk factors of gastric cancer (GC) independent of GC family history (GCFH), 2 studies were conducted using the database of the Hospital-based Epidemiological Research Program at Aichi Cancer Center: (i) a comparison of lifestyles between non-cancer cases with positive and negative GCFH status and (ii) a case-reference investigation of subjects with and without GCFH, treated separately. The first showed no significant variation of GCFH status with regard to smoking, drinking and most food habits. Multivariate analyses in the case-referent studies revealed odds ratios (ORs) for GC associated with habitual smoking of 2.78 (95% CI 1.22-6.28) for those with and 2.74 (95% CI 1.76-4.26) for those without GCFH. In individuals with GCFH, an independently lowered OR (0.52, 95% CI 0.27-0.99) was evident for frequent consumption of raw vegetables, whereas the opposite was noted for pickled vegetables (2. 39, 95% CI 1.28-4.45). No statistically significant interaction was found between GCFH and selected lifestyle items. In conclusion, our results suggest a limited influence of GCFH on risk factors for GC.

Adult↗
PubMed2000

Short-term effects of rapid pacing on mRNA level of voltage-dependent K(+) channels in rat atrium: electrical remodeling in paroxysmal atrial tachycardia.

BACKGROUND: Atrial fibrillation causes electrophysiological changes of the atrium, thereby facilitating its maintenance. Although the expression of ion channels is modulated in chronic atrial fibrillation, it is yet unknown whether paroxysmal atrial fibrillation can also lead to electrical remodeling by affecting gene expression. METHODS AND RESULTS: To examine the short-term effects of rapid pacing on the mRNA level of voltage-dependent K(+) channels, high-rate atrial pacing was performed in Sprague-Dawley rat hearts. Total RNA was prepared from the atrial appendages from 0 to 8 hours after the onset of pacing, and mRNA levels of Kv1.2, Kv1. 4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, erg, KvLQT1, and minK were determined by RNase protection assay. Among these 9 genes, the mRNA level of the Kv1.5 channel immediately and transiently increased, with bimodal peaks at 0.5 and 2 hours after the onset of pacing. Conversely, the pacing gradually and progressively decreased the mRNA levels of the Kv4.2 and Kv4.3 channels. The increase of Kv1.5 and the decrease of Kv4.2 and Kv4.3 mRNA levels were both rate dependent. In correspondence with the changes in the mRNA level, Kv1. 5 channel protein transiently increased in the membrane fraction of the atrium during a 2- to 8-hour pacing period. Electrophysiological findings that the shortening of the action potential produced by 4-hour pacing was almost abolished by a low concentration of 4-aminopyridine implied that the increased Kv1.5 protein was functioning. CONCLUSIONS: Even short-term high-rate atrial excitation could differentially alter the mRNA levels of Kv1.5, Kv4.2, and Kv4.3 in a rate-dependent manner. In particular, increased Kv1.5 gene expression, having a transient nature, implied the possible biochemical electrical remodeling unique to paroxysmal tachycardia.

Action Potentials↗
PubMed2000

NMR studies on functional structures of the AU-rich element-binding domains of Hu antigen C.

Hu antigen C (HuC) has three RNA-binding domains (RBDs). The N-terminal two, RBD1 and RBD2, are linked in tandem and bind to the AU-rich elements (AREs) in the 3'-untranslated region of particular mRNAs. The solution structures of HuC RBD1 and RBD2 were determined by NMR methods. The HuC RBD1 and RBD2 structures are quite similar to those of Sxl RBD1 and RBD2, respectively. The individual RBDs of HuC, RBD1 and RBD2 in isolation can interact rather weakly with the minimal ARE motif, AUUUA, while the didomain fragment, RBD1-RBD2, of HuC binds more tightly to a longer ARE RNA, UAUUUAUUUU. Chemical shift perturbations by the longer RNA on HuC RBD1-RBD2 were mapped on and around the two beta-sheets and on the C-terminal region of RBD1. The HuC RBD1-RBD2 residues that exhibited significant chemical shift perturbations coincide with those conserved in Sxl RBD1-RBD2. These data indicate that the RNA-binding characteristics of the HuC and Sxl didomain fragments are similar, even though the target RNAs and the biological functions of the proteins are different.

