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Biomedical subjects

M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 325 records · Page 18Linked to original sources
PubMed1992

N-acetylaspartylglutamate acts as an agonist upon homomeric NMDA receptor (NMDAR1) expressed in Xenopus oocytes.

The electrophysiological effects of N-acetylaspartylglutamate (NAAG), an endogenous peptide restrictively distributed in the central nervous system, were studied using Xenopus oocytes injected with RNAs transcribed from cloned glutamate receptor cDNAs. NAAG induced an inward current, dose dependently, in oocytes injected with RNA for an N-methyl-D-aspartate receptor subunit (NMDAR1). In contrast, the oocytes injected with RNAs for AMPA-selective glutamate receptors (GluR1, GluR3, GluR1+GluR2 and GluR2+GluR3) scarcely responded to NAAG, and the oocytes injected with RNA for kainate receptor (GluR6) did not respond to NAAG. The half-maximal response (ED50) value of NAAG on expressed NMDAR1 was 185 microM, which shows that NAAG is about 115-times less potent than L-glutamate (Glu), the ED50 of which value was 1.6 microM. The maximal current amplitude induced by NAAG was about 70% of that by Glu. NAAG-induced current in NMDAR1-injected oocytes was potentiated by glycine, dose-dependently antagonized by DL-2-amino-5-phosphonovaleric acid, and blocked by magnesium ions in a voltage-dependent fashion. These results suggest that NAAG is one of the endogenous agonists selective for NMDAR1.

2-Amino-5-phosphonovalerate↗
PubMed1992

Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA. N2,3-Ethenoguanine is known to cause G----A transitions during DNA replication in Escherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair of N2,3-ethenoguanine, we have prepared an N2,3-etheno[14C]guanine-containing DNA substrate by nick-translating DNA with [14C]dGTP and modifying the product with chloroacetaldehyde. E. coli 3-methyladenine DNA glycosylase II, purified from cells which carry the plasmid pYN1000, releases N2,3-ethenoguanine from chloroacetaldehyde-modified DNA in a protein- and time-dependent manner. This finding widens the known substrate specificity of glycosylase II to include a modified base which may be associated with the carcinogenic process. Similar enzymatic activity in eukaryotic cell might protect them from exposure to metabolites of vinyl chloride.

Acetaldehyde↗
PubMed1992

Inactivation of glibenclamide-sensitive K+ channels in Xenopus oocytes by various calmodulin antagonists.

In follicle-enclosed Xenopus oocytes, extracellular application of cromakalim (a K+ channel opener) or intracellular injection of cAMP induces the smooth outward K+ current which is inactivated by glibenclamide. We found that cromakalim- or cAMP-induced K+ currents in the oocytes were rapidly, reversibly and dose-dependently blocked by various drugs having a calmodulin antagonizing activity in common, namely, by a selective calmodulin antagonist (W-7), antipsychotics (trifluoperazine, chlorpromazine, haloperidol), an antidepressant (amitriptyline), a beta-adrenoceptor blocker (propranolol), a local anesthetic (lidocaine) and a calcium antagonist (prenylamine). W-7, trifluoperazine, chlorpromazine and prenylamine were relatively potent blockers. For example, IC50 values to block cromakalim (100 microM)-induced K+ currents were 12 microM for trifluoperazine and 16 microM for W-7, which were close to their IC50 values to inhibit Ca2+/calmodulin-dependent phosphodiesterase (an index of the potency of calmodulin antagonists). IC50 values to inhibit cAMP (20 pmol/oocyte)-induced K+ currents were 126 microM for prenylamine and 129 microM for chlorpromazine. The IC50 values of all drugs tested to block cromakalim or cAMP responses were significantly correlated with their calmodulin-antagonizing potencies. Isoproterenol-induced K+ currents in the oocytes were also dose-dependently inhibited by glibenclamide, W-7 and trifluoperazine. These results suggest the possibility that the activity of glibenclamide-sensitive K+ channels in follicle-enclosed oocytes are regulated by calmodulin or a calmodulin-dependent process.

