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Biomedical subjects

T Akizawa

Publications and source records attributed to T Akizawa.

At least 55 records · Page 3Linked to original sources
PubMed1998

Isolation and characterization of novel endogenous digitalis-like factors in the ovary of the giant toad, Bufo marinus.

We have previously described the structures of four novel unconjugated bufadienolides in the ovary of the toad, Bufo marinus. In this study, we report the separation and characterization of three novel bufadienolide conjugates. These compounds were purified by HPLC, and their structures were determined to be 11alpha, 19-dihydroxytelocinobufagin-3-(12-hydroxydodecanoic acid) ester, 11alpha,19-dihydroxytelocinobufagin-3-(14-hydroxy-7-tetra decenoic acid) ester, and 11alpha, 19-dihydroxytelocinobufagin-3-(14-hydroxytetradecanoic acid) ester on the basis of NMR and MS data. Numerous dicarboxylic acid esters of bufadienolides have previously been described, but the three bufadienolide conjugates described in this report differ from previously described esters in that they contain hydroxylated monocarboxylic acids. The function of these three conjugates is not known but they are, like bufotoxins, potent inhibitors of Na+, K+-ATPase and may play a developmental role in the differentiation of toad oocytes.

Animals↗
PubMed1998

Therapeutic apheresis for septic patients with organ dysfunction: hemoperfusion using a polymyxin B immobilized column.

A prospective clinical study was performed to evaluate a new method of treatment of endotoxin shock, a column containing polystyrene fibers with covalently bound immobile polymyxin B. Direct hemoperfusion using the column removes circulating endotoxin by adsorption. All of the patients studied, 37 in the treatment group and 33 in the control group, had endotoxemia and failure of 1 or more organs. The perfusion was performed 1-7 times per patient, 2 h/session. The survival rate was significantly higher in the treatment group (54%) than in the controls (36.4%). The mean plasma endotoxin concentration was significantly lowered by the treatment from 83.7 pg/ml before perfusion to 56.4 pg/ml immediately after and 28.5 pg/ml the day after the treatment, and the posttreatment level was much lower in those who survived (mean, 18.8 pg/ml) compared to those who died (mean, 88 pg/ml). Various parameters of cardiac function also improved after the treatment.

Adolescent↗
PubMed1998

Plasma exchange for thrombocytopenia in antiphospholipid syndrome: a case report.

The remarkable effect of plasma exchange (PE) was observed on thrombocytopenia in a patient with antiphospholipid syndrome (APS) associated with systemic lupus erythematosus. Three times PE led to recovery from severe thrombocytopenia refractory to treatment with corticosteroid, anticoagulant, and antiplatelet drugs accompanied by a decrease in the serum cardiolipin beta2 glycoprotein I antibody level. This result suggests that PE is a valuable therapeutic tool for refractory thrombocytopenia in APS.

Adult↗
PubMed1998

[Analysis of inspiratory flow-volume curve].

Inhaled antiasthmatic agents can be effective, but their efficacy depends on the delivery system and on inhalation technique, especially in metered-dose inhalers (MDI). To find out whether inhalation therapy for asthmatics had been performed correctly, we studied inspiratory flow in the slow matter in healthy subjects and bronchial asthma patients. About half of 30 patients studied inspired more than 1 L/sec inspiratory flow rate, especially in male. Inspiratory flow in the fast matter (peak inspiratory flow; PIF) is important in the use of dry powder inhaler, so we studied the correlationship between PIF and expiratory flow and volume parameters. PIF correlated with PEFR, FEV1 and FEV1% in female, FVC and %FVC in male, respectively. In summary, we must recognize slow inspiratory flow can not be gotten easily, and asthmatic patients always have to use MDI carefully in inspiratory flow. Some expiratory parameters of spirometry can accurately predict PIF. The analysis of inspiratory flow-volume curve gives us beneficial data in inhalation therapy.

Adult↗
PubMed1997

Chiral analysis of 3-methoxy-4-hydroxyphenylglycol in human urine.

