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Biomedical subjects

M Tsuboi

Publications and source records attributed to M Tsuboi.

At least 163 records · Page 9Linked to original sources
PubMed1986

Coordination structures and reactivities of compound II in iron and manganese horseradish peroxidases. A resonance Raman study.

Resonance Raman investigations on compound II of native, diacetyldeuteroheme-, and manganese-substituted horseradish peroxidase (isozyme C) revealed that the metal-oxygen linkage in the compound differed from one another in its bond strength and/or structure. Fe(IV) = O stretching frequency for compound II of native enzyme was pH sensitive, giving the Raman line at 772 and 789 cm-1 at pH 7 and 10, respectively. The results confirmed the presence of a hydrogen bond between the oxo-ligand and a nearby amino acid residue (Sitter, A. J., Reczek, C. M., and Terner, J. (1985) J. Biol. Chem. 260, 7515-7522). The Fe(IV) = O stretch for compound II of diacetylheme-enzyme was located at 781 cm-1 at pH 7 which was 9 cm-1 higher than that of native enzyme compound II. At pH 10, however, the Fe(IV) = O stretch was found at 790 cm-1, essentially the same frequency as that of native enzyme compound II. The pK value for the pH transition, 8.5, was also the same as that of native compound II. Unlike in native enzyme, D2O-H2O exchange did not cause a shift of the Fe(IV) = O frequency of diacetylheme-enzyme. Thus, the metal-oxygen bond at pH 7 was stronger in diacetylheme-enzyme due to a weaker hydrogen bonding to the oxo-ligand, while the Fe(IV) = O bond strength became essentially the same between both enzymes at alkaline pH upon disruption of the hydrogen bond. A much lower reactivity of the diacetylheme-enzyme compound II was accounted to be due to the weaker hydrogen bond. Compound II of manganese-substituted enzyme exhibited Mn(IV)-oxygen stretch about 630 cm-1, which was pH insensitive but down-shifted by 18 cm-1 upon the D2O-H2O exchange. The finding indicates that its structure is in Mn(IV)-OH, where the proton is exchangeable with a water proton. These results establish that the structure of native enzyme compound II is Fe(IV) = O but not Fe(IV)-OH.

Horseradish Peroxidase↗
PubMed1986

Interleukin 2 increases T lymphocyte membrane mobility before the rise in cytosolic calcium concentration.

Using stopped-flow fluorometry with three different fluorescence probes [2-[(1-pyrenyl-butyryl)oxy]stearic acid, chlortetracycline and Quin 2], we have studied initial stage of T lymphocyte activation after interleukin 2 (IL-2) binding to a specific cell-surface receptor. After IL-2 binding to cytotoxic T lymphocyte (IL-2-dependent mouse LC7 and CTLL-2 cells), membrane mobilities of the cells increased first (4.5 +/- 0.3 s-1 for LC7 and 3.8 +/- 0.2 s-1 for CTLL-2), then calcium was released from intracellular stores into the cytoplasm (1.6 +/- 0.1 s-1 for LC7 and 2.1 +/- 0.1 s-1 for CTLL-2), and lastly, calcium was transported from the external medium into the cytoplasm (1.3 +/- 0.1 s-1 for LC7 and 1.5 +/- 0.1 s-1 for CTLL-2). The slowest process, the calcium influx from the external medium, was suppressed in the presence of a calcium channel blocking agent (verapamil). These observations give us a new information to discuss a model in T lymphocyte activation after IL-2 binding to cell-surface receptors.

Aminoquinolines↗
PubMed1986

The structure of nucleosome core particles as revealed by difference Raman spectroscopy.

Raman spectra have been observed of nucleosome core particles (I) prepared from chicken erythrocyte chromatin, its isolated 146 bp DNA (II), and its isolated histone octamer (H2A+H2B+H3+H4)2 (III). By examining the difference Raman spectra, (I)-(II), (I)-(III), and (I)-(II)-(III), several pieces of information have been obtained on the conformation of the DNA moiety, the conformation of the histone moiety, and the DNA-histone interaction in the nucleosome core particles. In the nucleosome core particles, about 15 bp (A.T rich) portions of the whole 146 bp DNA are considered to take an A-form conformation. These are considered to correspond to its bent portions which appear at intervals of 10 bp.

