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Biomedical subjects

S Miwa

Publications and source records attributed to S Miwa.

At least 217 records · Page 12Linked to original sources
PubMed1989

[Pathophysiology and laboratory tests of hemolytic anemia: with special reference to erythroenzymopathies].

Since the discovery of glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase deficiency, erythroenzymopathies associated with hereditary hemolytic anemia have been extensively investigated. Kinetic and electrophoretic studies have shown that most erythroenzymopathies are caused by the production of a mutant enzyme. Single amino acid substitutions have been determined in G6PD and phosphoglycerate kinase variants by studies of the enzyme. Except for these two enzymes, it has been difficult to purify and to characterize the patient's enzyme because of the low protein contents in red blood cells. Recent advance in recombinant DNA technology has made possible the isolation of normal genomic DNA or cDNA for several enzymes. These results permit us to study the molecular basis of erythroenzymopathies at the nucleotide level. Single base substitutions have been identified in aldolase, triosephosphate isomerase, G6PD and adenylate kinase variants by the cloning and nucleotide sequence of the patients' genes. To date, all of the enzyme-deficient variants which have been investigated are caused by point mutations. An exception is a hemolytic anemia secondary to increased adenosine deaminase (ADA) activity. Red cell ADA activity increases on the order of a hundred-fold in affected individuals. The basic abnormality appears to result from overproduction of structurally normal enzyme due to abnormal translational efficiency.

Adenosine Deaminase↗
PubMed1988

Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma.

Two overlapping clones, covering the entire coding sequence of human M2-type pyruvate kinase (PK) cDNA, were isolated and sequenced. Nucleotide sequencing results showed that they contained the 109-bp 5'-untranslated region, the 1593-bp coding region and the 585-bp 3'-untranslated region. Nucleotide sequence homology was 90% and 69% with rat M2-type and L-type PK cDNA, respectively. In situ hybridization using the human M2-type PK cDNA probe disclosed that the gene for M2-type PK is located at band q22 on chromosome 15. Northern blot analysis with RNA from human hepatoma demonstrated that M2-type PK was predominantly expressed in hepatoma cells, whereas L-type PK was preferentially expressed in the non-tumor portion of the liver.

Amino Acid Sequence↗
PubMed1988

Genotypic analysis using a Y-chromosome-specific probe following bone marrow transplantation.

To monitor successful engraftment after bone marrow transplantation, we performed Southern hybridization analysis or dot blot analysis of DNA in a set of sex-mismatched cases using a Y-chromosome-specific DNA probe (pHY10). This method was extremely sensitive and rapid for checking which cells contain the Y-chromosome. Using this probe, analysis of cells from peripheral blood and bone marrow after transplantation demonstrated the usefulness of confirming engraftment of donor cells and of detecting mixed lymphohematopoietic chimerism.

Adult↗
PubMed1988

Elevated erythrocyte adenosine deaminase activity in a patient with primary acquired sideroblastic anemia.

We report a case of primary acquired sideroblastic anemia (PASA) associated with elevated erythrocyte adenosine deaminase (ADA) activity. The patient was an 85-year-old Japanese male. Analysis of the peripheral blood revealed pancytopenia, and the bone marrow findings showed marked ringed sideroblasts and chromosomal deletion (46XY, 11q-). The erythrocyte ADA activity was 17 times higher than that of normal control, the leukocyte ADA activity was within the normal range, and the plasma ADA activity was 2 times higher than the normal mean. The adenine nucleotides in the patient's erythrocytes were within normal range. According to starch gel electrophoresis, ADA isozyme of the patient was ADA 1. Western blotting showed an increased amount of ADA protein in the patient's erythrocytes. Southern blotting revealed no gene amplification or large structural change. Dot blot analysis of the reticulocyte mRNA showed no increase in the amount of ADA mRNA in the patient's reticulocytes compared with those of reticulocyte-rich controls. We considered that the mechanism of elevated ADA activity in this acquired defect was similar to that found in hereditary hemolytic anemia associated with ADA overproduction.

Adenine Nucleotides↗
PubMed1988

Genetic analysis of adenosine deaminase expression in adult T-cell leukemia.

We investigated the genetic basis for the increased adenosine deaminase (ADA) expression in adult T-cell leukemia (ATL). We found a correlation between the levels of ADA-specific mRNA and ADA immunoreactive protein in ATL. Southern blot analysis revealed no gene amplification or rearrangement of the ADA gene. These findings indicate that increased ADA expression in ATL cells reflects increased transcriptional activity for the ADA gene or increased stability of ADA mRNA.

Acute Disease↗
PubMed1988

Effects of hypoxia on contractile responses of rabbit aortic strips to transmural electrical stimulation.

