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Biomedical subjects

M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 379 records · Page 21Linked to original sources
PubMed1991

Doppler echocardiographic assessments of left ventricular diastolic filling in patients with amyloid heart disease.

Twenty-eight patients with amyloid heart disease, 9 with primary and 19 with familial amyloidosis, were examined by Doppler echocardiography to characterize transmitral flow velocity patterns and to assess restrictive ventricular hemodynamics. Six of the 28 patients had restrictive ventricular physiology, and the remaining 22 did not. Patients with a restrictive filling process had marked ventricular wall thickening with depressed fractional shortening. The transmitral flow velocity patterns in patients with restriction were characteristically manifested by the increased peak flow velocity and shortening of rapid diastolic filling, which was associated with a reduced flow velocity in atrial systole. In the 22 patients without restriction, the left ventricular filling patterns were subclassified as 1) filling patterns with the prolonged isovolumic relaxation time and reduced early diastolic filling, as well as enhanced atrial contribution to ventricular filling (16 patients with mild echocardiographic abnormalities), 2) normal filling patterns with normal echocardiograms (4 patients), and 3) normal filling patterns, with moderate ventricular wall thickening (2 patients). These findings suggested that restrictive ventricular hemodynamics can be characterized by the transmitral velocity pattern and that patterns of left ventricular diastolic filling are markedly varied depending on the magnitude of myocardial amyloid deposition in patients with amyloid heart disease.

Adult↗
PubMed1991

Correlative light and electron microscopic observations on ectopic neurons in the cerebellum of dreher mutant mouse.

The structure of ectopic neurons in the cerebellum of dreher mutant mouse was investigated by correlative light and electron microscopic observations. Tissue blocks were fixed in buffered aldehyde and embedded in a mixture of 2-hydroxypropyl methacrylate, Quetol 523, and methyl methacrylate. Sections at 0.4-0.5 microns in thickness were examined by electron microscopy after observation under a light microscope. By comparing the electron images with those of light microscopy in the same sites, the structures of ectopic cells were confirmed. Ectopic Purkinje cells were arranged with cell bodies that contained an oval, spherical or wrinkled nucleus without deep invagination and the thin layers of endoplasmic reticulum at the perinuclear regions. Granule cells were ectopically matured in the external granular layer and within the cluster at the cortical region. This method provides a useful procedure for understanding structures of the cerebellar neurons of the mutant.

Animals↗
PubMed1991

Stimulatory effect of romurtide on hematopoiesis in monkeys.

Repeated subcutaneous injections of romurtide (N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine, MDP-Lys(L18), muroctasin; CAS 78113-36-7), a synthetic muramyl dipeptide derivative, increased significantly the number of peripheral neutrophils and monocytes in a dose-dependent fashion in healthy cynomolgus monkeys (Macaca fascicularis). The number of platelets was also increased significantly in monkeys with repeated dosing of romurtide. After single dosing of romurtide (1 mg/head), neutrophils counts showed a marked increase within 24 h and at 120 h after romurtide injection. Monocytes counts were decreased transiently until 8 h, but were increased persistently from 48 to 120 h after injection. Lymphocytes counts were stable throughout the experimental period except for a significant decrease until 24 h. In addition, romurtide stimulated blood neutrophils and monocytes in vivo for enhanced chemiluminescence (CL) responses to opsonized Escherichia coli. When peripheral monocytes from monkeys were incubated with various concentrations of romurtide in vitro, production of colony-stimulating factors (CSFs), interleukin-1 (IL-1) and interleukin-6 (IL-6) by the cells was enhanced significantly, indicating that the augmenting effects of romurtide on the production of various monokines including CSFs by monocytes are involved in the mechanisms of hematopoiesis and enhanced CL generation by phagocytic cells in vivo.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1990

Positive and negative regulation of transcription by a cleavage product of Ada protein.

