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Biomedical subjects

T Miyake

Publications and source records attributed to T Miyake.

At least 433 records · Page 24Linked to original sources
PubMed1982

Partial purification and biological properties of thrombopoietin extracted from the urine of aplastic anemia patients.

Thrombopoietic (Tpo) and megakaryocyte-colony stimulating (Meg-CSF) activities were found in urinary extracts from patients with aplastic anemia. Preparative biochemical extractions were accomplished using Sephadex G-50 and DEAE-cellulose column chromatography. The biological activities of these extracts were assessed using not only an in vivo assay but were also examined in vitro employing the clonal development of megakaryocyte colonies. Both in vivo, as well as in vitro, biological activities were detected in the batch fraction which was stepwise eluted from DEAE-cellulose between 0.022 M NaCl in 0.016 M NaH2PO4 and 0.15 M NaCl in 0.05 M Na2HPO4 as a single fraction. When 0.4 mg of this fraction was injected daily into rats, a marked thrombopoiesis ensued producing an increase of 40% over initial platelet counts by 3 days after administration. This was followed by a decrease in platelets to a subnormal range by 21 days after the injection. Hemoglobin concentration gradually increased from 5% above initial value by day 7 to 20% above initial value by day 21. The effect of neuraminidase (NAse) on the properties of this extract was also examined. NAse-treated extracts, similar to the native extracts described above retained Tpo activity. Changes in megakaryocyte numbers in the spleen and bone marrow of rats were assayed with both the NAse-treated extract as well as with the native extract. A remarkable increase in megakaryocyte numbers, threefold above the normal count, was found in the spleens of rats given the native extract preparation; by contrast, however, no change was observed in splenic megakaryocyte numbers in rats given the NAse-treated extract. On the other hand, NAse-treated extract retained its ability to stimulate bone marrow megakaryocyte proliferation in the same rat. The urinary extract also revealed in vitro Meg-CSF activity with a specific activity of 31, 750 CFU-Meg colonies/mg of protein.

Anemia, Aplastic↗
PubMed1981

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.

Animals↗
PubMed1981

The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA.

Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.

Amino Acid Sequence↗
PubMed1981

Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.

A RNA molecule has been synthesized that is identical in sequence to Escherichia coli tRNAfMet except that it lacks the base modifications present in the E. coli tRNA. This was achieved by enzymatic joining of chemically synthesized oligonucleotides with chain lengths of 3-10 which were synthesized by the phosphodiester or phosphotriester method. First, quarter molecules of tRNA were constructed by joining of chemically synthesized fragments with RNA ligase. The 5'-quarter molecule (bases 1-20) served as an acceptor in joining reactions with the 3',5'-bisphosphorylated donor molecule (bases 21-34). The 5'-half molecule thus obtained was treated with phosphatase and joined to the 3'-half molecule which was prepared by ligation of the other quarter molecules (bases 35-60, acceptor; bases 61-77, donor) followed by 5'-phosphorylation with polynucleotide kinase. The synthetic tRNA was characterized by oligonucleotide pattern and was partially active in aminoacylation with E. coli methionyl-tRNA synthetase.

Base Sequence↗
PubMed1981

Thrombopoiesis- and megakaryocyte colony-stimulating factors in the urine of patients with idiopathic thrombocytopenic purpura.

The urinary extract from patients with severe, chronic idiopathic thrombocytopenic purpura (ITP) was capable of inducing significant thrombocytosis in rats in vivo and enhancing megakaryocyte colony formation in culture of mouse bone marrow cells. The apparent specific activity of megakaryocyte colony stimulating factor (MEG--CSF) in the ITP extract was approximately one-half of that of the urinary extract from patients with aplastic anaemia (AA). Daily injections of ITP extract did not cause an increase in Hb concentration, while rats receiving AA urinary extract revealed profound erythropoiesis 3 weeks later. In vitro assay of erythropoietin (EPO) failed to show significant EPO activity in the extract from patients with ITP. These findings excluded the possibility that the observed activities of thrombopoiesis-stimulating factor (TSF) and MEG--CSF in the ITP urinary extract are due to contaminating EPO. Urine of patients with severe ITP appears to be a good source of TSF and MEG--CSF.

Anemia, Aplastic↗
PubMed1981

Augmentation of specific cell-mediated immune responses to tumor cells in tumor-bearing rats pretreated wih the antileukemia drug busulfan.

Lethal growth of a syngeneic transplanted tumor (KMT-17) was inhibited in inbred WKA rats pretreated with the antileukemia drug busulfan (BU). However, the lethal growth of KMT-17 was not inhibited by pretreatment with cyclophosphamide, adriamycin, or ftorafur. With the Winn assay, spleen cells from BU-pretreated KMT-17-bearing rats (TBR) inhibited the growth of admixed KMT-17 cells more strongly than did spleen cells from BU-untreated TBR. The augmented tumor inhibitory activity of spleen cells was KMT-17-specific, and this activity was abrogated by in vitro treatment of spleen cells with anti-T serum and guinea pig complement. Augmentation of the immune response to KMT-17-associated antigen(s) in BU-pretreated TBR was also demonstrated in lymphocyte-mediated cytotoxicity, as detected by a 51Cr release assay and by a delayed-type hypersensitivity with a radioisotope footpad assay. Tumor regression in BU-pretreated rats was demonstrated to be mediated by the augmentation of T-cell-mediated immune responses to tumor-associated antigens. The tumor inhibitory effect of BU was abrogated by adoptive transfer with thymus cells from normal rats but not with those from BU-pretreated rats 1 day before tumor inoculation. The augmentation oif the antitumor immune responses by pretreatment with BU was suggested to be due to the fact that BU selectively inhibited the suppressor cells of their precursors.

Animals↗
PubMed1981

Chemical synthesis of 27-desamidosecretin gene by the polymer support method.

The polymer support method for the oligonucleotide synthesis has been developed and used for the chemical synthesis of a 27-desamidosecretin gene. The gene carrying Pst I recognition sites at both ends was built from 16 fragments and each of them was synthesized by a stepwise addition of protected nucleotides on a polymer support. The coupling yield of each step was almost 90% and after partial deblocking, oligonucleotides carrying trityl groups were purified by chromatography on a reverse phase column. The recombinant DNA containing this synthetic gene was expressed in bacterial cells.

Amino Acid Sequence↗