Animals↗
PubMed1992

Cytoarchitectonic abnormalities in hippocampal formation and cerebellum of dreher mutant mouse.

The laminated structures in the hippocampal formation and cerebellum of homozygous dreher mice were compared to their littermates and to C57BL/6J mice in Nissl- and myelin-stained preparations. In the dreher dentate gyrus, ectopic granule cells were situated in the molecular layer, and frequently there was either partial or complete absence of the infrapyramidal limb of the granule cell layer. In the dreher hippocampus, the cells of the pyramidal cell layer in area CA3 formed widely dispersed arrangements, and there were ectopically situated pyramidal cells in the stratum radiatum and stratum oriens. In the dreher cerebellum, 3 abnormal patterns were observed: (1) disruptions of foliation with normal cytoarchitectonic structure, (2) foliation with a mixture of normal laminated structure and abnormal laminated structure, and (3) almost complete absence of the cerebellum. In abnormal folia exhibiting the second or third pattern, islands consisting of agglomerations of both granule cells and Purkinje cells or just granule cells were observed. The neuronal heterotopias and cytoarchitectonic disorganization observed in the present study are apparently secondary to disruption of cell proliferation and neuronal migration produced directly or indirectly by the dreher mutation. In addition, the fact that the phenotypic abnormalities in homozygous dreher mice produces different abnormal morphologies in different specimens may be useful for analyzing the development of the hippocampal formation and cerebellum.

Animals↗
PubMed1992

Inhibition of development of Kaposi's sarcoma-related lesions by a bacterial cell wall complex.

In vitro and in vivo model systems for the study of human immunodeficiency virus (HIV)-associated Kaposi's sarcoma (KS) were used to evaluate compounds for their potential as therapeutic agents. A sulfated polysaccharide-peptidoglycan compound (SP-PG) produced by bacteria controlled the in vitro growth of acquired immunodeficiency syndrome (AIDS)-associated, KS-derived spindle-shaped cells (AIDS-KS cells) at noncytotoxic concentrations. Angiogenesis induced by AIDS-KS cells in the chicken chorioallantoic membrane assay was blocked by SP-PG, which also inhibited the vascular hyperpermeability response and the angiogenesis associated with the induction of KS-like lesions that develop after subcutaneous inoculation of AIDS-KS cells into nude mice. Suramin, pentosan polysulfate, and interferon alpha, which are currently in use for therapy of KS, were either less effective than SP-PG or much more cytotoxic, or both.

Acquired Immunodeficiency Syndrome↗
PubMed1992

MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis.

Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low. We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis. This error-avoiding process is catalysed by a protein encoded by the mutT gene of Escherichia coli, mutations of which increase the occurrence of A.T----C.G transversions 100 to 10,000 times the level of the wild type. Spontaneous oxidation of dGTP forms 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP), which is inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein specifically degrades 8-oxodGTP to the monophosphate. This indicates that elimination from the nucleotide pool of the oxidized form of guanine nucleotide is important for the high fidelity of DNA synthesis.

Bacterial Proteins↗
PubMed1992

Histological examination of surface repair tissue after successful osteotomy for osteoarthritis of the hip joint.

In this study biopsy specimens of the surface repair tissue from the weightbearing area of the hip joint were collected from 15 patients who had undergone successful valgus osteotomy, and these were examined histologically. In 6 of the 15 patients the specimens stained strongly with safranin-O in all three layers of the matrix. However, the repair tissue from another 6 patients showed strong staining only in the intermediate and deep layers, while that from 2 patients stained only in the deep layer. Specimens from 5 patients were stained for S-100 protein and positive cells were detected in all cases, demonstrating that the cells in the repair tissue were chondrocytes. However, there was no tide mark in the repair tissue and the arrangement of the cells differed from that in normal cartilage. Discontinuity of the subchondral bone was also noted. These findings make it likely that the repair tissue was of bone marrow origin.

Adult↗
PubMed1992

Regulatory elements for expression of the alkA gene in response to alkylating agents.