The 39,000 Mr Ada protein of Escherichia coli that carries two distinct methyltransferase activities and activity to promote transcription of the ada and the alkA genes is cleaved by a cellular proteinase. As a result, the 20,000 and the 19,000 Mr proteins are formed, which are derived from the N-terminal and the C-terminal halves of the protein, respectively. To elucidate the molecular mechanism of transcriptional control by Ada protein, the N-terminal 20,000 Mr protein was overproduced by manipulating the cloned ada gene. The protein possessed an activity to transfer a methyl group from the methylphosphotriester of the alkylated DNA to its own molecule and retained the potential to promote transcription of the alkA gene. The methylated form of the 20,000 Mr proteins binds to the proper alkA regulatory sequence, as does the intact Ada protein, and facilitates further binding of RNA polymerase to the promoter, thus forming an active transcription initiation complex. The non-methylated 20,000 Mr protein was incapable of binding itself or supporting RNA polymerase binding to the alkA promoter. When the 20,000 Mr protein was produced under the control of the lac promoter in E. coli and then exposed to a methylating agent, a considerable amount of 3-methyladenine-DNA glycosylase II, the product of the alkA gene, was formed. Thus, the results obtained in in vitro experiments were confirmed by the events observed in vivo. The methylated 20,000 Mr protein also binds to the ada promoter; however, it does not facilitate further binding of RNA polymerase to the promoter nor does it promote ada transcription in vitro. These findings indicate that the N-terminal half of Ada protein is mainly responsible for recognition of and binding to alkA and the ada regulatory sequence. The methylated 20,000 Mr protein occupies the same region of the ada promoter to which the intact Ada protein would bind, thereby suggesting that it acts as a repressor for expression of the ada gene. The ada transcription promoted by the Ada protein was greatly inhibited by the methylated, but not the non-methylated, form of the 20,000 Mr protein. In an in vivo system, formation of the 20,000 Mr protein leads to inhibition of transcription from the ada promoter. We suggest that termination of the adaptive response may come about by proteolytic cleavage of the Ada protein, the result being a loss of the activator as well as formation of the repressor for ada transcription.

Amino Acid Sequence↗
PubMed1990

Purification, structure, and biochemical properties of human O6-methylguanine-DNA methyltransferase.

The level of O6-methylguanine-DNA methyltransferase activity in a human cell line carrying a 1.1-kilobase cDNA fragment was about 50 times higher than that found in ordinary methyltransferase-proficient (Mer+) cell lines (Hayakawa, H., Koike, G., and Sekiguchi, M. (1990) J. Mol. Biol. 213, 739-747). Taking advantage of this overproduction, the enzyme was purified to apparent physical homogeneity and the physical and biochemical properties investigated. A single polypeptide with a molecular weight of approximately 25,000 was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most highly purified preparation. The Stokes radius of 22.5 A and the sedimentation coefficient of 2.0 S were obtained, from which the molecular weight of the native form of the enzyme was calculated to be 19,000. After digestion with lysyl endopeptidase, peptide fragments of the protein were isolated and sequenced. The amino acid sequences of these peptides and the amino acid composition of the protein were in good agreement with those deduced from the nucleotide sequence of the cloned cDNA. The purified enzyme catalyzed transfer of methyl groups from O6-methylguanine and O4-methylthymine, but not from methylphosphotriesters, of methylated DNA to the enzyme molecule.

Amino Acid Sequence↗
PubMed1990

Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA.

A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with [3H]methylnitrosourea. Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction. When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity. From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid. The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found. The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700. The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.

Base Sequence↗
PubMed1990

Interaction of Ada protein with DNA examined by fluorescence anisotropy of the protein.

