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Biomedical subjects

K Yorita

Publications and source records attributed to K Yorita.

12 recordsLinked to original sources

[Off-pump coronary artery bypass for old myocardial infarction associated with jejunotomy for obstructive ileus due to gall bladder stone; report of a case].

In non-cardiac operative cases with inflammatory digestive organ disease, bacterial translocation (BT) often results from non-enteral nutrition postoperatively. If coronary artery bypass grafting (CABG) is performed in the case having old myocardial infarction (OMI) and inflammatory digestive organ disease at first before non-cardiac operation, he seems vulnerable to have severe complications such as multiple organ failure due to systemic inflammatory response syndrome (SIRS) and preexisting BT postoperatively. We performed a off-pump CABG (OPCAB) for OMI associated with jejunotomy for obstructive ileus due to gall bladder stone. No complication was found in the postoperative course. We conclude that combined operation, non-cardiac surgery after OPCAB is worth considering in those cases. And we think OPCAB is better than conventional CABG in such cases, because cardiopulmonary bypass is known to ponder comparable damages to immune system, coagulation system and others.

Aged↗

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: conversion of FMN to 8-formyl-FMN.

Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of alpha-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH(3)- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.

Amino Acid Sequence↗

Abnormal immune function of hemopoietic cells from alymphoplasia (aly) mice, a natural strain with mutant NF-kappa B-inducing kinase.

Alymphoplasia (aly) mice, a natural strain with a mutant NF-kappa B-inducing kinase (NIK) gene, manifest a unique phenotype; they lack lymph nodes and Peyer's patches, have a disturbed spleen architecture, and exhibit defects in both Ab and cellular immune responses. Although a stromal defect caused by impaired lymphotoxin-beta receptor signaling accounts for their abnormal lymphoid organogenesis, the exact mechanisms underlying the development of immunodeficiency in aly mice are poorly understood. We therefore investigated the contribution of hemopoietic cells with the aly NIK mutation to the development of immunodeficiency. Transfer of aly/aly bone marrow cells into aly/+ mice resulted in poorly developed B cell follicles and lack of support for the development of germinal centers and isotype switching, indicating that the hemopoietic cells of aly mice contain an autonomous defect. However, follicular dendritic cell clusters were maintained in the spleens of these bone marrow chimeras, suggesting that the lack of follicular dendritic cell clusters in aly mice is probably due to the stromal defect. The aly mice lacked marginal zone B cells in their spleens, and aly/aly B cells showed an impaired proliferative response after in vitro stimulation. IL-2 production by activated T cells was also impaired. By contrast, the dendritic cells of aly mice exhibited grossly normal development and function. Supporting the concept of an autonomous cell defect, Rel protein expression was altered in aly/aly spleens. Thus, the aly NIK mutation affects hemopoietic cell function in an intrinsic fashion and, together with the stromal defect, may contribute to the development of immunodeficiency in aly mice.

Animals↗

On the interpretation of quantitative structure-function activity relationship data for lactate oxidase.

The native flavin, FMN, has been removed from the l-lactate oxidase of Aerococcus viridans, and the apoprotein reconstituted with 12 FMN derivatives with various substituents at the flavin 6- and 8-positions. Impressive linear relationships are exhibited between the sum of the Hammett final sigma(para) and final sigma(ortho) parameters and the redox potentials of the free flavins, and between the redox potentials of the free and enzyme-bound flavins. Rapid reaction kinetics studies of the reconstituted enzymes with the substrates l-lactate and l-mandelate show an increase in the reduction rate constant with increasing redox potential, except that, with lactate, a limiting rate constant of approximately 700 s(-1) is obtained with flavins of high potential. Similar breakpoints are found in plots of the rate constants for flavin N5-sulfite adduct formation and for the reaction of the reduced enzymes with molecular oxygen. These results are interpreted in terms of a two-step equilibrium preceding the chemical reaction step, in which the second equilibrium step provides an upper limit to the rate with which the particular substrate or ligand is positioned with the flavin in the correct fashion for the observed chemical reaction to occur. The relationship of rate constants for flavin reduction and N5-sulfite adduct formation with flavin redox potential below the observed breakpoint indicate development of significant negative charge in the transition states of the reactions. In the case of reduction by substrate, the results are consistent either with a hydride transfer mechanism or with the so called "carbanion" mechanism, in which the substrate alpha-proton is abstracted by an enzyme base protected from exchange with solvent. These conclusions are supported by substrate alpha-deuterium isotope effects and by solvent viscosity effects on sulfite binding.