Animals↗
PubMed1986

Salt induced B----A transition of poly(dG).poly(dC) and the stabilization of A form by its methylation.

Raman spectra of poly(dG).poly(dC) have been observed in aqueous solutions at various ionic strengths, [NaCl] = 0.03 to 4 M, and at different temperatures, 10 to 60 degrees C. At 30 degrees C, and at [NaCl] = 0.03 M, it was found to have a B-form (with O4'endo-anti guanosine and C2'endo-anti cytidine), whereas, at [NaCl] = 4 M, an A form (with C3'endo-anti guanosine and C3'endo-anti cytidine). At 30 degrees C and [NaCl] = 1 M, namely at an intermediate state, a fraction of this molecules was considered to have a "heteronomous A" form (with O4'endo-anti guanosine and C3' endo-anti cytidine). At 60 degrees C and [NaCl] = 1 M, it assumes the B form, and at 10 degrees C and [NaCl] = 1 M, the A form. Cytosine-5-methylation was found to cause a marked stabilization of the A form. Even at [NaCl] = 0.1 M (at 30 degrees C), a substantial portion of poly(dG).poly(dm5C) was found to have a heteronomous form, in which the dG atrand is in the B form and the dC an A form; it never assumes a complete B form.

Methylation↗
PubMed1986

Early transmembrane events in alloimmune cytotoxic T-lymphocyte activation as revealed by stopped-flow fluorometry.

We have studied early transmembrane events in mouse alloimmune cytotoxic T-lymphocyte (LC7, H-2b) activation by specific target cells (mouse mastocytoma P815, H-2d) and a mitogenic lectin, Con A, by using stopped-flow fluorometry with three different fluorescent probes. After binding to target cells (P815), cytotoxic T lymphocytes (LC7) first increased their membrane fluidity and, then, calcium was released from intracellular stores. After that, there was a calcium influx from the external medium into the T lymphocytes. This calcium influx was blocked by calcium antagonists (verapamil or diltiazem). The same sequence of events was also observed in the activation of T lymphocytes (LC7) by Con A and in the response of specific target cells (P815) after cytotoxic T lymphocytes (LC7) binding. Nonspecific (syngeneic) target cells (mouse lymphoma EL-4, H-2b) did not cause any early transmembrane events in cytotoxic T lymphocytes (LC7, H-2b).

Animals↗
PubMed1986

Sex difference in the effect of aspirin on intracellular Ca2+ mobilization and thromboxane A2 production in rat platelets.

The intracellular Ca2+ mobilization in thrombin-stimulated platelets was greater in male rats than in female rats. Thromboxane (TX) B2 production in male platelets was greater than that in female platelets. Aspirin suppressed Ca2+ mobilization in rat platelets, but the inhibitory effect of aspirin was more efficient in males than that in females. Aspirin inhibited TXB2 production, and this inhibitory effect of aspirin was stronger in male platelets than in female platelets. Castration decreased Ca2+ mobilization and TXB2 production and weakened the effect of aspirin on them. It is suggested that the sex difference in the antiplatelet effect of aspirin results from the difference in the inhibition of Ca2+ mobilization via the inhibition of TXA2 production in thrombin-stimulated rat platelets.

Animals↗
PubMed1986

Augmentation of catecholamine-stimulated [3H]GDP release in adipocyte membranes from exercise-trained rats.

The effects of exercise training on the catecholamine-stimulated [3H]GDP release in rat adipocyte membranes prelabeled with [3H]GTP and the adenylate cyclase activity were investigated. Exercise training significantly increased the release of [3H]GDP in response to (-)isoproterenol. The adenylate cyclase activity induced by a nonhydrolyzable guanine nucleotide analogue, Gpp(NH)p, was significantly greater in exercise-trained rats.