Adenosine↗
PubMed2000

Immunohistochemical detection of p53 and p21 proteins in canine testicular tumours.

The objective of this study was to detect by immunohistochemical means nuclear accumulations of p53 and p21 proteins in testicular tumours of dogs. Intense p53 protein nuclear labelling was shown by each of seven seminomas and one intermediate collision tumour. Moderate or intense immunoreactivity was shown by three Sertoli cell tumours. All the tumours also showed p21 nuclear reactivity in parallel with the p53 reactivity. In contrast, four Leydig cell tumours showed no detectable immunoreactivity. The results suggested that high levels of p53 accumulation were associated with the expression of wild-type p53, which was able to activate the transcription of the p21 gene. In addition, weak p53- and p21-nuclear reactivities were detectable in primary spermatocytes within normal seminiferous tubules.

Animals↗
PubMed2000

Selective inhibition of cyclooxygenase-2 with antisense oligodeoxynucleotide restricts induction of rat adjuvant-induced arthritis.

The effects of cyclooxygenase (COX)-2 antisense oligodeoxynucleotide (ODN) in induction of adjuvant-induced arthritis were investigated. Female Lewis rats were injected with Mycobacterium butyricum intradermally at the base of tails to induce arthritis. Synthetic 18 mer phosphorothioate ODNs corresponding to the translation initiation site of rat COX-2 mRNA were prepared. The antisense (AS), sense (S), and "scrambled" (Sc) ODNs were intraperitoneally administered. Arthropathy was evaluated with arthritis score, paw edema, and histological examination. Expression of COX-1 and -2 protein and mRNA were examined with immunostaining and reverse-transcription polymerase chain reaction, respectively. COX-2 AS ODN significantly suppressed induction of arthritis in a dose-dependent manner without severe adverse effects, whereas S and Sc ODNs did not show significant inhibitory effects. COX-2 mRNA and protein expression were also suppressed only by COX-2 AS ODN without any alteration of COX-1 expression. These data suggest that selective inhibition of COX-2 with AS ODN may have a therapeutic potency in the treatment of rheumatoid arthritis.

Animals↗
PubMed2000

Alteration of beta-catenin expression in esophageal squamous-cell carcinoma.

beta-catenin regulates cadherin-mediated cell-cell adhesion and also functions as a signaling molecule. In this study, we examined the expression pattern of E-cadherin, alpha-catenin and beta-catenin in 22 cases of esophageal squamous-cell carcinoma by Western-blot analysis. Expression of E-cadherin, alpha-catenin and beta-catenin was lower in carcinomas than in normal esophageal mucosa in 4 cases (18.2%) for E-cadherin, 6 cases (27.3%) for alpha-catenin and 9 cases (40.9%) for beta-catenin. Expression of beta-catenin was not always correlated with that of E-cadherin. Over-expression of beta-catenin was observed in 3 cases (13.6%). Of 3 cases that presented with over-expression of beta-catenin, 2 showed cytoplasmic staining by immunohistochemistry. Nuclear localization of beta-catenin was observed in one case that had higher beta-catenin level in tumor tissue (1.4-fold higher than normal mucosa). The genomic DNA sequences of the beta-catenin and the APC gene were analyzed. No mutation of the beta-catenin gene was observed in any cases. Silent mutation of the APC gene was found in all the cases that showed over-expression or nuclear localization of the beta-catenin protein. These results indicate that alterations of the cadherin-catenin complex may play an important role in a sub-set of esophageal carcinogenesis. Furthermore, it is suggested that beta-catenin over-expression is not caused by genetic alteration of either the beta-catenin or the APC gene.

Blotting, Western↗
PubMed2000

Nitric oxide-independent effects of nitric oxide donors on energy metabolism in erythrocytes.