Expression of the alkA gene in Escherichia coli is controlled by Ada protein, which binds to a specific region of the alkA promoter and enhances further binding of RNA polymerase holoenzyme to the complex. To determine the sequence recognized by the Ada protein, we introduced various base substitutions into the promoter region of alkA and examined their effects on expression of the gene, both in vivo and in vitro. Base changes within the sequence AAAGCAAA, located between positions -41 and -34 from the transcription initiation site, greatly decreased the frequencies of initiation of transcription. In footprinting experiments, the region containing this sequence was protected by the Ada protein and base changes within this sequence led to failure of binding of Ada protein to the promoter. It is likely that the Ada protein recognizes the AAAGCAAA sequence in the alkA promoter and binds to the region containing the sequence, thereby allowing ready access of RNA polymerase to the promoter. There are considerable differences between the mechanisms of action of Ada protein on the promoters of alkA and ada, even though the expression of both genes is positively regulated by Ada protein.

Alkylating Agents↗
PubMed1992

An overview on Takayasu arteritis.

Takayasu arteritis is a non-specific inflammatory disease of unknown etiology. It was first recognized as having a peculiar wreath-like arteriovenous anastomosis around the papillae of the retina by a Japanese ophthalmologist, Dr. M. Takayasu in 1908. A Japanese research committee reported more than 5,000 cases. For a supplement issue on Takayasu arteritis, this brief overview article has been written as an introduction to the disease.

Aorta, Thoracic↗
PubMed1992

Hypoxia-induced ANP secretion in subjects susceptible to high-altitude pulmonary edema.

We investigated the effects of acute hypoxia (10% O2) on plasma level of atrial natriuretic peptide (ANP) and pulmonary hemodynamics in five subjects with a history of high-altitude pulmonary edema (HAPE). Plasma renin activity and plasma levels of aldosterone, epinephrine and norepinephrine were also measured. The plasma ANP levels in HAPE-susceptible subjects rose significantly in response to 10% O2 (from 34.8 +/- 5.4 to 51.4 +/- 7.3 pg.ml-1; P less than 0.05), not associated with any increase in either atrial pressure. Compared with six control subjects, the rise of ANP level was greater in HAPE-susceptible subjects (16.6 +/- 4.4 vs 3.9 +/- 1.2 pg.ml-1; P less than 0.05). There was a significant positive correlation between the rise of ANP level and the increase of pulmonary arterial pressure. No significant difference was observed in any of other hormonal responses to acute hypoxia between the two groups. We interpret these results as indicating that the ANP secretory response to acute hypoxia in HAPE-susceptible subjects, which is not mediated by an increase in atrial pressure, may be greater than that in nonsusceptible subjects in association with a greater pressor response of pulmonary circulation.

Adult↗
PubMed1992

Characterization of a murine monoclonal antibody, 5D-4, reacting with pancreatic cancers and islets of Langerhans.

A murine monoclonal antibody (5D-4) was prepared by immunizing mice ip with human pancreatic cancer cell line (AsPC-1). The 5D-4 MAb reacted immunohistochemically with pancreatic and gastrointestinal tract tumors as well as pancreatic islets, and to a less extent with normal tissues. The 5D-4 MAb reacted not only with ca 50 KDa and 30 KDa solubilized protein from AsPC-1 cells by Western blot analysis but also with human insulin in a competition RIA. Double immunoperoxidase staining showed that the 5D-4 MAb cross-reacted with insulin but did not react with glucagon, somatostatin or pancreatic polypeptide. Immunoelectron micrograph of Langerhans island double-stained with the 5D-4 MAb and anti-insulin Ab revealed that the 5D-4 Mab recognized human insulin and ca 50 KDa and 30 KDa antigens in B-cells of islets of Langerhans. Thus, the 5D-4 Mab may be useful for the diagnosis of islet cell tumors as well as pancreatic cancers.

Animals↗
PubMed1992

Isolation and characterization of cDNA and genomic sequences for mouse O6-methylguanine-DNA methyltransferase.