Adenylyl Cyclases↗
PubMed1986

31P-NMR studies of energy metabolites in rat pancreas treated with trypsin inhibitor.

The energy metabolites and the composition of rat pancreas treated with trypsin inhibitor (T.I.) were investigated. T.I. treatment significantly induced the hypertrophy of pancreatic acinar cells and the increase in the amylase and sialic acid contents. However, when the pancreas was perfused by control solution or the solution containing acetylcholine (10(-7) M), the 31P-NMR spectra of energy metabolites in the pancreas treated with T.I. were similar to those of normal rats.

Acetylcholine↗
PubMed1986

Comparison of the inhibitory effects of aspirin and ticlopidine on platelet aggregation and Ca2+ mobilization in rat platelets.

To study the cause of the sex difference in the inhibitory effects of aspirin on platelet functions, we compared the effects of aspirin and ticlopidine in rat platelets. Aspirin showed the sex difference in the effects on the collagen-induced platelet aggregation and the malondialdehyde (MDA) production, but ticlopidine showed no sex difference in the effects on those. The C-AMP level in male rat platelets was the same as that in female rat platelets. Ticlopidine increased the C-AMP level in rat platelets, but aspirin did not. Aspirin suppressed the intracellular Ca2+ mobilization in male rat platelets more strongly than in female rat platelets. Ticlopidine suppressed the Ca2+ mobilization in both sexes equivalently. The above results suggest that the sex difference in the inhibitory effects of aspirin on rat platelet functions comes from the sex difference in the thromboxane (TX) production in rat platelets.

Animals↗
PubMed1986

A simple one-step hemolytic assay of the first component of human complement (C1) with polyethylene glycol treated human serum: details of the assay procedures and its application to the C1 activation study.

C1 was removed from human serum by polyethylene glycol (PEG) precipitation and the supernatant (C1 deficient serum, C1D) was used for assay of C1 hemolytic activity (C1D method). Serum concentration of PEG 6,000 ranging from 3.5-4% was determined to be suitable for preparation of C1D, but C1D prepared by PEG 4,000 were proved unsatisfactory for use. Oxidation of C1D by iodine treatment increased C1 activity by two-fold. The C1D method was comparable in sensitivity to the conventional method, and the correlation between the 2 methods was good (r = 0.94). The C1D method was shown to be a useful tool for the study of C1 activation, since the method specifically measured C1, but not C1 activity. A half-life (T1/2) of C1 activity in fluid phase at 37 degrees C was 20 min under physiologic conditions.

Complement Activation↗
PubMed1986

A Raman spectroscopic study on the sequence dependent conformations of DNA oligomers.

Eighteen kinds of oligodeoxyribonucleic acids have been examined to reveal their structures in aqueous solutions at different ionic strengths by Raman spectroscopy. The structures in solutions were found to be very polymorphic depending on their sequences as well as on the salt concentrations. At a low salt condition a DNA oligomer assumes a unique B form within a B family, for examples Ba, Bh, B', or Bn form. Amongst these DNA oligomers, d(CGCG)2 showed a salt induced Ba-Z transition, while d(GGGGCCCC)2 showed a salt induced Bh-A transition. DNA oligomers with AA/TT sequences were found to prefer B' form even at high salt condition. From comparing the structures of DNA oligomers in solutions with their crystal structures, it is safe to say that the crystal structure of a DNA oligomer is very similar to the structure in the high salt solution.

Base Sequence↗
PubMed1986

The activation of C5 in the fluid phase and in the absence of C3 through the classical pathway of the complement system.