Since 3-methoxy-4-hydroxyphenylglycol (MHPG) is a neutral metabolite from norepinephrine, it will be a diagnostic marker for mental diseases such as depression. For the development of an immunoassay, the natural enantiomer of MHPG would be required to prepare its antigen and to examine the specificity of the antibody. A natural enantiomer synthesized, however, has not been obtained so far. In this paper, we attempted to enantioseparate synthetic DL-MHPG and to assign D-enantiomer from the optical rotation of MHPG purified from human urine, because endogenous norepinephrine occurs as D-enantiomer which should metabolically generate D-MHPG. Enantioseparation conditions were tested using a Ceramospher Chiral RU-1 column (4.6 x 250 mm) at a flow rate of 0.5 ml/min. The resolution was adequate for the analysis and purification of synthetic DL- and the urinary MHPGs using methanol as a mobile phase and the column temperature at 0 degrees C, where DL-MHPG was detected as two peaks. The earlier peak (peak 1) showed (-) optical rotation, while the latter gave (+) optical rotation. After being treated with beta-glucuronidase, the normal human urine was extracted with ethyl acetate and then evaporated to dryness. The residue was suspended in water and the supernatant was analyzed and purified by a reversed phase column with a multi channel detector. A peak corresponding to MHPG was collected and concentrated to dryness. The pooled residues were dissolved in methanol and enantioseparated on the chiral HPLC. The urinary MHPG appeared as a single peak which was corresponded to the earlier peak of DL-MHPG and showing (-) optical rotation. Thus, the urinary MHPG was found to be D-(-)-MHPG. Then the absolute configuration of enantioseparated MHPGs were assigned to each optical rotation, judging from the chemical data and the metabolic pathway of the urinary D-MHPG. These enantiomers will be useful for studying on biochemistry and immunoassay.

Chromatography, High Pressure Liquid↗
PubMed1997

Development of enzyme immunoassay of 2'-deoxycytidine.

In order to study 2'-deoxycytidine (2'-dCyd) as a possible prognostic marker in cancer chemotherapy, an enzyme immunoassay (EIA) was developed. 2'-dCyd as a hapten was succinylated and two omicron-monosuccinyl-2'-dCyd's were purified by high performance liquid chromatography and identified by mass spectrometry and 1H-NMR. Two antigens were prepared by conjugating two omicron-succinyl derivatives with keyhole limpet hemocyanin (KLH) as a carrier. Both antigen produced specific antibodies to 2'dCyd in BALD/c mice. The spleen cells of one mouse immunized with 5'-omicron-succinyl-2'-dCyd-KLH were hybridized with myeloma cells. One monoclone selected produced a specific antibody. A convenient EIA was attained by using the monoclonal antibody.

Animals↗
PubMed1997

Flow injection analysis for measurement of activity of matrix metalloproteinase-7 (MMP-7).

A simple and convenient method for measuring the activity of a recombinant human matrix metalloproteinase 7 (MMP-7, matrilysin) was developed by flow injection analysis (FIA). For this method, purified recombinant MMP-7 zymogen expressed in E. coli and the substrate peptide (MOCAc-Pro-Leu-Gly-Leu-A2pr(DNP)-Ala-Arg-NH2) were used. Following the incubation of substrate peptide with activated r-proMMP-7, the resulting fluorescent product peptide (MOCAc-Pro-Leu-GLY) was monitored with a fluorescence detector (lambda ex 328 mm, lambda em 393 mm) without chromatographic separation. In this FIA system, the analysis time is 2 min and the standard curve is linear from 5 to 100 pmol of the product peptide injected. In order to use this FIA system as a method for screening inhibitors against MMP-7, the effects of CaCl2, EDTA and of the tissue inhibitor of metalloproteinase-1, and -2, were tested. A synthetic PRCGXPD-containing peptide (BS-10) was also observed to inhibit MMP-7 activity, with an IC50 value of 104 microM. Thus, it was concluded that the activity of r-MMP-7 can be reliably measured by the proposed system. Furthermore, to confirm the utility of this FIA system as a screening method, the inhibitory activity of the MMP-related substance in Joro spider (Nephilia clavata) venom was measured by this method. This inhibitory activity was observed in an extract of a venom diluted 1000-fold. Thus, the FIA method is not only simple and quick, but also sensitive enough to screen and analyze the inhibitory properties of a large number of test compounds.

Enzyme Inhibitors↗
PubMed1997

A novel bufadienolide, marinosin, in the skin of the giant toad, Bufo marinus.

We have identified a novel cardiac steroid, 11,19-epoxy-19-methoxytelocinobufagin, named marinosin (1), in the skin of the toad, Bufo marinus (L.) Schneider. The treatment of compound 1 with 50% CH3CN containing 0.1% trifluoroacetic acid yielded a 11 alpha-hydroxyhellebrigenin (2), which has not previously been isolated from animals or plants. The structures of both compounds were established from spectral data obtained by NMR and MS, which were compared with those of a reference bufadienolide, 11 alpha-hydroxytelocinobufagin. Compounds 1 and 2 have A/B cis and C/D cis configuration, which is characteristic of bufadienolides such as bufalin and marinobufagin. However, the stereo-structure of compound 1 was characterized by a boat form of the B ring, which is different from the chair form in typical bufadienolides such as compound 2. Compounds 1 and 2 both exhibited activity, as demonstrated by inhibition of Na+, K(+)-ATPase enzymatic activity and by inhibition of the binding of [3H]ouabain to Na+, K(+)-ATPase; however, marinosin (1) was as less effective inhibitor than 2, 11 alpha-hydroxyhellebrigenin. We have identified compound 2 in toad venom, but not in the skin.

