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Biomedical subjects

T Okayama

Publications and source records attributed to T Okayama.

At least 37 records · Page 2Linked to original sources

Evolution of genetic information flow from the viewpoint of protein sequence similarity.

As a course of inquiry into the evolution of genetic information flow, similarity relations of amino acid sequences between the proteins involved in translation, transcription and replication are investigated. The sequence data of these proteins are mostly accumulated from Escherichia coli, and the present investigation is carried out mainly on this organism by the FASTP program. This result reveals an interesting similarity linkage extending from ribosomal proteins to the proteins participating in translational elongation process and to the proteins in transcription and replication. Although the ribosomal proteins are of relatively short polypeptide chains, our systematic comparison between these proteins finds many similarity relations, being more than 100 in terms of "overlap", reducing them to about 14 elementary ribosomal proteins from which other ribosomal proteins would have diverged. Moreover, the proteins involved in translation, transcription and replication contain the regions similar to the elementary ribosomal proteins. In particular, some initiation and elongation factors in translation process are assigned to be similar to the elementary ribosomal proteins almost over the whole regions. To such an elongation factor Tu, the alpha and sigma 70 subunits of RNA polymerase and primase also show similarity in the wider regions than the individual ribosomal proteins, and they are shown to be fundamental for the similarity linkage extending to the other polypeptide chains involved in transcription and replication processes, although the latter polypeptide chains contain regions not similar to any ribosomal protein. This divergence pattern of similarity relations strongly suggests that the proteins involved in the contemporary genetic information flow DNA-->RNA-->protein have evolved from some elementary ribosomal proteins, first by gene fusion, in a primitive organism of the RNA-protein world, and then by the addition of the mechanism of domain shuffling from other genes in the DNA-RNA-protein world.

Amino Acid Sequence↗

Enzymic O-sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A-44.

The enzymic O-sulfation of Tyr residues in a recombinant hirudin variant-1 (rHV-1) and its analog in which Glu61 and Glu62 were replaced by Tyr, [E61Y, E62Y]rHV-1, was carried out by use of sulfotransferase isolated from an anaerobic bacterium from the human intestine, Eubacterium A-44. Although rHV-1 was not sulfated by this enzyme, the sulfation of [E61Y, E62Y]rHV-1 was observed, and three kinds of sulfated analog, whose C-terminal six amino acid residues were -PYY(SO3H)YLQ, -PYYY(SO3H)LQ, and -PYY(SO3H)Y(SO3H)LQ, were obtained. Among the sulfated hirudin analogs tested here, the Tyr62 and Tyr63 bisulfated [E61Y, E62Y]rHV-1 showed the strongest thrombin inhibition with the inhibition constant (Ki) of 0.0430 pM, followed by the Tyr63 monosulfated analog (Ki = 0.0593 pM) and the Tyr62 monosulfated one (Ki = 0.158 pM). The Tyr63 monosulfated analog and Tyr62 and Tyr63 bisulfated one were more potent inhibitors of thrombin than unsulfated rHV-1. The increase in affinity caused by sulfation was predominantly due to an increase in the association-rate constant.

Amino Acid Sequence↗

Synthetic dipeptide, N-stearoyl-D-Ser-L-Pro-OEt, induces release of tissue-type plasminogen activator in cultured cells and in experimental animals.

The tissue-type plasminogen activator (t-PA)-releasing action of synthetic dipeptides containing Gly, Ser or Pro was investigated. Among 10 dipeptides, Boc-L-Ser-L-Pro-OH and H-L-Ser-L-Pro-OH induced t-PA release in vitro, but the others were inactive. Since Boc-L-Ser-L-Pro-OH was more effective than H-L-Ser-L-Pro-OH, 7 related dipeptides with N-acylation were synthesized. Five of them enhanced the release of t-PA; N-stearoyl-L-Ser-L-Pro-OH (FK-5) had the greatest effect. Four compounds were further examined for activity to enhance the release of t-PA in rats. FK-5 produced a two-fold increase in fibrinolytic activity, and N-palmitoyl-L-Ser-L-Pro-OH (FK-4) also markedly enhanced the release of t-PA. Since FK-5 caused severe hemolysis, 7 analogues of FK-5 were synthesized. All of them enhanced the release of t-PA from melanoma (Bowes) cells. In rats, FK-5, N-stearoyl-D-Ser-L-Pro-OH (FK-8) and N-stearoyl-D-Ser-L-Pro-OEt (FK-10) enhanced the fibrinolytic activity two-fold. FK-5 and FK-8 also exhibited strong hemolytic activity, but FK-10 did not induce hemolysis. Therefore, FK-10 was examined in rabbits. After the injection of this compound, the fibrinolytic activity in the euglobulin fraction was markedly enhanced without accompanying hemolysis. Thus, FK-10 potently enhances fibrinolytic activity both in vitro and in vivo.