An enzyme, O6-methylguanine-DNA methyltransferase, is present in various organisms and plays an important role in repair of DNA damaged by alkylating agents. The enzyme transfers methyl groups from O6-methylguanine and other methylated moieties of the DNA to its own molecule. As a first step to construct animal models with altered levels of the enzyme activity, we cloned cDNA and genomic DNA sequences for mouse methyltransferase and elucidated their structures. The nucleotide sequence of the cDNA revealed an open reading frame comprising 211 amino acid residues. The mol. wt of mouse O6-methylguanine-DNA methyltransferase, calculated from the predicted amino acid sequence, was 22,400, and the methyltransferase protein of this size was present when the cDNA was expressed in methyltransferase-deficient human cells. The predicted amino acid sequence of the mouse methyltransferase exhibits an intense homology with those of human and bacterial counterparts. Using the cDNA as a probe, part of the mouse gene for methyltransferase was isolated. The gene consisted of at least four exons and spanned greater than 145 kb. Sequences around the exon/intron junctions for the mouse gene are almost the same as those for the human species.

Amino Acid Sequence↗
PubMed1992

Specific amino acid sequences required for O6-methylguanine-DNA methyltransferase activity: analyses of three residues at or near the methyl acceptor site.

O6-Methylguanine-DNA methyltransferase plays an important role in preventing tumor induction. To elucidate the significance of a highly conserved amino acid sequence of methyltransferase protein, amino acid substitutions were introduced by site-directed mutagenesis of cloned cDNA for human methyltransferase and the activity and stability of mutant forms of enzyme were examined. When cysteine-145, to which the methyl transfer occurs, was replaced by other amino acids, all of the mutants isolated showed the methyltransferase-negative phenotype. From one of the negative mutants, methyltransferase-positive revertants were isolated, all of which carried codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and cannot be replaced by other amino acids. Using this negative and positive selection procedure, analyses were extended to other residues near the acceptor site. At the histidine-146 site, four substitutions (phenylalanine, methionine, asparagine and glutamine) exhibited the positive phenotype but the levels of methyltransferase activity in these mutants were low. With valine-148 substitutions there were six types of positive revertants, among which mutants carrying isoleucine, cysteine and alanine showed significantly high levels of methyltransferase activity. Some mutant forms of cDNA were expressed in methyltransferase-deficient human cells, and the results obtained with Escherichia coli cells were confirmed.

Amino Acid Sequence↗
PubMed1992

Induced synthesis of O6-methylguanine-DNA methyltransferase in rat hepatoma cells exposed to DNA-damaging agents.

When the rat hepatoma cell line H4IIE was treated with DNA-damaging agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ultraviolet light and gamma-rays, the O6-methylguanine-DNA methyltransferase activity increased 2 to 3 times over the level seen in non-treated cells. SDS/polyacrylamide gel electrophoresis followed by fluorography revealed that a single species of methyltransferase protein with a molecular weight of 25,500 was present in both non-treated and treated cells. Northern blot analysis using a cloned rat cDNA as a probe revealed that the enzyme activity increased because transcription of the gene was enhanced. The level of enzyme activity increased within 48 h after UV irradiation and remained at a higher level for 150 h. Following UV irradiation, the cells become more resistant than the normal cells to MNNG.

Animals↗
PubMed1992

Effects of thromboxane synthase inhibition on tumor necrosis factor-induced lung injury in sheep.

