Biomedical subjects
Publications and source records attributed to I Yokota.
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An innovative chemically modified thrombin, succinylthrombin, was prepared by the treatment of thrombin with succinic anhydride. Succinylthrombin showed a remarkable reduction of fibrinogen clotting activity with a reduction of the potency for protein C (PC) activation. However, the degree of the remaining potency for PC activation was greater than the degree of the remaining clotting activity. The potency for PC activation was enhanced by the addition of thrombomodulin and calcium ion. The degree of the enhancement was approximately 6-times greater than that of acetylthrombin, which we previously suggested as an effective anticoagulant. The infusion of succinylthrombin into rabbits induced a prolongation of the activated partial thromboplastin time (APTT). The fibrinogen level was not affected even by the infusion of succinylthrombin of a dose which cause the prolongation of the APTT to the same extent as that following an infusion of thrombin. The factor Xa clotting time was also prolonged by the succinylthrombin without any significant drop in the number of the circulating platelet.
We assayed the rates of lactate production from [1-14C]pyruvate and decarboxylation of [1-14C]pyruvate in cultured skin fibroblasts from 8 patients with disorders of pyruvate metabolism and 16 control subjects. The disorders of pyruvate metabolism could be more readily detected by measuring the ratio between the rates of lactate production and pyruvate decarboxylation by cultured skin fibroblasts than by measuring either the rate in isolation.
Vitamin D-dependent rickets type II is a rare disease caused by a disorder of the receptor for 1, 25-dihydroxyvitamin D (1, 25(OH)2D). Several parameters of this receptor-effector system were investigated to obtain biochemical information on the presumed heterozygotes of vitamin D-dependent rickets type II in parents of five patients and in their age-matched controls. It was found that the serum concentrations of 1, 25-(OH)2D and 24,25-dihydroxy-vitamin D (24,25(OH)2D), and the ratio of 1,25-(OH)2D/24,25-(OH)2D differed significantly in the parents from those of the patients and the respective control groups. In the parents' cultured skin fibroblasts, the activity of 25-hydroxyvitamin D-24-hydroxylase induced by 10(-8) mol/L 1, 25-(OH)2D3 ranged from 50 to 82% of that of their controls (versus 1-13% of controls for the patients). The binding capacity of the parents' [3H]1, 25-(OH)2D3 to the nucleus was 38-54% of that of their control subjects (versus 7-27% of controls for the patients). The parents' values were thus in a range between those of the patients and the control groups. These findings suggest that, in the parents, a partial impairment of the receptor system for 1, 25-(OH)2D led to an imbalance of vitamin D metabolism, thus confirming that vitamin D-dependent rickets type II is an autosomal recessive inherited disease. Serum concentrations of 1, 25-(OH)2D and 24, 25-(OH)2D may provide useful parameters for detecting heterozygotes of this disease.
To explore the relationship between ribonucleotide reductase and immunodysfunction in adenosine deaminase deficiency, the effects of deoxyadenosine on ribonucleotide reductase in ADA-deficient lymphocytes was investigated. An assay system for ribonucleotide reductase in intact permeabilized lymphocytes was developed to approximate physiological conditions. The activity of cytidine diphosphate (CDP) reductase in resting but not in proliferating lymphocytes in culture was inhibited by 1 to 10 mumol/L deoxyadenosine. The resting cells were protected from the toxicity of 1 mumol/L deoxyadenosine by 5 mmol/L nicotinamide or 30 mumol/L deoxycytidine and from that of 10 mumol/L deoxyadenosine by 30 mumol/L deoxycytidine. These findings suggest that depletion of nicotinamide adenine dinucleotide might be the principal cause of death in resting lymphocytes with ADA deficiency. It is concluded that the mechanism of deoxyadenosine toxicity on non-replicating lymphocytes, which may not be mediated by ribonucleotide reductase inhibition, is closely related to the mechanism of immunodysfunction in patients with ADA deficiency.
Vitamin D-dependent rickets type II is a hereditary disease resulting from a defective vitamin D receptor. In three Japanese patients with vitamin D-dependent rickets type II whose fibroblasts displayed normal cytosol binding and impaired nuclear uptake of 1,25-dihydroxyvitamin D3, western, Southern, and northern analyses failed to disclose any abnormalities in vitamin D3 receptor protein and its gene. Exons 2 and 3 of the vitamin D receptor cDNA, which encode the DNA-binding domain consisting of two zinc fingers, were amplified by PCR and sequenced to identify the specific mutations in the vitamin D receptor gene. In the three patients and one normal control a T-to-C transition was found in the putative initiation codon, while this transition was not observed in another normal control. This finding suggested that an original initiation codon was located at positions 10-12 in the human vitamin D receptor cDNA sequence reported previously. In contrast, a unique G-to-A transition at position 140 in exon 3, resulting in substitution of arginine by glutamine at residue 47, was revealed only in these three patients. The arginine at 47 is located between two zinc fingers and is conserved within all steroid hormone receptors. Therefore, it is highly conceivable that this amino acid substitution is responsible for the defect of the vitamin D receptor in the patients. Single-strand conformation polymorphism analysis of amplified DNA confirmed that all patients were homozygous and that parents from one family were heterozygous carriers for this mutation.