Amino Acid Sequence↗

Vertebrate collagenase inhibitor. II. Tetrapeptidyl hydroxamic acids.

To develop a potent and specific collagenase inhibitor, a series of tetrapeptidyl hydroxamic acids were synthesized, based on the previous findings with tripeptidyl derivatives (Chem. Pharm. Bull., 38, 1007-1011, 1990). Among the series of tetrapeptidyl derivatives synthesized, R-Gly-Pro-Leu-Ala-NHOH and R-Gly-Pro-D-Leu-D-Ala-NHOH were found to be highly specific and potent inhibitors against vertebrate collagenase with an IC50 of 10(-6) M order, where R stands for Boc or acyl group. Analysis of their structure-activity relationships showed a characteristic feature of the substrate-binding site of collagenase as follows: 1) the S1 subsite forms a shallow hydrophobic pocket, although glycine residue corresponds to the subsite of the natural collagen substrate: 2) the S2 subsite constitutes a bulky pocket with less requirement for hydrophobicity: 3) the S3 subsite preferentially accommodates Pro residue: and 4) the accommodation of the P4-P1 subsites of peptidyl collagenase inhibitor to the S4-S1 subsites is required to form a tight binding of its hydroxamic acid moiety to the zinc ion at the catalytic site of the enzyme. The introduction of an enantiometric dipeptide unit, D-Leu-D-Ala, to the P2-P1 subsites demonstrated an increased binding capacity to the extended S4-S1 subsites of collagenase, thus providing proteinase-resistant inhibitor.

Amino Acid Sequence↗

Transmission of HTLV-I to rabbits via semen and breast milk from seropositive healthy persons.

Four rabbits inoculated intravenously with milk cells from 4 post-partum women seropositive for human T-cell leukemia virus type I (HTLV-I) and one rabbit inoculated with semen cells from a seropositive healthy man seroconverted for HTLV-I after 3-5 weeks but no seroconversion occurred in 2 rabbits inoculated with milk cells from a seronegative mother or with heated (56 degrees C, 30 min) milk cells from a seropositive mother. Attempts were made to isolate HTLV-I from peripheral blood lymphocytes harvested 5-15 weeks after cell inoculation and cultured in the presence of interleukin-2. An HTLV-I-carrying lymphoid cell line of rabbit origin was established from a rabbit inoculated with milk cells. Another long-term culture, derived from a rabbit inoculated with semen cells, also expressed HTLV-I antigens and harbored virus particles. Furthermore, transfusion of 20 ml of blood from all 5 seroconverted rabbits, but not from the 2 seronegative ones, caused seroconversion in normal recipient rabbits after 4-6 weeks.

Animals↗

Vertebrate collagenase inhibitor. I. Tripeptidyl hydroxamic acids.

A series of tripeptidyl analogues carrying hydroxamic acid residue at the C-terminus of the molecule were synthesized, and their inhibitory activities against vertebrate collagenase and other metalloenzymes including bacterial collagenase were examined. Both Z-Pro-Leu-Ala-NHOH and Z-Pro-D-Leu-D-Ala-NHOH showed highly specific and potent inhibitory activity against tadpole and human skin collagenases with an IC50 of 10(-6) M order.

Amino Acid Sequence↗

A light and electron microscopic study of calcitonin gene-related peptide in the rat caudate putamen.

Calcitonin gene-related peptide (CGRP)-like immunoreactive (CGRP-IR) structures have been studied in the rat caudate putamen using avidin-biotin peroxidase immunohistochemistry. Immunoreactivity was found in the axons of this nucleus but not in the perikarya. CGRP-IR fibers were most densely concentrated along the ventral border and in the caudal portion of the rat caudate putamen. CGRP-IR fibers were sparsely distributed throughout the rest of this nucleus. Almost all immunoreactive boutons which contained small clear vesicles had formed asymmetrical synapses. Postsynaptic targets included dendritic spines and shafts. Asymmetrical synapses in the caudate putamen are supposed to be extrinsic in origin. These observations, together with the results of other investigations, suggest that CGRP-IR boutons form synapses with spiny striatal neurons, which, most likely, are medium-sized spiny projecting neurons. Moreover, evidence indicates that these boutons are of extrinsic origin.

Animals↗

Neuropeptide Y-immunoreactive neurons receive synaptic inputs from dopaminergic axon terminals in the rat neostriatum.

Double immunocytochemistry using peroxidase-antiperoxidase and protein A-gold was performed to determine whether neuropeptide Y (NPY) immunoreactive neurons receive synaptic inputs from catecholaminergic axon terminals in the rat neostriatum. Tyrosine hydroxylase-immunoreactive axons were found to be in synaptic contact with the somas and proximal dendrites of NPY-immunoreactive neostriatal neurons. These latter neurons were medium-sized and had indented nuclei, and thus were thought to be medium aspiny interneurons. Thus nigrostriatal dopaminergic neurons may monosynaptically influence striatal NPY neurons.

Animals↗