We made use of enhancement of fluorescence anisotropy of protein upon DNA binding to analyze interactions between Ada protein and DNA. Ada protein is a DNA repair enzyme that also acts as a transcription regulator. The isotropic fluorescence was not significantly affected upon interaction with DNA and could not be used as a signal for detection of the binding. The anisotropy did became larger because the binding to DNA reduces diffusion of the protein. The change was reproducible and independent of protein concentration and also independent of the degree of saturation of DNA with the protein when DNA was large; these values can readily be converted to the proportion of the complexed protein. The binding parameters were then determined by direct comparison between experimental and theoretical variations of anisotropy, with increasing concentrations of DNA. The theoretical variations were computed by considering the overlap of potential binding sites on the DNA lattice [McGhee & von Hippel (1974) J. Mol. Biol. 86, 469-489]. Binding does not seem to occur in a cooperative manner. The number of base pairs covered by a protein monomer was 7 +/- 1; this number is independent of the salt concentration. The equilibrium association constant decreased from 4 X 10(7) to 3 X 10(5) M-1 for an increase of NaCl concentration from 0.1 to 0.2 M, thereby indicating the possible involvement of ionic interactions between phosphate groups of DNA and the protein.

Base Sequence↗
PubMed1990

Molecular mechanisms of replicational fidelity in Escherichia coli.

DNA polymerase III holoenzyme is responsible for chromosomal DNA synthesis in Escherichia coli and seems to be a major determinant of the fidelity of replication of this organism. Among ten different subunits of the holoenzyme, the alpha subunit, encoded by the dnaE gene, has a polymerase activity, while the epsilon subunit, encoded by the dnaQ gene, is a proofreader with a 3'-5' exonuclease activity. Using poly(dA)/oligo(dT)20 as a template-primer, misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and exonucleolytic editing of those mispairs by the epsilon subunit were investigated. When the polymerization reaction was performed with the alpha subunit, dCMP and dGMP but not dAMP were misincorporated. This would suggest that the polymerase might have a base-selecting function to avoid dA:dA mispairing. A subassembly of the DNA polymerase III consisting of alpha, epsilon, and theta subunits misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit may correct the dC:dA but not the dG:dA mispair. Addition of a protein encoded by the mutT gene, defects of which cause AT to CG transversions in vivo, diminished the misincorporation of dGMP onto poly(dA) template by the alpha subunit. A dGTPase activity was associated with the MutT protein. The significance of the dGTPase activity in the prevention of dG:dA mispairing is discussed.

Bacterial Proteins↗
PubMed1990

Estimation of pulmonary hypertension by perfusion lung scintigraphy: gravitational effect of postural changes between the lateral decubitus positions.

To estimate pulmonary hypertension in patients with various heart diseases, we devised a new method using perfusion lung scintigraphy with technetium-99m-labelled macroaggregated albumin. In this method, changes in the distribution of pulmonary perfusion caused by gravitational effects, namely, changes in the total count ratios of the right lung against the left lung between right and left lateral decubitus positions (rt/lt), were assessed in 62 patients and in 10 normal subjects. The rt/lt ratios were calculated as indices of the above changes. They correlated significantly with mean pulmonary arterial pressure (mPAP) (gamma = -0.62, P less than 0.001), pulmonary capillary wedge pressure (gamma = -0.63, P less than 0.001) and pulmonary arteriolar resistance (gamma = 0.50, P less than 0.001) in all subjects. In 17 patients with valvular heart diseases, the ratio correlated significantly with mPAP (gamma = -0.84, P less than 0.001). In 10 patients with various heart diseases, the U/S ratio, i.e. the index of changes in the count ratios of the upper field against the lower field for the right lung following postural change from the upright to the supine position, was also obtained as well as the rt/lt ratio. The latter evidenced a better correlation with mPAP (gamma = -0.90, P less than 0.001) than the former (gamma = -0.64, P less than 0.05). We conclude that this method is valuable as a noninvasive approach for the estimation of pulmonary hypertension.

Female↗
PubMed1990

Effect of cyclic guanosine monophosphate on hypoxic and angiotensin-II-induced pulmonary vasoconstriction.