Unsensitized guinea-pig erythrocytes (Egp) were lysed by a combination of eight isolated, human-derived complement components, Cls, C4, C2, C5, C6, C7, C8 and C9 (Cls-C9exC3), even in the presence of anti-C3. It was determined that a factor was generated in the reaction mixture of Cls, C4, C2, C5 and C6, which had a lytic activity against Egp when C7, C8 and C9 were added. The lytic factor was similar to C56 in the following properties: the activity of the lytic factor decreased when incubated with C7 prior to its reaction with Egp, the lytic factor did not bind to Egp by itself but it did bind in the presence of C7, EDTA did not have any inhibitory effect on the lytic factor, and the activity of the lytic factor was lost by treatment with anti-C5 or anti-C6 but not by treatment with anti-C4. Furthermore, C5a, a cleavage product of C5, was clearly detected in the reaction mixture of Cls, C4, C2 and C5. These findings indicate that C5 can be activated proteolytically into C5a and C5b in the fluid phase solely by the classical pathway C3 convertase, C42, without any participation of C3.

Animals↗
PubMed1986

C3-independent immune haemolysis: mechanism of membrane attack complex formation.

The isolated active complex of C5 and C6, C56, was found to bind to EAC142 in the absence of C3 or C7, and to form a unique intermediate, EAC14256, which is susceptible to lysis by the addition of C7, C8 and C9. Further studies revealed that C56 alone could bind to EAC142 but not to E, EA, EAC1 or EAC4, nor to EAC14 in the absence of C7, that the C56 binding to EAC142 was highly dependent on temperature and on the ionic strength of the buffer, and that the degree of EAC14256 formation from EAC142 and C56 depended on the amount of C2 on EAC142 and on the amount of added C56. These findings suggest that C2 or C42 on EAC142 may be an acceptor for C56. In addition, C56 appears to bind to EAC142 much more efficiently than to unsensitized erythrocytes, even in the presence of C7. Thus, binding of C56 to EAC142 is likely to be an initial step of membrane attack complex formation in C3-independent immune haemolysis.

Animals↗
PubMed1985

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy.

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.

DNA↗
PubMed1985

Early transmembrane events in tumour cell responses observed by stopped-flow fluorometry.

Early transmembrane events of tumour cells (mouse myeloma X5563 and lymphoma RDM4) after binding of a monoclonal antibody against mouse MHC antigen and a mitogenic lectin, Con A, were examined by stopped-flow fluorometry with 3 different fluorescent probes. The results showed that membrane fluidities of the cells increased first after binding of anti H-2Kk monoclonal antibody (11-4.1), then calcium was released from intracellular stores into the cytoplasma, and lastly calcium influx occurred from the external medium into the cytoplasma. While Con A only induced calcium influx from the external medium into the cytoplasma.

Animals↗
PubMed1985

Resonance Raman study of cytochrome b562-o complex, a terminal oxidase of Escherichia coli in its ferric, ferrous, and CO-ligated states.

Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli, has been studied by resonance Raman spectroscopy in its air-oxidized, dithionite-reduced, and reduced and CO-ligated states. In the reduced state, with a 406.7-nm excitation, there appeared 1494 and 1473 cm-1 lines, indicating that low spin and high spin components are included in the cytochrome b562-o complex. For the air-oxidized protein, resonance Raman lines were observed at 1372, 1503, and 1580 cm-1 with a 413.1-nm excitation, indicating that there is a ferric low spin heme. In addition, a weak but appreciable Raman line was observed at 1480 cm-1 assignable to a ferric high spin heme. Accordingly, it was concluded that low spin and high spin components are included in the cytochrome b562-o complex in the reduced and the air-oxidized states. In the CO-ligated state, with a defocused laser beam of 413.1 nm, two Raman bands assignable to the Fe-CO stretching mode have been observed at 489 and 523 cm-1, as a major and a minor component, respectively. When the laser beam was focused upon the sample to cause a photodissociation of CO from the heme moiety, the intensity of the major band at 489 cm-1 was reduced as expected. On the other hand, the minor band at 523 cm-1 remained still obvious. It was suggested that the cytochrome b562-o complex may have an additional anomalous site for CO that is resistant to photodissociation.

Carbon Monoxide↗