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is an inborn error of fatty-acid oxidation that is characterized by fasting intolerance and recurrent episodes of hypoglycemic coma which can be fatal. Its incidence is one of the highest among genetic metabolic disorders. Using a modified PCR and NcoI digestion method, we have surveyed 46 additional, unrelated MCAD-deficient patients for a prevalent mutation, an 985A-to-G transition (985A----G), that we previously identified in nine MCAD-deficient patients. Among the total of 55 studied, 44 were homozygous and 10 were heterozygous for the 985G allele, whereas one did not carry this mutant allele, indicating that the prevalence of the 985G allele is 89.1%. Furthermore, we identified five other types of mutation: one each in three of the compound heterozygotes and two in the single non-985G patient. An RFLP study of 12 985G-homozygotes showed that all 24 alleles fell into a single haplotype. A questionnaire regarding the ethnic and national origin of their patients was sent to all referring investigators. All 41 patients for whom this information was provided were Caucasians. Of 29 patients whose country of origin was specified, 19 and five were from the British Isles and Germany, respectively. These data suggest that 985A----G may have occurred in a single person in an ancient Germanic tribe.
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In our previous study of eight glutaric acidemia type II (GAII) fibroblast lines by using [35S]methionine labeling and immunoprecipitation, three of them had a defect in the synthesis of the alpha-subunit of electron transfer flavoprotein (alpha-ETF) (Ikeda et al. 1986). In one of them (YH1313) the labeling of the mature alpha-ETF was barely detectable, while that of the precursor (p) was stronger. In another (YH605) no synthesis of immunoreactive p alpha-ETF was detectable. In the third cell line (YH1391) the rate of variant p alpha-ETF synthesis was comparable to normal, but its electrophoretic mobility was slightly faster than normal. In the present study, the northern blot analysis revealed that all three mutant cell lines contained p alpha-ETF mRNA and that their size and amount were comparable to normal. In immunoblot analysis, both alpha- and beta-ETF bands were barely detectable in YH1313 and YH605 but were detectable in YH1391 in amounts comparable to normal. Sequencing of YH1313 p alpha-ETF cDNA via PCR identified a transversion of T-470 to G. We then devised a simple PCR method for the 119-bp section (T-443/G-561) for detecting this mutation. In the upstream primer, A-466 was artificially replaced with C, to introduce a BstNI site into the amplified copies in the presence of G-470 from the variant sequence. The genomic DNA analysis using this method demonstrated that YH1313 was homozygous for T----G-470 transversion. It was not detected either in two other alpha-ETF-deficient GAII or in seven control cell lines. The alpha-ETF cDNA sequence in YH605 was identical to normal.
99mTc-ECD SPECT was performed in 19 patients with cerebrovascular disease, and location, extent, and severity of the lesions on 99mTc-ECD SPECT were compared with those on 123I-IMP SPECT. The initial brain uptake was 5.5 +/- 0.7% of the injected dose at 10 minutes after injection, 5.3 +/- 1.3% at 90 minutes, and clearance from the brain is slow. The distribution in the brain was changed, especially washout from the thalamus was slower than that from other regions. The count ratio of perfusion defect to normal area (D/N) on 99mTc-ECD SPECT was unchanged over the time, and had no significant differences from that on 123I-IMP SPECT. 99mTc-ECD SPECT was superior in detection of the lesion in the basal ganglia, and showed the images with superior spatial resolution due to physical characteristics of 99mTc. However, mild ischemic lesion and peri-infarct area was not clearly visualized, while 123I-IMP SPECT could demonstrate these lesions with better contrast.
Thrombin was acetylated by treatment with acetic anhydride, and the potency for protein C activation and the fibrinogen clotting activity of the resultant acetylthrombin were investigated in vitro and in vivo. The acetylthrombin retained an amidolytic activity to the synthetic substrate, S-2238, and reduced both of the potency for protein C activation and the clotting activity. The potency for protein C activation (0.47% of that of thrombin) was retained more than the clotting activity (0.015% of that of thrombin). The enzymatic activities of acetylthrombin were in inverse proportion to the molecular weights of the substrates, S-2238, protein C, and fibrinogen. Similarly, the inhibitory activity on acetylthrombin was dependent on the molecular weights of the inhibitors, Thromstop, I-2581, hirudin, and antithrombin-III. When acetylthrombin (5000 units/kg body weight) was infused into rabbits, the activated partial thromboplastin time was prolonged to the same extent as that following infusion of thrombin (125 units/kg), but the fibrinogen level was not decreased in contrast to the large decrease following infusion of thrombin. It is suggested that acetylthrombin activates protein C without clotting fibrinogen in vivo.