In order to study the roles of nitric oxide (NO) in various biological events, several types of NO-releasing agents have been extensively used. Although both NO and its donors and/or their decomposed products may have biological activities, most of the cellular responses to these donors have been postulated to reflect NO-dependent events. Among the various NO donors, 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-l-triazene (NOC7), 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), S-nitrosoglutathione, S-nitrosocysteine (CysNO), and related nitrosothiols are commonly used agents. To investigate the biological activities of these donors and their decomposed products, we tested their effects on energy metabolism in erythrocytes. When incubated with freshly prepared erythrocytes, NOC7, Cys-NO, and their decomposed products, but not NO and its oxidized metabolites, nitrite and nitrate, decreased cellular ATP levels. Although SIN-1 generates both NO and superoxide radical thereby forming peroxynitrite (ONOO-), this donor had no appreciable effect on cellular ATP levels, even in the presence of superoxide dismutase. These results indicate that NOC7 and CysNO and/or their decomposed product(s), but not NO and its oxidized metabolites, are responsible for the decrease in cellular ATP levels. Thus, the effects of not only NO and its oxidized metabolites (NO2, NO3 ), but also NO donors and their decomposed products, should be taken into account when attempting to understand the mechanism of biological responses induced by NO donors.

Adenosine Triphosphate↗
PubMed2000

Vzg-1/lysophosphatidic acid-receptor involved in peripheral pain transmission.

The nociception by intraplantar (i.pl.) lysophosphatidic acid (LPA) injection was significantly, but partially blocked when mice received intrathecal (i.t.) antisense oligodeoxynucleotide treatment for the vzg-1 type LPA-receptor. The residual LPA-nociception observed under the condition of pertussis toxin-treatment, which is expected to block presynaptic contribution, was abolished by diphenhydramine (i.pl.), an H1-type histamine receptor antagonist. Taking into account that vzg-1 mRNA was detected in the dorsal root ganglion by RT-PCR method, these findings suggest that the LPA-induced nociception is attributed to the mechanism through vzg-1 receptor on nociceptor endings, and to that through unidentified LPA-receptor on peripheral, possibly mast cells.

Anesthetics, Local↗
PubMed2000

14-3-3tau associates with a translational control factor FKBP12-rapamycin-associated protein in T-cells after stimulation by pervanadate.

Proteins of the 14-3-3 family can associate with and/or modulate the activities of a variety of proteins, such as protooncogene and oncogene products, Cdc25 phosphatases and phosphatidylinositol 3-kinase, and thus are implicated in regulation of signaling pathways and the cell cycle. We report here that treatment of Jurkat T-cells with an inhibitor of protein tyrosine phosphatase, pervanadate, induces the association of 14-3-3tau with a translational control factor, FKBP12-rapamycin-associated protein (FRAP), with significant latter's autophosphorylation. Coimmunoprecipitation of various mutants of FRAP coexpressed with 14-3-3tau in COS-7 cells revealed that 14-3-3tau binds to the C-terminal side of FRAP at unknown site(s) different from the predicted binding motifs to date.

14-3-3 Proteins↗
PubMed2000

Sp1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT).

Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. The human telomerase catalytic subunit (hTERT) is a rate limiting determinant of the enzymatic activity of human telomerase. In the previous study, we identified the proximal 181 bp core promoter responsible for transcriptional activity of the hTERT gene. To identify the regulatory factors of transcription, transient expression assays were performed using hTERT promoter reporter plasmids. Serial deletion assays of the core promoter revealed that the 5'-region containing the E-box, which binds Myc/Max, as well as the 3'-region containing the GC-box, which binds Sp1, are essential for transactivation. The mutations introduced in the E-box or GC-box significantly decreased transcriptional activity of the promoter. Overexpression of Myc/Max or Sp1 led to significant activation of transcription in a cell type-specific manner, while Mad/Max introduction repressed it. However, the effects of Myc/Max on transactivation were marginal when Sp1 sites were mutated. Western blot analysis using various cell lines revealed a positive correlation between c-Myc and Sp1 expression and transcriptional activity of hTERT. Using fibroblast lineages in different stages of transformation, we found that c-Myc and Sp1 were induced to a dramatic extent when cells overcame replicative senescence and obtained immortal characteristics, in association with telomerase activation. These findings suggest that c-Myc and Sp1 cooperatively function as the major determinants of hTERT expression, and that the switching functions of Myc/Max and Mad/Max might also play roles in telomerase regulation.

Base Sequence↗