Animals↗
PubMed1997

Incidence and clinical characteristics of hypoparathyroidism in dialysis patients.

The incidence and clinical characteristics of hypoparathyroidism (Hypo) were evaluated in 8188 hemodialysis (HD) and 1207 CAPD patients treated in 65 hospitals or clinics in Japan. Hypo was defined by an intact parathyroid hormone (PTH) level below 160 pg/ml, which corresponded to the low normal limit of intact PTH to maintain a normal osteoblastic surface in 40 bone biopsy specimens of Japanese dialysis patients, and patients were classified into two groups: absolute Hypo (A-Hypo), intact PTH < 60 pg/ml, and relative Hypo (R-Hypo), 60 pg/ml < or = intact PTH < 160 pg/ml. A total of 2537 (31.0%) and 2736 (33.4%) HD patients were classified into A- and R- Hypo, and 401 (31.3%) and 379 (31.4%) CAPD patients occupied A- and R-Hypo, respectively. A high incidence of Hypo was observed in the HD patients with diabetes mellitus (DM) and old age (> or = 70 years old) compared with that of a nationwide epidemiological report for dialysis patients by Japanese Society for Dialysis Therapy. Hypo patients who were treated by CAPD had a background of being younger, having a shorter duration on dialysis, and were less frequently diagnosed with DM than in those Hypo patients on HD. Bone pain and metastatic calcification were observed in approximately 20% and 25% of Hypo patients, respectively. No difference was observed in the background factors and the prevalence of signs and symptoms between the A- and R-Hypo groups, regardless of the mode of treatment (HD or CAPD). These results suggest that a very high incidence and specific backgrounds (DM and aging) of Hypo exist in Japanese dialysis patients.

Adult↗
PubMed1996

Processing of a precursor of 72-kilodalton type IV collagenase/gelatinase A by a recombinant membrane-type 1 matrix metalloproteinase.

Membrane-type 1 matrix metalloproteinase that is associated with the proteolytic activation of progelatinase A was expressed as a recombinant fusion protein in Escherichia coli. The recombinant enzyme cleaved the propeptide sequence of gelatinase A in a sequence-specific manner. A mutant progelatinase A that has a substitution of Asn(66)-Leu to Ile-Val was not processed at all. The processing was blocked by tissue inhibitor of metalloproteinases-2 or BB-94 but not by tissue inhibitor of metalloproteinases-1. Thus, membrane-type 1 matrix metalloproteinase is a direct activator of progelatinase A without requiring additional proteases.

Animals↗
PubMed1996

rHuEPO enhances the production of plasminogen activator inhibitor-1 in cultured endothelial cells.

The direct effects of recombinant human erythropoietin(rHuEPO) on coagulation and fibrinolysis factors were evaluated in a cultured endothelial cell (EC) system. Confluent quiescent ECs were incubated with or without 5.0 U/ml rHuEPO for 1, 6, and 18 hours, and supernatant concentrations of plasminogen activator inhibitor-1 (PAI-1): antigen (Ag), tissue plasminogen activator and thrombomodulin, and supernatant activities of tissue factor pathway inhibitor and von Willebrand factor were measured. The results showed that only PAI-1 levels were increased by the presence of rHuEPO. In order to assess the effect of rHuEPO on PAI-1 production by EC more precisely, confluent ECs were incubated with various doses of rHuEPO (0, 1.0, 2.5, 5.0, 10.0 U/ml) for 1, 6, 12, and 18 hours, and PAI-1:Ag concentrations in the supernatants of media were measured. PAI-1:Ag in the supernatants were increased by the presence of rHuEPO at all incubation times (P < 0.01) and the increase in PAI-1:Ag was dependent on rHuEPO concentration. The increases in PAI-1:Ag by 5.0 U/ml rHuEPO were comparable to those by 0.1 U/ml tumor necrosis factor-alpha, 1.0 microgram/ml lipopolysaccharide, and 0.5 U/ml thrombin. The increase in PAI-1:Ag by rHuEPO was suppressed by pre-incubation with 10 micrograms/ml cycloheximide (P < 0.01) or 0.2 microgram/ml actinomycin D (P < 0.01). These results indicate that rHuEPO directly stimulates PAI-1 production in cultured EC via de novo protein and RNA syntheses.

Cells, Cultured↗
PubMed1996

Effect of 22-oxacalcitriol on hyperparathyroidism of dialysis patients: results of a preliminary study.

