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Biomedical subjects

M Kozaki

Publications and source records attributed to M Kozaki.

At least 19 recordsLinked to original sources
PubMed1994

Purification and analysis of a flavoprotein functional as NADH oxidase from Amphibacillus xylanus overexpressed in Escherichia coli.

The gene encoding the Amphibacillus xylanus flavoprotein has been cloned into pTTQ18 and overexpressed in Escherichia coli. The recombinant enzyme has been purified to homogeneity yielding 15 mg of pure enzyme/liter of cell culture. Recombinant flavoprotein is fully active and has an absorption spectrum identical to that of the enzyme purified from A. xylanus. The N-terminal sequence analysis and analytical gel filtration data confirm the structural identity of recombinant and A. xylanus enzymes. The Km value for oxygen and the Km value for NADH are 1.7 mM and 33.3 microM, respectively. In the presence of free additional FAD, however, the Km value for oxygen decrease dramatically. The NADH oxidase activity is accelerated markedly in the presence of additional FAD. The intracellular free FAD concentration of A. xylanus is calculated about 13 microM. This FAD concentration would be enough to accelerate the NADH oxidase activity of flavoprotein in cells of A. xylanus. Two-electron reduction of the enzyme FAD by the strong reductant dithionite occurs during the total uptake of 6 electrons. Such behavior usually indicates the presence of non-flavin redox centers. The high degree of homology between this enzyme and alkyl hydroperoxide reductase F52a protein and thioredoxin reductase suggests that these centers are the redox-active disulfide adjacent to the FAD and another disulfide, which is able to slowly interchange with the redox-active disulfide. The presence of two disulfides has been demonstrated.

Amino Acid Sequence↗
PubMed1994

Clinical significance of glucocorticoid pharmacodynamics assessed by antilymphocyte action in kidney transplantation. Marked difference between prednisolone and methylprednisolone.

A number of studies have demonstrated the impact of glucocorticoid response of peripheral lymphocytes on kidney allograft survival, suggesting that the better the glucocorticoid selection, the better the clinical outcome. However, individual differences in pharmacodynamics of clinically important glucocorticoids have not been taken into account. Four glucocorticoids (hydrocortisone, prednisolone, methylprednisolone, and dexamethasone) were examined for their ability to suppress in vitro blastogenesis of mitogen-stimulated PBL obtained from 122 chronic renal failure (CRF) patients waiting for renal transplantation and 98 healthy volunteers. Concentrations of steroids that gave 50% inhibition of lymphocyte blastogenesis (IC50) were determined individually in order to compare steroids and subject groups. Graft outcomes in 36 kidney transplant recipients treated with prednisolone were compared retrospectively with the prednisolone pretransplant IC50 values. Lymphocyte response to each glucocorticoid showed wide deviations among the subjects. Prednisolone IC50 values of the CRF patients showed the largest deviation, ranging from 1.0 to 10,000 micrograms/L. Thus, a significantly large population of the CRF patients (26.2%), when compared with the healthy subjects (4.1%) showed a marked decrease in lymphocyte response to prednisolone (P < 0.01). The binding capacity and affinity of lymphocyte glucocorticoid receptors did not differ significantly between the responders and nonresponders, suggesting that steroid resistance is a post-receptor event. The antilymphocyte potency of prednisolone assessed by IC50 of the steroid was less than that of hydrocortisone, whereas methylprednisolone was > 12-fold superior to prednisolone. After kidney transplantation, CRF patients who showed impaired preoperative lymphocyte response to prednisolone had a significantly high incidence of acute allograft rejection under prednisolone/CsA therapy (P < 0.01). It is concluded from these results that methylprednisolone could be of benefit to prednisolone-resistant recipients, who can be identified by the preoperative lymphocyte culture.

Adolescent↗
PubMed1993

Comparison of adrenal functions in kidney transplant recipients with different long-term immunosuppressive treatments--prednisolone and azathioprine versus prednisolone and cyclosporine.