To clarify the effects of hypoxia on adrenergic transmission, we examined the contractile responses of isolated rabbit aortic strips to electrical stimulation, the concentration-response relationships for noradrenaline and KCl, and the electrical stimulation-evoked overflows of total [3H] and [3H]noradrenaline from strips preloaded with [3H]noradrenaline in media equilibrated with gas mixtures containing various concentrations of O2. Contractile responses to electrical stimulation were completely inhibited by tetrodotoxin and alpha-adrenoceptor antagonists such as phentolamine and phenoxybenzamine, but were not affected by indomethacin. When the concentration of O2 in the gas mixture was decreased from 95% to 20%, the contractile responses to electrical stimulation remained unchanged, but as the concentration of O2 was further decreased, the responses were inhibited concentration-dependently. At 0% O2, the response was inhibited by about 80% when compared with control values obtained at 95% O2, and the electrical stimulation-evoked overflows of total [3H] and [3H]noradrenaline into the superfusates were decreased by about 55%. At 0% O2, the concentration-response curve for exogenous noradrenaline was shifted to the right about 50-fold and the maximum response was decreased by 25%. The maximum contractile responses of aortic strips from animals pretreated with reserpine or 6-hydroxydopamine to high KCl were decreased slightly (about 15%). These results suggest that inhibition of adrenergic transmission under hypoxic conditions is mainly the result of a decrease in the stimulus-evoked release of noradrenaline and of a decrease in the affinity of alpha-adrenoceptor for noradrenaline and/or inhibition of signal transduction mechanisms, although hypoxia also causes a slight decrease in the contractility of vascular smooth muscle.

Animals↗
PubMed1988

A new glucose-6-phosphate dehydrogenase variant (G6PD Tsukui) associated with congenital hemolytic anemia.

A new glucose-6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was found in a 20-year-old Japanese male who showed mild hemolysis after an upper respiratory tract infection. The patient had been noted to have jaundice and reticulocytosis several times before this episode. The enzyme activity of the variant was 1.5% of normal. The enzymatic characteristics were slow anodal electrophoretic mobility, high Km G6P, normal Km NADP, decreased heat stability, and a normal pH optimum. From these results, the enzyme was considered to be a new class 1 variant and was designated G6PD Tsukui.

Adult↗
PubMed1988

A nonisotopic method for determination of the in vivo activities of tyrosine hydroxylase in the rat adrenal gland.

A rapid and reliable method for determination of in vivo activities of tyrosine hydroxylase in the rat adrenal gland is presented. This method involves determining the rate of accumulation of 3,4-dihydroxyphenylalanine (Dopa) in the adrenal gland after decarboxylase inhibition by NSD 1015, using HPLC with electrochemical detection after purification of the acid-deproteinized tissue extract with Bio-Rex 70 columns followed by alumina batch method. Purification of the sample with alumina adsorption alone, a method usually used for purification of catecholamines and Dopa, was ineffective: epinephrine and norepinephrine, which are present in high concentrations, interfered with an accurate determination of Dopa, and dopamine, which is retained strongly on the reverse-phase column, interfered with a rapid analysis. Purification with Sephadex G-10 columns followed by alumina adsorption was also ineffective. After purification with columns of weak cation-exchange resins such as Bio-Rex 70 or Amberlite CG-50 followed by alumina adsorption, most of the epinephrine and norepinephrine was removed and dopamine was eliminated. Thus a rapid and accurate determination of Dopa could be made. Of the two cation exchangers, Bio-Rex 70 was more effective. Accumulation of Dopa in the adrenal gland was linear up to 30 min after administration of NSD 1015 and a plateau was reached with doses over 10 mg/kg. Using this method, we investigated the effects of immobilization stress, reserpine, and hypoxia on in vivo activities of tyrosine hydroxylase in the adrenal gland.

Adrenal Glands↗
PubMed1988

Thyroxine surge in metamorphosing flounder larvae.

The tissue concentration of thyroxine (T4) in larval flounder (Paralichthys olivaceus) was studied at various stages of their metamorphosis using a specific radioimmunoassay developed for samples obtained by quantitative extraction of the hormone from fish eggs and larvae. T4 concentrations were below the limit of detection in extracts of fertilized eggs and larvae until the end of prometamorphosis. Several dorsal fin rays continued growing from premetamorphosis until the end of prometamorphosis. The hormone became detectable at the onset of metamorphic climax when the elongated dorsal fin rays stopped growing. A sharp increase in tissue T4 concentration was observed in midclimax of metamorphosis, and the high level of T4 (11-13 ng/g body weight) was maintained until the end of the climax. The period of elevated T4 levels coincided with resorption of the elongated dorsal fin rays. The right eye completed translocation from the right to the left side of the body during metamorphic climax. The tissue T4 level declined to about a half of the peak value during postclimax, and then increased moderately in later adult-type juveniles. The results of an additional experiment, in which fertilized eggs and newly hatched larvae were treated with T4 and/or thiourea, suggested the presence of negative feedback regulation of pituitary-thyroid axis, implying an involvement of thyroid hormones even in early premetamorphic larvae. These results suggest that metamorphic climax is induced by a surge of thyroid hormone, and that thyroid hormone may also regulate development before and after the metamorphosis.