Apoenzymes↗

L-lactate oxidase from Aerococcus viridans crystallized as an octamer. Preliminary X-ray studies.

Crystals of flavoenzyme L-lactate oxidase from Aerococcus viridans (LOX) have been obtained that diffract to 3.0 A resolution (P2(1)2(1)2(1), a = 118.4 A, b = 138.4 A, c = 194.6 A). Crystallographic studies suggest that the enzyme may exist as an octameric form with non-crystallographic two- and four-fold axes in the center of the octamer. The four-fold axis makes the tetramer tight, and the tetramers lie upon one another by the two-fold axis.

Chromatography, Gel↗

On the reaction mechanism of L-lactate oxidase: quantitative structure-activity analysis of the reaction with para-substituted L-mandelates.

The rate constants for reduction of the flavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3-7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of alpha-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the alpha-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

Binding Sites↗

Conversion of L-lactate oxidase to a long chain alpha-hydroxyacid oxidase by site-directed mutagenesis of alanine 95 to glycine.

A mutant form of L-lactate oxidase (LOX) from Aerococcus viridans in which alanine 95 was replaced by glycine was constructed as a mimic of L-lactate monooxygenase but proved instead to be a mimic of the long chain alpha-hydroxyacid oxidase from rat kidney. A95G-LOX keeps oxidase activity with L-lactate at the same level as wild type LOX but has much enhanced oxidase activity with longer chain L-alpha-hydroxyacids, alpha-hydroxy-n-butyric acid, alpha-hydroxy-n-valeric acid, etc., and also the aromatic alpha-hydroxyacid, L-mandelic acid. Kinetic analysis of the activity with these substrates indicates that the reduction of the enzyme bound flavin by substrates is the rate-limiting step in A95G-LOX. The affinity of pyruvate for the reduced enzyme is increased, and sulfite binding to the oxidized enzyme is weaker in A95G-LOX than in native enzyme. Wild type LOX stabilizes both the neutral and anionic flavin semiquinones with a pKa of 6.1, but A95G LOX stabilizes only the anionic semiquinone form. These results strongly suggest that the environment around the N5-C4a region of the flavin isoalloxazine ring is changed by this mutation.

Alanine↗

Gluconeogenesis in perfused chicken kidney. Effects of feeding and starvation.

1. Starvation for 48 hr doubled the rate of gluconeogenesis from lactate and pyruvate in perfused chicken kidney, but did not change the rate of production of glucose from malate, succinate, or alpha-ketoglutarate. 2. Amino-oxyacetate and D-malate inhibited the production of glucose from lactate and from pyruvate by 55% in each case. Quinolinate reduced the production of glucose from lactate and from pyruvate by 50% in both fed and starved chickens, but had no effect on the production of glucose from intermediates in the citric acid cycle. 3. Starvation increased the rate of formation of mitochondrial phosphoenolpyruvate from pyruvate, but had no effect on the rate of formation of mitochondrial phosphoenolpyruvate from malate.

Animals↗

Distribution of glycolysis and gluconeogenesis in perfused chicken kidney.

Regional metabolism of glucose in chicken kidneys was studied in kidneys perfused with either an arterial system or a portal system. Provided that kidneys were perfused with oxygenated buffer solution at a flow rate of 5 ml/min per gram of kidney in both perfusion systems, oxygenation of the kidney was achieved, as judged by the rates of O2 uptake and the formation of lactate, gluconeogenesis, and the ratio of lactate to pyruvate. The rate of formation of lactate and pyruvate during glucose metabolism, in the presence or absence of KCN, was markedly higher with the arterial system than with the portal system. The rate of gluconeogenesis was equal with both perfusion systems but the rate was modulated by the type of substrate used. With succinate as substrate, ouabain inhibited glucose production and O2 uptake in both perfusion systems. With lactate and pyruvate as substrate, ouabain had no effect on glucose production in both perfusion systems, whereas the inhibition of O2 uptake by ouabain was greater with the arterial system than with the portal system. From the viewpoint of the accepted morphological components of the blood supply, these results suggest that chicken renal gluconeogenesis occurs in the cortex and that glycolysis occurs in medullary structures.

Aerobiosis↗