Adverse effects of cyclosporine on the adrenal cortex have been documented in animal experiments, but nothing has been reported in human subjects. Endogenous cortisol in peripheral blood was monitored for three years after transplantation, with 30 kidney recipients on two different immunosuppressive treatments. In the azathioprine group, 16 patients were treated with coadministration of prednisolone at an initial dose of 120 mg/day. In the cyclosporine group, 14 patients were also treated with prednisolone, using an initial dose of 60 mg per day. Short ACTH stimulation tests were performed to reconfirm the results obtained by basal cortisol monitoring. During the first year following transplant, cortisol concentrations in the cyclosporine group were higher, though not significantly so, than those in the azathioprine group, in accordance with cumulative amounts of prednisolone administered. At three years, however, the mean cortisol concentrations in the azathioprine group were 2-3 times higher than those in the cyclosporine group (P < 0.05). All patients in the azathioprine group responded well to ACTH, whereas 4 patients out of 14 in the cyclosporine group showed continuous severe suppression without considerable response to ACTH (P < 0.01). In conclusion, we would like to suggest that adrenocortical toxicity of long-term cyclosporine use may appear one year after transplant, resulting in chronic suppression of the adrenal cortex, and, accordingly, difficulty in further reduction of prednisolone use.

Adrenal Glands↗
PubMed1993

Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148.

Amino Acid Sequence↗
PubMed1993

A flavoprotein functional as NADH oxidase from Amphibacillus xylanus Ep01: purification and characterization of the enzyme and structural analysis of its gene.

Amphibacillus xylanus Ep01, a facultative anaerobe we recently isolated, shows rapid aerobic growth even though it lacks a respiratory pathway. Thus, the oxidative consumption of NADH, produced during glycolysis and pyruvate oxidation, should be especially important for maintenance of intracellular redox balance in this bacterium. We purified a flavoprotein functional as NADH oxidase from aerobically growing A. xylanus Ep01. The A. xylanus enzyme is a homotetramer composed of a subunit (M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH as the preferred electron donor and exhibits no activity with NADPH. The flavoprotein gene of A. xylanus Ep01 was cloned by using a specific antibody. The amino acid sequence of 509 residues, deduced from the nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhimurium and NADH dehydrogenase from alkalophilic Bacillus sp. strain YN-1, respectively. Bacillus spp. have a respiratory chain and grow well under aerobic conditions. In contrast, Amphibacillus spp., having no respiratory chain, grow equally well under both aerobic and anaerobic conditions, which distinguishes these two genera. Salmonella spp., which are gram-negative bacteria, are taxonomically distant from gram-positive bacteria such as Bacillus spp. and Amphibacillus spp. The above findings, however, suggest that the flavoprotein functional as NADH oxidase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase diverged recently, with only small changes leading to their functional differences.

Amino Acid Sequence↗
PubMed1992

Staphylococcus piscifermentans sp. nov., from fermented fish in Thailand.

New coagulase-negative staphylococci were isolated from fermented fish in Thailand. These organisms were differentiated from other Staphylococcus species on the basis of DNA relatedness and biochemical characteristics. Staphylococcus piscifermentans sp. nov. is described, and the type strain is strain SK03 (= NRIC 1817 = JCM 6057 = TISTR 824).

Animals↗
PubMed1992

Promotion of lymphocyte blastogenesis by hemodialysate of chronic renal failure.

Effects of hemodialysate of patients with chronic renal failure (CRF) on blastogenesis of human peripheral blood lymphocytes in the presence of concanavalin A or phytohemagglutinin was investigated. Hemodialysates from 11 CRF patients significantly promoted lymphocyte blastogenesis when compared with control dialysis fluids (p less than 0.01). The strongest activity (39% promotion of the lymphocyte blastogenesis) was observed with a hemodialysate obtained at 0.5 h after beginning dialysis. The activity decreased thereafter. On the contrary, the blastogenesis-promoting activity in plasma decreased significantly after hemodialysis (p less than 0.01, n = 11). To further confirm the presence of low-molecular-weight factor(s) in CRF, ultrafiltration of plasma obtained from CRF and healthy subjects was conducted using a membrane filter with a molecular cutoff of 3,000 D. The filtrate of CRF plasma significantly promoted lymphocyte blastogenesis when compared to that of healthy subjects (p less than 0.01). Heat treatment (100 degrees C, 40 min) did not abolish the activity of the hemodialysate. None of the drugs taken by the patients nor creatinine accumulated in CRF promoted the lymphocyte blastogenesis. Chromatographic analysis of a hemodialysate demonstrated several peaks which were absent in the control dialysis fluid. These results showed the presence of a novel lymphocyte-stimulating factor(s) in CRF, which is heat stable and has a low molecular weight (less than 3,000 D).

Allopurinol↗