Intermittent high dose administration of calcitriol or alfacalcidol is effective in suppressing secondary hyperparathyroidism in chronic dialysis patients, however calcaemic action of these vitamin D derivatives is a major obstacle. 22-Oxacalcitriol (OCT) has been reported to have less calcaemic action than calcitriol, while preserving a comparable suppressive effect on parathyroid hormone (PTH) secretion. This preliminary study was conducted to examine the effects of OCT on secondary hyperparathyroidism in chronic dialysis patients. OCT was administrated intravenously immediately after every haemodialysis session three times a week for 12 weeks to three haemodialysis patients with secondary hyperparathyroidism. An initial dose of OCT of 5.5 micrograms/haemodialysis session was increased stepwise by 5.5 micrograms/haemodialysis up to 22 micrograms/haemodialysis according to the suppression of PTH and calcaemic action. OCT was discontinued for at least a week when serum calcium adjusted to albumin concentration measured just before haemodialysis exceeded 11.5 mg/dl. Marked reduction in plasma PTH, alkaline phosphatase and tartrate-resistant acid phosphatase was observed in all three patients. Although the dose of OCT was increased to 22 micrograms/haemodialysis in one patient, the final dose of OCT remained 5.5 micrograms/haemodialysis in the other two patients because of hypercalcaemia. It is concluded that OCT is highly effective in suppressing PTH in dialysis patients with secondary hyperparathyroidism. Hypercalcaemia may be a major factor which limits the use of OCT, though it may occur with higher doses of OCT than those of calcitriol usually given to suppress PTH hypersecretion.

Calcitriol↗
PubMed1996

Dialysis membranes in the year 2000: performance and biocompatibility.

Among many substances accumulated or generated by renal failure, much attention has focused on the pathogenetic effects of advanced glycation end-product (AGE)-bound proteins such as AGE-modified beta 2-microglobulin, and bioactive substances such as inflammatory cytokines. In order to remove these substances efficiently, dialysis membranes in the year 2000 should have a higher molecular cut-off point and a sharper cut-off curve, with less formation of concentration polarization, or a greater adsorbing capacity for target substances with less ensuing protein gel layer. Adequate supplementations for depleted substances by more efficient dialysis will be necessary as at present with carnitine and vitamins. Further, from now until the year 2000, membranes in which biocompatibility nearly corresponds to that of capillary endothelium, and safe and economical purification systems of dialysate should be realized.

Biocompatible Materials↗
PubMed1996

Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization.

Human matrix metalloproteinase-7 (MMP-7 = matrilysin) was overproduced in Escherichia coli as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines. Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution. The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner. The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7. Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7. The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions. The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases-1 and -2, MMP-specific inhibitors. The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state. Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors.

Amino Acid Sequence↗
PubMed1996

Heterogeneity and lability of endogenous digitalis-like substances in the plasma of the toad, Bufo marinus.

Three major groups of endogenous digitalis-like substances (EDLS) have been identified in the plasma of the toad, Bufo marinus. One group of compounds, present in fresh plasma, is composed of chromatographically homogeneous polar conjugates, principally bufadienolide 3-sulfates, which exhibit relatively weak Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitory activity. A second and larger group of compounds, also found in fresh plasma, includes chromatographically heterogeneous conjugates, which are effective inhibitors of Na(+)-K(+)-ATPase; these compounds possess properties similar to those of bufotoxins. The third group of EDLS consists of free unconjugated bufadienolides, which are also effective Na(+)-K(+)-ATPase inhibitors. These unconjugated bufadienolides are present in relatively low concentrations in fresh toad plasma, but appreciable quantities are enzymatically generated from conjugates (believed to consist principally of bufotoxins) during the in vitro incubation of plasma. We suggest that the extent to which circulating polar EDLS are enzymatically deconjugated in vivo may be important in the regulation of the digitalis-sensitive Na(+)-K(+)-ATPase of toad brain, the only known digitalis-sensitive Na(+)-K(+)-ATPase in the toad.

Animals↗
PubMed1996

Marinoic acid, a novel bufadienolide-related substance in the skin of the giant toad, Bufo marinus.

We found a novel substance, 3 beta-hydroxy-11,12-seco-5 beta, 14 beta-bufa-20,22-dienolide-11,14-olide-12-oic acid (1), which we called marinoic acid, in the skin of the toad, Bufo marinus. The structure was established from NMR and MS data. Like bufadienolides, marinoic acid contained an A/B ring structure in the cis configuration and a D/alpha-pyrone ring structure, but the structure of the C ring differed considerably from that of bufadienolides. Marinoic acid exhibited biological activity, as demonstrated by inhibition of Na+, K(+)-ATPase enzymatic activity, and by inhibition of [3H]ouabain binding to the digitalis receptor site on Na+, K(+)-ATPase, although marinoic acid was a less effective inhibitor than typical bufadienolides. Although marinoic acid cannot be classified as a bufadienolide, its chemical structure and its Na+, K(+)-ATPase inhibitory activity suggest that it is bufadienolide-related.

Animals↗