Three patients with vitamin D-dependent rickets type II were given massive doses of 1 alpha-hydroxyvitamin D3 for 29 to 36 months and their calcium-phosphate balance was studied during treatment and one month after cessation of treatment. During treatment fasting hypercalciuria was observed in patient 1 and an increased rate of calcium excretion after calcium loading in patients 1 and 2. In these patients, calcium excretion was parallel with the serum 24,25-dihydroxyvitamin D concentration, which may reflect the level of receptor for 1,25-dihydroxyvitamin D. These findings suggested that the responsiveness to 1,25-dihydroxyvitamin D improved during long-term treatment of these two patients with vitamin D-dependent rickets type II.
We sequenced polymerase chain reaction (PCR)-amplified variant medium chain acyl-CoA dehydrogenase (MCAD) cDNAs in cultured fibroblasts from three MCAD-deficient patients. In all three patients, an A to G transition was identified at position 985 of the coding region. Since no appropriate restriction sites for detecting this point mutation were found, we devised a PCR method that amplifies an 87-bp fragment from position 955. In the 5' primer encompassing positions 955 to 984, A-981 was artificially substituted with C. With the presence of C-981 and G-985, an Nco I restriction site is introduced in the mutant copies. When cDNA or genomic DNA from fibroblasts of nine MCAD-deficient patients were tested with this method, the copies from all of them completely cleaved into two shorter fragments by Nco I, indicating their homozygosity for the A----G-985 transition. In contrast, the copies from all eight controls remained intact. Thus, this A----G-985 transition is the single prevalent mutation causing MCAD deficiency, a highly unusual feature for any genetic disorder. The PCR/Nco I digestion method is suitable for the diagnosis of MCAD deficiency.
A method for assay of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) activity in phytohemagglutinin (PHA)-stimulated lymphocytes was applied to determine whether vitamin D-dependent rickets type II (VDDR II) is hereditary. In normal lymphocytes incubated with PHA for 3 days, maximal and half-maximal responses of 24-hydroxylase were observed after exposure to 10(-8) mol/L and (1.3 +/- 0.4) x 10(-9) mol/L 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], respectively. These responses were similar to those of cultured skin fibroblasts. In contrast, after exposure to 10(-8), 10(-7), and 10(-6) mol/L 1,25-(OH)2D3, no 24-hydroxylase activity was detected in cells from patients with VDDR II, and intermediate activity was observed in cells from their parents. These findings indicated the presence of an intracellular receptor-effector system for 1,25-(OH)2D3 in peripheral lymphocytes. Heterozygotes of VDDR II could be identified, and autosomal recessive inheritance of the disease was demonstrated. Detection of heterozygotes of this disease was not possible by assay of inhibition of thymidine incorporation, another marker of the function of 1,25-(OH)2D3 in PHA-stimulated lymphocytes. Therefore, assay of 24-hydroxylase induction reflected the receptor status more closely than assay of inhibition of DNA biosynthesis. The assay of 24-hydroxylase activity in PHA-stimulated lymphocytes described here will be useful for diagnosis of VDDR II and study of families of patients with this disease.
A minor component separated by HPLC from egg-white lysozyme was found to have a different substrate specificity from the main component, lysozyme. However, it did not show any difference in molecular weight and immuno-cross reactivity from lysozyme. There was no evidence based on our study that this minor component is an artificially-produced substance.
Coronary artery territories were evaluated precisely by Bull's-eye and unfolded map generated from myocardial SPECT. In addition, unfolded maps including apical information were also developed. The coronary artery territories were determined by 54 cases of typical single vessel disease (more than 75% stenosis) with myocardial infarction and angina pectoris. There were 12 of right coronary artery disease, 24 of left anterior descending coronary artery disease, 5 of diagonal coronary artery disease, and 13 of left circumflex artery disease. Each diseased region was summed and normalized using the standard pattern (21 cases of normal male and 13 cases of normal female). Apical information was simply obtained using the anterior and posterior count from short and long axial image. Then, their data were added to the unfolded map. In conclusion, unfolded map was proven to be useful for the determination of coronary artery territory, since this map expressed the extent and site of myocardial damage precisely compared to Bull's-eye. The unfolded map to contain the apical information was also useful for the evaluation of left anterior descending coronary artery involvement.