Animals↗
PubMed1988

Regional difference in the kinetics of choline acetyltransferase in brains of neurologically normal elderly people and those with Alzheimer-type dementia.

In order to clarify the biochemical changes which occur in the brains of patients suffering from Alzheimer-type dementia (ATD), the kinetics of choline acetyltransferase (ChAT) were measured in 8 or 11 regions of post-mortem brains of 6 patients with non-neurological diseases (control subjects) and 8 patients with ATD. In the control subjects, Michaelis constants (Km) of ChAT for choline were lower in the caudate nucleus and putamen than in other regions examined, whereas Km values for acetyl-CoA in these two regions were higher. Maximal velocities (Vmax) for the control subjects were higher in the caudate nucleus and putamen than in the other regions studied. Km values for choline of ATD patients were higher than those of control subjects in all regions except the amygdala and substantia innominata (innominate); however, Km values for acetyl-CoA of ATD patients were higher than those of control subjects only in the caudate nucleus, putamen and thalamus. Vmax values of ATD patients were lower than those of control subjects in all the regions of brains with ATD. These results suggest that the binding site of ChAT for choline is sensitive to the pathological process of ATD, whereas the binding site for acetyl-CoA is resistant. Based on the presence or absence of variations of Km values, we have classified brain regions into 3 types: highly sensitive, somewhat sensitive or resistant to the pathological process of ATD.

Aged↗
PubMed1988

Cytogenetic study of chronic lymphocytic leukemia in ten Japanese patients with a case of the same chromosome abnormality both in T and B cells.

Peripheral blood lymphocytes from ten patients with chronic lymphocytic leukemia (CLL) stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM), Epstein-Barr virus (EBV), and T-cell growth factor were studied cytogenetically. Eight of ten cases were clinically and immunologically B-cell CLL, and two cases were T-cell CLL. Chromosome analysis revealed that one patient, diagnosed as B-cell CLL, had a clonal abnormality in the leukemic cells. The abnormal karyotype was 46,XY,+3,-15,t(10;19)(q22;p13),t(11;14) (q13;q32),13q+,17q-. The frequencies of the abnormal cell stimulated with PHA, PWM, and EBV were 50%, 13%, and 100%, respectively. Finding the same clonal abnormality after stimulation with three different mitogens suggests that the factor responsible for the development of CLL might affect the stem cell common to T-cell and B-cell lymphocytes. In cells with this abnormal karyotype, chromosome 13q+ seems to have a homogeneous staining region. In the present series of B-cell CLL, no trisomy 12 known to be the specific chromosome abnormality was observed.

Adult↗
PubMed1988

Expression of L- and M-type pyruvate kinase in human tissues.

Pyruvate kinase (PK) has four isozymes (L, R, M1, M2) that are encoded by two different genes of PK L and M. Differential splicing produces L- and R-type PK mRNA and M1- and M2-type PK mRNA from the PK L gene and the PK M gene, respectively. The nucleotide sequences of the 3'-noncoding region are the same between the L- and the R-type PK and between the M1- and the M2-type PK. We isolated 3'-noncoding sequences of human L- and M2-type PK cDNA to construct L-type and M-type PK specific probes. With these probes, we performed Northern blot analysis of the RNA samples extracted from human tissues. Northern blot analysis showed that both kidney and liver had mRNAs that hybridized with both the L and M probes. Small intestine, skeletal muscle, brain, testis, and lung mRNAs hybridized only with the M probe. Our probes are considered useful for the detection of the types of PK isozymes expressed in small amounts, which are very difficult to detect using the conventional PK polyacrylamide gel electrophoretic method.

DNA Restriction Enzymes↗
PubMed1988

Human liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells.

Pyruvate kinase (PK) has four isozymes (L, R, M1, M2) that are encoded by two different genes. Among these isozymes, abnormalities of liver (L)-type PK is considered to be associated with hereditary nonspherocytic hemolytic anemia in humans. We isolated and determined the full-length sequence of human L-type PK cDNA. The cDNA contains 1629 base pairs encoding 543 amino acids, 68 base pairs of 5'-noncoding sequence, and 734 base pairs of 3'-noncoding sequence. The similarity between human and rat L-type PK was 86.9% at the nucleotide sequence level and 92.4% at the amino acid sequence level. The full-length L-type PK cDNA was placed under the promoter of simian virus 40 and introduced into monkey COS cells. Human L-type PK activity was detected in the extract of COS cells by the classical PK electrophoresis method.

Amino Acid Sequence↗