We examined, in isolated blood perfused rat lungs, the effect of the cell permeable 8-bromo derivative of cGMP on pulmonary vasoconstriction induced by either alveolar hypoxia or angiotensin II. 8-Bromo cGMP dose-dependently reduced both hypoxia-(IC50 = 2.2 X 10(-5) M) and angiotensin-II-induced pulmonary vasoconstriction (IC50 = 5.0 X 10(-5) M). This effect of 8-bromo cGMP on pulmonary vasoconstriction was not affected by cyclooxygenase blockade. M & B 22948 (0.1 mM), an inhibitor of cGMP-phosphodiesterase, reduced synergistically with 8-bromo cGMP the hypoxia or angiotensin-II-induced vasoconstriction. The cGMP-phosphodiesterase inhibitor M & B 22948, by itself, selectively reduced hypoxia-induced vasoconstriction, suggesting a modulating effect of endogenous cGMP during hypoxic vasoconstriction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1990

Effects of vanillin and o-vanillin on induction of DNA-repair networks: modulation of mutagenesis in Escherichia coli.

Vanillin and its isomer o-vanillin have an effect on the adaptive and SOS responses, as well as mutagenesis, induced in Escherichia coli by N-methyl-N-nitrosourea (MNU) and UV irradiation, potentiating in some cases and suppressing in others. o-Vanillin markedly inhibited the MNU-induced adaptive response, while both vanillins potentiated the UV-induced SOS response. These phenomena appear to be responsible for the comutagenic or antimutagenic role of these chemicals in MNU and UV mutagenesis.

Benzaldehydes↗
PubMed1990

GABAB receptors expressed in Xenopus oocytes by guinea pig cerebral mRNA are functionally coupled with Ca2(+)-dependent Cl- channels and with K+ channels, through GTP-binding proteins.

Transmembrane currents induced by (-)-baclofen (BAC), a specific agonist of the gamma-aminobutyric acid-B (GABAB) receptor, in Xenopus oocytes injected with guinea pig cerebral mRNA were electrophysiologically and pharmacologically characterized under a voltage-clamp condition. The oocytes injected with mRNA acquired responsiveness to BAC and showed two types of currents at a holding potential of -50 mV. One was the slow and smooth inward current which had a short latency and associated with a decrease in membrane conductance, and its amplitude was decreased by hyperpolarization and increased by depolarization. The other was the large fast oscillatory inward current with a long-latency, which was decreased in amplitude by depolarization and reversed at -26 mV. Both currents were not blocked by bicuculline but were depressed by 2-hydroxysaclofen (2-OH-SAC), though the smooth current was less sensitive to 2-OH-SAC; about 40% blockade at the 2-OH-SAC concentration capable of abolishing the oscillatory current. The smooth current was depressed by Ba2+. The oscillatory current was time-dependently attenuated and almost abolished by intracellularly injected pertussis toxin (PTX), while the smooth current was not depressed by this toxin even when the oscillatory current was nearly abolished. The intracellular injection of GTP-gamma-S into oocytes attenuated both oscillatory and smooth currents. These results suggest the possibility that GABAB receptors expressed in Xenopus oocytes by cerebral mRNA are functionally coupled with two signal transduction systems, one is the opening of Ca2(+)-dependent Cl- channels mediated by PTX-sensitive GTP-binding protein(s) and the other is the closure of K+ channels through PTX-insensitive GTP-binding protein(s).

Animals↗
PubMed1990

Dopamine receptors expressed in the Xenopus oocytes injected with bovine striatal mRNA.

The response to dopamine (DA) of Xenopus oocytes injected with bovine striatal mRNA was electrophysiologically and pharmacologically investigated under a voltage-clamped condition. In oocytes injected with bovine striatal mRNA and then treated with collagenase (denuded oocytes), DA applied by superfusion dose-dependently induced oscillatory inward currents with a long latency and an ED50 value of 12.0 microM. mRNA non-injected denuded oocytes did not respond to DA up to concentrations of 1 mM. The reversal potential of DA-induced currents was about -25 mV, indicating that DA increased a Cl(-)-conductance. DA-induced currents were easily desensitized by repeated applications of DA. Haloperidol and SCH-23390 abolished DA-induced currents, while propranolol and mianserin showed no effect. Furthermore, SK&F-38393, a specific agonist of the DA D1 receptor, induced similar oscillatory currents as DA did. These results suggest the possibility that functional D1 and D2 DA receptors were co-expressed in Xenopus oocytes by injection of bovine striatal mRNA.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1990

Phototoxic potential of quinolone antibacterial agents in Balb/c mice.