To examine the role of thromboxane (Tx) A2 in the pathogenesis of acute lung injury caused by tumor necrosis factor alpha (TNF), we tested the effects of OKY-046, a selective thromboxane synthase inhibitor, on pulmonary hemodynamics, lung lymph balance, circulating leukocytes, arterial blood gas analysis, and TxA2 (as TxB2) and prostacyclin (as 6-keto-prostaglandin F1 alpha) levels in plasma and lung lymph after TNF infusion in awake sheep. Infusion of human recombinant TNF (3.5 micrograms/kg) into a chronically instrumented awake sheep caused a transient increase in pulmonary arterial pressure (Ppa). The Ppa peaked within 15 min of the start of TNF infusion from 23.3 +/- 1.1 (SE) cmH2O of baseline to 42.3 +/- 2.3 cmH2O and then decreased toward baseline. The pulmonary hypertension was accompanied by transient hypoxemia, peripheral leukopenia, and the increases in TxB2 in plasma and lung lymph. These changes were followed by an increase in flow of protein-rich lung lymph, consistent with an increase in pulmonary microvascular permeability. OKY-046 significantly prevented the rises of Ppa and TxB2 concentrations in plasma and lung lymph during early phase after TNF infusion. OKY-046, however, did not attenuate the increase of lung lymph flow, transient hypoxemia, and leukopenia. From these data, and by comparison with our previous studies of OKY-046-pretreated sheep during endotoxemia, we conclude that TxA2 has an important role of the increase in the early pulmonary hypertension, but it is not related to the early hypoxemia, leukopenia, and lung lymph balances in TNF-induced lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Myocardial uptake of 111In monoclonal antimyosin Fab in detecting doxorubicin cardiotoxicity in rats. Morphological and hemodynamic findings.

BACKGROUND: The therapeutic value of doxorubicin (DOX) is limited by its cardiotoxicity, which is dose dependent. To improve the detection of such myocardial damage, this study was designed to determine whether the 111In antimyosin antibody Fab could serve as a marker for cardiotoxicity in treated versus control rats on the basis of comparative morphological and hemodynamic findings. METHODS AND RESULTS: DOX was administered by intravenous injection to rats at a dose of 3 mg.kg-1.week-1 for 5 weeks. Three weeks after the final injection, an intravenous dose of 111In antimyosin, 740 kBq (20 microCi), was administered, and tissue distribution of the radioisotope (percent kilogram dose per gram) was assessed in 48 hours. Myocardial uptake of radioactivity by both ventricles was more prominent in the DOX-treated rats than in control rats (p < 0.001). The heart-to-blood and heart-to-lung uptake ratios were markedly higher in the treated rats than in controls (p < 0.001). As the severity of the myocardial damage increased, there was a progressive increase in myocardial uptake. There was a strong correlation between the severity of myocardial damage and the ventricular end-diastolic pressure (r = 0.84 and r = 0.83 in the left and right ventricles, respectively). On microscopic immunoautoradiography of the DOX-treated heart, there was a specific immunolocalization of the radiotracer in the injured myocytes but no radioactivity in the control myocytes. CONCLUSIONS: 111In antimyosin antibody appears to be a useful immunoradiotracer in detecting cardiac damage induced by DOX administration and in assessing the severity of cardiotoxicity. These data reinforce the clinical observation that myocardial imaging using 111In antimyosin Fab is able to provide information to guide the course of patients receiving DOX treatment.

Animals↗
PubMed1992

Recombinant-human superoxide dismutase attenuates endotoxin-induced lung injury in awake sheep.

Oxygen radicals have been implicated in the pathogenesis of acute lung injury associated with clinical and experimental sepsis. With the use of endotoxin infusion as an in vivo model of sepsis we studied the effect of recombinant-human superoxide dismutase (r-hSOD; 4,200 U/mg), an enzyme that catalyzes the dismutation of superoxide anion, on both the physiologic and biochemical lung changes in awake sheep. Sheep (n = 11) were prepared for chronic measurement of pulmonary hemodynamics and lung fluid balance. Paired experiments were performed in seven of the animals in which they received either endotoxin (1 microgram/kg) alone or in combination with r-hSOD in random order. An additional four sheep received r-hSOD without the lipopolysaccharide. Intravenous infusion of r-hSOD (a loading dose of 12,600 U/kg followed by a maintenance dose of 14,700 U/kg/h for 7 h) resulted in substantial SOD activity, measured by electron spin resonance spectrometry, both in plasma and in lung lymph, and attenuated the expected changes in pulmonary arterial pressure and lung lymph flow after administration of endotoxin. When administered without endotoxin, r-hSOD produced no perceptible change in pulmonary hemodynamics and lung fluid balance. These data suggest that superoxide anion plays an important role in endotoxin-induced lung injury in sheep.

Animals↗