The phototoxic potentials of quinolone antibacterial agents were investigated in Balb/c strain mice. The mice were orally administered nalidixic acid (NA), enoxacin (ENX), ofloxacin (OFLX), ciprofloxacin (CPFX), lomefloxacin (LMFX) and DR-3355 (s-isomer of OFLX), and immediately exposed to ultraviolet-A (UVA) for 4 h (21.6 joules/cm2). The ears were examined for overt damage, as a major phototoxic parameter, 0, 24 and 48 h after irradiation ended. At doses of 200 mg/kg, LMFX, NA and ENX caused marked cutaneous phototoxic reactions on the ears, whereas CPFX, OFLX and DR-3355 caused none. At 800 mg/kg, however, CPFX, OFLX and DR-3355 also caused phototoxic reactions on the ears. These phototoxic changes were characterized grossly by erythema, and histopathologically by edema and infiltration of inflammatory cells, especially neutrophils, into the connective tissue surrounding the cartilage. The 50% erythema-inducing doses of LMFX, ENX, NA, OFLX, DR-3355 and CPFX were calculated at 19, 102, 143, 553, 619 and 741 mg/kg, respectively. Thus, the phototoxic potencies of the quinolones tested were: LMFX greater than ENX, NA greater than OFLX, DR-3355, CPFX.

4-Quinolones↗
PubMed1990

Increased levels of beta-human atrial natriuretic peptide-like immunoreactivity in chronically overloaded atrial tissue.

The levels and molecular form of atrial natriuretic peptide-like immunoreactivity (ANP-LI) in human atrial tissue were investigated. The levels of right atrial ANP-LI were significantly higher in mitral disease than in other cardiac or noncardiac diseases. The increased ANP-LI was mainly accounted for by an increase in beta-human ANP-LI (beta-hANP-LI). Both total ANP-LI and beta-hANP-LI levels were associated with the presence of atrial fibrillation and with increased atrial pressure. Plasma beta-hANP-LI levels were also increased in mitral disease. These results suggest that human atrium with hemodynamic overloads is characterized by increased tissue levels of ANP and by a shift to the beta-hANP form.

Adult↗
PubMed1990

Analysis of immunologic mechanisms of high natural killer cell activity in tuberculous pleural effusions.

We found natural killer (NK) activity to be high in tuberculous pleural effusions from which Mycobacterium tuberculosis was cultured. In this study, we investigated and compared the mechanisms of this NK activity in tuberculous pleural effusions and peripheral blood. Cytotoxicity was augmented in tuberculous pleural effusion mononuclear cells (PEMNC) and peripheral blood mononuclear cells (PBMNC) by culture with purified protein derivative (PPD). Prior to culture with PPD, there were many more interleukin-2 receptors (IL-2R, p55) on Leu 11+ (CD16+) cells in tuberculous PEMNC than in tuberculous PBMNC. After 5 days of culture with PPD, the numbers of IL-2R increased in both PEMNC and PBMNC. PPD-induced cytotoxicity was inhibited by the addition of anti-IL-2R monoclonal antibody (anti-IL-2R mAb). Interferon-gamma (IFN-gamma) production was abundant in the supernatants of cultured tuberculous PEMNC and PBMNC, but very little interleukin-2 (IL-2) and IFN-alpha and -beta were produced. Considerably more IFN-gamma was produced in tuberculous PEMNC than in tuberculous PBMNC, and PPD-induced cytotoxicity was inhibited by anti-IFN-gamma monoclonal antibody (anti-IFN-gamma mAb). Cell surface analysis after PPD-induced cytotoxicity was performed by complement-mediated cytolysis. Leu 11+ cells exhibited essential cytotoxicity in the induction and effector phases, but neither OKT4+ (CD4+) nor OKT8+ (CD8+) cells displayed such cytotoxicity. These data suggest that Leu 11+ cells in tuberculous PEMNC are activated by M. tuberculosis through IFN-gamma and that IL-2R plays an important role in the activation of NK cells.

Adult↗